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Practical applications of in vitro techniques to forage germplasm

T.J. Ruredzo and Jean Hanson
Forage Genetic Resources Unit
International Livestock Centre for Africa (ILCA)
P. O. Box 5689, Addis Ababa


Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References


Abstract

Lack of seeds for collection, conservation, multiplication and distribution of forage germplasm is a major constraint to greater availability and utilisation of germplasm of some forage species. In Vitro culture techniques can be applied to overcome these constraints. Procedures are being developed at ILCA for the collection and conservation of the forage grasses, Cynodon and Digitaria, and the multiplication of these and browse species. The results and their current and potential application to increase availability of selected forage germplasm from the ILCA genebank are discussed.

Introduction

The genetic resources activities of collection, multiplication, storage and dissemination are conventionally carried out using seeds. However, some vegetatively propagated species seldom produce viable seeds and other species only produce seeds after several years. Other outbreeding species produce heterogeneous seeds which do not represent the original genotype. Until recently the only means of collecting these materials was by cuttings, tillers or whole plants which are bulky and short-lived. Conservation of these species was in field genebanks, where the material is maintained in the vegetative state requiring considerable space, careful management and plants are at risk from pests, diseases and natural disasters.

The development of in vitro methods for genetic resources has been possible due to recent advances in in vitro culture. Many species have been successfully collected (IBPGR, 1984), cultured (Sharp et al, 1984, Ammirato et al, 1984), multiplied (Hussey, 1983) and some maintained for long periods in vitro (Withers, 1980). In vitro methods use less space and fewer pests inputs than conventional methods and cultures are protected from pests and diseases, including viruses. In genetic resources, in vitro technology can also be used for disease elimination giving rise to higher yielding plants which are subject fewer quarantine restrictions and for creating variability in genotypes through adventitious regeneration. Work in progress at ILCA on appropriate methods of collecting Digitaria decumbens, conserving Cynodon and Digitaria species and multiplying these former species and Leucaena leucocephala, Erythrina brucei and Sesbania sesban in vitro is reported. Their potential use in forage genetic resources is discussed.

Materials and methods


Materials
Surface sterilisation
Inoculation
Incubation
Rooting
Transfer to soil


Materials

The plant species used in this work and their ILCA (accession) numbers are given in Appendix 1. The grasses and Leucaena were harvested from plots at the Zwai Seed Multiplication Site of ILCA whilst the other legumes were collected at ILCA headquarters.

Surface sterilisation

Leaves were removed to reveal axillary buds from stem cuttings.

The stem cuttings used in the collection experiment were then washed for 30 minutes in 1.09/l of Halazone water purifying tables in the open air.

Stem cuttings used in the rest of the experiments were divided into cuttings of one node each and the rest of the work was carried out in a laminar flow cabinet. The cuttings were washed thoroughly in distilled water, surface sterilized by dipping in 90% alcohol, washed in sterile water, and shaken in locally available bleach (formula unknown) for five minutes followed by at least five washes in sterile distilled water.

Inoculation

Stem cuttings for the collection experiment were divided into nodal cuttings and inoculated straight onto medium containing five different combinations of Benlate with Rifamycine under non-aseptic conditions.

Nodal cuttings used for other experiments were prepared for culture by cutting off the severed edges exposed to the sterilizing agents. Axillary buds and meristems (2-3 leaf primordia) were dissected asceptically in a laminar flow cabinet with the aid of a stereoscopic microscope at a magnification of X10.

Green pods with mature but green seeds of Sesbania sesban were washed in tap water and opened to remove the seeds in the laminar flow cabinet without surface sterilization for research on adventitious regeneration of Sesbania. The embryos were squeezed out of the seed coat and divided into embryo axes, cotyledons and hypocotyls for culture.

Excised nodal cuttings, buds and meristems were placed on a culture medium solidified with 0.7% (w/v) agar in glass test tubes which were covered with cotton wool plugs and aluminium foil. The culture media used were based on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962). The media were denoted; MS3, 1/2MS3, MSS, MSSG, and MS2 (Appendix 2). The media were supplemented with 3% (w/v) sucrose, 1.07 × 10-7M naphthalene acetic acid (NAA), 2.22 × 10-7 M benzylamino purine (BA) and 1.44 × 10-7M gibberrelic acid III. All media were autoclaved for 15 minutes at 121°C after adjusting the pH to 5.6.

