Articles

Conservation of coconut (Cocos nucifera L.) biodiversity in Sri Lanka

Method for identifying the genetic polymorphism of esterase enzymes in ecotypes of Pennisetum glaucum

Common names of the sweetpotato (lpomaea batatas) in the Americas

Insect pollination and isolation requirements in tomato collections (Lycopersicon esculentum Mill.)

Use of computer databases to manage an in vitro collection of potato germplasm

Genetic resources of Podophyllum hexandrum Royle, an endangered medicinal species from Garhwal Himalaya, India

Genetic contamination by windborne pollen in germplasm-regeneration plots of smooth bromegrass

Genetic resources of patented seeds available to the public worldwide

Articles

Conservation of coconut (Cocos nucifera L.) biodiversity in Sri Lanka

L. Perera, R.R.A. Peries and W.M.U. Fernando

Coconut Research institute, Lunuwila, Sri Lanka

Summary

The progress in research toward the conservation of coconut biodiversity in Sri Lanka and the utilization of germplasm for the development of improved cultivars is reviewed. Both random and biased collections were made but preference was given to collecting populations that exhibit characteristics of economic importance. A total of 39 accessions has been collected to date and conserved in duplicate in ex situ genebanks. Consideration of long-term physiological adaptation of the palms led to development of in situ genebanks to produce planting material suitable for low-input sustainable agriculture systems. Procedures to quantify the between- and within-population variability among coconut populations using quantitative variation and molecular methods are also being investigated.

Introduction

Coconut is the most widely grown plantation crop in Sri Lanka, occupying about 416 000 ha of the total area of about 6.5 million ha. It is important in the daily diet of the average Sri Lanka, and has an estimated per capita consumption of 110 nuts/year. Around 80% of the estimated total production is consumed domestically. About 135 000 persons are directly or indirectly employed in the coconut sector which includes processing, trade and other related activities (CDA 1993).

The available coconut germplasm in Sri Lanka is categorized into three distinct varieties: typica, nana and aurantiaca (Liyanage 1958). Characteristics of different forms of these three varieties are shown in Figs. 1 and 2. The typica or tall type has been the most widely exploited. It is generally cross-pollinating, does not have a reliable method of vegetative propagation and has a long generation interval.

Systematic collecting and conservation of coconut germplasm were initiated in Sri Lanka around 1984 (Wickramaratne 1984). Previously there had been only a traditional varietal collection that was planted in 1940 and 1960. This collection was comprised of eight forms of the typica variety, three forms of the nana variety and two forms of the aurantiaca variety (Table 1) and the pool of elite palms at the Isolated Coconut Seed Garden (ISG) (Liyanage 1961ab).

Systematic genetic conservation was needed because of genetic erosion in coconut, which occurs at the rate of about 1% per annum of the area under coconut cultivation (Peries 1993). Exotic germplasm for breeding is not available because of the risk of introducing lethal diseases. The main reason for this high rate of genetic erosion is rapid industrialization and urbanization in the traditional coconut growing areas. Further, natural disasters such as cyclones and droughts have caused severe losses in valuable coconut genetic material amounting to about two million

coconut palms in the east coast area of Sri Lanka in 1978 and nearly 200 000 palms in the southern part of the country during 1988-89. The introduction of improved coconut varieties (Liyanage 1956, 1957) into the national replanting programme of the country has led to the gradual replacement of existing biodiversity over time.

- Fig. 1. Shapes of different forms of coconuts found in Sri Lanka: Forms of typica variety: A1-typica, A2-Kamandala, A3-bodiri, A4-navasi, A5-ran thembili, A6-gon thembili, A7-pora pol, A8-dikiri pol; forms of nana variety: B1-dwarf green, B2-dwarf yellow, B3-dwarf red; forms of aurantiaca variety; C1-thembili (King coconut), C2-navasi thembili (Liyanage 1958).

- Fig. 2. Longitudinal sections of nuts of coconut forms illustrated in Fig. 1. The legend is the same as that for Fig. 1 (Liyanage 1 958).

Table 1. Varieties and forms of coconut in Sri Lanka (according to Liyanage 1958)

Variety	Breeding habit	Forms
Typica (tall)	Out-breeding	Typica
		Bodiri
		Ran thembili
		Gon thembili
		Nawasi
		Pora pot
		Kamandala
		Dikiri pol
Nana (dwarf) (kundira)	In-breeding	Pumilla (dwarf green)
		Eburnea (dwarf yellow)
		Regia (dwarf red)
Aurantiaca	In-breeding	Thembili (king coconut)
		Nawasi thembili

Collecting: physiological adaptation and preferential selection

Although large populations of cross-pollinating species are likely to contain a large proportion of the total genetic variation, particularly for quantitative characters, it is unlikely that any single population will contain all of the genetic variation. The total genetic variation of a species is therefore likely to be fractionated over populations as the impact and direction of natural selection varies from one to another, due to environmental variation and genetic drift (Lawrence and Rajanaidu 1985).

It is reported that coconut existed in Sri Lanka as far back as 101-77 BC. However, growing coconut on a plantation scale commenced only after foreign invasion of the country during 1855. There is a great deal of circumstantial evidence to suggest that the coconut palm adapts itself to pests, disease and climatic factors associated with a particular location when grown over a period of time (Foale 1991). Although Sri Lanka is a small island, there is a great deal of climatic variation within the country which would generate a wide range of variation among the coconut populations, particularly the survival capabilities, depending on the diversity of the agro-ecology.

Evidence suggests that the coconut plantations in Sri Lanka, especially the large ones, have undergone preferential selection for various characters such as nut yield, shape, colour and size. In some instances, even the weight of husked nut and thickness of the kernel have been considered in the selection of mother palms over several generations. Therefore it can be reasonably assumed that populations in different environments and under different management are physiologically adapted to their respective agro-ecologies and thus have the capacity to represent diverse gene pools.

Under the circumstances, a part of the collecting programme was directed at representative samples from phenotypically different coconut populations. Historical information available in estates was reviewed and specific populations were identified for initial collecting rather than collecting at random from the entire island.

Exotic material

Prior to implementation of quarantine regulations to control the accidental introduction of coconut diseases, some coconut growers imported certain forms of coconut (Wickramaratne 1987; Peries 1991). Collecting activities are being focused on locating and purifying exotic material that has been built up through introduction and has become naturalized under different agro-ecological conditions in Sri Lanka.

Drought tolerance

Because of adverse weather and the resulting fluctuations in coconut yield, drought tolerance became a priority in coconut breeding in the mid-1980s. During 1988-89, the country, especially the southern part, experienced a prolonged drought that resulted in a loss of about two million coconut palms. In these areas, as well as in other areas such as Puttalam and Kurunegala situated in the periphery of the main coconut triangle where frequent droughts are common, certain populations and even individual palms maintained their productivity despite the severe water deficit. Such naturally adapted palms and/or populations were identified and representative samples collected as part of the germplasm conservation activity.

Other distinguished palms populations

The collecting programme also focused on individual palms and populations with phenotypes uncommon to the known diversity. The brown dwarf of the nana variety (Peries 1991) was one such interesting collection and spicata types (Wickramaratne 1987) were also included in this category.

Conservation: ex situ genebanks

Collected material has been conserved in ex situ genebanks representing two different environments (Table 2). To date these ex situ collections contain a total of 39 accessions comprising 13 traditionally classified varieties and forms, 21 tall accessions of the variety typica and four dwarf accessions including two exotic dwarf types.

Table 2. Current (after 1984) status of germplasm collection in Sri Lanka

Type	Accession name	Size of population/location_		Remarks_
		B/E	PRS
Tall
	Moorock	84	82	ET
	Palugaswewa	60	85	ET
	Pitiyakanda	86	85	ET
	Clovis	85	85	NE
	Namalwatta	79	95	LEI/DT
	St. Annes	-	85	ET
	Margaret	-	85	NE
	Kasagala	-	85	LEI/DT
	Debarayaya	-	87	LEI/DT
	Akurassa	84	89	NE
	Ambakelle special	78	91	IV
	Melsiripura	-	91	ET
	Mangala Eliya	-	86	ET
	Goyambokka	-	90	LEI/DT
	Keenakelle	-	86	ET
	Maliboda	-	87	ET
	Horakelle	-	83	ET
	Walahapitiya	-	85	ET
	Wellawa	84	79	DT
	Ambakelle tall	86	-	IV
	Goluwapokuna	-	90
	West African tall	3	-	Exotic
Dwarf
	Kundasale	-	88	ET
	Braune (dwarf brown)	-	89
	Cameroon red dwarf	6	86	Exotic
	Brazilian green dwarf	5	20	Exotic

^_B/E=Bandirippuwa estate in wet-intermediate zone, PRS=Poththukulama Research Station in dry-intermediate zone.

^_ET=Ecotype, LEI=Low external-input system, DT=Putative drought tolerance, IV=lmproved variety, NE=Exotic, now naturalized.

In situ genebanks

In situ collecting was initiated according to the ecobank concept proposed by Peries (1993). The rationale of the in situ genebank is that the populations in a particular agro-ecology, subjected to mass selection over several generations by ordinary farmers and/or through natural selection, are distinguishably different from each other in survival capabilities associated with physiological adaptation, although they may be morphologically similar. Therefore the different populations that exist within the country contain beneficial genes that give the palms the much needed survival capability under a given farming system. Evidence suggests that 'improved' material introduced into these agro-ecologies may not be able to adapt rapidly enough to thrive under the same suboptimal agro-ecological systems.

Under this genebank programme, populations which are putatively adapted physiologically to have a high-yield potential are identified and conserved in situ after very poor yielders are eliminated from the population (Zobel and Talbert 1984). It is proposed that areas having similar agro-ecological conditions be replanted with seedlings from these in situ genebanks.

Evaluation

Without a detailed understanding of the genetic structure of the species and its populations, it is impossible to adopt a sampling strategy that captures a desired level of genetic variation. Because the genetic structure of coconut has not been studied adequately, the present collecting strategy has been drawn up to collect one nut from 50 to 100 palms per population for collection purposes (Carpio 1990) regardless of the fraction of total genetic variation among populations and among families within population components. Therefore, as a first step, a systematic trial was designed to exploit the between-population variation and the between- and within-family variation to develop suitable collecting strategies for our conservation programme in addition to evaluation and characterization methods.

The systematic germplasm evaluation trial was begun in 1994 using nine selected germplasm accessions (Moorock, Maliboda, Clovis, St. Annes, Kasagala, Debarayaya, Margaret, Ambakelle special and Ambakelle tall) that were already in ex situ genebanks (Perera and Peries 1993). The seedlings were planted in a completely randomized design, consisting of 15 palms per accession and five progenies per palm, representing half-sib families from each palm (15 x 5 = 75).

Because the interaction between genotype and environment on the important quantitative characters of coconut appears to be reasonably high, the genetic variation could be masked by environmental factors, making evaluation of genotypes based on phenotype inconclusive. As a result, methods of evaluating material at the molecular level are regarded to be more accurate. Restriction fragment length polymorphism (RFLP) and isozyme analysis of coconut have been given priority in the Sri Lankan research programmes.

Protocols to extract leaf isozymes, which were unsuccessful in earlier studies (Carlos 1980; Meunier 1992) because of polyphenol interferences, have been established. Recent studies have shown that the level of polymorphism shown by leaf isozymes is high compared to levels in haustorium tissues. A high level of polymorphism was detected at the esterase loci among the individuals of improved tall and tall x tall (CRIC 60), compared to the ordinary tall known as plus palm [selected mother palms), suggesting that differentiation among populations based on allelic composition may be possible (Fernando 1995).

Utilization

The analysis of the vegetative data of the progeny of selected crosses at ISG has shown that genetic gain due to crossing is very low, i.e. H²_b was 0.15 (Fernando 1995). Thus, response to further selection may be limited in the highly selected pool at ISG. Hence, it was important to test the potential combining ability of seed palms at ISG with different pollen sources in order to broaden the genetic base and to test the drought tolerance and the yield potential of a cross. Four germplasm accessions, Moorock, Debarayaya, Kasagala and St. Annes, which were shown to be distinctive by multivariate discriminant analysis of the accession data, have been crossed to a selected pool of proven high and stable yielders at the ISG; at Ambakelle (Fernando and Perera 19C)3). The seedlings are now under nursery evaluation and are expected to be planted in late 1995 in multilocation trials, especially in areas where droughts are frequent.

Future work

Collecting and conserving more populations from areas which are still unexplored within the country has been proposed. Evaluation and characterization will be done in the field as well as in the laboratory using molecular methods (isozymes and RFLPs). Physiologically adapted material will be identified and conserved in situ. Accessions will be tagged for important characters such as nut yield, weight of husked nut, adaptability and stability so that they can be used in breeding programmes to develop varieties with high yields and stability for different localities in the country.

References

Carlos, C.B. 1980. Biochemical status of Cocos nucifera L., MSc thesis University of Birmingham, UK.

Carpio, C.B. 1990. Collecting strategies. Training manual of IBPGR short-term training course on collection, conservation and characterization of coconut genetic resources, Philippines. 25 November - 9 December 1991.

CDA.1993. Sri Lanka coconut statistics. Coconut Development Authority

Foale, M.A. 1991. Coconut Genetic Diversity: Present knowledge and future research needs. Proceedings of the IBPGR workshop on coconut genetic resources, Cipanas, Indonesia. 8-11 October 1991. p 4653.

Fernando, W.M.U. 1992. Report of the Genetics and Plant Breeding Division. In Report for 1992, Coconut Research institute, Sri Lanka.

Fernando, W.M.U. 1995. Unpublished data.

Fernando, W.M.U. and L. Perera. 1993. Report of the Genetics and Plant Breeding Division. In Report for 1993, Coconut Research institute, Sri Lanka.

Lawrence, M.J. and N. Rajanaidu. 1985. The genetic structure of natural populations and sampling strategy. Proceedings of the international workshop on oil palm germplasm and utilization, Selangor, Malaysia. 26-27 March, 1985. pp. 15-26.

Liyanage, D.V. 1956. Intra specific hybrids in coconuts. Bulletin No. 7. Coconut Research institute, Ceylon.

Liyanage, D.V. 1957. Annual Report of Coconut Research institute of Sri Lanka for 1957.

Liyanage, D.V. 1958. Varieties and forms of the coconut palm grown in Ceylon. Ceylon Coconut Q. 9:1-10.

Liyanage, D.V. 1961a. The use of isolated seed gardens for coconut seed production. Ceylon Coconut Q. 12:245-252.

Liyanage, D.V. 1961b. Annual Report of Coconut Research institute of Sri Lanka for 1961.

Meunier, J. 1962. Genetic diversity in coconut isozyme electrophoresis. In Proc. of the IBPGR workshop on coconut genetic resources Cipanas, Indonesia 8-11 October 1992. International Crop Network series & IBPGR, Rome 1992.

Perera, L. and R.R.S. Peries. 1993. Report of the Genetics and Plant Breeding Division. In Report for 1993, Coconut Research institute, Sri Lanka.

Peries, R.R.A. 1991. Report of the Genetics and Plant Breeding Division. In Report for 1991, Coconut Research institute, Sri Lanka.

Peries, R.R.A. 1993. Improving the efficiency of the low input coconut agro-ecology, through conservation and utilization of biodiversity. Proceedings of the 10th IFOAM conference, LincoIn University, New Zealand 1]-16 December, 1994. p. 122.

Wickramaratne, M.R.T. 1984. Report of the Genetics and Plant Breeding Division. In Report for 1984, Coconut Research institute, Sri Lanka.

Wickramaratne, M.R.T. 1987. Report of the Genetics and Plant Breeding Division. In Report for 1987, Coconut Research institute, Sri Lanka.

Zobel, B. and J. Talbert. 1984. Applied Forest Tree Improvement. John Wiley and Sons, inc., USA.

Résumé

Conservation de la biodiversité de la noix de coco (Cocos nucifera L.) à Sri Lanka

Le présent article examine les progrès de la recherche concernant la conservation de la biodiversité de la noix de coco a Sri Lanka et l'utilisation du matériel génétique pour la mise au point de cultivars améliores. Les récoltes ont été faites a la fois au hasard et systématiquement, mais la préférence a été donnée a la récolte de populations présentant des caractéristiques d'importance économique. Au total, 39 accessions ont été récoltées jusqu'ici et des doubles vent conserves dans des barques de gènes ex situ. L'examen de l'adaptation physiologique a long terme des cocotiers a conduit a la création de barques de gènes in situ en vue de produire du matériel végétal adapte aux systèmes d'agriculture durable à faible apport d'intrants. Sont également étudiées des procédures pour quantifier la variabilité entre les populations et a l'intérieur d'une population de noix de coco a l'aide de méthodes de variation quantitative et de techniques moléculaires.

Resumen

Conservación de la biodiversidad del coco (Cocos nucifera L.) en Sri Lanka

Se pasa revista en este trabajo a los avances realizados en la investigación pare la conservación de la biodiversidad del coco en Sri Lanka y el empleo de germoplasma pare el desarrollo de cultivares mejorados. Se han hecho colecciones tanto aleatorias como sesgadas aunque se dio preferencia a la recogida de poblaciones que mostraran características de importancia económica. Se han recogido en total 39 ejemplares hasta la fecha, que se han conservado por duplicado en bancos de genes ex situ. El estudio de la adaptación fisiológica a largo plazo de las palmas dio lugar al desarrollo de bancos de genes in situ pare producir material de siembra adecuado a los sistemas de un agricultura sostenible a base de bajos insumos. También se están investigando los procedimientos pare cuantificar la variabilidad entre poblaciones y dentro de ellas en el sector del coco, empleando pare ello métodos de variación cuantitativa y moleculares.

Method for identifying the genetic polymorphism of esterase enzymes in ecotypes of Pennisetum glaucum

Didier Balma¹, Claude. André St-Pierre², Jean Collins and Jean Guy Parent³

¹ institut d'Etudes et de Recherches Agricoles, 01 BP 476 01, Ouagadougou, Burkina

Faso

² Département de Phytologie, Faculté des Sciences de l'Agriculture et de l'Alimentation, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4

³ Service de Phytotechnie de Québec, Complexe Scientifique 2700, rue Einsten, Sainte-Foy, Quebéc, Canada G1P 3W8

Summary

Sixty landraces of pearl millet (Pennisetum glaucum (L.)) representing genetic variability of eight countries of West Africa, including Cameroon, were used to compare the amplitude of genetic polymorphism. Original seed samples and fresh leaf samples harvested from the greenhouse were screened for _- and _-esterase by polyacrylamide gel electrophoresis. For all 60 accessions, seed samples exhibited more banding patterns than leaf material. The number of isozyme bands ranged from a minimum of 17 to a maximum of 24. We observed four zones as 0 to 2, 2 to 5, 8 to 12 and 1 to 4 isozyme profiles named EST 1, EST 2, EST 3 and EST 4, respectively. Principal component analysis and similarity index studies revealed a total absence of esterase enzymatic variability or small differences among landraces, mostly those collected in eastern and south-central Burkina Faso. The similarity index average ranged from 90 to 100%. Evidence is presented to support the hypothesis that the semi- and late-maturing genotypes collected in areas with greater amounts of rainfall could arise from the same complex of cultivated populations. These populations acquired regional adaptation during their evolution after several modifications imposed by the limitation of the geneflow which could have occurred only among earlier genotypes. However, large esterase enzymatic variability was found among earlier landraces collected from northern and central Burkina Faso, including those of the other countries of West Africa. These results, on one hand, showed the possible different sources of enzymes from the wild progeny through the West Africa region and, on the other hand, confirmed the possibility of different origins of the species' domestication. However, the technique has potential for practical application; subsequent studies should refine the procedure and discover other pearl millet enzyme systems based on corresponding allele number that might reveal desirable agronomic characters, useful parent genotypes for breeding programmes, or samples to be conserved in genebanks.

