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Bar-Joseph, M. & Loebenstein, G. 1973. Effects of strain, source plants and temperature on the transmissibility of citrus tristeza virus by the melon aphid. Phytopathol., 63: 716-720.
Bar-Joseph, M., Raccah, B. & Loebenstein, G. 1977. Evaluation of the main variables that affect citrus tristeza virus transmission by aphids. Proc. Int. Soc. Citricult., 3: 958-961.
Bridges, G.D., Youtsey, C.O. & Nixon, R.R. 1965. Observations indicating psorosis transmission by seed of carrizo citrange. Proc. Fla. Hort. Soc., 78: 48-50.
Calavan, E.C. & Bové, J.M. 1989. Ecology of Spiroplasma citri. In R. F. Whitcomb & J.G. Tully, eds. The mycoplasmas, p. 425485. New York, Academic Press.
Cameron, J.W. & Frost, H.B. 1968. Genetics, breeding and nucellar embryony. In W. Reuther, L.D. Batchelor & H.J. Webber, eds. The citrus industry, vol. II. Anatomy, physiology, genetics and reproduction, p. 325-370. Berkeley, Div. Agric. Sci., Univ. Calif.
Capoor, S.P., Rao, G. & Viswanath, S.M. 1967. Diaphorina citri Kuwayama, a vector of the greening disease of citrus in India. Indian J. Agric. Sci., 37: 572-576.
Celino, S.C., Cortez, E.R. & Salibe, A.A. 1966. Diaphorina citri Kuwayama, the insect vector for the leaf mottle virus of citrus in the Philippines. Manila, Bureau of Plant Industry.
Chagas, C.M. & Rossetti, V. 1984. Transmission of leprosis by grafting. In Proc. 9th Conf: IOCV p. 215-217. Riverside, Univ. Calif.
Chiavegato, L.G. & Salibe, A.A. 1984. Transmissibility of leprosis symptoms by Brevipalpus phoenicis to young citrus plants under laboratory conditions. In Proc. 9th Conf IOCV, p. 218-221. Riverside, Univ. Calif.
Childs, J.F.L. 1965. The question of seed transmission of cachexia-xyloporosis virus. In Proc. 3rd Conf: IOCV, p. 90-94. Gainesville, Univ. Fla. Press.
Duran-Vila, N., Cambra, M., Pina, J.A., Ballester, J.E. & Navarra, L. 1988. Virus content and growth patterns of callus cultured in vitro from healthy and virus-infected citrus species. In Proc. 10th Conf: IOCV, p. 310-321. Riverside, Univ. Calif.
Fos, A., Bové, J.M., Lallemand, J., Saillard, C., Vignault, J.C., Ali, Y., Brun, P. & Vogel, R. 1986. La cicadelle Neoaliturus haematoceps (Mulsant et Rey) est vecteur de Spiroplasma citri en Méditerranée. Ann.lnst.Pasteur/Microbiol., 137A:97-107.
Frost, H.B. 1926. Polyembryony, heterozygosis and chimeras in citrus. Hilgardia, 1: 365-402.
Frost, H.B. & Soost, R.K. 1968. Seed reproduction: development of gametes and embryos. In W. Reuther, L.D. Batchelor & H.J. Webber, eds. The citrus industry, vol. II. Anatomy, physiology genetics and reproduction p. 290-324. Berkeley, Div. Agric. Sci., Univ. Calif.
Fulton, R.W. 1966. Transmission of tatter-leaf virus from cowpea to citrus. Phytopathol., 56: 575.
Garnier, M. & Bové, J.M. 1983. Transmission of the organism associated with citrus greening disease from sweet orange to periwinkle by dodder. Phytopathol., 73: 1358-1363.
Garnsey, S.M. 1974. Mechanical transmission of a virus that produces tatterleaf symptoms in Citrus excelsa. In Proc. 6th Conf: IOCV, p. 137-140. Berkeley, Div. Agric. Sci., Univ. Calif.