Embryo-derived explants of Sesbania were cultured on agar solidified MS3 medium supplemented with 10-6 and 10-5 benzyl aminopurine.

Incubation

Cultures for the collection experiment were incubated under ambient conditions in the laboratory.

Cultures were kept for normal growth in an incubator between 25 and 29°C and illuminated for about twelve hours by white fluorescent light (36uEM-2s-1 PAR). After four to six weeks some well established cultures were transferred to incubators which were kept at 15°C and 5°C for slow growth conservation. These were observed monthly and ten cultures were sampled and put back in the normal growth incubator after every 100 days.

Rooting

Where there was no spontaneous rooting, shoots were rooted by dipping their bases in 10-6 M indole-3-butric-acid (IBA), inoculating on fresh medium and incubating them under normal growth conditions.

Transfer to soil

Cultures of C. aethiopicus (ILCA 2006 and 6624) and C. dactylon (ILCA 13828 and 13831) were retrieved from the agar solidified medium, washed with distilled water to remove agar and transferred to sterilized vermiculite, sterilized forest soil and unsterilized forest soil in clear plastic boxes and kept in the normal growth incubators. After two weeks the cultures were transferred to unsterilized forest soil in plastic pots and covered with plastic bags to maintain a high relative humidity. The humidity was kept high by frequent watering. The pots in plastic bags were transferred to the greenhouse after two weeks in the normal growth incubator. The plastic bags were opened after two weeks in the greenhouse and finally removed after another week.

The procedure of using unsterilized soil has been modified into a minimal facility method which is successful for the transfer of cultures to untreated soil. The cultures are transferred to unsterilized forest soil in pots, covered with plastic bags and kept in an uncontrolled environment greenhouse throughout their establishment.

Results


Collection
Initiation and multiplication
Slow growth conservation
Adventitious regeneration
Establishment in Soil


Collection

55% of Digitaria decumbens cuttings collected under minimal facility conditions and cultured on MS2 medium supplemented with 1.5g/l Benlate and 0.19/l Rifamycine in the open air were successfully recovered without contamination after eight weeks (Plate 1). It was also observed that some cuttings grew in spite of visible bacterial contamination. Whilst fungal contamination was only 5% in this, the best treatment, bacterial contamination was high at 40%.

Initiation and multiplication

In vitro cultures of all the accessions of grasses and legumes were initiated from axillary buds, meristems and nodal cuttings in all media tested. Whilst cultures were successfully initiated in the different media, the accessions showed preference for different media irrespective of the species (Table 1). Both grasses and legumes have been successfully multiplied using nodal cuttings of the in vitro cultures.

Slow growth conservation

Established cultures were kept under slow growth conditions for up to 300 days. Retrieved cultures were successfully reestablished under normal growth conditions except for Digitaria decumbens (ILCA 9729) which did not survive 5° for 200 days (Table 2).

Adventitious regeneration

Adventitious shoots were recovered from embryo-derived cotyledons (19% and 24%) and hypocotyls (35% and 62%) of S. sesban cultured on MS3 medium supplemented with the two highest levels (10-6 and 10-5 of BA respectively (Plate 2). In addition to caulogenesis from these explants, some embryo axes cultured on these media formed multiple shoots.

Establishment in Soil

In vitro cultures of two accessions of C. aethiopicus (ILCA 2006 and 6624) were successfully transferred to sterilized vermiculite without any plant mortality. 67 and 92% of C. dactylon (ILCA 13828 and 13831) and 38% of C. aethiopicus (ILCA 6624) were successfully established in sterilized forest soil. More significantly, 67 and 83% of C. dactylon (ILCA 13828 and 13831 respectively) and 50% of C. aethiopicus (ILCA 6624) were successfully established in unsterilized forest soil directly from in vitro conditions (Plate 3).

Plate 1. Rooted plantlets Digitaria decumbens from in vitro collection (8 weeks)

Plate 2. Adventitious regeneration from hypocotyl tissue of Sesbania sesban.