Introduction

Interest in collecting the genetic diversity available in cultivated pearl millet, its wild relatives and wild species began 20 years ago. Several institutes started to evaluate the genetic diversity of these collections. These evaluations sometimes aimed at examining the biological structure of the species, the information on elements of their evolution and their domestication. Now, the process will have to evolve toward the use of the genetic resources by plant breeders and rationalization of the management of the genebanks to minimize the conservation of redundant samples, which is sometimes useless and costly.

Genetic polymorphism is based on a certain number of genes coding for 8-12 enzymes, including the _-esterase (EST) which has already been obtained in pearl millet. Bono (1973) showed the similarity between malian and burkinabe pearl millets, and Tostain et al. (1987) showed the great diversity between Nigerian pearl millets from eight enzymes studied seed to seed and separated on starch gel and whose _-esterases were separated on polyacrylamide. According to Sarr et al. (1988), the relative efficiency of each pollen source is determined by gene-specific alleles coding for isozymes as well as for esterase and alcohol dehydrogenase.

Esterase activity can be measured easily from extracts of plants. The esterase zymogrammes contain a characteristic pattern of a large number of strips (sometimes more than ten) controlled by several loci; these esterase isozymes that are distinct molecular models of only one enzyme acting on the _-naphthylacetate and the _-naphthylacetate are esterase carboxyls (EC 3.1.1.1) (Cubadda and Quattrucci 1974). They have been used often to study the intra- and inter-specific polymorphism (Brown and Weir 1983). One of the advantages of using isozymes to study polymorphism is that a range of enzyme loci on one individual can be studied easily using a small quantity of material with minimum preparation and cost (Kidambi et al. 1990).

Our objective was to use electrophoresis to determine, rapidly and inexpensively, the existence of genetic polymorphism in esterase enzymes of 60 ecotypes of Pennisetum glaucum species collected in eight countries of West Africa, including Cameron

Materials and methods

Sixty ecotypes selected at random from the 110 ecotypes used for the evaluation of agromorphological characters were used (Table 1). The seeds of pearl millet collected in West Africa (Fig. 1) derive directly from the genebank in Ottawa, Canada. Ground seeds and leaves of young plants cultivated in pots in greenhouses were used.

Table 1. List of ecotypes studied

No.	CIN1	Origin2	No.	CIN1	Origin2	No.	CIN1	Origin2
1	50	Nigeria	21	2851	CBF	41	3209	NBF
2	87	Nigeria	22	2861	CBF	42	3669	EBF
3	724	Cameroon	23	2864	CBF	43	3671	EBF
4	733	Cameroon	24	3141	CBF	44	3672	EBF
5	1968	Mali	25	3144	NBF	45	3674	EBF
6	1114	Mali	26	3145	NBF	46	3676	EBF
7	1273	Senegal	27	3152	NBF	47	3679	EBF
8	1309	Senegal	28	3156	NBF	48	36B1	EBF
9	1501	Niger	29	3157	NBF	49	3683	EBF
10	1510	Niger	30	316B	NBF	50	3687	EBF
11	2001	Niger	31	3176	NBF	51	3690	EBF
12	2034	Benin	32	3180	NBF	52	3693	EBF
13	2812	CBF	33	3183	NBF	53	3699	EBF
14	2816	CBF	34	3187	NBF	54	3702	SCBF
15	2821	CBF	35	3193	NBF	55	3705	SCBF
16	2830	CBF	36	3201	NBF	56	3708	SCBF
17	2837	CBF	37	3202	NBF	57	3710	SCBF
18	2841	CBF	38	3204	NBF	58	3716	SCBF
19	2844	CBF	39	3206	NBF	59	3751	SCBF
20	2846	CBF	40	3207	NBF	60	3769	SCBF

¹ CIN=Collector identification number assigned to samples stored in genebank at Ottawa.

² CBF=central Burkina Faso; NBF=northern Burkina Faso; EBF=eastern Burkina Faso; SCBF=south-central Burkina Faso

- Fig. 1. Collecting area of pearl millet in countries of West Africa

In a mortar on ice, 0.35 g of seeds (about 45 seeds on average) was ground with 1.2 ml of extraction stopper [Tris(hydroxymethyl)aminomethane (0.1M, pH 7.5)] containing 1 mM sodium ethylenediaminetetraacetate (EDTA), 10 mM magnesium chloride, 10 mM potassium chloride, 100 mg/ml polyvinyl pyrrolidone of 40 000 average molar mass (Cousineau and Donnelly 1989) and 0.1 % 2-mercaptoethanol (Gottlieb 1981). The paste was made homogeneous and then decanted into a 1.5-ml conical tube and centrifuged for at least 15 min at 13 000g. The liquid floating on the surface was recovered and transferred into another conical tube which was stored at 70°C until used. The purpose of making the paste homogeneous was to liberate soluble proteins and preserve them in solution.

Green leaves (0.30 g) newly collected from ten plants 24 weeks old were introduced into a conical tube on ice. Silica carbide (carborundum, grains 320) was added to facilitate grinding. The tissues were ground with a glass rod that just fit into the conical tube. The centrifugation, recovery and conservation of liquid were similar to those for the seeds.

Thirty minutes before prefocalization, the tubes containing the liquid were placed on a mixture of water and ice (0°C) to thaw. We have since used a Fisherbiotech apparatus, FB 1001, to thaw the extracts. The apparatus was pre-cooled to 10°C; electricity was controlled by a 2197 LKB Bromma regulator.

A LKB polyacrylamide gel with dimensions of 245 x 110 x 1 mm was cut to 205 x 110 x 1 mm and placed on a rest previously moistened with kerosene to favour thermic conductivity. Electrolysis bands LKB 280 x 6 x 1 mm carefully cut to the gel dimension were dipped in anodic (0.5M acetic acid) or cathodic (0.5M NaOH) solutions. After being lightly sponged, they were put in contact with the electrodes on the gel. The apparatus was set to function for 15 min of prefocalization at a contact current of 20 out 200 V.

On a prefocalized gel, a silicon applier pierced with holes every 2 mm was placed 1 cm from the anode and the samples (2.5 ml) were put into the holes. The migration was conducted at a constant power of 11 W allowing free fluctuation of the electric tension. After 30 min. the silicon applier was removed and the excess liquid carefully sponged away. Then the electrophoresis continued for 2 hours.

After electrophoresis the gel was put into a specific colouring, consisting of 50 ml phosphate solution (0.2 M, pH 6.4), 50 mg _-naphthylacetate and 50 mg _-naphthylacetate in 5 ml acetone, 100 mg of Flast Blue RR salt (hemisalt of zinc chloride benzoylamino-4-dimethoxy-2.5-benzendiazonium), according to the method of Cheliak and Pitel (1985). This solution was incubated with shaking in darkness at 37°h for 2 h.

The gel was transferred into decolouration solution (26% methanol and 7% acetic acid) taking care to keep it in the dark. For drying, the gel was rinsed with distilled water and dipped into 10% glycerol for 30-45 minutes. It was then covered with a plastic sheet previously dipped into the glycerol solution to prevent air pockets, and left in darkness at room temperature, protected from dust, for at least 3 days.

Each band of isozyme activity was identified with a capital letter going in alphabetical order following the isoelectric point from the lowest to the highest. The numeric values 0, 1, 2 and 3 correspond respectively to the absence or the presence, on a semiquantitative scale, of the visually observed band intensity: 0 = absent, 1 = slightly coloured, 2 = fairly coloured, 3 = very coloured.

The similarity between various profiles was expressed by the similarity index. The percent similarity was calculated for the different ecotypes following the procedure of Whitney et al. (1968):

Percent similarity = [(Number of pairs of similar bands) / (Total number of bands in both ecotypes)] x 100

Results and discussion

The difference between the enzymatic profiles observed in the ecotypes concerns the presence or the absence, the intensity of the bands and their isoelectric position (Figs. 2 and 3). The symbol used by Kahler and Allard (1970) permits identification of the esterase band according to its position on the zymogramme. It was possible to identify in the 60 populations a total of 24 bands that are very distinct at pH 4 0 6 4 These bands of esterase isozymes are named in alphabetical order A to X. The total number of isozyme bands varies between 17 and 24. Cubadda and Quattrucci (1974) enumerated 17 in barley. Isozymes A, B. M and X are common to the 60 ecotypes (Fig. 4). With the allogame species any population can often be representative of the whole species because of a more frequent allele for a given gene as Gottlieb (1981) indicates for the subspecies Staphnomeria exigua and as Kidambi et al. (1990) notes for the species Onobrychis.

- Fig. 2. Isozyme banding patterns obtained with ground seeds from a single ecotype

Fig. 3. Isozyme banding patterns obtained with individual seed from ecotype 3141

The 24 bands are distributed in four zones named EST 1, EST 2, EST 3 and EST 4. There are 2 isozymes in EST 1, 26 in EST 2, 8-12 in EST 3 and 1-4 isozymes in EST 4. As to the intensity of the bands or the designation of the different esterase zones, we are reluctant to comment because of the lack of measurement of the quantities or the enzymatic activities.

In the zone of EST 1, there are two bands for all ecotypes. Band B is more voluminous and more intensely coloured than band A in almost all of the ecotypes.

The EST 2 zone includes isozyme bands of different relative intensities and the number varies between 2 (ecotype 2851) and 5. The isozymes E, F. G and H are present in all ecotypes except in ecotype 2851. They are particularly coloured in ecotype 2864. Bands C and D are very intense for ecotypes 2830, 3687, 724, 733, 3702 and 3710.

The zone of EST 3 includes the most active part whose number of bands varies between eight (2851) and 12. For all ecotypes except ecotypes 1273, 2861, 1501 and 3699, the most intense band is L. The number of bands varies mainly in foreign ecotypes and in those from northern Burkina Faso.

Though less complex than the EST 3 zone, the EST 4 zone possesses some very intensely coloured bands and a great variability. The number of bands varies between one and four. The isozymes T. V and W respectively are present at only 13%, 4% and 67%. The band T. present in most ecotypes, is a very intense pink colour, except in 724, 733, 1114, 2837, 2844, 3152, 3176, 3202, 3207, 3209, 3683, 3690, 3699, 3710 and 3769 where it is also smaller. Trigui (1984), Tostain and Riandey (1984) and Trigui et al. (1986) showed that for esterases separated on starch gel, some bands are differently coloured in grey by anaphthylacetate or in pink by B-naphthylacetate. They reported that five bands of esterase are observed in leaves with a- or p-naphthylacetate as a substratum and that of the pluri-allelic loci, the most outstanding would be the locus EST E possessing 7 alleles.

- Figure. 1, Programmes showing the banding patterns of 60 pearl millet ecotypes for esterase. Isozymes 2, 3-5, 8-12 and 1-4 are in EST 1, EST 2, EST 3 and EST 4 respectively.

Figure 4 shows the main characteristics of the electrophoresis profiles. The variability expressed in the EST 4 zone is by far the most important, followed by zone EST 3.

We previously showed the discrimination produced by the enzymatic esterase system among the 60 ecotypes. In general, the esterase system showed less polymorphism within the 60 ecotypes when we used several seeds or several leaves at the same time. The observation of three ecotypes with a particular colour and band position showed a very high intra-ecotype variability, that is ecotypes 50, 2830 and 3141 (Fig. 3).

Figure 5 gives the results of the principal component analysis (PCA) of the isozyme banding patterns. The four main axes that we have not reported here account for 26, 20, 16 and 15% respectively, giving a cumulated total of 77% of variance, demonstrating the association among several alleles (Tostain et al. 1987) and the presence of strongly associated groups. This analysis is based mainly on the presence or the absence of bands. As such it is phenotypical and its interpretation will not take into account genetic control.

- Fig. 5. Graphic representation of the principal component analysis according to the number of isozyme banding patterns.

The PCA graph shows that there are three ecotype groups which can present a certain phenotypical diversity on the plan 1 - 2 for the extracts of seeds: these consist of foreign ecotypes and of those of northern and central Burkina Faso. The point of maximum diversity would be in the middle of the group of the ecotypes of central Burkina Faso: this indicates that the greatest genetic diversity would be within the ecotypes as Tostain et al. (1987) stipulate. They show the existence of the greatest diversity within the ecotypes of the north (high Sahelian zone) in relation to the centres of origin of the species. Our results do not contradict those of Tostain et al. (1987), because as drought advances toward the southern Sahara, the local farmers tend to sow early ecotypes. That can explain the increasingly rapid displacement of the ecotypes of the north toward the central and southern parts of the country. The further we go toward the zone where semi-late and late varieties are cultivated, the more the enzymatic variability is reduced.

To estimate the variation of the bands of esterase isozymes between the different ecotypes studied, we calculated the percentage of similarity between the ecotypes of the same area of prospection, between 60 ecotypes, then between ecotypes known to be different between themselves. For the ecotypes collected from Burkina Faso: from the centre of the country, the similarity indication varies between 53% and 100% with averages as high as 71.8% and 93.7%; for those from the north, the east and the south central, the similarity indication varies individually between 82.0 and 100% with averages between 83% (3176) and 99.2%. Only the ecotypes of the central region are all different from one another. Ecotype 2851 was the most distinct, having only 53% of similarity. The graphic representation of these ecotypes on the PCA plan 1 - 2 gave a great distribution of those of central Burkina Faso. The discriminating esterase systems show that:

· two of the northern ecotypes, 3145 (93.4%) and 3176 (91.7%) have average similarity indications less than 95%, which gives two distinct ecotypes plus an outcrossed population constituted by the remaining ecotypes of the same area of prospection;

· of the ecotypes of eastern region, only the ecotype 3699 (92.9%) possesses an average less than 95%: we keep from this group the distinct ecotype and an outcrossed population constituted by the rest of the ecotypes

Table 2. Similarity indices for esterases in 23 ecotypes collected from Burkina Faso and from other countries of West Africa (in %)

Ecotype	87	724	733	1068	1114	1273	1309	1501	1510	2001	2034	2812	2837	2851	2861	2864	3141	3145	3176	3204	3699	3769	Mean
50	88	90	90	93	95	90	90	93	88	84	93	95	93	78	79	90	93	93	91	95	88	77	89.4
87	-	97	95	90	97	97	97	95	87	68	85	86	84	73	74	87	90	90	88	95	84	89	88.0
724	-	100	93	95	95	95	97	90	72	88	88	87	71	78	90	93	93	90	98	87	92	89.9
733	-	93	95	95	95	97	97	72	88	88	87	71	78	90	93	93	90	98	84	92	93.4
1068	-	93	88	88	90	98	81	95	100	95	74	86	93	100	95	93	95	90	89	91.5
1114	-	90	95	93	90	79	93	93	88	78	79	95	93	95	91	98	88	87	90.9
1273	-	95	92	85	67	83	88	87	65	78	85	93	88	90	93	87	86	87.1
1309	-	92	85	72	88	83	82	71	72	90	88	88	86	93	82	86	86.9
1501	-	92	74	85	86	89	73	74	87	90	90	88	95	84	89	88.4
1510	-	83	98	93	92	71	83	90	98	93	90	93	87	79	88.9
2001	-	86	82	80	87	75	78	81	77	74	76	69	76	76.9
2034	-	93	90	74	81	80	95	91	88	98	85	82	88.1
2812	-	90	76	82	93	95	96	93	95	86	83	89.3
2837	-	79	86	87	95	90	88	90	84	80	87.4
2851	-	53	76	74	70	67	74	73	62	72.3
2861	-	93	86	82	84	86	74	76	79.0
2864	-	93	88	86	93	82	79	87.5
3141	-	95	93	95	90	87	91.4
3145	-	98	95	90	87	89.9
3176	-	93	88	80	87.7
3204	-	90	82	91.8
3699	-	86	84.4
3769	-	83

· among the south central ecotypes, there is only the ecotype 3751 which possesses an average of 83%: there would be therefore this sample and the rest to constitute a distinct ecotype and a new outcrossed population.

For all ecotypes of Burkina Faso, we can, without any extrapolation of these results, envisage the following restructuring:

· eleven ecotypes of central Burkina Faso;

· three ecotypes and two new samples as outcrossed populations;

· one ecotype and a new outcrossed population in eastern Burkina Faso, as well as in the south central region. There may be a total of 19 samples instead of 48 (31 %) to be conserved in the genebank.

The indications of similarity for all ecotypes considered to be different are reported in Table 2: the ecotypes of Burkina Faso that have an indication of similarity less than 95% are compared with those of foreign countries. The values vary from 62 to 100% with averages between 72.3 and 91.8%. Ecotype 2851 has the lowest average (72.3%) followed by 2001 (76.9%).

In all the populations studied there are two esterase enzyme groups with great variability: the group of foreign ecotypes and the group of central Burkina Faso ecotypes. The other groups have a minor enzyme diversity. There is some similarity between isozymatic, morphological and geographical distributions.

The ecotypes of south central and eastern Burkina Faso constitute the majority of homogeneous groups and enzymatically are much less variable. The diversity observed among foreign ecotypes is not surprising because the geographical distances between the countries are sufficiently large to produce these differences in pearl millet. However, it is difficult to explain the net amount of diversity among the ecotypes of the north and the remaining ecotypes of the other regions of Burkina Faso by geographical variations. Nevertheless, it is known that the ecotypes of the south central and the eastern region are semi-late genotypes that are sensitive to the photoperiod. The ecotypes of the central region would constitute a transitory zone toward the north, and would therefore have a greater diversity; but this diversity is not very visible on the enzymatic plan. This cannot be explained by environmental factors.

The ecotypes of the northern region could come from several domestications and from several wild parents growing along the southern Sahara (Tostain et al. 1987). The diversity among the ecotypes of the north could be explained by their proximity to the Nigerian pearl millets known as the most varied of the Sahelian region, and thus would have benefited from a flow of genetic exchanges. These ecotypes are also at the crossroads of the domestication centres recommended by Harlan (1971).

The semi-late and the late varieties, which are probably the result of a migration of early varieties from the north to the south, could have become more specialized to conform to a rainy and regular climate. However, the populations of early pearl millets could also be the consequence of a long migration process through arid and even rainy regions of West Africa.

The discordance of esterasic distances comes probably from the fact that enzymes remain neutral in the interaction between genotype and environment. The differences observed at the level of the esterase showed that out of 60 ecotypes studied, 30 can be considered similar. To complete the morphological data, redundant samples can be detected. For Fraleigh (1989), grouping samples according to genetic resemblance allows the constitution of a reduced representative collection (idea of core collection). The samples that form the groups can be maintained together in a strategy of 'dynamic conservation' (Pernès et al. 1984). These sources of information are not only necessary for the rationalization of the conservation of the genetic resources, but they are also a means for breeders to have at their disposal the available genetic variability. Though the observation of esterasic similarity indicates great genetic proximity among the majority of the ecotypes collected, the isozyme bands present sufficiently distinct profiles to permit ecotype differentiation, and the intensity of the bands can assist in their identification.

Conclusion

These results show that genetic evaluation of allogame populations requires the study of phenotypical and enzymatic variations to optimize genetic diversity of the cultivated forms. The revelation of genetic determinism of esterase isozymes and the use of electrophoresis to analyze other pearl millet enzymes could permit better understanding of the genetic variability of the cultivated species. However, this method needs to be developed so that the genetic control of isozymes can be explained. In support of multivariate analysis methods, enzymatic discrimination permits visualization of the formation of new outcrossed populations even genetically richer and less redundant for germplasm conservation. However, the genetic scope of this phenotypical diversity must be checked by studying the crossing between the collected and already evaluated ecotypes, in particular enzymatic polymorphism and restriction fragment length polymorphism. It is also necessary to find the cause of the regional distribution of the diversity of the ecotypes of West Africa. That knowledge will shed light on the evolution of pearl millet species, the content of the genetic richness that will permit improvment of the prospection techniques, the identification and the sampling of the ecotypes in their primary and secondary differentiation areas in order to have better strategies of collecting and of conservation of genetic resources in these regions of Africa.