Garnsey, S.M. & Jones, J.W. 1967. Mechanical transmission of exocortis virus with contaminated budding tools. Plant Dis. Rep., 51: 410-413.
Garnsey, S.M. & Muller, G.W. 1980. Efficiency of mechanical transmission of citrus tristeza virus. In Proc. 10th Conf: IOCV, p. 46-54. Riverside, Univ. Calif.
Garnsey, S.M. & Timmer, L.W. 1980. Mechanical transmissibility of citrus ringspot virus isolates from Florida, Texas and California. In Proc. 8th Conf: IOCV, p. 174-179. Riverside, Univ. Calif.
Garnsey, S.M. & Weathers, L.G. 1972. Factors affecting mechanical spread of exocortis virus. In Proc. 5th Conf IOCV, p. 105-111. Gainesville, Univ. Fla. Press.
Harris, K.F. & Maramorosch, K. 1980. Vectors of plant pathogens. New York, Academic Press.
Hermoso de Mendoza, A., Ballester-Olmos, J.F. & Pina Lorca, J.A. 1984. Transmission of citrus tristeza virus by aphids (Homoptera, Aphididae) in Spain. In Proc. 9th Conf IOCV, p. 23-27. Riverside, Univ. Calif.
Hermoso de Mendoza, A., Ballester-Olmos, J.F., Pina Lorca, J.A., Serra, J. & Fuerte, C. 1988. Differences in transmission efficiency of citrus tristeza virus by Aphis gossypii using sweet orange, mandarin or lemon trees as donor or receptor host plants. In Proc. 10th Conf: IOCV, p. 62-64. Riverside, Univ. Calif.
Kaloostian, G.H., Oldfield, G.N., Pierce, H.D., Calavan, E.C., Granett, A.L., Rana, G.L. & Gumpf, D.J. 1975. Leafhopper natural vector of citrus stubborn disease. Calif. Agric., 29: 14-15.
Lallemand, J., Fos, A. & Bové, J.M. 1986. Transmission de la bactérie associée à la forme africaine de la maladie du "greening" par le psylle asiatique Diaphorina citri Kuwayama. Fruits, 41(5): 341-343.
Liu, H.Y., Gumpf, D.J., Oldfield, G.N. & Calavan, E.C. 1983. Transmission of Spiroplasma citri by Circulifer tenellus. Phytopathol, 73(4): 582-585.
Martinez, A.L. & Wallace, J.M. 1967. Citrus leaf-mottle yellows disease in the Philippines and transmission of the causal virus by a psyllid: Diaphorina citri. Plant Dis. Rep., 51: 692-695.
Massonie, G., Garnier, M. & Bové, J.M. 1976. Transmission of Indian citrus decline by Trioza erytreae Del Guercio, the vector of South African greening. In Proc. 7th Conf IOCV, p. 18-20. Riverside, Univ. Calif.
McClean, A.P.D. 1957. Tristeza virus of citrus: evidence for absence of seed transmission. Plant Dis. Rep., 41: 281.
McClean, A.P.D. & Oberholzer, P.C.J. 1965. Citrus psylla, a vector of the greening disease of sweet orange. S. Afr. J. Agric. Sci., 8: 297-298.
Meneghini, M. 1946. Sobre a natureza e transmisiblade da doença "tristeza" dos citrus. O Biológico, 12: 285-287.
Nault, L.R. & Rodriguez, J.G. 1985. The leafhoppers and planthoppers New York, Wiley.
Oldfield, G.N. & Kaloostian, G.H. 1979. Vectors and host range of the citrus stubborn disease pathogen, Spiroplasma citri. In Proc. ROC- US Cooperative Science Seminar on Mycoplasma Diseases of Plants, p. 119-124.
Olson, E.O. 1965. Evidence that xyloporosis virus does not pass through the seeds of Palestine sweet lime. In Proc. 3rd Conf IOCV, p. 86-89. Gainesville, Univ. Fla. Press.