Plate 3. Establishment of Cynodon aethiopicus cultures in soil.

Table 1. Establishment of cultures from different explants on 3 media.

Species

Accession Number

Medium

Percentage establishment




Axillary bud cultures

Meristems cultures

Nodal cutting cultures

C. dactylon








13828

MS3

66

77

53


MSSG

60

-



MSS

84

80

-

13829

MS3

33

40

-


MSSG

15

-

-


MSS

9

60

-

13831

MS3

56

60

-


MSSG

64

-

-

C. aethiopicus






2006

MS3

50

-

93


MSSG

23

-

-


MSS

74

-

-

6624

MS3

76

43

-


MSSG

91

-

-


MSS

90

100


D. decumbens

 

9729

MS3

60

-

75


MSS

66

40

-

D. smutsii


6611

MS3

17

-

-


MS3

4

-

-

L. leucocephala


11662

MS3

-

-

90


1/2MS3

-

-

35

E. bruceii

 

-

MS3

-

-

75


1/2MS3

-

-

42

S. sesban

 

10865

MS3

-

-

21


1/2MS3

-

-

58

Table 2. Survival of cultures after retrieval from slow growth storage.


Species


Accession


Medium


Temperature (%C)

% survival after storage

100

200

300 days

C. dactylon





13831

MSS

15

100

100

100


MSS

5

100

100

-


MS3

15

60

100

100

13828

MSS

15

90

82

-


MS3

15

100

100

-

C. aethiopicus

6624

MS3

15

90

50

-

D. decumbens

9729

MS3

5

60

0

-

So far an eighty percent success rate has been achieved using the minimal facility method for transferring in vitro cultures to soil. Plants derived from in vitro field collection and slow growth conservation have been directly transferred to untreated soil using this method.

Discussion

In vitro collection methods have already been developed for other crops. Using minimal facility in vitro field collection methods, Altman et al (1987) reported 83% success for wild species of Gossypium whilst Assy Bar et al (1987) reported 90% success for coconut, after keeping the cultures for four Weeks. Great care must be taken in interpreting results because the best treatment at four weeks became heavily contaminated after eight weeks using minimal facility methods for the collection of D. decumbens. Time factors are therefore important since collection missions can be for very long periods of time.

Over the past two decades it has been shown that even within species, different genotypes can have different requirements for optimum growth in vitro (Evans et al, 1981). In the grass and browse species used here, medium preferences were observed in the establishment of different accessions. Since genetic conservation intends to capture the whole gene pool of an accession it is important that the conditions under which optimum numbers of explants and accessions can be established as cultures be determined and adopted. Work is in progress to evaluate the possibility of in vitro selection.

Obligate bacteria, fungi and viruses can be eliminated from in vitro cultures by using meristem culture together with thermotherapy (Kartha, 1986). These techniques rely on the establishment of cultures from meristems with three or fewer leaf primordia. Thermotherapy techniques will be eventually developed for the species which were successfully initiated from meristems in this work.

Growth suppression using low temperatures is the most promising approach to in vitro conservation. The temperate forage grass species Lolium, Festuca, Dactylis and Phleum can tolerate 2-4ºC (Dale, 1978, 1980); even so, some tropical species such as Ipomea batatas. (Alan, 1979) and Manihot esculentum (Roca, 1978) cannot tolerate temperatures below 15°C. Despite the varied temperature tolerance of accessions, the grasses used here show good recovery rates for up to 300 days at 15°C. Further investigations are necessary to adequately establish survival rates at lower temperatures.

In vitro multiplication of forage grasses and legumes has been routinely carried out in this work using nodal cuttings which give rise to genetically stable plants. It may be hastened by using adventitious regeneration forming masses of shoots from parts of the plant without meristems. Apical axillary meristems can also be induced to form multiple shoots. Whilst multiple shoots from preexisting meristems and plants that form adventitiously directly on the explant are usually genetically identical to the mother tissue, those from indirect adventitious regeneration through a callus stage have been found to be genetically heterogeneous in some species (Scowcroft, 1984). Of particular interest is the genetic enhancement of desirable agronomic characters in some of these so called somaclonal variants when compared to the mother plants (Larkin and Scowcroft, 1981; Scowcroft, 1984). Work is in progress at ILCA to root and establish adventitious regenerants from cotyledons and hypocotyls of S. sesban in soil for eventual evaluation.