References

Bono, M.1973. Contribution à la morphosystématique des Pennisetum annuels cultivés pour leurs grains en Afrique occidentale francophone. Agron. Trop (Paris) 28:229-356.

Brown, A.H.D. and B.S. Weir. 1983. Measuring genetic variability in plant populations. Pp. 219-239 in Isozymes in plant genetics and breeding, Part A. (S.D. Tanksley and T.J. Orton, eds.). Elsevier, Amsterdam, Oxford.

Cheliak, W.M. et J.A. Pitel. 1985. Techniques d'électrophoresè sur gel d'amidon des enzymes d'essences d'arbres forestiers. Institut forestier national de Petawawa, service canadien des forêts. Rapport d'information PI - X - 42F, 47 pp.

Cousineau, J.C. and D.J. Donnelly. 1989. Identification of raspberry cultivars in vivo and in vitro using isoenzyme analysis. Hort. Sci. 24:490-492.

Cubadda, R. and E. Quattrucci. 1974. Separation by gel electrophocusing and characterization of wheat esterases. J. Sci. Food Agric. 25:417-422.

Fraleigh, B. 1989. Importance des barques de resources phytogénétiques. Amélioration et Protection des Plantes Vivrières Tropicales (Claude André Saint-Pierre et Yves Demarly Resp. Sci.). Ed. AUPELF-UREF. John Libbey Eurotext. Paris. pp. 13-18.

Gottlieb, L.D. 1981. Electrophoretic evidence and plant populations. Prog. Phytochem. 7:1-46.

Harlan, J.R. 1971. Agricultural origins: Centers and non-centers. Science 14:468-474.

Kahler, A.L. and R.W. Allard. 1970. Genetics of isozyme variants in barley. I. Esterases. Crop Sci. 10:444-448.

Kidambi, S.P., J.R. Mahan, A.G. Matches, J.J. Burke and R.R. Nunna. 1990. Genetic variability for esterase enzyme in Onobrychis species. Theor. Appl. Genet. 80:433436.

Pernes J. et al., 1984. Gestion des ressources génétiques des plantes: Tome I et Tome 11. Monographies - Agence de Coopération Culturelle et Technique, Paris.

Sarr, A., M. Sandmeier and J. Pernes. 1988. Gametophytic competition in pearl millet, Pennisetum typhoides (Stapf et Hubb.). Genome 3:924-929.

Tostain, S. et M.F. Riandey. 1984. Polymorphysme et déterminisme génétique des enzymes de mil (Pennisetum glaucum L.). Etude des alcools deshydrogénases, catalases, endopeptidases et estérases. L'Agron. Trop. 39(4):335-345.

Tostain, S., M.F. Riandey and L. Marchais. 1987. Enzyme diversity in pearl millet (Pennisetum glaucum). Theor. Appl. Genet. 74:188-193.

Trigui, N.1984. La variabilité génétique des mils (Pennisetum typhoides (Bur.) Stapf & Hubb.) de Tunisie. Etude biométrique et analyse du polymorphisme enzymatique. Thèse 3 e cycle (Developpement et amélioration des végétaux), Univ. Paris Xl - Orsay, 125 p.

Trigui, N., M. Sandmeier, M. Salanoubat et J. Pernes.1986. Utilisation des données enzymatiques et morphologiques pour l'etude des populations et de la domestication des plantes: I. Séparation et identification génétique d'isozymes chez le mil (Pennisetum typhoides Bur. Stapf & Hubb.).

Whitney, P. J., J.G. Vanghum and J.B. Heale. 1968. A disc electrophoretic study of the proteins of Verticillium albo-atrum, V. dahliae and Fusarium oxysporium with reference to their taxonomy. J. Exp. Bot. 19:415-426.

Résumé

Méthode d'identification du polymorphisme génétique des enzymes de l'estérase dans les écotypes de Pennisetum glaucum.

Soixante variétés locales de mil chandelle (Pennisetum glaucum (L.)) représentant la variabilité génétique de huit pays d'Afrique de l'Ouest, dont le Cameroun, ont été utilisées pour comparer l'amplitude du polymorphisme génétique. On a testé des échantillons de semences originaux et des échantillons de feuilles fraîches récoltes en pépinière pour détecter l'estérase a et _ par electrophorese en gel de polyacrylamide. Pour les 60 obtentions, les échantillons de semences ont affiche davantage de spectres de bande que le matériel feuillu. Le nombre de bandes d'isozymes allait d'un minimum de 17 à un maximum de 24. Nous avons observe quatre zones: 0 3 2, 2 a 5, 8 à 12, et 1 à 4 profils d'isozymes dénommes respectivement EST 1, EST 2, EST à et EST 4. L'analyse en principales composantes et des études de l'indice de similitude ont révère une absence totale de variabilité dans les enzymes de l'esterase ou de petites différences entre variétés locales, principalement celles récoltées dans l'est et le centre-sud du Burkina Faso. La moyenne de l'indice de similitude allait de 90 a 100 %. On a démontre que les génotypes 3 maturation intermédiaire et tardive récoltes dans des zones a plus forte pluviosité pourraient venir du même ensemble de populations cultivées. Ces populations se vent adaptes aux conditions locales durant leur évolution apres plusieurs modifications imposées par la limitation du flux de gènes qui pourrait s'y être produite seulement parmi les premiers génotypes. Toutefois, une forte variabilité te des enzymes de l'estérase a été relevée parmi les premières variétés locales récoltées dans le nord et le centre du Burkina Faso, y compris celles des autres pays d'Afrique de l'Ouest. Ces résultats ont d'une part montre les différentes sources possibles des enzymes provenant de descendants sauvages en Afrique de l'Ouest et ont d'autre part confirme la possibilité que la domestication de l'espèce ait diverges origines. Néanmoins, la technique pourrait avoir des applications pratiques; des études complémentaires devraient peaufiner la procédure et découvrir d'autres systèmes d'enzymes du mil chandelle fondes sur le nombre d'allèles correspondent qui pourraient révéler des caractères agronomiques souhaitables, des génotypes directement utilisables comme parents pour les programmes d'amélioration ou des échantillons 3 conserver dans des barques de gènes.

Resumen

Método para identificar el polimorfismo genético de las enzimas de esterasa en ecotipos de Pennisetum glaucum.

Para comparar la amplitud del polimorfismo genético se utilizaron 60 variedades de mijo perla (Pennisetum glaucum (L.)), que representaban la variabilidad genética de ocho países de Africa occidental, incluido Camerún. Para la esterasa alfa y beta se seleccionaron, mediante electrofóresis del gel de poliacrilamida, muestras de semillas originales y de hojas verdes recogidas en invernadero. Para todos los 60 ejemplares, las muestras de semillas mostraron mas pautas de distribución en bandas que el material foliar. El numero de bandas isozimas oscilaban de un minimo de 17 a un máximo de 24. Observamos cuatro zonas de perfiles de isozimas de 0 a 2, 2 a 5, 8 a 12 y 1 a 4, que denominamos EST 1, EST 2, EST 3 y EST 4, respectivamente. De un análisis de los componentes principales y de estudios de los índices de similaridad, resultó una ausencia total de variabilidad en las enzimas de esterasa o pequeñas diferencias entre las especies, sobre todo en las recogidas en la zona oriental y centromeridional de Burkina Faso. La media de los índices de similaridad fluctuó del 90 a 100% Se aportan también pruebas que apoyan la hipótesis de que los genotipos de madurez tardía y mediana recogidos en zonas con mayores volúmenes d e pluviosidad ad podrían proceder del mismo conjunto de poblaciones cultivadas. Estas poblaciones adquirieron una adaptación a la región durante su evolución tras varíes modificaciones impuestas por la limitación del flujo genético que había podido darse solo entre los genotipos anteriores. No obstante, se hallo una gran variabilidad de enzimas de esterasa entre las especies anteriores recogidas de las regiones septentrional y central de Burkina Faso, inclusive en las de los otros paises de Africa occidental. Por una parse, estos resultados mostraban las posibles fuentes distintas de enzimas respecto de la progenie silvestre en toda la región de Africa occidental y, de la otra, confirmaba la posibilidad de distintos orígenes en la domesticación de las especies. Sin embargo, esta técnica encierra posibilidades de aplicación practica; en los estudios que se hagan en adelante debería perfeccionarse el procedimiento y descubrir otros sistemas de enzimas del mijo perla basados en un numero de alelos correlativos que tal vez revelen caracteres agronómicos convenientes, o aporten genotipos parentales útiles pare programas de fitogenética, o muestras pare su conservación en los bancos de genes.

Common names of the sweetpotato (lpomaea batatas) in the Americas

Fermín de la Puente¹, Daniel F. Austin² and Jaime Díaz³

¹ Departmento de Recursos Genéticos, Centro internacional de la Papa, Apartado Postal 1558, Lima, Perú

² Department of Biological Sciences, Florida Atlantic University, Boca Raton, FL 33431, USA

³ Centro internacional de Mejoramiento de Maíz y Trigo, Apdo. Postal 6-641, Mexico 06600, D.F. Mexico

Summary

The different common names given to the sweetpotato and its cultivars in different Latin American and Caribbean countries are summarized. The origins of these names in each country were studied and found to be related to physical traits, people regions, localities and uses. In general the common names are determined by a single criterion.

Introduction

In 1985 the Centro internacional de la Papa (CIP) began to form a genebank of the sweetpotato (Ipomoea batatas (L.) Lam.), commonly known as camote, batata or boniato, with material collected in Latin America and the Caribbean. Toward this goal a number of systematic excursions were organized in different localities and habitats in Latin America and the Caribbean. These excursions did not target specific ethnic groups, but sought all people who were growing and using sweetpotatoes. Both Amerindian and non-indigenous populations were sampled. Data sheets with predetermined questions were used to obtain both ethnobotanical and horticultural data about the plants.

As of May 1992, some 16 countries had been explored, and collections made of 5395 accessions of cultivated and wild germplasm. Of the germplasm collected, some 3422 (63.4%) accessions were of the cultivated species, 171 (3.2~o) were of wild Ipomoea batatas and 1807 (33.4%) were of wild species. The cultivated germplasm was collected in Argentina, Bolivia, Brazil, Colombia, Dominican Republic, Ecuador, Honduras, Jamaica, Mexico, Nicaragua, Panama, Paraguay, Peru and Venezuela.

Within the cultivated germplasm samples there were different common names for the cultivars and cultivated strains. These names varied among the countries, states, departments, provinces, municipalities, towns and villages according to the political divisions of each country. The origins of the common names are related to harvesting and cooking customs, and to the dominant morphological characteristics of the plants and fleshy roots. This paper summarizes the broad patterns among the common names currently used.

Common names

The following common names, along with numerous orthographic variants (Austin 1988), are most frequently used for the cultivated plants in Latin American and Caribbean countries (Table 1): batata, camote, boniato, sweetpotato, palate doce, batata doce, chaco, apichu, kumara, is, chin, mabi and yety. These names are derived from local dialects and /or languages. and may have the same meanings when translated. For example, both batata and camote are used for the plants in different Spanish-speaking regions. Austin (1988) made no attempt to list all common names for the species as had Yen (1974). Instead, Austin (1988) examined linguistic relationships between the principal lineages of names, presented evidence that batata and camote are cognates, and concluded that the current use of these two names is related to historical distribution. In Cuba the same plants are called boniato by Spanish-speakers. Sweetpotato, patate dôce and batata dôce are English, French and Portuguese versions respectively of the same concept.

Table 1. Common names of sweetpotato in Latin American and Caribbean countries and possible origin of the names

Common name	Language/origin	Countries of usage
Batata	Chibchan->Spanish	Argentina, Bolivia, Colombia, Dominican Republic, Ecuador, Nicaragua, Panama, Paraguay, Puerto Rico, Venezuela
Camote	Nahuatl->Spanish	Bolivia, Chile, Costa Rica, El Salvador, Guatemala, Honduras, Mexico, Panama, Peru, Uruguay
Boniato	?Taino->Spanish	Cuba, Uruguay
Sweetpotato	English->sweet+batata	Jamaica, St. Vincent
Palate dôce	French->bataba+sweet	Haiti
Batata dôce	Portuguese->bataba+sweet	Brazil
Chaco	Arawakan?	Venezuela
Apichu	Quechuan	Bolivia, Peru
Kumara	Quechuan	Peru
Is, sis, chin	Mayan	Guatemala
Camotli	Nahuatl	Mexico
Mabi	Cariban	Honduras
Kuwas	?	Panama
Yety	Tupí-Guananí	Argentina, Paraguay

Our research has found that many additional names are used. For example, Table 2 shows the countries that have the most diversity in common names. Some names are widely used but others are more regional (Table 3). No attempt has been made to analyze the more regional names.

Table 2. Latin American countries with the greatest diversity of common names of sweetpotato

Country	No. of names
Peru	394
Argentina	115
Ecuador	66
Colombia	58
Bolivia	55
Honduras	50
Dominican Republic	28
Paraguay	26
Brazil	25

Table 3. Common names with the widest geographic distribution in Latin American and Caribbean countries

Common name	Country used
Amarillo	Argentina, Bolivia, Colombia, Ecuador, Honduras, Mexico, Panama, Paraguay, Republic, Venezuela
Blanco	Argentina, Bolivia, Brazil, Colombia, Ecuador, Honduras, Mexico, Paraguay, Peru, Dominican Republic
Colorado	Argentina, Colombia, Ecuador, Honduras, Panama, Paraguay, Peru, Dominican Republic
Morada	Argentina, Bolivia, Colombia, Honduras, Mexico, Panama, Peru, Dominican Republic
Rosado	Argentina, Colombia, Honduras, Mexico Dominican Republic
Camote papa	Argentina, Ecuador, Peru, Honduras, Mexico
Camote rosado	Bolivia, Honduras, Peru

Names of cultivate

Most common names may be grouped according to one predominant criterion. However, some names are more complex and are based on more than one trait. Simple names are typically based on a single criterion: morphological, physiological and culinary characteristics; production capacity; geographic origin; person who introduced the cultivar; morphological similarity with other cultivars.

Vegetative parts are the basis for some simple names. In Peru the name apichu is applied to plants with a trailing habit, and kumara to those which have a compact habit. This usage is of long-standing historical application in spite of comments to the contrary by Brand (1971). Some plants named for leaf shape include pate de pave, pie de gallina, hoja de pato, pate de gallo, pate de rana, sies pumas, and hoja redonda.

Names given because of skin colouration are: blanco, amarillo, morado, Colorado, rosado, rojo, negro and matizado. Among names based on root shapes are: blanco alagrada, colorada chica, amarilla redonda, amarilla grant and menudita. Typical names based on flesh colour are: blanco, amarillo, morado, blanco matizado an anaranjada.

Flesh colour also is the basis for names: camote used for cultivars with yellow flesh and batata for thou with white flesh. This association is continued by English speakers who call the yellow-fleshed roots 'yams' and the white-fleshed roots 'sweetpotatoes'. Common names based on cooking qualities include: yema de huevo, amarillo dulce, blanco sal, cambray dulce, morado harinoso, camote de mesa, camote coche and camote de sopa.

Names based on vegetative period (physiology) are tresmesino, noventero, cuarentón, cuarenta días and se meses.

Other names are based on production capacity. Thee include tumba burro, cargamento, rompe piedra, siete bold and amarillo rompe costal.

A few names based on geographic origin are Americana, Brasileira, Chileno, Cubano, Italiano, Japone Haitiana, Venezolana, Peruano, Serranita, Costeña; Chiclayana, Bogotana, Piurana, Trujillana, Paramonguino Mochera, Santa Fesina, Limeña, Torre blanca and Paulista.

Common names based on the person who made the intro auctions are Vargas, Valdivia, Pedrito, Remigio and Blank Salazar.

Names based on the similarity of the cultivar to other cultivated species, including traits like shape of the storage roots, skin colour, flesh colour and taste include remolacha; camote papa, camote yuca, batata abobora manicera, zapallo, batata zahanoria and camote oca.

Composite names generally refer to two traits of the cultivar such as blanco papa (white skin and flavour of Solanum potato), cargamontn dulce (good production an, sweet taste), morado harinoso (red skin and flour-like texture), colorada grande (red skin and large storage roots) roxa pintada (reddish skin and spotted flesh), morado mochero (reddish skin and flesh), amarillo rompe-costal (yellow flesh and good production), blanco matizado, (white flesh matrix with coloured spots), blanco grand (white skin and large storage roots), amarilla pequeña (yellow skin and small storage roots). Reasons for applying composite names to these plants probably vary al though most of these names distinguish between two similar local varieties.

Conclusions

The origins of the common names are related to characteristics of the cultivars, origin of movement, people who introduced the cultivar into a particular locality, and the strain's similarity to other cultivated plant species. Countries with the largest diversity of common names for the cultivars are Peru, with 394 different names, and Argentina with 115. Other surveyed countries have less than 100 common names in use for the sweetpotato.

In spite of the abundant variety of common names, comparisons of our findings with those presented in earlier studies (e.g. Yen 1974) show a marked decline in the number of names. We believe that this decline is linked with both the extirpation and extinction of local languages and the introduction of 'improved' lineages of sweetpotatoes. Both of these phenomena lead to biodiversity loss within the cultigen. Another difference between our data and those presented by Yen (1974) is that we did not target indigenous populations for census. However, we suspect that had less impact on our data than general biodiversity loss. The decline we found appears to be real and undoubtedly reflects the homogenization of human populations and cultigens that is in progress around the world. The numbers and types of geographical names in current usage support this interpretation.

Acknowledgements

The authors acknowledge financial support from IPGRI (Z. Huaman and F. De la Puente, principal investigators), CIP, USDA (D.F. Austin, principal investigator, Grant No. 586659-1-102) and the National Geographic Society (D.F. Austin, principal investigator, Grant No. 4478-91).

References

Austin, D.F. 1988. Taxonomy, evolution and genetic diversity of sweet potatoes and related wild species. P. 27-60 in Exploration, Maintenance and Utilization of Sweet Potato Genetic Resources, Proceedings of the First Sweet Potato Planning Conference, Centro Internacional de la Papa, Lima, Peru.

Brand, D.D. 1971. The sweet potato: an exercise in methodology. Pp. 343365 in Man Across the Sea (C.L. Riley et al., eds.). University of Texas Press, Austin.

Yen, D.E. 1974. The Sweet Potato and Oceania. Bishop Museum Press, Honolulu. pp. 1-389.

Résumé

Noms commune de la patate douce (Ipomoea batatas) dans les Amériques

Le présent article récapitule les différents noms commune donnés à la patate douce et à ses cultivars dans les pays d'Amérique latine et des Caraïbes. On a étudié l'origine de ces noms dans chaque pays et découvert qu'ils ont trait aux caractères physiques, aux populations, aux régions, aux localités et aux usages. En général, les noms commune vent déterminés par un seul critère.

Resumen

Nombres vulgares de la batata (Ipomoea batatas) en las Américas

Se hace un resumen de los distintos nombres vulgares dados a la batata y a sus cultivares en distintos países de América Latina y el Caribe. Se estudiaron los orígenes de esos nombres en cada país y se llegó a la conclusión de que guardan relación con las características físicas, la población, las regiones, las localidades y los usos. En general, los nombres vulgares están determinados en función de un único criterio.

Insect pollination and isolation requirements in tomato collections (Lycopersicon esculentum Mill.)

Th. Gladis¹, K. Hammer¹, H.H. Dathe² and H. Pellmann³

¹ Institut fur Pflanzengenetik und Kulturpflanzenforschung Genebank, Corrensstr. 3, D-06466 Gatersleben, Germany

² German institute of Entomology, Schicklerstr. 5, D-16225 Eberswalde, Germany

³ Department of Zoology, Leipzig University, Talstr. 33, D-04103 Leipzig, Germany

Summary

Cross-pollination in tomatoes in countries outside their natural distribution is a problem during ex situ seed regeneration in germplasm collections of wild and cultivated taxa. Isolation of different populations during the flowering period is one of the best methods to avoid hybridization. Bees and hover flies are efficient native pollinators of Lycopersicon species in Central Europe.