Reuther, W., Batchelor, L.D. & Webber, H.J., eds. 1968. The citrus industry, vol. 11. Anatomy, physiology, genetics and reproduction. Berkeley, Div. Agric. Sci., Univ. Calif.
Roistacher, C.N. & Bar-Joseph, M. 1957. Transmission of citrus tristeza virus by Aphis gossypii and by graft inoculation to and from Passiflora species. Phytophylactica, 19: 179-182.
Roistacher, C.N. & Bar-Joseph, M. 1984. Transmission of tristeza and seedling yellows-tristeza virus by Aphis gossypii from sweet orange, grapefruit and lemon to Mexican lime, grapefruit and lemon. In Proc. 9th Conf: IOCV, p. 9-18. Riverside, Univ. Calif.
Roistacher, C.N., Calavan, E.C. & Blue, R.L. 1969. Citrus exocortis virus - chemical inactivation on tools, tolerance to heat and separation of isolates. Plant Dis. Pep., 53: 333-336.
Roistacher, C.N., Nauer, E.M. & Wagner, R.L. 1980. Transmissibility of cachexia, dweet mottle, psorosis, tatterleaf and infectious variegation viruses on knife blades and its prevention. In Proc. 8th Conf: IOCV, p. 225-229. Riverside, Univ. Calif.
Roistacher, C.N., Nauer, E.M., Kishaba, A. & Calavan, E.C. 1980. Transmission of citrus tristeza virus by Aphis gossypii reflecting changes in virus transmissibility in California. In Proc. 8th Conf IOCV, p. 76-82. Riverside, Univ. Calif.
Schneider, H. 1968. The anatomy of citrus. In W. Reuther, L.D. Batchelor & H.J. Webber, eds. The citrus industry, vol. 11. Anatomy, physiology, genetics and reproduction, p. 1-85. Berkeley, Div. Agric. Sci., Univ. Calif.
Smith, K.M. 1977. Plant viruses. London, Chapman and Hall.
Tanaka, H. & Imada, J. 1974. Mechanical transmission of satsuma dwarf, citrus mosaic, navel infectious mottling and Natsudaidai dwarf to herbaceous hosts. In Proc. 6th Conf: IOCV, p. 141-145. Berkeley, Div. Agric. Sci., Univ. Calif.
Timmer, L.W. & Garnsey, S.M. 1980. Natural spread of citrus ringspot virus in Texas and its association with psorosis-like diseases in Florida and Texas. In Proc. 8th Con/: IOCV, p. 167-173. Riverside, Univ. Calif.
Toxopeus, H.J. 1936. Die Zuchtung von unterlagen für Citrus sinensis Osb. immun gegen Phytophthora parasitica, die Ursache der "gum-disease" in Java. Zuchter, 8: 1-10.
Vogel, R. & Bové, J.M. 1980. Pollen transmission to citrus of the agent inducing cristacortis and psorosis young leaf symptoms. In Conf: IOCV, p. 188-190. Riverside, Univ. Calif.Proc. 8th
Weathers, L.G. & Harjung, M.K. 1967. Dodder transmission of citrange stunt virus -a virus associated with tatterleaf of citrus. Plant Dis. Pep., 51: 629-630.
Whitcomb, R.F. & Tully, J.G. 1989. The mycoplasmas, vol. V. New York, Academic Press.