A successful in vitro genetic resources programme also requires minimal facility techniques for reestablishment of cultures in soil. The results obtained at ILCA have been very significant because plantlets have been established in soil with very high success rates using minimum facilities.

Acknowledgements

The authors would like to acknowledge the financial support of the International Board for Plant Genetic Resources for this project.

References

Alan, J.J. 1979. Tissue culture for storage of sweet potato germplasm. PhD. thesis, University of Birmingham, Birmingham, U.K.

Altman, D.W., Fryxell, P.A. and Howell, C.R. 1987. Development of a tissue culture method for collecting wild germplasm of Gossypium. Plant Genetic Resources Newsletter, 71:14-15.

Ammirato, P.V., Evans, D.A., Sharp, W.R. and Yamada, Y. (eds) 1984. Handbook of Plant Cell Culture, Vol. 3, Crop Species. Macmillan Publishing Company, New York.

Assy Bar, B., Durand-Gasselin, T. and Pannetier, C. 1987. Use o zygotic embryo culture to collect germplasm of coconut (Cocos nucifera L.). Plant Genetic Resources Newsletter, 71:4-10.

Dale, P.J. 1978. Meristem tip culture in herbage grasses. International Association for Plant Tissue Culture. Calgary Meeting. Abstract No. 1206, 106. Calgary, Canada.

Dale, P.J. 1980. A method for in vitro storage of Lolium multiflorum Lam. Ann. Bot. 45:497-502.

Evans, D.A. Sharp, W.R. and Flick, C.D. 1981. Growth and behaviour of cell cultures: Embryogenesis and Organogenesis. In: T.A. Thorpe (ed), Plant Tissue Culture. Methods and Applications in Agriculture. pp. 45-113, Academic Press, New York.

Hussey, G. 1983. In vitro propagation of horticultural and agricultural crops. In: Plant Biotechnology. S.H. Mantell and H. Smith (eds): pp. 111-138. Cambridge University Press, Cambridge.

IBPGR. 1984. The potential for using in vitro techniques for germplasm collection. Rome.

Kartha, K.K. 1986. Production and indexing of disease-free plants. In: Plant Tissue Culture and its Agricultural Applications. L.A. Withers and P.G. Alderson (eds): pp. 219-238, Butterworths, London.

Larkin, P.J. and Scowcroft, W.R. 1981. Somaclonal variation - a novel source of variability from cell culture for plant improvement. Theor. Appl. Genet. 60:197-214.

Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and biossays with tobacco tissue cultures. Physiol. Plant. 15:473-497.

Roca, W.M. 1978. Report: Genetic Resources Unit, CIAT, Colombia. Scowcroft, W.R. 1984. Genetic Variability In Tissue Culture: Impact On Germplasm Conservation and Utilisation. A technical report commissioned by The In Vitro Storage Committee. IBPGR, Rome.

Sharp, W.R., Evans, D.A., Ammirato, P.V. and Yamada, Y. (eds): 1984. Handbook of Plant Cell Culture. Vol. 2. Macmillan Publishing Company. New York.

Withers, L.A. 1980. Tissue Culture Storage for Genetic Conservation. IBPGR Technical Report, IBPGR, Rome.

Appendix 1. Plant species used in the experiments

Species

ILCA accession number

Cynodon dactylon

13828, 13819 and 13831

Cynodon aethiopicus

2006 and 6624

Digitaria decumbens

9729

Digitaria smutsii

6611

Sesbania sesban

10865

Erythrina brucei

-

Leucaena leucocephala

11662

Appendix 2. Culture media used

MS3

MS medium without IAA or kinetin and with 3% sucrose

1/2MS3

medium diluted to half the normal concentration maintaining 3% sucrose

MSS

Salts of MS medium with myo-inositol, thymine. HCI and 3% sucrose

MSSG

MSS medium with glycine

MS2

MS medium without IAA or kinetin and with 2% sucrose


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