Introduction

Modern tomato varieties are pure breeds (monomorphic), predominantly self-pollinated, and there is assumed to be a very small amount of cross-pollination caused by insects. The outcrossing rate is correlated with the evolutionary age of the variety, i.e. with the position of the style within the flower. However, most wild tomato species (Lycopersicon spp.) and primitive types of cultivated or weedy plants, potato-leafed cultivars or forms with double blossoms, beefsteak tomatoes etc., are often long styled (Ashworth 1991). This paper reports observations on a large tomato collection maintained at the Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK) genebank, Gatersleben, Germany. The implications for genebank work and breeding (isolation requirement) are discussed.

Multiplication of tomato collections under Central European conditions is considered a very easy task for cultivated material (Esquinas-Alcazar 1981). It only requires sowing or planting the accessions side by side, and harvesting them separately. The highest published crossing rate in Central Europe is 2%. Only in tropical areas may the cross-pollination rate attain 47% (Hackbarth 1960). Therefore, in northern countries the problem of foreign pollination is largely neglected and tomatoes are often treated as self-pollinating crops in breeding work and germplasm collections. Experience with other self-pollinating crops shows that there may be a large variation with respect to outcrossing tendencies, e.g. barley (Hammer 1975), and genebanks must develop specific methods to avoid unwanted intercrossings. Intensive entomological studies (Gladis 1989) and research on the floral biology of each plant population are preconditions for the work with entomophilous plants.

Observations

The IPK Gatersleben genebank maintains a living collection of more than 3050 tomato populations, i.e. varieties, mutants, unnamed populations, weeds and wild species. Each year about 300 accessions (average) are grown in reproduction fields and greenhouses for determination, multiplication and comparison of identity. A simple method is used to detect spontaneously occurring crosses, the so called disjunctive growing system as proposed by Lehmann and Mansfeld (1957): neighbouring populations carry specific morphological characters, sometimes making it easy to distinguish them and to detect outcrossings in the following generation.

Various characters in flower morphology were observed within one variety or population, within one plant, and even within one raceme (Fig. 1) regarding the length of the floral style. This has not yet been explained. Other observations after multiplication steps of genebank material led us to look for outcrosses after each seed-growing season, e.g. In 1976 a new variation within four older genebank accessions, out of 522 populations grown that year, was found and documented. Very often newly introduced material shows a high degree of heterozygosity when grown for the first time. This is attributed to occasional natural cross-pollination in southern countries (Lehmann and Schwanitz 1965). Lines derived from original material or self-pollinated species are maintained with higher priority than offspring of old varieties. They are always selected in the direction of the parental generation or characters of primary growth.

- Fig. 1. Various floral characters were observed within one tomato plant, and even within one raceme

Selection steps used to eliminate plants derived from outcrossings are always well documented, as the following examples illustrate: accession T 910/85 (first regeneration in 1995), in 1986 split into three different types (T 910/86, T 982/86, T 1025/86). In 1991 and 1992, two additional types derived from T 1025 (T 1206/91, T 1318/92). The original material (T 910) was collected in Italy in the Cosenza province in the region of Calabria in 1993. The indeterminate growing local variety with fruits of the San Marzano type led to additional morphotypes after ex situ reproduction steps at Gatersleben. This splitting seems to be more common in some years than in others. Comparable findings resulted from L. pimpinellifolium accessions, grown within the L. esculentum block. The following generations contained several intermediate plant types with different hair densities and fruit characters, which might originate from intercrossings: T 1127/88-93 segregated into T 1208, T 1209, T 1266, T 1279, T 1280 and T 1281). Characterization data, descriptions and herbarium sheets, altogether a complete reference file that includes photographs of the whole collection, may help with decisions about doubtful populations and duplications in many cases, but not in all. To study the responsible pollinators, field observations were carried out on insect densities in the tomato reproduction field in August 1993. That year was characterized by very inclement weather conditions for seed production as well as for flower-visiting insects. After a long period of low temperatures near the ground, 17-18 Aug were days with temperatures higher than 30°C at 5 cm above ground, the highest atmospheric pressure in the month (7.67 x 10^-2 - 7.72 x 10^-2 MPa), sunshine throughout the day, and less wind than other days. These two days were used to observe and to collect insects visiting tomato flowers.

Results

During the same period, the collecting behaviour of insects on tomato flowers was different from that concurrently observed on more attractive flowers (i.e. Apiaceae, Asteraceae, Brassicaceae, Campanulaceae, Fabaceae, Lamiaceae, Scrophulariaceae). Aggregations of hover flies, mainly Episyrphus balteatus, could be found peacefully feeding on these families. Solitary bees and honeybees collecting alone or jointly, often solitary bumblebees (seldom two or more workers on sunflower, Helianthus annuus L.), or females of larger Megachile species, being very aggressive to other insects on the same flower (erected abdomen with visual stinger) were also seen. The highest degree of intolerance is expressed by males of Anthidium manicatum. This common solitary bee mainly visits flowers of Fabaceae, Lamiaceae and some Scrophulariaceae. The males own and defend restricted areas where only females of the same species are allowed to expropriate the flowers of preferred plants. They also mate there. Competitors, whether capable of defending themselves or not, are turned out or killed. The predominance of different visitors on the same flower of, for example, Telekia speciosa (Asteraceae) or Verbascum sp. (Scrophulariaceae) followed more or less the same sequence (not seen in the case of Anthidium). In contrast to these observations, insects were never found fighting for Solanum or Lycopersicon flowers or those of related genera. The few insects collected on tomato flowers are listed in Table 1.

Table 1. Insects collected on tomato flowers (L. esculentum, L. pimpinellifolium), 17-18 Aug 1993

I. Hymenoptera, Apoidea:	Months of activity	Remarks	Sol1
0:1_ Andrena bicolor (F.)	Apr May, July-Aug	Polylectic	-
0:l_ Apis mellifera L.	Feb-Dec	Polylectic	+
0:3_ Bombuspascuorum (Scop.) Polylectic	Mar.Nov	Polylectic	-
0:1_ Bombus sylvarum (L.)	Apr-Aug	Polylectic	-
0:1_ Bombus terrestris (L.)	Mar-Oct	Polylectic	+
II. Diptera, Syrphidae:
0:5_ Sphaerophoria scripta (L.)	Mar-Oct	Polylectic	-

_Sex ratio, male:female (1:2 means 1 male and 2 females)

_ 0,1 indicates 1 worker of a social species.

¹ Sol +/reported as visitors on solanaceous flowers, yes/no

Discussion

Teppner (1993) observed Hylaeus gibbus as buzz pollinators of Lycopersicon flowers. Independent of the insect species, only females or workers, but never males, were found on tomato flowers. However, males of bumblebees were simultaneously present in other places. The females always have a greater need for pollen food than males, because of either their high egg production rate (hover fly) or their collecting larval food (bees).

All of the mentioned species are well-known as indefatigable flyers and pollinators. The observed behaviour of these insects on tomato flowers is quite different from their well-known behaviour on flowers of other plant families. Figs. 2 and 3 show how efficiently they collect tomato pollen. All insects stayed a very short time, had a swift and irresolute collecting behaviour and avoided distances far more than those for more attractive flowers. A comparable situation was seen in large fields planted for onion seed production (Allium cepa) near Quedlinburg, with Helophilus trivittatus (Syrphidae, Eristalinae) as one of the pollinating insects in the 1980s. In contrast, honeybees and bumblebees behaved normally there.

From the insect's view, availability of pollen is the most limiting factor for development and abundance of the following generation. Only this food is rich in proteins. Usually, there is more nectar that is easily available from flowers, extrafloral nectaries, as well as from the excrements of aphids.

Flowers of the Solanum type are oligandrous pollen flowers with 'modern', strictly melittophilous specialization (Vogel 1978). They developed from oligandrous nectar flowers, which sometimes show a rudimentary, nonfunctional nectary. The powdery pollen is released in very small portions by vibrations caused by the visitors and has to be mixed with nectar for gathering. Besides bumblebees, only one solitary bee species, Anthophora quadrimaculata as described by Westrich (1989, 1990), collects pollen from flowers of Solanum nigrum in Germany.

- Fig. 2. Single pollen grain within the cone of anthers

- Fig. 3. Pollen loads of two bumblebee workers show how efficiently they collect tomato pollen

These less attractive flowers are visited by a few workers of social bees, i.e. honeybees and bumblebees, and very few other solitary bee species possessing a type of innate skill. According to Symon (1979), these bees "have learned" to vibrate the flowers by shivering their indirect flight muscles during direct stamina! contact when collecting pollen. This explanation is not completely convincing because buzzing may also be observed in other situations. It should not be expected that only inexperienced individuals are working, because these few observed are specialists. If such hungry insects could gather all food from one flower or one plant, they would never move to other plants of the same species. Due to their poor attractiveness, Lycopersicon flowers are more dependent on bee visits than are other plants. Fruit set, fruit size and seed set are considerably increased by frequent insect visits. This explains the use of bumblebees (mainly Bombus terrestris) and solitary bees (e.g. the alfalfa leafcutter bee, Megachile rotundata), to successfully pollinate isolated vegetable tomatoes grown in greenhouses (Holm 1986, Free 1993).

Symon (1979) refers to worldwide observations of in sect species, mainly bees of several families but also hover flies (the first record of Syrphidae was in 1883), feeding on solanaceous flowers: white, blue and yellow are the most preferred flower colours for pollen-collecting insects. "White, blue or yellow are the common colours of Solanum flowers and the conspicuous yellow cone of anthers is one of the most consistent features of Solanum flowers over hundreds of species." in this respect the genus Lycopersicon does not present an exception.

Only one hover fly species was found frequently feeding on tomato flowers. Sphaerophoria scripta is a common and polyphagous species, but it has never been observed on Solanaceae in Europe (De Buck 1990). Buzzing (Nickol 1991) and buzz pollination are also described for hover flies, especially in Solanaceae (D'Arcy 1991). In contrast to pollen-collecting bees, the flies are pollen eaters. The observed species is very small and it only shows this behaviour if the individual's head is fixed while feeding within the floral organs. A very similar reaction was found in Eristalis spp., Myathropa and Lathyrophthalmus (Syrphidae, Eristalinae) when they were caught for individual marking.

Out of several possible sources of genetic variation, hybridization is most likely responsible for phenotypic variation occurring in nature and under cultivation (Rick 1958). Bijlsma et al. (1986) found cases of nonrandom mating in an open-pollinated maize population. Their mixed mating model shows a kind of pollen selection for genetic distance: "Analysis of allelic frequencies in the pollen that produced seed on detasselled plants established that different maternal plants sampled genetically different populations of pollen from the outcross pollen pool." This has not yet been detected for tomatoes, but it cannot be excluded for this crop and would, if proven, mean that cross-pollination of less near related sources within a plant species has a higher chance of resulting in germination and fertilization.

Conclusion

Knowledge of the respective breeding system as well as the pollen vectors and their behaviour is necessary for preserving the crop plant diversity ex situ. Maintaining these genetic resources, as well as breeding work, must block evolutionary processes, including domestication, at precisely defined levels and must not develop it in an advantageous direction. It will be necessary to measure the rates of natural crossing (monitoring) in applied research studies, e.g. by using male-sterile mutants as markers in regions with breeding material or to detect the pollination efficiency of insects. An important and elementary method in genebank work has to be applied to this part of the whole collection for regeneration purposes, i.e. isolating tomatoes during their flowering period. Another possibility for outdoor growth is controlling and marking each flower for position of the style and to only harvest the marked fruits. The method of disjunctive growing fails in the case of maintaining large tomato collections ex situ and should not be applied in the future because of the described collecting behaviour of the observed insects.

Acknowledgements

The authors thank Dr K. Adler for the opportunity to use the scanning electron microscope. To Mr F. Bondonga-Mboyo and Mr F. Camara, School for Tropical Agriculture in Altenburg, we want to express our thanks for their help ful assistance in the field work and to Mr B. Askari for proofreading the manuscript.

References

Ashworth, S. 1991. Seed to seed. Seed saving techniques for the vegetable gardener. Seed Saver Publ. Decorah, lowa, 222 pp.

Bijlsma, R., R.W. Allard and A.L. Kahler. 1986. Nonrandom mating in an open-pollinated maize population. Genetics 12:669-680.

D'Arcy, W.G. 1991. The Solanaceae since 1976, with a review of its biogeography. Pp 75-137 in Solanaceae III, Taxonomy, Chemistry, Evolution. (J.G. Hawkes, R.N. Lester, M. Nee and N. Estrada eds.). Royal Bot. Gardens Kew.

De Buck, N. 1990. Bloembezoek en bestuivingsecologie van Zweefvliegen (Diptera, Syrphidae) in het bijzonder voor België. Ed. Inst. Royal Sci. Nat. Belg., 167 pp.

Esquinas-Alcazar, J.T. 1981. Genetic resources of tomatoes and wild relatives-a global report. IBPGR Rome, 65 pp.

Free, J.B. 1993. Insect pollination of crops. 2nd ea., Academic Press Harcourt Brace Jovanovich Publ. London, San Diego, New York, Boston, Sydney, Tokyo, Toronto, 684 pp.

Gladis, Th. 1989. Die Nutzong einheimischer insekten (Hymenopteren und Dipteren) zur Bestäubung von Kulturpflanzen in der Genbank Gatersleben. Kulturpflanze 37:79-126.

Hackbarth, J. 1960. Spontane Fremdbestäubung bei Selbstbefruchtern. Vortr. Pflanzenzücht., DLG-Verlag Frankf./M. 6:140-151.

Hammer, K. 1975. Die Variabilität einiger Komponenten der Allogamieneigung bei der Kulturgerste (Hordeum vulgare L s l.). Kuturpflanze 23: 167-130.

Holm, S.N. 1986. Bestovning af tomater med lucernebladskaerebier. Gartner Tidende 8:254-255.

Lehmann, Chr. O. and R. Mansfeld. 1957. Zur Technik der Sortimentserhaltung. Kulturpflanze 5:108-138.

Lehmann, Chr.O. and F. Schwanitz. 1965. Ein Beitrag zur Kenntnis der Formenmannigfaltigkeit der Kulturtomaten (L. esculentum, Mill.) Mittelamerikas. Kulturpflanze 13:545-585.

Nickol, M. 1991. Beitrag zur Kenntnis von Scaeva pyrastri L. (Diptera: Syrphidae). Mainzer Naturw. Archiv 29:205-213.

Rick, C.M. 1958. The role of natural hybridization in the derivation of cultivated tomatoes of western South America. Econ. Bot. 12:346 367.

Symon, D.E. 1979. Sex forms in Solanum (Solanaceae) and the role of pollen collecting insects. Pp. 385-397 in The biology and taxonomy of the Solanaceae. (J.G. Hawkes, R.N. Lester and A.D. Skelding eds.), Linn. Soc. Symp Ser. 7.

Teppner, H. 1993. Die Tomate. Verwandtschaft, Geschichte, Blütenökologie in Amerika - Zur Entdeckung - Kulturpflanzen Lebensraum Regenwald. Katalog zur Ausstellung Schloßmusseum Linz, 13.10.1992 - 14.3.1993: 189-211.

Vogel, St. 1978. Evolutionary shifts from reward to deception in pollen flowers. Pp. 89-96 in The pollination of flowers by insects (A.J. Richards ed.). Linn. Soc. Symp. Ser. 6.

Westrich, P.1989, ]990. Die Wildbienen Baden-Wurttembergs. 2. ea., 2 Vols., Ulmer Stuttgart, 972 pp.

Résumé

Pollinisation par les insectes et besoins d'isolement dans les collections de tomates (Lycopersicon esculentum Mill.)

La pollinisation croisée chez les tomates dans les pays hors de leur aire de répartition naturelle pose problème durant la régénération ex situ des semences dans les collections de matériel génétique de taxons sauvages et cultivées. L'isolement de plusieurs populations durant la période de floraison est une des meilleures méthodes pour éviter l'hybridation. Abeilles et syrphes vent de bons pollinisateurs indignées de l'espèce Lycopersicon en Europe centrale.

Resumen

Polinización por insectos y exigencias de aislamiento en las colecciones de tomate (Lycopersicon esculentum Mill.)

La polinización cruzada de los tomates en países fuera de su distribución natural constituye un problema durante la regeneración de las semillas ex situ en las colecciones de germoplasma de taxones silvestres y cultivados. Para evitar la hibridización, uno de los mejores métodos consiste en aislar las diferentes poblaciones durante el período de floración. En Europa central, las abejas v las moscas que revolotean son buenos polinizadores naturales de las especies de Lycopersicon.

Use of computer databases to manage an in vitro collection of potato germplasm

Debabrata Sarkar and Prakash S. Naik

Biotechnology Laboratory, Division of Genetics & Plant Breeding, Central Potato Research institute, Shimla 171 001, H.P. India

Summary

A relational database has been created to manage an active in vitro collection of potato germplasm. This replaces a database management system (for conventional genebank) used to manage the in vitro collection. In the model described, the data are recorded so that the regeneration date of any accession can be scheduled interactively in accordance with other information. For modeling, the dBASE III PLUS software package is used. A number of application programmes written in a dBASE III PLUS environment help with interactive retrieval and updating the data. Details of any record can be accessed through the relationship defined in the model. This user-friendly model can be used by persons without much computer knowledge. The flexible nature of the system makes it adaptable to changing requirements for in vitro potato germplasm management.

Introduction

Conservation and international exchange of disease-free potato germplasm have been greatly facilitated by recent advances in plant tissue culture techniques. An in vitro collection of potato germplasm has been maintained in India at the Central Potato Research institute in Shimla for ten years. A database management system (DBMS) developed for the conventional germplasm collection had been expanded to manage this in vitro repository. This system relied partly on manually recorded data, which was found to be inadequate for proper data validation, and flexible storage and retrieval. This is mainly due to the differences in the conservation methods of these two systems.

The in vitro method of germplasm conservation (minimal or slow-growth storage) differs from the conventional approach in several ways:

· in the conventional genebank, the 'seeds' (tubers) are stored at low temperature and are not given regular care. The accessions are routinely multiplied in glasshouses and fields every year. An in vitro system involves frequent transfers to fresh media, constant vigilance and control of storage conditions. Thus, this system provides a heavy monitoring burden to the curator of the culture collection.

· The foremost requirement of short-term tissue culture conservation is to schedule the regeneration date of cultures. Because genotypes differ widely in their response to in vitro storage (Westcott 1981), minor delays in regeneration frequently result in complete loss of culture viability. A careful balance must be maintained to avoid general loss of viability, and, worse, selective loss of viability that could lead to genetic drift (Withers 1989).

· Because of small sample size, the population in tissue cultures becomes 'tight' (Withers 1991) which, theoretically, subjects the cultures to higher chances of mutational hazards. Therefore, it is essential for accessions to be checked periodically for genetic integrity and managed accordingly (Dodds 1988a).

The above observations demonstrate the need for a separate management system for an in vitro repository. Development of a new system required the formation of a relational database with the provision for interactive cultural tracing capability.

Computer assistance for maintaining plant propagation records using a CODASYL type database management system with a FORTRAN interface was developed during the 1970s (Clarke and Caserio 1979). Special databases have been constructed from bibliographies on isozyme analysis (Simpson and Withers 1986) and the in vitro conservation of temperate fruit species (Stushnoff and Fear 1985). An in vitro conservation database has been generated at Nottingham University, UK, using the FAMULUS database management system (Withers et al. 1990). However, these data base management systems were developed for documentation and information exchange regarding in vitro storage techniques and associated conservation problems. The need for a computer assisted management system for running the in vitro repository has been reiterated by many workers (Chavez et al. 1988, Dodds 1988ab, Jarret 1988). However, detailed published research on such in vitro management systems is lacking.