Chapter 4: Techniques for the detection and identification of virus and virus-like citrus pathogens
Visual inspection and its limitations
Indicator plants: Biological indexing
Laboratory
indexing
Bibliography
Visual inspection and its limitations
As indicated in Chapter 2, a given citrus species can be susceptible to certain pathogens and tolerant of others (see Tables 5 and 6). For instance, sweet orange is susceptible to the pathogens of scaly bark psorosis and gummy bark, among others, but tolerant of those of cachexia and exocortis, to mention only two diseases. However, a field tree is most frequently grafted on a rootstock; for example, a sweet orange tree grafted on Poncirus trifoliata rootstock. If the tree is infected by scaly bark psorosis, gummy bark, cachexia or exocortis, these pathogens will be uniformly distributed throughout the scion and rootstock of the tree. However, symptoms of scaly bark and gummy bark will be shown only by the susceptible sweet orange scion while symptoms of exocortis will affect only the susceptible P. trifoliata rootstock. Hence, symptoms of three diseases can be seen by visual inspection. Two will affect the sweet orange scion: bark scaling as a result of the psorosis agent and the presence of gum in the bark because of the gummy bark agent. One will affect the P. trifoliata rootstock: bark scaling caused by the exocortis viroids. However, cachexia infection will remain undetected, as both the sweet orange scion and the P. trifoliata rootstock are tolerant of the cachexia viroid. The tree is therefore a symptomless carrier of the cachexia pathogen. This example illustrates that visual inspection is most often ineffective for the detection of all the pathogens present, and the pathogens of which the tree is tolerant must be detected by other means. Even those pathogens to which the tree is susceptible are not always detected by visual inspection. the tree might be too young for psorosis and/or exocortis symptoms to be expressed, for instance. In a cool climate, symptom expression of exocortis takes longer than it does under hot conditions. Inversely, psorosis young leaf flecking is present only in cool weather. The time required for symptom expression in certain hosts is longer with mild strains of certain pathogens than with severe strains. Symptoms sometimes fade away and are seen only at certain times of the year for short periods. The leaf flecking and oak-leaf patterns associated with psorosis, concave gum, cristacortis and impietratura are only seen in young, immature leaves from the spring or autumn flush of growth when the weather is cool. These so-called psorosis young leaf symptoms fade as the leaf matures.
These examples show that visual inspection must be supplemented by indexing, i.e. experimental detection of the virus and virus-like pathogens. The experimental techniques used for indexing are essentially of two types: i) those that are based on the use of the most susceptible indicator plants (biological indexing); and ii) those that directly detect the pathogen or the pathogen's constituents through a range of methods (laboratory indexing), such as electron microscopy, electrophoretic analysis, serological reactions, molecular probes, gene amplification by polymerase chain reaction (PCR) or culture of the agent (e.g. Spiroplasma citri) when possible.
Indicator plants: Biological indexing
Table 12 lists the major virus and virus-like citrus pathogens as well as the indicator plants used for their detection. For instance, tristeza virus is indexed on small-fruited acid lime (Citrus aurantifolia) seedlings. A bud, a piece of bark, a piece of leaf or a budwood stick from a candidate tree is graft-inoculated on to a lime seedling. If the inoculum contains the tristeza virus, the indicator lime seedling will become systemically infected and will then show the specific symptoms of tristeza: leaf vein clearing (Fig. 128) and stem pitting. The presence of these symptoms on the inoculated lime seedling proves that the candidate tree (from which the graft inoculum was taken for indexing) is infected with tristeza virus. Generally, several identical indicator plants are graft-inoculated with tissue from the candidate tree to take into account the possibility of uneven distribution of the agent in the candidate tree. Uninoculated indicator plants must be kept as negative controls. Indicator plants that have been graft-inoculated with tissue inoculum known to be infected with the relevant pathogen agent are used as positive controls. In the case of blight, successful graft transmission of the agent was achieved by approach-grafting pieces of roots from affected trees on to roots of healthy Valencia late sweet orange trees on rough lemon rootstock. Table 12 also lists non-rutaceous herbaceous plants that can be infected through mechanical inoculation of their leaves. As shown in the table, it takes only a few days to obtain a positive reaction on these mechanically infected herbaceous plants. In contrast, it might take several years for the symptoms to appear on citrus indicator plants. Of course, a good indicator plant is one that shows specific symptoms within a short time -a few weeks to a few months. For instance, the best indicator plant for the exocortis viroid complex is Etrog citron selection "861 S1". Symptoms are obtained within weeks or months. Prior to the discovery of the Etrog citron test, P. trifoliata and Rangpur lime were used as indicator plants and the test took from several months to several years. Similarly, the cachexia test on Parson's Special mandarin is much faster than the previous test which used Orlando tangelo.