Here we describe a flexible database management system for monitoring day-to-day activities of the in vitro potato germplasm collection. The system has been developed to meet the specific storage needs of potato microplant tissue cultures.

In vitro conservation model

Our experience led us to the conclusion that in vitro conservation can be modelled by corresponding data changes in the computer. The objective is not to store the information as such, but to give the system an interactive approach. The system is structured to assist with scheduling the regeneration date of each accession exactly in accordance with information stored in conservation events. For modelling, we use the dBASE III PLUS (1986) software package. Various application programmes have been written in dBASE III PLUS to suit to our specific requirements.

The present model, which may require modifications based on individual experience, incorporates recording of the following:

· Accession passport: The data on accession passport include the history and background of each accession.

· in vitro notes: These provide information about the in vitro response of each accession.

· in vitro events: These record conservation events of each accession during in vitro storage.

Details of the recorded data and database file(s) structure are presented in Table 1.

In tissue culture conservation, the in vitro storage period of potato microplants depends on in vitro response and percent regeneration of cultures. The in vitro response of a culture is determined on the basis of an overall visual preference score on a decreasing 1-3 scale. We have observed over the years that irrespective of in vitro response, any potato accession can be conserved up to one year in culture. Therefore, the in vitro life expectancy of a culture is calculated on the basis of its in vitro response and percent regeneration. In vitro life expectancy is defined as:

In vitro life expectancy = (REVIVAL / IVRES) + 365

where REVIVAL = Percent regeneration of culture, and IVRES = in vitro response on a 1-3 scale

The regeneration date of any accession (culture) is determined by its in vitro life expectancy.

In vitro management

A relational database was created to manage an in vitro conservation system. Three separate files were constructed; two for recording static or semi-static information such as accession passport and in vitro notes, and one for recording in vitro events, which are subject to change. Thus, each accession (culture) has three separate records. The record constructed for the accession passport file (ACESPSRT.DBF) contains the history of the accession. We designate this record as 'static', because its contents never change. The in vitro notes record (INVTNOTE.DBF) maintains the in vitro response and repository status of the accession. This record is known as 'semistatic', because its contents may be changed in the event of special needs. For example, sometimes we may increase the number of set (NOSET) or number of culture tubes per set (NOCTSET_) or change the location of the culture or set (LOCATSET_) for a particular accession. For monitoring day-to-day in vitro conservation work, a third record for each accession is the in vitro events file (INVTEVNT.DBF). We denote this record as 'changeable', because its contents are updated periodically following changes in data fields. Table 2 presents these three records for an accession (culture).

All three files are linked together by two common fields: culture number (CULTNO) and CP number (CPNO). The files have been indexed mainly on these two fields, which helps in the interactive tracing of cultures. Each file has separate view, query, catalogue and report modes. This facilitates working with a number of database files simultaneously at different working areas of the dBASE III PLUS system. The input to the databases is in a form that is as close as possible to the manually recorded data. For each field, a number of alternative expressions (character or numeric) have been established to describe that field. Fuller descriptions of abbreviated terms or expressions, i.e. code number, meaning of the code, meaning of the term, characteristics, are stored in a separate dictionary file. This dictionary file allows the user to access the meaning of any abbreviated term in the model. The database structure also permits easy flow of information to and from the database management system of the conventional genebank. The relationship between different database files and fields is shown in Fig. 1.

Table 1. Structure of in vitro conservation databases of potato with the details data recorded in each file.

Field Name_	Type_	Width	Description of information content
I. Accession passport (ACESPSRT.DBF)
CPNO	N	5	Identification number of potato accessions received at CPRI
CULTIVNM	C	15	Name or code of the cultivar
ORIGIN	C	3	The country from where the accession was received
SPPGR	C	10	Name of the species for wild or primitive cultivars; otherwise the entry denotes group
PHYTOSANNO	N	10	Statement number of the phytosanitary certificate issued by the donor country
CULTNO	N	5	Number assigned during in vitro culture initiation
PSSRTCOM	C	50	Passport comments include valuable information regarding the accession
II. In vitro notes (INVTNOTE.DBF)
IVRES	N	1	Numerical grading of in vitro response to minimal growth storage (1=good, 2=moderate and, 3=poor)
REVIVAL	N	3	Percent regeneration of cultures
IVMICPP	N	6	In vitro microtuber production potentiality, assigned by giving the average number and weight of microtubers per 250-ml flask
STORMICR	N	1	Numerical grading of the storage capacity of microtubers (1=good, 2=moderate and 3=poor)
NOSET	N	1	Number d sets i.e. owes d accessions maintained
NOCTSET	N	1	Number of culture tubes per set (3 microplants per set)
LOCATSET	N	3	Location of sets in the repository by numerical coding
CULTCOM	C	50	Culture comments include in vitro characteristics of the microtubers followed by name of the scientist who made the observations
III. In vitro events (INVTEVNT.DBF)
GENERNO	N	2	Number of times a clone has been subcultured or revived following initial culture establishment
INCBDTSET -	D	8	The date on which the cultures are established on minimal medium for conservation
REVDTSET-	D	8	The date on which the cultures will be revived
PLANTHLTH	C	10	Plant health, denoted by the presence or absence of potato virus (Y or N). A "Y" should be followed by the name of the virus in a single letter coding
MICROPROP	C	6	Micropropagation status denotes whether the conserved accession is in rapid multiplication stage (Y or N); if "Y", how many culture tubes (CT) or flasks (F) have been incubated
EVENTNM	C	4	Event name describes the normal condition (norm), death (deth), accidental destruction (ache) and microorganisimal contamination of cultures (cons)
SOMAINTGR	C	8	Somaclonal integrity is expressed by indicating the culture generation on which the test has been conducted and the methodology (RFLP/RAPD) employed. The result of the test is denoted by a positive or negative sign
CULTEXCHNG	C	12	Culture exchange to other research institute or commercial compare (Y or N) is designated by abbreviated code followed by the mode of exchange, i.e. test tube plant (TP) or microtuber (MT)
EVENTCOM	C	50	Comments on events include observations made by the curator of the culture collection followed by name of the curator

_Field name ending with a dash (-) denotes that depending on the need, multiple fields can be constructed.

_C=character; N=numeric; D=date.

Table 2. Typical records of an accession in the databases of in vitro collection of potato germplasm

Static record		Semi static record		Changeable record
Field	Entry	Field	Entry	Field	Entry
CPNO	3368	IVRES	2	GENERNO	1
CULTIVNM	CIP 382178.14	REVIVAL	80	INCBDTSET1	09J28/94
ORIGIN	PER	IVMICPP	10/120	INCBDTSET2	10/12/94
SPPGR	Tuberosum	STORMICR	3	REVDTSET1	07/28/95
PHYTOSANNO	PS-215-94	NOSET	2	REVDTSET2	08/12//95
CULTNO	360	NOCTSET1	8	MICROPROP	Y 50F
PSSRTCOM	Re:08/229; adapt-short; A. Landeu	NOCTSET2	8	EVENTNM	Norm
		LOCATSET1	252	PLANTHLTH	N
		LOCATSET2	322	SOMAINTGR	1 RFLP
		CULTCOM	C: violet D: 2m M: 60d P.S.Naik	CULTEXCHNG	N
				EVENTCOM	Microplant etiolation, root no. Iess; D. Sarkar

In addition to default commands, a number of application programmes have been developed in the dBASE III PLUS system. The menu-driven main programme consists of three subprogrammes with suitable help modes.

CHECKMASTER: This programme is used for checking the mistakes in database files and was developed because some types of data are prone to typing mistakes during input. It consists of a series of subprogrammes, one for each field in the model. The programme first asks the user to select a field and then scans all records for that particular field. If any mistake is detected, it prompts the user to correct it. For example, there are four possible entries-for the EVENTNM field in the in vitro events record. These are "norm", "cont", "acde" and "deth", which describe normal condition, contamination, accidental destruction and death of the cultures, respectively. Entries other than these four are treated as illegal. The subprogramme for EVENTNM field is given in Programme 1.

EDITMASTER: This programme is used for editing or updating the records in database files. Given the choice of a particular record number or culture number or CP number, the programme prompts the user to edit or update. Each field of a record is displayed sequentially. Until a legal expression is entered, the next field of the record is not displayed for editing or updating. After all of the fields in a record are edited, the programme asks for the next record number or culture number or CP number. A transcript of the programme, which edits files based on record number, is presented in Programme 2.

DATEMASTER: As has been pointed out earlier, the scheduling of the regeneration date for cultures on the basis of incubation date is most important in the management of the in vitro repository. Usually, the regeneration date of any accession is determined according to in vitro notes specific for that accession. DATEMASTER calculates in vitro life expectancy of each accession from in vitro notes (see in vitro conservation model), and automatically validates the regeneration date (Programme 3). However, unwarranted developments in some in vitro events may require resetting incubation as well as regeneration dates. Using DATEMASTER, automatic validation of incubation and regeneration dates is possible following corresponding changes in the in vitro events data fields. For example, if a number of cultures (accessions) become contaminated, the EVENTNM fields of those cultures in the in vitro events database file are immediately edited. Subsequently, those cultures are revived from either a duplicate set of current generation or, in extreme case, from the materials conserved in conventional genebank. DATEMASTER asks for the new incubation date of these new cultures and sets the regeneration date accordingly (Programme 4). This greatly reduces the burden of time-consuming editing efforts. We have also observed that the input of data to the database is very prone to typing mistakes. Frequently due to typing error, regeneration date precedes incubation date. This programme finds the records with errors and validates the regeneration dates in agreement with incubation dates.

Programme 1. A CHECK MASTER subprogramme * This subprogramme corrects the EVENTNM field in the vitro events database file CLEAR SET TALK OFF TEXT : *Help mode ENDTEXT mcheck=" " DO WHILE .NOT . mcheck $ "YN" : *Option loop for checking eventnm field ENDDO USE invtevnt _The in vitro events database file SET FIELD TO; eventnm _Event name field DO WHILE .NOT. EOF() _Checking all records SET FILTER TO; eventnm<>"norm".AND.eventnm<>"cont".AND.eventnm<>"acde".AND.; eventnm<>"deth" GO TOP EDIT SKIP _Skip a new record ENDDO SET TALK ON USE

- Fig. 1. The relationship among different database files in the in vitro conservation management system (the files are indexed on the underlined fields).

Programme 2. An EDITMASTER subprogramme * This subprogramme edits/updates records in the in vitro events database file CLEAR SET TALK OFF TEXT : _Help mode ENDTEXT medit=" " DO WHILE.NOT.medit $ "N" : _Option loop for editing records ENDDO CLEAR USE invtevnt _The in vitro events database file row=20 columnn=25 nextrecord=" " DO WHILE.NOT.next record $ "N" @ row, column SAY "Next record:" GET nextrecord PlCTURE "!" READ CLEAR IF nextrecord="Y" STORE RECNO() TO recnbr @ 18,25 SAY "Enter record number:":GET cpno PICTURE "9999" READ GO recnbr CLEAR @ 4,25 SAY "CPNO:" GET cpno PICTURE "9999" RANGE 1000, 8668 READ @ 8,25 SAY "EVENTNM: GET eventnm PICTURE "AAAA" READ DO WHILE .NOT. eventnm $ "normcontacdedeth" eventnm="AAAA" @ 8,34 eventnm READ ENDDO : _A series of loops for other fields ENDIF ENDDO SET TALK ON USE

In our laboratory, the incubation date of any culture is updated in the database using EDITMASTER on the same day the culture has been subcultured or revived on fresh minimal growth medium. EDITMASTER has a provision for not accepting any date for INCBDTSET_ field other than current date. This provision eliminates the possibility of entering the wrong incubation date.

Discussion

Flexible data storage and retrieval procedures are essential for the development of any genebank information system(Yndgaard 1982). In this model, any information stored in the databases can be easily retrieved interactively. Any type of data can be accessed through the relationship defined in the model. The data about in vitro events meet the main requirements of monitoring the in vitro cultures. Major in vitro events are interactively related to in vitro notes. In vitro life expectancy of cultures helps to schedule the regeneration date of each accession. Furthermore, the meaning of any abbreviated term used in the model can be accessed by displaying the dictionary at any time during an interactive session with the database.

Programme 3. A DATEMASTER subprogramme. * This subprogramme assets in automatic validation of regeneration date on the basis of in vitro notes SET TALK OFF SELECT A USE invent *The in vitro events database file SELECT B USE invtnote *The in vitro notes database file SELECT A DO WHILE .NOT. EOF *Scheduling of regeneration [revival] dates for all the records SELECT B STORE revival TO mrevival STORE ivres TO mivres invtflex=(mrevival/mivres)+365 *invtflex is in vitro life expectancy of cultures SKIP STORE invtflex TO minvtlflex minvtlflex=INT(invtlflex) SELECT A STORE incbdset TO mincbdset STORE revdtset: TO mrevdtset mrevdtset=mincbdtset+minvtlflex REPLACE revdtset WITH mrevdtset SKIP ENDDO SET TALK ON USE

To make the model more flexible and user-friendly, different application programmes have been developed. These can be used by anyone who has no prior computer experience. These programmes can update the records interactively with other information stored in the model without using dBASE III PLUS commands. These application programmes also prevent unwanted trespassing of data. They safeguard against errors in data maintenance, which is one of the most important considerations in any in vitro germplasm collection (Dodds 1988b). In the future, modifications in the in vitro conservation methodology may produce different management requirements. Because of its flexible nature, the model can be adapted to such changes.

In an in vitro repository, hundreds of cultures are revived or subcultured every month. This generates a huge volume of secondary data that can be managed using these programmes. To gain the advantages offered by these programmer, the voluminous amount of information must be handled carefully and data entry must be systematic and cautious. Wrong information fed to the databases may upset the in vitro management of germplasm accessions. Though the system has adequate provisions for safeguarding against illegal data entries, it can not discriminate among legal expressions. To avoid this, the system should be customized so that it will first ask for the record or culture or CP numbers that are to be updated, and then prompt the user to select the field and input the expression accordingly. Thus, for identical data entries for a large number of records, the user would make an entry only one time. This will not only minimize the chances of input error, but also relieve the users from time-consuming editing efforts. We are trying to incorporate this aspect into our programmed.

Programme 4. A DATEMASTER subprogramme. * This subprogramme updates/edit date entry in the in vitro events database file SET TALK OFF TEXT : *Help mode ENDTEXT update=" " DO WHILE .NOT. update $ "YN" : *Option loop for updating dates ENDO CLEAR USE invtevnt *The in vitro events database file indate=CTOD(" / / ") STORE indate+365 TO mdate @ 10,20 SAY Enter new incubation date: "GET indate PICTURE (" / / ") READ DO WHILE .NOT. EOF() *Updating all records IF eventnm="cont" REPLACE incbdtset WITH indate ENDIF IF incbdtset=indate REPLACE revdtset WITH mdate ENDIF IF revdtset<incbdtset REPLACE revdtset WITH mdate ENDIF : *Other IF..ENDIF clauses SKIP ENDDO SET TALK ON USE

Copies of the programme can be obtained from the Director, Central Potato Research institute, Shimla 171 001, India, for a modest handling charge.

Acknowledgements

The authors are thankful to Dr G.S. Shekhawat, Director, Central Potato Research institute for providing necessary facilities, and to Dr P.C. Gaur, Head, Division of Genetics & Plant Breeding and Dr N.P. Sukumaran, Head, Division of Crop Physiology & Biochemistry for critically reviewing the manuscript.

References

Chavez, R., W.M. Roca and J.T. Williams. 1988. The IBPGR-CIAT pilot in vitro genebank. Genome 30(Suppl. 1):488.

Clarke, P.A. and B. Caserio. 1979. A genealogical database for plant propagation records. Euphytica 28:785-792. dBASE III PLUS.1986. Programming with dBASE III PLUS.2. Ashton-Tate.

Dodds, J.H. 1988a. Review of in vitro propagation and maintenance of sweet potato germplasm. Pp. 185-192 in Exploration, maintenance and utilization of sweet potato genetic resources: Report of the first sweet potato planning conference 1987. International Potato Centre, Lima, Peru.

Dodds, J.H. 1988b. Status of the in vitro potato collection at CIP and new approaches for long term conservation. Pp. 79-87 in Strategies for the conservation of potato: Genetic Resources IV (XXIX Planning Conference 1987). International Potato Centre, Lima, Peru.

Jarret, ILL. 1988. The US sweet potato germplasm repository. Pp. In Exploration, maintenance and utilization of sweet potato genetic resources: Report of the first sweet potato planning conference 1987. International Potato Centre, Lima, Peru.

Simpson, M.J.A. and L.A. Withers. 1986. Characterization of plant genetic resources using isozyme eiectrophoresis: A guide to the literature. IBPGR, Rome, 120p

Stushnoff, C. and C.D. Fear.1985. The potential use of in vitro storage of temperate fruit germplasm: A status report. AGPG: IBPGR/ 85/213. IBPGR, Rome, 21p.

Westcott, R.J. 1981. Tissue culture storage of potato germplasm. 1. Minimal growth storage. Potato Res. 24:331-342.

Withers, L.A. 1989. In vitro conservation and germplasm utilization. Pp. 309-334 in The use of plant genetic resources (A.H. Brown, D.R. Marshall, O.H. Frankel and I T. Williams, eds.). Cambridge University Press, Cambridge.

Withers, L.A. 1991. Biotechnology and plant genetic resources conservation. Pp. 273-297 in Plant Genetic Resources. Conservation and Management: Concepts and Approaches (R.S. Paroda and R.K. Arora, eds.). IBPGR, New Delhi.

Withers, L.A., S.K. Wheelans and I T. Williams. 1990. In vitro conservation of crop germplasm and the IBPGR databases. Euphytica 45:9-22.

Yndgaard, F. 1982. A documentation system for Nordic genebank. IBPGR Plant Genetic Resources Newsletter 49:34-36.

Résumé

Utilisation des bases de données informatiques pour gérer une collection in vitro de matériel génétique de pomme de terre

Une base de données relationnelle a été mise en place pour gérer une collection active in vitro de matériel génétique de pomme de terre. Elle remplace le système de gestion de bases de données (pour une banque de gènes traditionnelle) utilisé pour gérer une collection in vitro. Dans le modèle décrit, les données sont enregistrées de manière à ce que la date de régénération de n'importe queue accession puisse être fixée de manière interactive en accord avec d'autres informations. Pour la modélisation, le progiciel dBASE III PLUS a été utilisé. Un certain nombre de programmes d'application écrits dans un environnement dBASE III PLUS aide au recouvrement interactif et à la mise à jour des données. Des détails de chaque entrée sont fournis moyennant le rapport défini dans le modèle. L'utilisation de ce modèle convivial ne nécessite pas beaucoup de connaissances en informatique. Etant de nature flexible, le système peut être adapté aux besoins changeants de la gestion de matériel génétique de pomme de terre in vitro.

Resumen

Utilización de bases electrónicas de dates para la ordenación de una colección in vitro de germoplasma de la papa

Se ha creado una base de dates relacional pare una colección active in vitro de germoplasma de la papa. Sustituye a un sistema de ordenación de bases de datos (pare bancos convencionales de genes) utilizada pare una colección in vitro. En el modelo aquí presentado se registrar los datos de suerte que puedan programarse interactivamente los dates de regeneración de cualquier accesión de acuerdo con otros dates. Para la modelización, se utiliza el conjunto de programas dBASE III PLUS. Varios programas de aplicación escritos en un entorno dBASE III PLUS ayudan a la recuperación interactiva de los datos y a su actualización. A través de la relación definida en el modelo puede tenerse acceso a detalles de cualquier registro. Este modelo de fácil utilización puede ser empleado por personas sin grandes conocimientos de informática. El carácter flexible del sistema lo trace adaptable a las diversas necesidades pare el manejo de germoplasma de la papa in vitro.

Genetic resources of Podophyllum hexandrum Royle, an endangered medicinal species from Garhwal Himalaya, India

S.K. Bhadula¹, Anoop Singh¹, H. Lata¹, C.P. Kunlyal¹ and A.N. Purohit²

¹ G.B. Pant institute of Himalayan Environment and Development, Garhwal Unit, PO Box 92, Srinagar (Garhwal) 246 174, U.P., India

² High Altitude Plant Physiology Research Centre, PO Box 14, Srinagar (Garhwal) 246174, lop., India

Summary

Podophyllum hexandrum Royle is a herbaceous and rhizomatous species of great medicinal importance that has endangered status in India. It is distributed in very restricted pockets in the Himalayan zone at altitudes ranging from 2000 to 4000 m a.s.l. Several lignans have been isolated from its rhizomes, the most important being podophyllotoxin which has cytotoxic and antitumour properties and has been used in the treatment of certain forms of cancer. In the recent past, the frequency of this species in nature has declined considerably because of exploitation to meet the ever-increasing demand of pharmaceutical companies. In the natural habitat, seed germination and seedling establishment are very poor and propagation is mostly through rhizomes. Because the species is already endangered, and exploitation of its underground parts continues to exceed the rate of natural regeneration, it needs immediate attention for conservation. Studies of its population biology and genetic diversity are important for successful development of conservation strategies. This paper describes the characteristics of various populations of P. hexandrum collected from Garhwal Himalaya and presents future conservation strategies for this important species.

Materials and methods

Field surveys were conducted in different alpine and subalpine ranges of Garhwal Himalaya, namely, Kedarnath, Tungnath, Chopta, Valley of Flowers, Ghangaria and Dayara (Fig. 1) to determine the distribution pattern and population polymorphism of P. hexandrum. Demographic data and representative herbarium specimens were collected from all locations. Propagules (underground parts and seeds) of all populations were collected and planted under nethouse conditions at Srinagar (550 m) and Tungnath (3600 m). The morphological variations among and within natural populations were also recorded.

Seed germination was tested under laboratory conditions. Seeds from the freshly collected berries were extracted and washed thoroughly in distilled water to remove the fruit pulp, which seems to inhibit germination. The seeds were air dried at room temperature, stored at 40C and used for germination studies within a month after collection. For each population, 25 seeds were soaked in sterile distilled water and sown in petri dishes with two layers of moist Whatman filter paper No. 1. The seeds were kept at various light and temperature conditions. In some cases where no germination was observed after a long period, the seed coats from the micropylar side were removed to enhance germination.

The seeds of individual populations and their morphological variants were analyzed for esterase isozyme and protein patterns to determine whether there are biochemical variations among and within populations besides apparent morphological variations. The methods described by Bhadula and Sawhney (1987) and Sawhney and Bhadula (1987) were followed for esterase and protein-profile analysis respectively.

Results and discussion

Distribution and population size

The regions of Garhwal Himalaya, India, surveyed from 1993 to 1995 during studies of the distribution pattern of P. hexandrum are shown in Fig. 1. In the subalpine forests, P. hexandrum was found as an under-canopy plant, whereas in high altitudes, it was found near boulders and sometimes in open meadows. Compared with several other endangered or threatened species from this region, e.g. Nardostachys jatamansi, Picrorhiza kurrooa and Aconitum atrox the natural population of P. hexandrum were distributed in very restricted and small pockets. As Table 1 shows, population sizes in different localities varied from 40 plants (Valley of Flowers) to 700 plants (Dayara). Thus, P. hexandrum exhibits a small population size in nature. It is worth mentioning that the number of plants in all of these populations is decreasing and some of the populations that we have observed since 1982 in the Kedarnath and Tungnath regions have virtually disappeared, mainly due to anthropogenic activities and overexploitation.

- Fig. 1. The regions of Garhwal Himalaya, India, surveyed for the study of the distribution pattern of P. hexandrum.

Table 1. Distribution pattern of Podophyllum hexandrum in Garhwal Himalaya

Region	Altitude (m.a.s.l.)	Climatic zone	Number of morphological variants within no. of population plants	Population size (total)
Kedarnath	3700	Alpine	6	200
Tungnath	3300	Alpine	2	70
Valley of Flowers	3000	Timberline	4	40
Chopta	2800	Subalpine	5	60
Ghangaria	3000	Subalpine	5	400
Dayara	2300	Subalpine	4	700

Inter- and intra-population variations

Chatterjee (1952, 1953) reported plants having 35-40 cm stem length with generally two and occasionally three leaves 10-20 cm long and 20-25 cm wide. Our survey indicates a much wider range in plant height and leaf size. With some exceptionally large forms, plant height generally varied from 20 to 50 cm and leaf size varied from 7 to 25 cm in length and 20 to 40 cm in width. As a general rule, plant height and leaf size decreased with increasing altitude. This is a common phenomenon and has been reported in other species (Clausen et al. 1940; Billings and Mooney 1968; Bhadula and Purohit 1994).

Our regular observations of these areas revealed that the plants growing in different locations in Garhwal Himalaya show considerable morphological variations in plant height, leaf characteristics, fruit weight, seed weight and colour, and other traits. Therefore, the plants of each locality were considered to represent a different population. Within a single population, however, morphological variations in plant height as well as in the number, shape, size, robing and pigmentation of leaves were observed. Differences in fruit weight and seed weight and colour were also seen. Thus, because of intra-population variations, the germplasm of individual morphological variants of each population was collected and analyzed separately. Plants of a population were classified into different groups based on the shape, size, robing and number of leaves (three, two or one leaf in fruiting stage) and on the basis of seed colour. Thus, six morphological variants in the Kedarnath population, five each in the Chopta and Ghangaria populations, four each in the Valley of Flowers and Dayara and two in the population from Tungnath were identified. Within each population, individual plants differing in leaf characteristics as well as in seed colour ranging from black, brown to pink were observed. Fig.2 shows some of the morphological variations in leaf characteristics of P. hexandrum populations and their individual plants collected from Chopta range (A-E) and Ghangaria (F-I).

- Fig.2. Inter- and intra-population leaf polymorphism in P. hexandrum: A-E are leaves of five variants from the Chopta population; F is a giant form from Ghangaria; G-l are three different leaf forms (in fruiting stage) from the Ghangaria population.

Biochemical studies indicate that there are considerable inter- and intra-population variations in esterase isozyme and polypeptide patterns. In particular, esterase isozymes are excellent markers of different populations and in most cases, intra-population variants as well.

In general, fruit weight decreased and the number of seeds per fruit increased with increasing altitude. Also, variation was observed in seed weight and colour. The ecophysiological significance of this variation is not clear. Further studies, including chemical analysis of seed coat components, should help with understanding the seed coat's relationship to germination or adaptational mechanisms with respect to particular environmental conditions.

Seed germination

Studies on seed germination showed that seeds collected from alpine areas germinated best at 25°C temperature, 16/8 hours light/dark. Under such conditions, 90-100% of the alpine seeds germinated. However, different populations and variants of the same population collected from alpine areas differ in the time needed to initiate and complete germination. The seeds of timberline and subalpine populations showed very poor and delayed germination (sometimes over 200 days). Scarification of seeds was found to enhance germination of such seeds. However, a maximum of 27% seed germination was observed even after scarification. Attempts to overcome constraints in the germination of subalpine seeds are in progress. Seed germination behaviour of individual seed types should provide useful information about the relationship between seed coat colour and viability. This work has already been initiated in our laboratory

Seed germination in this species is very. poor under natural conditions. The main reasons seem to be a postharvest ripening requirement (Nautiyal et al. 1987) and a hard seed coat, especially in temperate populations where germination takes several months. It was interesting to note that whereas the seeds of most (but not all) temperate populations showed hypogeal germination, all of the alpine populations undergo epigeal germination. The adaptive significance of such variation in germination behaviour of alpine and temperate populations is not clear.

An interesting phenomenon of pseudomonocotyly in this species has been reported by Purohit and Nautiyal (1988). We also have seen fused cotyledons which appear like a single cotyledon in some germinating seeds, but such observations are rare.

It is worth mentioning that the morphological and biochemical variations do not seem to be due only to differences in growing conditions (environmental) of the natural habitat. These differences were maintained when plants from various locations were grown under similar environmental conditions at Srinagar (550 m altitude, under nethouse conditions). However, plants grown at this altitude did not look exactly like those in the natural habitat. Studies on phenotypic plasticity might elucidate the advantage or adaptive significance of particular characters with respect to certain environmental conditions. A germplasm collection (seed stock and plants of various populations) is currently being maintained in our laboratory and studies on its genetic diversity are being conducted.

In general, rare or geographically restricted species are known to possess a low level of genetic diversity (Kress et al. 1994). Although it may be too early to draw a conclusion, our results indicate that a fairly high level of genetic diversity exists in P. hexandrum. From the above mentioned variations, it is clear that this species represents P. hexandrum complex. It should also be noted that habitat destruction due to overgrazing, environmental degradation and changing ecological conditions may be considered to be contributing factors for the species' endangered status. The mass-scale uprooting of this species to meet the ever increasing demand for podophyllotoxin is the major cause of its de creasing population size and therefore, the species may be considered a 'man-made endangered species'. Although the species exhibits a high degree of natural population polymorphism and does not seem to be under genetic threat, if its overexploitation continues at the present rate, it will certainly face extinction in the near future. For conservation biologists, to lose even a single population would mean losing biodiversity of the species; therefore, immediate conservation measures must be taken to preserve this economically important species.

Conservation strategies

The over-exploitation of P. hexandrum from the Himalayan region has posed a potential threat to this species. Thus, it is important to initiate attempts for its in situ conservation. There are, however several problems, e.g. the rate of exploitation far exceeds the rate of natural regeneration, seed germination and seedling survival under natural habitat are very poor, overgrazing and illegal exploitation. Nevertheless, conservation attempts should be initiated immediately. Since this species has a high cash value and commercial demand, cultivation packages need to be developed so that farmers in high-altitude areas can grow it. Once the commercial demand is fulfilled using cultivated raw material, the pressure on the natural habitat automatically will be reduced and the natural regeneration will improve. Additional survey work is needed to discover and study more populations in other localities and the germplasm of individual populations must be conserved.

We have started a demonstration garden for the cultivation of this species in the Garhwal Himalayan region. We have also begun reintroducing rhizome cuttings and laboratory-tested viable seeds in nature to replenish the depleted stocks. Multiplication by vegetative propagation using rhizome cuttings treated with various growth regulators has been successfully achieved in our laboratory. In addition to in situ conservation methods, ex situ conservation strategies need to be developed and used for the species' mass-scale multiplication and reintroduction into the natural habitat. Callus cultures derived from leaves have also been established and attempts to initiate multiple shoots are in progress. The application of biotechnology to improve the active ingredients for cultivation of desired populations should also help conservation efforts.

Acknowledgments

The financial assistance from the Department of Biotechnology, Government of India, is gratefully acknowledged. The authors are thankful to S. Nautiyal and R. Shekar for field assistance.

References

Bhadula, S.K. and A.N. Purohit. 1994. Adaptational strategies of plants at high altitudes and future prospects for the conservation of biodiversity. Adv. Plant Sci. Res. 1:1-24.

Bhadula, S.K. and V.K. Sawhney. 1987. Esterase activity and isozymes during the ontogeny of stamens of male fertile Lycopersicon esculentum Mill, a male sterile stamen less-2 mutant and low temperature-reverted mutant. Plant Sci. 52:187-194.

Billings, W.D. and H.A. Mooney. 1968. The ecology of arctic and MacRae, W.D. and G.H.N. Towers. 1984. Biological activities of alpine plants. Biol. Rev. 43:481-529

Chatterjee, R. 1952. Indian Podophyllum. Economic Bot. 6:342-354

Chatterjee, R. 1953. Rec. Bot. Sur. Indict 16:43-51

Clausen, J., D.D. Keck and W.M. Hiesey. 1940. Experimental studies on the nature of species. I. Effect of varied environments of Western North American plants. Carnegie inst. Washington Publ. No. 520, pp. 1-452.

Holthuis, J.J.M. 1988. Etoposide and teniposide. Bioanalysis, metabolism and clinical pharmokinetics. Pharm. Weekbl. (Sci) 10:101-116.

Kress, W.J., G.D. Maddox and C.S. Roesel. 1994. Genetic variation and protection priorities in Ptilimnium nodosum (Apiaceae), an endangered plant of the Eastern United States. Conserv. Biol 8:271-276.

MacRae, W.D. and G.H.N. Towers. 1984. Biological activities of lignans. Phytochemistry 23:1207-1220.

Nautyial, M.C., A.S. Rawait, S.K. Bhadula and A.N. Purohit. 1987. Seed germination in Podophyllum hexandrum. Seed Res. 15:206-209

Purohit, A.N. and M.C. Nautiyal. 1988. Inhibitory effect of cotyledons on plumule development in two alpine rosettes. Can. J. Bot. 66:205-206.

Richter, A.U. Strausfeld and R. Knippers. 1987. Effects of VM-26 (teniposide), a specific inhibitor of type II topoisomerase, on SV-40 DNA replication in vivo. Nucleic Acids res. 15:3455-3468.

Sawhney, V.K. and S.K. Bhadula. 1987. Characterization and temperature regulation of soluble proteins of male sterile tomato mutant. Biochem. Genet. 25:717-728.

Résumé

Ressources génétiques de Podophyllum hexandrum Royle, espèce médicinale menacée d'extinction dans l'Himalaya de Gharwal, Inde

Podophyllum hexandrum Royle est une espèce herbacée et rhizomateuse de grande importance médicinale qui est menacée d'extinction en Inde. Elle est répartie dans des zones très limitées de l'Himalaya à des altitudes allant de 2000 à 4000 m au-dessus du niveau de la men Plusieurs substances ont été isolées de ses rhizomes, la plus importante étant la podophyllotoxine qui a des propriétés cytotoxiques et anticancéreuses et a été utilisée pour traiter certaines formes de cancer. Ces derniers temps, la présence de cette espèce dans la nature a considérablement diminué en raison d'une exploitation intense due à la demande de plus en plus forte de la part des industries pharmaceutiques. Dans son aire naturelle la germination des graines et l'établissement des plants vent très faibles et la multiplication se fait principalement par les rhizomes. Du fait que cette espèce est déjà menacée et que l'exploitation de ses parties souterraines continue de dépasser le taux de régénération naturelle, il est urgent de s'occuper de sa conservation. Il importe d'étudier la biologic et la diversité génétique de sa population afin d'élaborer avec succès des stratégies de conservation. Cet article décrit les caractéristiques des diverges populations de P. hexandrum récoltées dans l'Himalaya de Gharwal et présente des stratégies de conservation futures pour cette importante espèce.

Resumen

Recursos genéticos de Podophyllum hexandrum Royle, una especie medicinal amenazada de extinción del Himalaya de Garkwal, en la india

El Podophyllum hexandrum Royle es una especie herbácea y rizomatosa de gran importancia medicinal que está amenazada de extinción en la india. Se halla distribuida en bolsas muy reducidas de la zona de los Himalayas a altitudes que van de los 2000 a los 4000 metros sobre el nivel del mar. Se han aislado varios lignanos de sus rizomas, siendo el más importante la podofilotoxina, que tiene propiedades citotóxicas y antitumorales y que se ha empleado en el tratamiento de algunas formas de cáncer. Ultimamente, la frecuencia de esta especie en la naturaleza ha bajado mucho debido a su explotación pare satisfacer una demanda cada vez mayor por parte de las compañías farmacéuticas. En su hábitat natural, la germinación de la semilla y el arraigo de las plántulas son muy deficientes y la propagación se realiza casi siempre a través de los rizomas. Debido a que esta especie corre ya peligro de extinción, y a que la explotación de sus partes subterráneas sigue superando el ritmo de regeneración natural, es menester prestar inmediata atención a su conservación. Para un buen desarrollo de las estrategias de conservación es importante realizar estudios sobre su biología de población y su diversidad genética. En este trabajo se describen las características de varias poblaciones de P. hexandrum recogidas en la zona del Himalaya de Garhwal y se exponen pare el futuro estrategias de conservación de una especie tan importante.

Genetic contamination by windborne pollen in germplasm-regeneration plots of smooth bromegrass

R.C. Johnson¹, V.L. Bradley¹ and R.P. Knowles²

¹ USDA-ARS Plant Germplasm and Testing Unit, Washington State University, Pullman, WA 99164-6402, USA; Email: [email protected]

² Agriculture Canada, 107 Science Crescent, Saskatoon, Saskatchewan, Canada

Summary

Hybridization among cross-pollinating accessions is a serious concern for seed-regeneration efforts at germplasm-management sites. Estimates of genetic contamination from windborne pollen in grass-regeneration plots at the Western Regional Plant introduction Station (WRPIS), Pullman, WA, USA, were made using strains of dominant pubescent and recessive glabrous smooth bromegrass (Bromus inermis Leyss.). In 1988 and 1989, contamination measured from a central pubescent row to glabrous rows spaced 1.5 m apart averaged 17% at 1.5 m, but only 0.5% at 9 m distance. In 1991 and 1992 experiments, contamination in plots adjacent and parallel to pubescent plots averaged 15.7% at 3 m distance. Contamination in plots located 1.5 m from the ends or 2.1 m diagonally from pubescent plots averaged only 4.8%, but ranged from 0.7% to 13.7%. In 1995, bromegrass marker plots integrated into WRPIS seed-regeneration nurseries at two locations resulted in average contamination of 4.2% at distances between 22 and 27 m. Distance and the abundance of non-contaminating pollen appeared to determine the extent of contamination. For regeneration plots at WRPIS, isolation distances of 22 to 27 m resulted in a relatively low level of contamination.

Introduction

The USDA-ARS Western Regional Plant introduction Station (WRPIS), Pullman, WA, USA, maintains approximately 12 000 accessions of forage and turf grass germplasm, representing more than 800 species, that are distributed to researchers worldwide. The original seed samples of these largely heterogenetic, wind-pollinated species received at WRPIS are usually of insufficient quantity for distribution to scientists. Seed quality may also be poor and therefore not optimal for distribution or long-term storage. For these reasons an initial seed regeneration is usually required before accessions can be distributed. As seed stocks or seed viability diminish over time, additional seed regeneration is required. It is critical that the genetic profiles of regenerated samples resemble those of original samples as closely as possible, but cross-fertilization by windborne pollen can be a major source of genetic mixing during regeneration of wind-pollinated species. Previous work has focused on isolation distances for maintaining cultivar purity of wind-pollinated grasses in large plots or fields. Jones and Newell (1946) used petroleum jelly-covered slides to trap pollen at various distances and heights and found a rapid decrease in pollen loads with distance from the source. Nevertheless, 1 %, of the pollen grains trapped at the source were found at a distance of 300 m, which was equivalent to thousands of pollen grains per square metre. Although Jones and Newell (1946) estimated pollen dispersal, they did not measure actual hybridization between plants. Griffiths (1950), working with perennial ryegrass (Lolium perenne L.) and an anthocyanin genetic marker, found that contamination associated with windborne pollen decreased rapidly with distance, especially as increasing quantities of non-contaminating pollen became available. Nevertheless, some contamination did occur even at distances up to 900 m. Knowles and Ghosh (1968) estimated genetic contamination by pollen in smooth bromegrass (Bromus inermis Leyss.) with a dominant yellow-leaved mutant marker. Average contamination of plots was 9.6 1.0% and 0.2% for isolation distances of 1, 61, and 183 m, respectively. Copeland and Hardin (1970) estimated outcrossing rates in Lolium with a dominant fluorescent marker in ryegrass roots. For plants spaced 3 m apart, outcrossing diminished from 36% at 3 m distance from the pollen source to 4% at 30 m distance. In studies with reed canarygrass (Phalaris arundinacea L.), crested wheatgrass [Agropyron cristatum (L.) Gaertner], and smooth bromegrass, Knowles (1983) found average contamination of plots 50 m apart to be less than 5%

Results from the work cited above indicate large isolation distances (,,200 m) or regeneration in cages or chambers (Tyler 1982) may be necessary to eliminate genetic contamination from pollen in outcrossing grasses. Germplasm managers, however, are often faced with a backlog of accessions of the same species in need of regeneration. Sufficient land for large isolation distances, or the resources for large-scale chamber or cage regeneration, may not be available. Estimates of genetic contamination in small plots designed for seed regeneration of wind-pollinated grass accessions under field conditions are needed. Our objective was to estimate genetic contamination by pollen in smooth bromegrass seed-regeneration plots at the WRPIS.