In spite of the progress made in identifying new, fast-reacting indicator plants, techniques based on the direct detection of the pathogens or their constituents have recently emerged. Table 13 lists the four main techniques for the detection of specific citrus pathogens. With time, more and more of the virus and virus-like pathogens will be characterized and this will probably lead to the development of additional laboratory indexing methods. The four methods currently available are:
TABLE 12 Indicator plants for the detection of the major virus and virus-like agents of citrus
- electron microscopy of the phloem-restricted prokaryotes (Spiroplasma citri, witches' broom MLO, rubbery wood MLO, greening BLO);
- culture of S. citri and observation of the cultured spiroplasma by dark field microscopy; or
- sPAGE and staining of the viroid RNAs of cachexia and exocortis.
Some of these methods are not yet routine indexing techniques. For instance, the MAs against the greening BLO turn out to be highly specific and they will only detect the homologous serotypes of the BLO. Hence, electron microscopy detection of the BLO is still the most reliable technique. Eventually, serological techniques such as ELISA will be replaced by the more sensitive molecular hybridization techniques and ultimately by PCR. While the MAs against the greening BLO are too specific to be used in routine indexing, they have been most useful as research tools in the purification of the BLO by immunoaffinity chromatography. The purified BLOs are now available for DNA extraction and the DNA can be cloned in Escherichia cold and sequenced, thus opening up the way for the detection of the greening BLO by molecular hybridization and PCR.
Finally, it should be noted that more and more strains of virus and virus-like pathogens of citrus are being discovered and that diseases thought to be caused by a single pathogen now appear to involve several such agents. For instance, many strains of tristeza virus have been detected, but detection of these strains, mild or severe, requires the joint use of indicator plants and ELISA, showing that indexing on indicator plants and serological techniques are complementary. A strain of tristeza virus from kumquat induces no symptoms whatsoever on acid lime, the normal indicator plant for the virus. Only ELISA is able to detect the strain. One last example concerns exocortis. indexing various isolates of exocortis on Etrog citron gave a whole range of symptoms on the indicator plants, leading to the belief that there were mild, moderate and severe strains of the same exocortis viroid RNA molecule (CEV viroid RNA). Electrophoretic analysis of the viroid RNAs present in the various exocortis isolates has revealed that, besides CEV viroid RNA, other viroid RNAs are present and also produce a reaction on Etrog citron (Tables 2 and 3, p. 11 - 12). Thus, the combined use of Etrog citron as an indicator plant and of the electrophoretic analysis of viroid RNAs results in a new understanding of exocortis as a disease caused by a complex of various viroid RNAs rather than only one.
Bar-Joseph, M. & Loebenstein, G. 1970. Leaf flecking on indicator seedlings with citrus in Israel. Plant Dis. Rep., 54: 643646.
Bar-Joseph, M., Garnsey, S.M., Gonsalves, D., Moscovits, D.E., Clark, M. F. & Loebenstein, G. 1979. The use of enzyme-linked immunosorbent assay for detection of citrus tristeza virus. Phytopathol., 69: 190- 195.
Bové, J.M. 1980. Spiroplasma citri identification. In J.M. Bové & R. Vogel, eds. Description and illustration of virus and virus-like diseases of citrus, p. 22. A collection of colour slides. Paris, IRFA SETCO-FRUITS.
Calavan, E.C., Frolich, E.F., Carpenter, J.B., Roistacher, C.N. & Christiansen, D.W. 1964. Rapid indexing for exocortis of citrus. Phytopathol., 54: 1359-1362.
Calvert, L.A., Lee, R.F. & Hiebert, E. 1988. Characterization of the RNA species of citrus variegation virus with complementary DNA clones. In Proc. 10th Conf: IOCV, p. 327-333. Riverside, Univ. Calif.
Childs, J.F.L. et al., eds. 1968. Indexing procedures for 15 virus diseases of citrus trees. Washington, DC, Agricultural Research Service, United States Department of Agriculture.
Christie, R.G. & Edwardson, J.R. 1986. Light microscopic techniques for detection of plant virus inclusions. Plant Dis., 70: 273-279.
Dodds, J.A. & Bar-Joseph, M. 1983. Double-stranded RNA from plants infected with closteroviruses. Phytopathol., 73(3): 419-423.
Dodds, J.A., Jarupat, T., Lee, J.G. & Roistacher, C.N. 1987. Effects of strain, host, time of harvest and virus concentration on double-stranded RNA analysis of citrus tristeza virus. Phytopathol., 77(3): 442-447.
Duran-Vila, N., Pina, J.A., Molina, M.l. & Navarro, L. 1991. A new indexing method for cachexia. In Proc. 11th Conf IOCV, p. 224-229. Riverside, Univ. Calif.
Flores, R. 1988. Detection of citrus exocortis viroid in natural and experimental citrus hosts by biochemical methods. In Proc. 10th Conf IOCV, p. 192-196. Riverside, Univ. Calif.
Garnier, M., Martin-Gros, G. & Bové J.M. 1987. Monoclonal antibodies against the bacterium-like organism associated with citrus greening disease. Ann. Inst. Pasteur/Microbiol., 138: 639-650.
Garnier, M., Zreik, L. & Bové, J.M. 1991. Witches' broom disease of lime trees: transmission of the mycoplasma-like organism (MLO) to periwinkle and citrus, production of monoclonal antibodies for the detection of the MLO. In Proc. 11th Conf. IOCV, Riverside, Univ. Calif.
Garnsey, S.M., Bar-Joseph, M. & Lee, R.F. 1981. Application of serological indexing to develop control strategies for citrus tristeza virus. Proc. Int. Soc. Citricult., 1: 448-452.
Garnsey, S.M., Gonsalves, D. & Purcifull, D.E. 1979. Rapid diagnosis of citrus tristeza virus infections by sodium dodecyl sulfate [SDS]-immunodiffusion procedures. Phytopathol., 691: 88-95.
Gillings, M.R., Broadbent, P. & Gollnow, B.I. 1988. Biochemical indexing for citrus exocortis viroid. In Proc. 10th Conf. IOCV, p. 178-187. Riverside, Univ. Calif.
La Rosa, R., Albanese, G., Azzaro, A., Sesto, F. & Domina, F. 1988. Suitability of nucleic acid analysis to diagnose viroid infections in citrus. In Proc. 10th Conf. IOCV, p. 188-191. Riverside, Univ. Calif.
Martin-Gros, G., Garnier, M., Iskra, M.L., Gandar, J. & Bové, J.M. 1987. Production of monoclonal antibodies against phloem-limited prokaryotes of plants: a general procedure using extracts from infected periwinkles as immunogen. Ann. Inst. Pasteur/Microbiol., 138: 625-637.
Roistacher, C.N. 1991. Graft-transmissible diseases of citrus. handbook for detection and diagnosis. Rome, FAO. 286 pp.
Roistacher, C.N., Blue, R.L. & Calavan, E.C. 1973. A new test for cachexia. Citrograph, 58: 261-262.
Roistacher, C.N., Calavan, E.C., Blue, R.L., Navarro, L. & Gonzales, R. 1977. A new more sensitive citron indicator for detection of mild isolates of citrus exocortis viroid (CEV). Plant Dis. Rep., 61: 135139.