Materials and methods

Knowles (1980) used mass selection to develop pubescent and glabrous smooth bromegrass strains and showed that the pubescent trait was partially dominant. Continued development of these strains has produced pubescent stocks with nearly complete dominance over glabrous stocks. Fertilization of glabrous plants by pollen from dominant pubescent strains results in seeds that produce pubescent seedlings.

1988-89 tests

In an initial test, seeds of the pubescent strain S-8753 (PI 557 438) and of the glabrous strain S-9179 (PI 557 439) were planted in flats under greenhouse conditions and transplanted as described by Johnson and Bassett (1991). Seedlings that emerged were thinned to one per pot, determined to be true to type (glabrous or pubescent) and maintained in the greenhouse for about 1 month. For transplanting, plants were placed in a furrow approximately 0.25 m wide and 0.1 m deep made by a V-blade attached to a lawn tractor. Each plot consisted of 20 plants spaced 0.5 m apart within each row with 1.5 m between rows. Six east-west rows of the glabrous strain were established on each side of a single, centrally placed pubescent row. Transplanting was completed on 15 April 1988. Plots were irrigated each week from late May until early July when irrigation was increased to twice weekly until harvest. At harvest, panicles in each row were cut with a hand sickle, placed in paper bags, dried, threshed and cleaned. Thus, seeds from plants within a plot u ere bulked in this and subsequent tests for estimates of average plot contamination. Seeds produced in 1988 and in 1989 were used to grow seedlings under greenhouse conditions for counts of pubescence. Leaves of at least 200 seedlings at the two- to three-leaf stage were examined, and percent pubescence calculated.

1991-92 tests

In 1991, replicated plots were established to test how row direction, a single border row of cereal rye (Secale cereale L.) and plot position affected pollen contamination. Plots were arranged in north-south and east-west directions at the Central Ferry Research Farm, WA. Each plot consisted of 60 transplants arranged in rows of 30 plants spaced 0.3 m apart and with 0.3 m between plants within rows. A central pubescent plot surrounded by glabrous plots was established as shown in Figs. 1 and 2. The experiment was a randomized complete block with two replications in a splitplot arrangement. The main plots were row direction (east-west or north-south) and in 1992 the presence or absence of a cereal rye barrier (not shown in Fig. 2). Subplots were plot position within row direction and the cereal rye barrier treatments. The blocks were separated by approximately 625 m and the subplots within a block by at least 50 m.

The plants were established and maintained as described for the 1988-89 test, except that the glabrous strain S-9077 (PI 576 975) was used instead of S-9179. It was found by bagging panicles in greenhouse tests at Saskatoon, Saskatchewan, Canada, that S-9077 had essentially no self-fertility. There was, however, a potential for 30% self-fertility in the glabrous strain S-9179, which could lead to underestimation of pollen contamination.

East-west rows	1991 a)
3.0 (9.9)	15.7 (23.3)	2.7 (9.5)
2.2 (8.5)	97.7 (81.3)	2.2 (8.5)
3.2 (10.3)	17.5 (24.7)	2.7 (9.5)
North-south rows	1991 b)
6.5 (14.8)	12.7 (20.9)	7.7 (16.1)
13.7 (21.7)	98.0 (81.9)	60 (14.2)
3.5 (10.8)	10.5 (18.9)	3.5 (10.8)

Fig. 1. Percent genetic contamination by pollen in smooth bromegrass field plots in 1991 with different row directions. The central plot, a dominant pubescent strain (dotted line), was surrounded by plots of a glabrous recessive strain (solid lines). The number in parentheses is the percentage data subjected to an arc-sine transformation in degrees used for statistical comparisons among plots. The LSD₀₀₅ for comparisons among plot positions was 6.6 degrees. Each plot was 9 m long and spaced 3 m apart with 1.5 m between plot ends.

East-west rows	1992 a)
2.7 (9.5)	21.2 (27.3)	5.0 (12.9)
5.7 (13.8)	98.7 (83.5)	2.5 (9.1)
4.0 (11.5)	18.5(25.5)	3.2(10.3)
North-south rows	1992 b)
9,5 (17.9)	19.7 (26.3)	10.5 (18.9)
6.7 (15.0)	98 2 (82 3)	5.7 (13.8)
2.5 (9.1)	10.2 (18.6)	0.7 (4 8)

Fig. 2. Percent genetic contamination by pollen in smooth bromegrass field plots in 1992 with different row directions. The central plot, a dominant pubescent strain (dotted line), was surrounded by plots of a glabrous recessive strain (solid lines). The number in parentheses is the percentage data subjected to an arc-sine transformation in degrees used for statistical comparisons among plots. The LSD₀₀₅ for comparisons among plot positions was 8.6 degrees. Each plot was 9 m long and spaced 3 m apart with 1.5 m between plot ends.

Sufficient seed was collected from smooth bromegrass plots in the summer of 1991 for estimates of pollen contamination. Because there was no barrier row in the summer of 1991, only plot direction (east-west or north-south rows) was analyzed as main plots, with the position effects as the subplots. Panicles from each plot were harvested in bulk for estimates of pollen contamination as described for the 1988-89 test. Seeds from each plot were used to grow seedlings in a greenhouse for counts of pubescence. Work in 1988-89 showed that there was little difference in percent contamination between counts of 200 and 100 seedlings, so pubescent seedlings were determined on 100 plants per plot.

On 11 Oct 1991 and in designated main plots, a continuous single row of winter annual cereal rye (not shown in Fig. 2) was seeded at 50 seeds/m on both sides of, and parallel with, the pubescent central plot. There were 1.5 m between the cereal rye and the bromegrass marker plots. During plant development in the spring the height of the cereal rye plants was always greater than the height of the bromegrass plants. Seeds from the bromegrass plots were harvested 14-15 July 1992 as described for the 198889 tests. Seedings were grown in a greenhouse and percent pubescent seedlings determined on 100 plants per plot.

1994-95 tests

Based on 1988-89 and 1991-92 results that suggested substantial reductions in pollen contamination with relatively small isolation distances, grass-regeneration nurseries at WRPIS were configured as shown in Figs. 3 and 4. These nurseries included regeneration plots of hundreds of accessions and numerous grass species. Accessions were arranged in strips four (Fig. 3) or five plots wide (Fig. 4). Each accession within a strip, represented by a single line in the figures, was established at a minimum distance of 22 m from a potentially hybridizing accession. Along with surrounding plots, plots of the pubescent bromegrass strain S-8753 (P1 557 438) and glabrous strain S-9077 (P1 576 975) were transplanted at Central Ferry on 21 April 1994 and at Pullman on 4 May 1994. At Central Ferry (Fig. 3) bromegrass plots were replicated three times and at Pullman (Fig. 4) they were replicated twice in randomized complete blocks. Each block was isolated from other bromegrass plants by at least 50 m. Individual accessions consisted of 60 transplants as described for 1991-92 tests. The area between the strips was planted with either wheat (Triticum aestivum L.) or barley (Hordeum vulgare). Thus isolation distance was established both within the strips, which consisted of individual accessions of various species, and across the cereal border. There were ten strips established at Central Ferry, each 90 m long, which were irrigated as described above. At Pullman, two strips each 275 m long were established and grown under dryland conditions.

- Fig 3. Percent genetic contamination by pollen in smooth bromegrass plots arranged within regeneration nurseries at Central Ferry, WA, 1995. The bold solid lines represent glabrous plots and the dotted line the pubescent plot. The number in parentheses is the percentage data subjected to an arc-sine transformation in degrees used for statistical comparisons among plots. The LSD₀₀₅ for comparing differences in plot position was 7.1 degrees. Plots were 9 m long and spaced 1.5 m apart

- Fig 4. Percent genetic contamination by pollen in smooth bromegrass plots arranged within regeneration nurseries at Pullman, WA, 1995. The bold solid lines represent glabrous plots and the dotted line the pubescent plot. The number in parenthesis is the percentage data subjected to an arc-sine transformation in degrees used for statistical comparisons among plots. The LSD₀₀₅ for comparing differences in plot position was 6.6 degrees. Plots were 9 m long and spaced 1.5 m apart.

There was no significant bromegrass seed production the first year of planting in 1994. Bromegrass marker plots were harvested in bulk on 13 July 1995 at Central Ferry and on 4 Aug 1995 at Pullman. Counts of pubescence were made as described above on 100 seedlings per plot.

The arc-sine transformation was calculated for each percent pubescence value (Steel and Torrie 1980) and analyses of variance conducted on transformed data for the 1991-92 and the 1994-95 experiments. The F-test for treatment effects was declared significant at P<0.05 and an LSD at P=0.05 calculated for multiple comparisons.

Results and discussion

1988-89 tests

In both 1988 and 1989, genetic contamination associated with windborne pollen attenuated rapidly with distance from the central contaminant row (Table 1). As expected, contamination was generally highest in rows adjacent to the central pubescent row (rows 6 and 8). In 1988 there was also considerable contamination in row 4 (15%), which was in the direction of the prevailing south to north wind. At 9 m distance, 1% or less contamination was observed both years. The rapid reduction in contamination with distance is consistent with results reported by Griffiths (1950) for perennial ryegrass, Knowles and Ghosh (1968) for bromegrass, and Copeland and Hardin (1970) for ryegrasses. The distances Griffiths (1950) and Knowles and Ghosh (1968) evaluated for genetic contamination by windborne pollen were much greater than those shown in Table 1, although those of Copeland and Hardin (1970) were similar.

The extent of pollen contamination in Table I might be underestimated because of the 30% potential for self-fertilization associated with the glabrous strain S-9179. Despite this, the high selfing rate observed in bagged panicles would likely be considerably lower under open-pollination, since pollen from other plants would be available for hybridization. Moreover, at distances greater than 4.5 m, pollen contamination was so low that even a 30% increase would result in a relatively small percentage increase. Even with the potential for some error in the estimates, the data showed that with relatively small isolation distances, low levels of genetic contamination may be possible during regeneration of wind-pollinated grasses.

1991-92 tests

In the 1991 experiment the row direction effect on percent contamination was significant at P=0.05. Seedlings from the eight glabrous rows situated east-west averaged 6.1% contamination, which was significantly less than the 8.0% average for the eight glabrous north-south rows (Fig. 1). In east-west rows, contamination values in the two rows adjacent and parallel to the central pubescent row, 15.7% and 17.5%, were significantly greater than values in other surrounding rows (Fig la). In north-south rows there also tended to be high levels of contamination in rows adjacent and parallel to the pubescent row. The highest contamination value of 13.7% was, however, observed in an end-row position treatment (Fig. 1b). The reason for the greater average contamination value and somewhat different distribution of contamination values in north-south than east-west rows in 1991 was not clear. The prevailing wind was in the south to north direction, which may have been a factor. Random, differential pollen movement could also occur in association with local atmospheric eddies.

In 1992 (second-year plants), the presence of the cereal rye barrier row did not affect percent contamination, so data from plots with and without the barrier row were averaged in Fig. 2. It is not clear, however, how distance alone and distance combined with additional barrier rows would interact to affect pollen contamination. Unlike in 1991, the row-direction effect on pollen contamination was not significant. Similar to east-west rows in 1991, the two rows adjacent and parallel to the central pubescent row had significantly more contamination than the surrounding rows (Fig. 2a). And as in 1991, this was true to a lesser extent in north-south rows (Fig. 2b). Nevertheless, the contamination values over both years in plots adjacent and parallel to the pubescent row averaged 15.7% compared to 4.8% for the other surrounding rows.

1994-95 tests

At Central Ferry in 1995, there were no significant differences among the glabrous treatment positions in pollen contamination, and contamination values in the glabrous plots averaged 3.7% (Fig. 3). Results at Pullman were similar to those at Central Ferry. No differences in pollen con lamination values among glabrous treatment positions were significant and the five-plot average value of gla-brous plots was 4.7% (Fig. 4).

Griffiths (1950) reported that as the abundance of non contaminating pollen increases, fertilization by contaminating pollen is reduced. As the distance from the contra pubescent row increased (Table 1) the amount of non-con laminating pollen from glabrous lines would also increase in other words, it is likely that more contamination would have occurred at 9 m, for example, if there had been no intervening glabrous rows. This effect may also have beer a factor for the results in Figs. 1 and 2, in which contamination levels were often quite low at small distances from the pubescent plots. In 1995 (Figs. 3 and 4), glabrous plots wee not only isolated from the pubescent pollen source but to a considerable extent from other glabrous plants. This is likely a major reason why the contamination at 2227 m was often comparable to contamination at the much closer distance in Figs. 1 and 2.

Table 1. Test of lateral movement of pollen in smooth bromegrass. pubescent dominant strain was planted in row 7 with 6 rows of a recessive glabrous strain on both sides

Row	Distance	% Pubescent seedling
	(m)	1988	1989	Mean
1	9.0	0	0	0.0
2	7.5	2	1	1.5
3	6.0	2	3	2.5
4	4.5	15	1	8.0
5	3.0	14	10	12.0
6	1.5	15	21	18.0
7 (pubescent)	0.0	98	94	96.0
8	1.5	19	13	16.0
9	3.0	7	6	6.5
10	4.5	7	4	5.5
11	6.0	2	2	2.0
12	7.5	3	1	2.0
13	9.0	1	1	1.0

A further reduction of pollen contamination at »22 m distance would likely be possible by growing three rows of each accession, and harvesting only the central row. The outside rows would act to increase the abundance of desirable pollen and help to dilute and 'filter' foreign pollen. This procedure would, however, have some drawbacks. The additional plants and rows would require additional land and management resources per accession. Moreover, the quantity of viable seed is often limiting for new accessions needing regeneration. Using plants from a limited population for bordering, which would lead to a small effective population size during regeneration, could result in large changes in the genetic profile of a given accession (Franker et al. 1995).

Resource limitations and the backlog of accessions needing regeneration at many repositories, including WRPIS, result in difficult choices. If there is zero tolerance for contamination, required regeneration procedures would necessitate greatly decreasing the number of accessions regenerated and made available for distribution. Although pollen contamination in other grass species should be tested, we have shown that a relatively low level of genetic contamination, averaging less than 5%, appears possible through modest isolation distances.

Acknowledgment

Thanks to Douglas Rains and Brenda Kuznicki for their fine technical assistance.

References

Copeland, L.O. and E.E. Hardin. 1970. Outcrossing in the ryegrasses (Lolium spp.) as determined by fluorescence tests. Crop Sci. 10:254257.

Frankel, O.H., A.H.D. Brown and J.J. Burdon. 1995. The conservation of plant biodiversity. Cambridge University Press, Cambridge, UK.

Griffiths, D.J. 1950. The liability of seed crops of perennial ryegrass (Lolium perenne) to contamination by windborne pollen. J. Agric. Res. 40:1938.

Johnson, R.C. and L.M. Bassett. 1991. Carbon isotope discrimination and water use efficiency in four cool-season grasses. Crop Sci. 31:157-162.

Jones M. D. and L.C. Newell. 1946. Pollination cycles and pollen dispersal in relation to grass improvement. Res. Bull. 148, University of Nebraska College of Agriculture, Agricultural Experiment Station, LincoIn, NE, USA.

Knowles, R.P. 1980. Seedling pubescence as a genetic marker in smooth bromegrass (Bromus inermis Leyss.). Can. 1. Plant Sci. 60:1163-1170.

Knowles, R.P. 1983. Tests of isolation requirement in three perennial grasses. Can. J. Plant Sci. 63:927-933.

Knowles, R.P. and A.N. Ghosh. 1968. Isolation requirements for smooth bromegrass, Bromus inermis Leyss., as determined by a genetic marker. Agron.). 60:371-374.

Steel, R.G.D. and J.H. Torrie. 1980. Principles and procedures of statistics. 2nd ed. McGraw-Hill, New York.

Tyler, B.F. 1982. Practical aspects of a regeneration scheme in forage grasses. Pp. 69-78 in Seed regeneration in cross-pollinated species. (E. Porceddu and G. Jenkins, eds.). A.A. Balkema, Rotterdam the Netherlands.

Résumé

Contamination génétique par le pollen transporté par le vent dans les parcelles de régénération du matériel génétique du brome inerme

Parmi les accessions allogames, l'hybridation est un problème sérieux pour les activités de régénération des semences sur les sites de gestion du matériel génétique. Des estimations de la contamination génétique par le pollen transporté par le vent dans les parcelles de régénération de cette graminée à la Western Régional Plant introduction Station (WRPIS), Pullman, WA, Etats-Unis, ont été faites en utilisant des souches de brome inerme pubescentes dominantes et glabres récessives (Bromus inermis Leyss.). En 1988 et 1989, la contamination mesurée en partant d'une ligne pubescente centrale jusqu'à des lignes glabres espacées de 1,5 m était en moyenne de 17 % à 1,5 m, mais seulement de 0,5 % à une distance de 9 m. Dans des expériences menées en 1991 et 1992, la contamination dans des parcelles adjacentes et parallèles aux parcelles pubescentes était en moyenne de 15,7 % à une distance de 3 m. La contamination dans des parcelles situées a 1,5 m des extrémités ou à 2,1 m en diagonale des parcelles pubescentes était en moyenne de 4,8 % seulement, mais allait de 0,7 % à 13,7 %. En 1995, des parcelles de brome sous marquage intégrées dans des pépinières de régénération des semences du WRPIS sur deux sites ont révélé une contamination moyenne de 4,2 % à des distances allant de 22 à 27 m. La distance et l'abondance du pollen non contaminant semblaient déterminer l'étendue de la contamination. Pour les parcelles de régénération au WRPIS, les espacements de 22 à 27 m donnaient un niveau de contamination relativement bas.

Resumen

Contaminación genética por polen de transmisión eólica en parcelas de regeneración del germoplasma de bromo inerme

La hibridización entre accesiones de polinización cruzada constituye una gran preocupación en los esfuerzos de regeneración de semillas en los sitios de ordenación de germoplasma. Se han efectuado estimaciones sobre contaminación genética por polen llevado por el viento en parcelas de regeneración herbácea en la Western Regional Plant introduction Station (WRPIS), Pullman, WA, EE.UU., empleando estirpes de ejemplares dominantes glabrosos, pubescentes y recesivos de bromo inerme, llamado también cebadilla perenne (Bromus inermis Leyss.) En 1988 y 1989, la contaminación medida desde un surco pubescente central hasta surcos glabrosos, con un espaciamiento de 1.5 m, promedio un 17% a 1.5 m, pero sólo un 0.5% a 9 m de distancia. En los experimentos realizados en 1991 y 1992, la contaminación en parcelas adyacentes y paralelas a parcelas pubescentes promedió un 15.7% a 3 m de distancia. La contaminación en parcelas ubicadas a un 1.5 m de los extremos o a 2.1 m en sentido diagonal respecto de las parcelas pubescentes promediaron sólo un 4.8%, aunque oscilando 0.7% al 13.7% En 1995, las parcelas trazadoras de bromo inerme integradas en los viveros de regeneración de semillas de la WRPIS, en dos lugares, arrojaron una contaminación media del 4.2% a distancias comprendidas entre los 22 y 27 m. Diríase que la distancia y la abundancia de polen no contaminante determinaba el grado de contaminación. Para las parcelas de regeneración existentes en la WRPIS, las distancias de aislamiento de 22 a 27 m daban un nivel relativamente bajo de contaminación.