Saillard, C., Garcia-Jurado, O., Bové, J.M., Vignault, J.C., Moutous, G., Fos, A., Bonfils, J., Nhami, A., Vogel, R. & Viennot-Bourgin, G. 1980. Application of ELISA to the detection of Spiroplasma citri in plants and insects. In Proc. 8th Conf: IOCV, p. 145-152. Riverside, Univ. Calif.
Vela, C., Cambra, M., Sanz, A. & Moreno, P. 1988. Use of specific monoclonal antibodies for diagnosis of citrus tristeza. In Proc. 10th Conf: IOCV, p. 55-61. Riverside, Univ. Calif.
Villechanoux, S., Garnier, M., Renaudin, J. & Bové, J.M. 1992. Detection of several strains of the bacterium-like organism of citrus greening disease by DNA probes. Curr. Microbiol., 24: 89-95.
Chapter 5: Production of citrus clones free of virus and virus-like pathogens
Visual inspection and indexing
Production of nucellar clones
Shoot-tip
grafting
Bibliography
The works of Murashige et al. (1972) and Navarro, Roistacher and Murashige (1975) have led to the technique of shoot-tip grafting and its use in recovering citrus clones free of virus and virus-like agents. Over the last ten years, shoot-tip grafting has become the major, if not the only, technique used for producing citrus material free of graft-transmissible pathogens. Before the era of shoot-tip grafting, the production of citrus material free of disease agents followed three approaches: selection by visual inspection and indexing, the production of nucellar clones and recovery of virus-free material from infected plants by thermotherapy.
Visual inspection and indexing
In this technique, outstanding candidate trees were selected by visual inspection and were then indexed on indicator plants for the major virus and virus-like pathogens. The candidate trees were mainly old-line clones, i.e. clones that had lost their juvenile characters long ago. Only a small percentage of candidate trees were found to be free of all the agents for which they were indexed. A tree selected by this technique becomes a "mother tree". The progeny trees, propagated from buds of the mother tree, will have the same horticultural properties as the mother tree and, in particular, they will have no juvenile characters if the mother tree is an old-line clone. This is the major advantage of the "inspection indexing" technique over the "nucellar clone" technique where juvenile characters affect the nucellar trees for several years.
The nucellar clone technique is based on the fact that most virus and virus-like pathogens of citrus do not pass through the seed (see Chapter 3) and that most commercial citrus species and varieties have not only a sexual hybrid embryo resulting from the development of the pollinated ovule, but also several somatic, asexual embryos resulting from the development of nucellar cells into nucellar embryos.
Nucellar cells are somatic cells with 2n chromosomes and they have the same chromosomal composition as the mother tree. Thus, the development of a nucellar embryo yields a nucellar seedling plant identical to the mother plant, i.e. true to type. In fact, there is some variability among the various nucellar seedlings and only those with the most desirable properties would be kept to yield nucellar clones. In the production of nucellar clones, pollination has to be controlled. Flowers of the mother tree will be pollinated with Poncirus trifoliata pollen. As the inheritance of the trifoliate leaf character is dominant, the seedling from the sexual embryo will have trifoliate leaves; the nucellar seedlings will have no trifoliate leaves and can thus be easily identified. In their early years, nucellar trees show the typical juvenile characters of vegetative vigour, thorniness, upright growth, slowness in fruiting, abnormal fruit characters and alternate bearing. The juvenile condition tends to decrease with time and, in general, ten to 15 years are necessary to evaluate nucellar clones.
Commercial nucellar clones have been produced for the major commercial citrus species, including sweet orange, mandarin, grapefruit and lemon. Some of the better known nucellar clones were produced by Frost (1938). It should be remembered that shaddock, citron, Clementine, King mandarin, Meyer lemon and Tahiti lime are monoembryonic and have no nucellar embryos. However, techniques have been developed to induce nucellar embryos artificially in monoembryonic species. Rangan, Murashige and Bitters (1968) were the first to induce formation of such embryos in vitro. Esan (1973) made an extensive study of adventive embryogenesis in monoembryonic species. Juarez, Navarro and Guardiola (1976) obtained nucellar plants of several clementine cultivars.
Even in the case of seedless polyembryonic varieties, nucellar plants can still be obtained. In certain varieties, seeds and, hence, nucellar embryos can be easily induced by cross-pollination but, in other varieties, such as the navel sweet orange, megasporocite degeneration makes it difficult to obtain any seed. This problem can be avoided by culturing, in vitro, unpollinated and unfertilized ovules before they are aborted. In this way, Navarro and his co-workers obtained nucellar plants of several navel cultivars by culturing whole ovules excised from ovaries at the flower bud stage and from developing fruits.
White (1934) was the first to show, at the Rockefeller Institute in New York, that root tips of tomato plants infected with TMV were free of the virus. Limasset, Cornuet and Gendron (1949) at INRA in Versailles, suggested that shoot tips could also be free of viruses. This hypothesis was confirmed by Morel and Martin (1952, 1955) who showed, also at INRA in Versailles, that shoot tips or meristems could be cultured in vitro into plants on special media, and that these plants would be free of viruses. The first plant to be freed of viruses in this way was a dahlia hybrid, then potato, carnation and strawberry. Today, many cultivated plants have been "regenerated" by shoot-meristem culture. Unfortunately, meristems of woody plants such as citrus do not grow, or grow only poorly, on the media devised for herbaceous plants. This is why Murashige et al. (1972), at Riverside, California, had the idea of replacing the medium by a small, test-tube grown citrus rootstock. The meristem was placed on the decapitated rootstock where it bound to the rootstock by a graft union and, being adequately fed by the rootstock, grew into a shoot. The technique has been carefully standardized by Navarro and his co-workers in Spain. The rootstock used is Troyer citrange, a trifoliate hybrid of P. trifoliata. The shoot grown from the meristem is not trifoliate and is easily detected. Once the shoot is well developed, the small, test-tube plantlet (rootstock and shoot) is transplanted to soil. Alternatively, when difficulties are encountered in the transplant process, the plantlet can be grafted on a vigorous, one-year-old rootstock seedling in the greenhouse.
Shoot-tip grafted plants have all the characteristics of the mother tree from which the shoot tips have been taken. Contrary to nucellar plants, they have no juvenile characters and they are able to flower and produce fruit within a year after grafting. The shoot-tip grafting process has been so carefully worked out by Navarro and his co-workers that it can easily be reproduced, even in laboratories without much experience. However, shoot-tip grafting in itself is only part of the whole procedure. It must be followed by the indexing of every plant grown from a shoot tip. Indeed, some shoot-tip grafted plants are still found to be infected by virus and virus-like pathogens. This is because, for the shoot tip to take and grow on the rootstock, it has to be of a minimum size. Normally, the grafted shoot tip comprises the meristem and two to three leaf primordia (0.10 to 0.15 mm). If the shoot tip is too small, it will not grow. If it is too large, it will not be free of pathogens. Conditions for indexing the shoot-tip grafted plants are at least as important as the shoot-tip grafting laboratory itself. They must be grown free of nutritional deficiencies and pests, under controlled greenhouse or screenhouse conditions. They must be of the highest possible quality so as to express even the mildest symptoms. The gradual replacement of indicator plants by serological and other techniques will probably make the indexing procedure simpler. Tristeza virus is already indexed by ELISA; cachexia and exocortis viroids by PAGE; and stubborn by ELISA as well as culture of the spiroplasma. Hopefully, similar techniques will soon be available for other citrus pathogens. In the Mediterranean area, Spain has entirely regenerated its citrus species and varieties through shoot-tip grafting and indexing. Only clones that are free of virus and virus-like pathogens are now propagated by nurseries. Similarly, many disease free citrus clones are available from Corsica (France). The Corsican disease-free citrus collection is being duplicated in Martinique while Morocco has also started a shoot-tip grafting programme and produces many nursery trees free of infectious agents.
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