Genetic resources of patented seeds available to the public worldwide

Shung Chang Jong and Jeannie M. Birmingham

American Type Culture Collection, Rockville, MD 20852, USA

Summary

The American Type Culture Collection (ATCC) is an internationally recognized patent depository for living biological material, whenever a deposit of such material is required by patent offices in the United States and other countries as a part of the disclosure in a utility patent application. The ATCC stores the germplasm while a patent is pending, makes it available to the public when the patent issues, and safeguards it during the life of the patent. More than 100 patented seeds of various plant varieties are on deposit with the ATCC and are available for general distribution worldwide.

Patent protection for plants

The most versatile type of protection for novel plant inventions in the US is the utility patent. Not only specific varieties, but also seeds, plant varieties having the same traits or functional properties, plant parts (e.g., fruits, nuts and flowers), plant genes, gene fragments, nucleic acids, polypeptides, proteins, processes of producing plant varieties and hybrids can be claimed. However, where the enablement of a plant invention cannot be achieved reliably from the written disclosure in a utility patent application, a deposit of plant material in a patent depository may be required by the United States Patent and Trademark Office (USPTO) to fulfill disclosure requirements.

Seeds deposited at the ATCC

The American Type Culture Collection (ATCC), a nonprofit repository in Rockville, MD, USA, accepted its first patent-related deposit in 1949 and was approved as the first international Depository Authority (IDA) under the Budapest Treaty in 1981. It accepted its first seed deposit in 1985. Since that time, more than 100 seed deposits of various plant varieties have been made. Terms of deposit and other necessary matters are arranged by the ATCC as required by patent offices of the US or other countries. The plant material must be available for 30 years from date of deposit, or for a period of at least five years after the most recent request for a sample, whichever is longer.

The USPTO allows a depositor to restrict the availability of a seed deposit until a US patent issues. If a depositor does not choose to restrict availability during the patent application process, the seeds are made available to the public from the time of deposit. However, all restrictions on the availability of the deposited seeds must be irrevocably removed upon the granting of the patent.

The fact that new plant varieties can receive patent protection and be a source of potential profit to the inventor has greatly increased patent activity. On one hand, access to the plant germplasm protected by utility patents may be temporarily restricted. On the other hand, the grant of protection, by its very nature, promotes disclosure of new and useful material and is beneficial to everyone. The ATCC stores this valuable germplasm while a patent is pending, makes it available to the public when the patent issues, and safeguards it during the life of the patent.

Table 1 lists patented seeds that are currently available from the ATCC. The cost for a packet of 25 seeds is US$80 for non-profit US and Canadian institutions and US$126 for other US and foreign institutions. Also included is the name of the depositor, the strain designation, and the US patent in which it is cited.

For more information on released patented seeds, contact Dr S.C. long, Mycology and Botany Collection, American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852-1776, USA (tel: 1-301-231-5564; fax: 1301-816-4365; Email: [email protected].

To place an order, write to the ATCC Sales Department at the above address for an account number. Thereafter, orders can be placed by phone (1-800-638-6597) or fax (1301-816-4361). For additional sales information, Email [email protected] or access ATCC's home page on the World Wide Web at http://www.atcc.org/.

Table 1. Patented seeds currently available from the ATOP

Common name	Scientific name	ATCC number	Depositor	US patent
Alfalfa	Medicago sativa	ATCC 40616	VISTA	Pending; fungal resistant
Bean	Phaseolus vulgaris	ATCC 40262	NPI AgService Corp. NPIAgS2	US Pat. 4,769,512; low pod detachment force
Bean	Phaseolus vulgaris	ATCC 40263	NPI AgService Corp. NPIAgS1	US Pat. 4,769,512; low pod detachment force
Cabbage	Brassica oleracea	ATCC 40376	Wisconsin Alumni Res. Fnd.
	var. BBC-1		PHW 220441m60 BBC, dwf1	US Pat. 5,107,064; unique dwarf for laboratory use
Coffee	Coffea arabica	ATCC 75261	KraH Foods, inc. LS 1992	US Pat. 5,436,395; high yield, low caffeine
Coffee	Coffee arabica	ATCC 75262	KraH Foods, inc. LA 2154	US Pat. 5,436,395; high yield, low caffeine, drought resistant
Corn	Zea mays	ATCC 4018O	DeKalb-Pfizer Genetics OK 524	US Pat. 4,629,819; hybrid
Corn	Zea mays	ATCC 40182	DeKalb.Pfizer Genetics 78010	US Pat. 4,654,466; inbred
Corn	Zea mays	ATCC 40225	DeKalb-Pfizer Genetics HBA1	US Pat. 4,594,810; inbred
Corn	Zea mays	ATCC 40229	DeKalb-Pfizer Genetics KD672	US Pat. 4,607,453; inbred
Corn	Zea mays	ATCC 40301	Pioneer Hi.Bred intl. Inc. 3471	Pending
Corn	Zea mays	ATCC 40413	Pioneer Hi-Bred intl. Inc. PHV78 inbred. [JP87V78C20C PM]	US Pat. 4,812,599; inbred
Corn	Zea mays	ATCC 40414	Pioneer Hi-Bred intl. Inc. PHK05. [CA87K05020C]	US Pat. 4,806,669; inbred
Corn	Zea mays	ATCC 40415	Pioneer Hi-Bred intl. Inc. PHR25 [CA87R25C10 LF]	US Pat. 4,806,652; inbred
Corn	Zea mays	ATCC 40416	Pioneer Hi-Bred intl. Inc. PHK29 [J6K29C PM]	US Pat. 4,812,600; inbred
Corn	Zea mays	ATCC 40499	Iowa State Univ. Ae-5180 stand B70	US Pat. 5,004,864; high amylose starch
Corn	Zea mays	ATCC 40507	Univ. Minnesota 2167-9/2160-154 SZ-12	US Pat. 5,162,602; herbicide tolerant
Corn	Zea mays	ATCC 40508	Univ. Minnesota 2167-9/2160-154 H1	US Pat. 5,162,602; herbicide tolerant
Corn	Zea mays	ATCC 40519	United AgriSeeds inc. 139/39-Bulk	US PaL 5,306,864; enhanced response b anther culture
Corn	Zea mays	ATCC 40520	United AgriSeeds inc. 139/39-DH	US PaL 5,306,864; enhanced response b anther culh~re
Corn	Zea mays	ATCC 40721	Holden's Fndn. Seeds, inc. LH195	US Pat. 5,059,745; inbred
Corn	Zea mays	ATCC 40722	Holden's Fndn. Seeds, inc. LH204	US Pat. 5,285,003; inbred
Corn	Zea mays	ATCC 40724	Holden's Fndn. Seeds, inc. LH210	US Pat. 5,276,262; inbred
Corn	Zea mays	ATCC 40725	Holden's Fndn. Seeds, inc. LH211	US Pat. 5,387,743; inbred
Corn	Zea mays	ATCC 40782	Holden's Fndn. Seeds, inc. LH163	US Pat. 5,285,001; inbred
Corn	Zea mays	ATCC 40784	Holden's Fndn. Seeds, inc. LH206	US Pat. 5,304,712; inbred
Corn	Zea mays	ATCC 40844	Wilson Hybrids, inc. WIL500	US Pat. 5,082,993; high protein and Iycine production
Corn	Zea mays	ATCC 40923	Holden's Fndn. Seeds, inc. LH181	US Pat. 5,304,713; inbred
Corn	Zea mays	ATCC 40925	Holden's Fndn. Seeds, inc. LH212	US Pat. 5,276,260; inbred
Corn	Zea mays	ATCC 40926	Holden's Fndn. Seeds, inc. LH213	US Pat. 5,276,259; inbred
Corn	Zea mays	ATCC 75072	Pioneer Hi-Bred intl. Inc. PHJ33	US Pat. 5,097,093; inbred
Corn	Zea mays	ATCC 75073	Pioneer Hi-Bred intl. Inc. PHK35	US Pat. 5,095,174; inbred
Corn	Zea mays	ATCC 75074	Pioneer Hi-Bred intl. Inc. PHW20	US Pat. 5,097,096; inbred
Corn	Zea mays	ATCC 75075	Pioneer Hi-Bred intl. Inc. PHM10	US Pat. 5,097,095; inbred
Corn	Zea mays	ATCC 75076	Pioneer Hi-Bred intl. Inc. PHN37	US Pat. 5,082,991; inbred
Corn	Zea mays	ATCC 75077	Pioneer Hi-Bred intl. Inc. PHP02	US Pat. 5,082,992; inbred
Corn	Zea mays	ATCC 75078	Pioneer Hi-Bred intl. Inc. PHP60	US Pat. 5,097,094; inbred
Corn	Zea mays	ATCC 75079	Pioneer Hi-Bred intl. Inc. PHR62	US Pat. 5,097,092; inbred
Corn	Zea mays	ATCC 75089	Plant Science Res. Inc. C28-SKO-13-46/48	US Pat. 4,581,373; elevated tryptophan
Corn	Zea mays	ATCC 75113	Holden's Fndn. Seeds, inc. LH164.	US Pat. 5,304,714; inbred
Corn	Zea mays	ATCC 75122	Wisconsin Alumni Res. Fnd.	US Pat. 5,331,108; gltl-1 mutant allele
Corn	Zea mays	ATCC 75134	Molecular Genetics Res. Inc. XA17	US Pat. 4,761,373; herbicide resistant
Corn	Zea mays	ATCC 75155	Holden's Fndn. Seeds, inc. LH216	US Pat. 5,276,263; inbred
Corn	Zea mays	ATCC 75157	Holden's Fndn. Seeds, inc. LH197	US Pat. 5,304,716; inbred
Corn	Zea mays	ATCC 75158	Holden's Fndn. Seeds, inc. LH198	US Pat. 5,304,717; inbred
Corn	Zea mays	ATCC 75159	Holden's Fndn. Seeds, inc. LH199	US Pat. 5,304,715; inbred
Corn	Zea mays	ATCC 75166	Ciba-Geigy Corp., CG00637	US Pat. 5,457,276; inbred
Corn	Zea mays	ATCC 75199	Dekalb Plant Genetics DK451	US Pat. 5,451,705; hybrid
Corn	Zea mays	ATCC 75205	Pioneer Hi-Bred intl. Inc. PHN73	US Pat. 5,157,208; inbred
Corn	Zea mays	ATCC 75220	Pioneer Hi-Bred intl. Inc. PHP55	US Pat. 5,159,134; inbred
Corn	Zea mays	ATCC 75221	Pioneer Hi-Bred intl. Inc. PHR63	US Pat. 5,159,132; inbred
Corn	Zea mays	ATCC 75222	Pioneer Hi-Bred intl. Inc. PHV37	US Pat. 5,159,133; inbred
Corn	Zea mays	ATCC 75254	Pioneer Hi-Bred intl. Inc. PHN82	US Pat. 5,157,206; inbred
Corn	Zea mays	ATCC 75312	Holden's Fndn. Seeds, inc. LH159	US Pat. 5,276,267; inbred
Corn	Zea mays	ATCC 75314	Holden's Fndn. Seeds, inc. LH172	US Pat. 5,276,266; inbred
Corn	Zea mays	ATCC 75364	Holden's Fndn. Seeds, inc. LH167	US Pat. 5,304,726; inbred
Corn	Zea mays	ATCC 75367	Holden's Fndn. Seeds, inc. LH217	US Pat. 5,304,727; inbred
Corn	Zea mays	ATCC 75425	Pioneer Hi-Bred intl. Inc. PHJ65 [WF89J65A20N]	US Pat. 5,220,114; inbred
Corn	Zea mays	ATCC 75426	Pioneer Hi-Bred intl. Inc. PHJ90 [CA89J9OA1 DC]	US Pat. 5,245,125; inbred
Corn	Zea mays	ATCC 75493	Pioneer Hi-Bred intl. Inc. PHR31	US Pat. 5,276,265; inbred
Corn	Zea mays	ATCC 75548	Pioneer Hi-Bred intl. Inc. PHBW8	US Pat. 5,285,004; inbred
Corn	Zea mays	ATCC 75557	Pioneer Hi-Bred intl. Inc. PHK74. [CA9OK74A20C]	US Pat. 5,347,080; inbred
Corn	Zea mays	ATCC 75565	Holden's Fndn. Seeds, inc. LH225	US Pat. 5,416,255; inbred
Corn	Zea mays	ATCC 75610	Pioneer Hi-Bred intl. Inc. PHT47. [UT9OT47C1OC]	US Pat. 5,304,719; inbred
Corn	Zea mays	ATCC 75611	Pioneer Hi-Bred intl. Inc. PHHV4	US Pat. 5,304,720; inbred
Corn	Zea mays	ATCC 75616	Holden's Foundation Seeds, inc., LH168	US Pat. 5,457,275; inbred
Corn	Zea mays	ATCC 75617	Holden's Foundation Seeds, inc., LH169	US Pat. 5,304,720; inbred
Corn	Zea mays	ATCC 75618	Holden's Foundation Seeds, inc., LH185	US Pat. 5,416,261; inbred
Corn	Zea mays	ATCC 75619	Holden's Foundation Seeds, inc. LH186	US Pat. 5,416,262; inbred
Corn	Zea mays	ATCC 75641	Pioneer Hi-Bred intl. Inc. PHEW7	US Pat. 5,354,941; inbred
Corn	Zea mays	ATCC 75642	Pioneer Hi-Bred intl. Inc. PHEM9	US Pat. 5,354,942; inbred
Corn	Zea mays	ATCC 75677	Pioneer Hi-Bred intl. Inc. PHK56. [JP88K56A1 OC]	US Pat. 5,347,081; inbred
Corn	Zea mays	ATCC 75692	Pioneer Hi-Bred intl. Inc. PHFA5	US Pat. 5,387,755; inbred
Corn	Zea mays	ATCC 75693	Pioneer Hi-Bred intl. Inc. PHMKO	US Pat. 5,365,014; inbred
Corn	Zea mays	ATCC 75694	Pioneer Hi-Bred intl. Inc. PHRE1	US Pat. 5,416,254; inbred
Corn	Zea mays	ATCC 75695	Pioneer Hi-Bred intl. Inc. PHGV6	US Pat. 5,347,079; inbred
Corn	Zea mays	ATCC 75721	Pioneer Hi-Bred intl. Inc. PHHB9	US Pat. 5,367,109, inbred
Corn	Zea mays	ATCC 75747	DeKalb Genetics Corp. LIBC4	US Pat. 5,424,483, inbred
Corn	Zea mays	ATCC 75748	DeKalb Genetics Corp. FBLL	US Pat. 5,424,483; inbred
Corn	Zea mays	ATCC 75749	Pioneer Hi-Bred intl. Inc. PHTM9. [WL9OTM9A1 AN]	US Pat. 5,349,119; inbred
Corn	Zea mays	ATCC 75769	Pioneer Hi-Bred intl. Inc. PHGW7	US Pat. 5,387,754; inbred
Corn	Zea mays	ATCC 75962	Pioneer Hi-Bred intl. Inc. PHT11	US Pat. 5,434,346, inbred
Corn	Zea mays	ATCC 97034	Pioneer Hi-Bred intl. Inc. PHR03	US Pat. 5,436,390, inbred
Cress, mouse-ear	Arabidopsis thaliana	ATCC 75042	C. Somerville & Y. Poirier T4-2A	Pending
Cress, mouse-ear	Arabidopsis thaliana	ATCC 75043	C. Somerville 8 Y. Poirier S8-1-2A	Pending
Cress, mouse-ear Arabidopsis thaliana		ATCC 75044	C. Somerville & Y. Poirier RedD-3A	Pending
Lettuce, pnckly	Lactuca serriola	ATCC 40815	Idaho Res. Fndn. DCT-R-PL	US Pal. 5,198,599; herbicide resistant
Pepper	Capsicum annuum	ATCC 75141	Rogers NK Seed Co. NVH 3074	US Pal. 5,440,069 orange-colored fruit
Pepper	Capsicum annuum	ATCC 75975	Rogers NK Seed Co. 0434; 90-3083	US Pat. 5,440,069 orange-colored fruit
Rape	Brassica napus	ATCC 40277	Allelix inc. 84-5-0012	US Pat. 4,751,347; transfer of cytoplasmic elements
Rape	Brassica napus	ATCC 40278	Allelix inc. tr/p-	US Pat. 4,751,347; transfer of cytoplasmic elements
Safflower	Carthamus tinctonus	ATCC 75807	SeedTec international, inc. dN101	US Pat. 5,436,386; hybrid
Safflower	Carthamus tinctorius	ATCC 75808	SeedTec international, inc. dN102	US Pat. 5,436,386; hybrid
Soybean	Glycine max	ATCC 75466	Stine Seed Farm inc. 9202709	US Pat. 5,304,728; hybrid
Soybean	Glycine max	ATCC 75467	Stine Seed Farm inc. 9211713	US Pat. 5,304,729; hybrid
Spruce, white	Picea glauca	ATCC 75614	Forgene, inc. 4614-10-4 X 1888-10-1	US Pat. 5,304,725; hybrid
Spruce, white	Picea glauca	ATCC 75615	Forgene, inc. 4596-10-4 X 4612-8-1	US Pat. 5,304,725; hybrid
Tobacco	Nicotiana tabacum cv. Xanthi	ATCC 40904	Amoco Corp. plant 14-8	US Pats. 5,349,126 8 5,365,017; sterol accumulation

Tomato	Lycopersicon esculentum	ATCC 40450	DNA Plant Tech. Corp. 101-33	US Pat. 5,438,152; disease resistant; high pigment;
Tomato	Lycopersicon esculentum	ATCC 40506	DNA Plant Tech. Corp. 103-114	US Pat. 5,438,152; disease resistant; high pigment; reduced scar size
Tomato	Lycopersicon esculentum	ATCC 40460	LSL, inc. BR-201	US Pat. 4,843,186; hybrid; long storage life
Tomato	Lycopersicon esculentum	ATCC 40461	LSL, inc. BR-214	US Pat. 4,843,186; hybrid; long storage life
Wheat, mutant	Triticum aestivum	ATCC 40994	American Cyanamid Co. FS1	US Pat. 5,369,022; herbicide resistant
Wheat, mutant	Triticum aestivum	ATCC 40995	American Cyanamid Co. FS2	US Pat. 5,369,022; herbicide resistant
Wheat, mutant	Triticum aestivum	ATCC 40996	American Cyanamid Co. FS3	US Pat. 5,369,022; herbicide resistant
Wheat, mutant	Triticum aestivum	ATCC 40997	American Cyanamid Co. FS4	US Pat. 5,369,022; herbicide resistant

Résumé

Ressources génétiques de graines sous brevet à la disposition du public dans le monde entier

L'Amerrissant Type Culture Collection (ATCC) est un dépôt de brevets de réputation mondiale pour le matériel biologique vivant, qui sert les bureaux de brevets des Etats-Unis et d'autres pays. L'ATCC stocke le matériel génétique quand un brevet est en suspens, le met à la disposition du public quand le brevet est délivré et le conserve durant la vie du brevet. Plus de 100 variétés de graines sous brevet de diverges espèces végétales déposées à l'ATCC peuvent être fournies au public dans le monde entier.

Resumen

Recursos genéticos de semillas patentadas a disposición de todo el público

La American Type Culture Collechon (ATCC) es un banco depositario de material biológico vivo, reconocido internacionalmente, en los caves en que las oficinas de patentes de los Estados Unidos y de otros países exigen el depósito de ese material como parte de la divulgación de la aplicación general de una patente. La ATCC almacena el germoplasma mientras está en trámite una patente, lo pone a disposición del público cuando se autoriza la patente y lo conserve durante la vida de ésta. En la ATCC se hallan depositadas más de 100 semillas patentadas de diversas variedades de plantas, que éstan listas pare su distribución general en todo el mundo.