R. K. Arora Department of Horticulture, Haryana Agricultural University, Hisar , India

Transcription

1 Proceedings of the Global Citrus Germplasm Network Appendix 7 In Situ Conservation of Biological Diversities in Citrus R. K. Arora Department of Horticulture, Haryana Agricultural University, Hisar , India Abstract. There is a huge biodiversity in Citrus on the planet. During evolution, a remarkable diversity has developed. The germplasm has not been collected thoroughly for want of proper survey and utilization goals. The indigenous germplasm and potential varieties with respect to physiographic conditions of growing area on a global basis needs to be conserved in situ, and collected for the expansion of gene banks for utilization in the future. The North Eastern part of the Himalayas, particularly the Garo and Nilgiri hills of India, is definitely the homeland of unchanged forms of lime (C. aurantifolia), pummelo (C. grandis Osbek), citron (C. medica L.), and maurtius papeda (C. hystrix D. C.). The wild form of C. helimii is still to be found. The trees of C. medica are bushy with spiny branches, leaves dark green but lighter on the underside with small flowers with reddish tint in 4-10 clusters. The fruit are oval with coarse skin, yellow in colour. Juice is sour and bitter. The major factors complicating delimitation of species is the prevention of variability normally resulting from hybridization, segregation, and mutation because of adventive nucellar embryony and lack of information on chromosomes, morphology, and the genetic basis of the observed variability. Conservation of DNA using frozen material like anthers and pollens should be considered. Molecular and tissue culture techniques in a biotechnological programme need to be further adopted. Motivation of fruit growers to conserve the germplasm in situ in the orchards and the dooryards is necessary under a secondary expansion programme along with the enactment of legislation to prevent the shifting of germplasm without the recommendation of a technical committee. A remarkable diversity in citrus has developed due to natural hybridization and cultivation since ancient times after Australia was separated from Asia (Swingle, 1943) some thirty millions years ago. There is a general agreement that monsoon regions of Asia particularly between West Pakistan and North & South Central China in the Himalyan regions are the places of origin of most of the citrus species. Indian Wild Orange (C. indica Tan), C. assamensis (Ada Jamir) of Eucitrus, C. ichangensis (Inchang papeda), Khasi Papeda (C. latipes Tan), and Melanesian Papeda (C. macropterd) of Papeda appear indigenous to Assam in India. Moreover, C. medica (Citron), C. limon (lemon), and C. aurantium (Sour Orange) have their origin in India. Rai et al (1996) conducted a survey in different regions of the Darjeeling hills of East India. They indicated that Citron (C. medica L.), Pummelo, and sour orange have various local names and uses. There was a long period of progressive evolution of the genus citrus. It has become difficult to ascertain the centres of origins of most of the citrus cultivars because of natural hybridization and cultivation since ancient times. Both interspecific and intergeneric hybrids have made the identification of citrus species more difficult. These hybrids have become stabilized leading to production of overlapping types. Moreover, the germplasms have not been collected thoroughly for want of proper survey. Cooper and Chapot (1977) have indicated the probable places of origin as listed in Table 1.

2 Following are the characteristics of some of the important rare citrus species of Asia. 1) C. medica (Citron): Trees are bushy with spiny branches, leaves dark green but lighter on the underside. Small flowers with redish tint, 4-10 per cluster. Fruit oval with coarse skin, yellow in colour, juice sour and bitter. 2) C. indica (Indian Wild orange): It still grows in Garo hills of Meghalaya (INDIA). It may be useful for breeding purpose. 3) C. hystrix (Kansi): It is the hybrid of citrus macropetra with pummelo which occurred in Philippines. The leaves are broadly ovate to eleptical, pointed at the tip, broadly acute at the base. Monoembryonic stamens (20-23), free ovary, oblate fruit with a smooth lemon-yellow colored peel. 4) C. ichangensis: The Ichang papeda is cold resistant but an evergreen species. It is used as a rootstock for breeding purpose. The Yuzu, a natural hybrid between C. ichangensis and C. reticulata, is cultivated in China and Japan. 5) C. macroptera (Satkara): The Melanesian Papeda grows wild in India, China and Thailand. It is vigorous, resistant to citrus canker and immune to wither tip. Swingle and Reece (1967) considered it as a promising rootstock and useful for breeding new rootstocks.

3 Importance of Conservation In citrus, the genetic diversity is diminishing very fast because of a number of factors like displacement of the natural gene pool by selection and introduction into the intensive horticulture of a limited gene pool. These conditions of control of environment such as fertilization, use of more suitable soils, controlled irrigation, more suitable temperatures, non-shading and plant protection measures have created conditions in which certain varieties may be grown over large parts of the world without stresses that would promote natural selection. This monoculture has increased the danger of crop losses from the outbreak of pest and diseases, which is continuously increasing. Deforestation for various purposes has brought down the diversity in citrus. Pummelo and sweet orange erosion have been indicated in Indonesia and Malaysia. It is, therefore, very important to protect and preserve the valuable primitive and wild gene populations of citrus and allied genera which are threatened with extinction. The wild form of C. helimii is still to be found. Conservation of indigenous spp. of citrus as a genetic source is very important for studies in evolution, genetics, physiology, biochemistry, and plant breeding for improving adaptability, yield potential, and fruit quality. Conservation of germplasm is useful in the development of new cultivars. Secondly, these are used for the improvement of existing cultivars. Thirdly these indigenous species can be future varieties. The International Plant Genetic Resource Institute (IPGRI) is actively maintaining the citrus germplasm through its network all over the world (Table 2). Methods of Conservation In Situ Conservation. Maintenance of germplasm in wild population without disturbing the natural ecosystem is in situ conservation. It is very important for a vegetatively propagated crop like citrus where seeds are short lived. Cost is the limiting factor for such conservations. The Garo hills in India have been declared as a natural biosphere where a number of wild citrus spp. grow. Protected Gene Sanctuaries. In protected gene sanctuaries, species of primary gene pool like C. medica, C. aurantium, C. grandis, C. limon, C. aurantifolia, C. sinensis, and C. reticulates and secondary gene pool species i. e. C. macroptera, C. hystrix, C. ichangensis, all the species of Fortunella and Poncirus trifoliata and some tertiary gene pools of species of other genera are conserved. Such types of conservations are important to develop and evaluate varieties from the rare species. For example, grapefruit, Rangpur Lime, Citron, Pummelo, and probably

4 hybrids of mandarins have developed naturally. Secondly, natural evolution continues i. e. strains of natural varieties like Rough lemon, trifoliate orange, flying dragon, and Rich 16-6 may be natural mutations. These sanctuaries are important sources of resistance against various biotic and abiotic stresses. Farmers field. The most important aspect of conservation is to maintain the germplasm of indigenous species. Generally these rare species are uprooted by the farmers as being uneconomical. These citrus species can be maintained through motivation of fruit growers to conserve the germplasm in situ in the orchard and door yards of edible types by giving suitable incentives and compensations. In the secondary expansion programme, fruit growers should be prevented from shifting and uprooting of citrus germplasm without the recommendation of a technical committee under the enactment of legislation. Ex- Situ Conservation. This refers to the Conservation of species of provenances outside of their natural range in planted stands or as seed, pollen, or tissue. This is the invaluable method of Conservation where species are under heavy pressure in their natural ranges or where the species may not be of present interest to their native countries but are of importance elsewhere. There are a number of methods of ex-situ conservation. 1) Field Gene bank 2) Seed storage 3) Pollen storage 4) In vitro methods a. Cryopreservation b. Minimal growth maintenance (tissue culture) c. Use of Molecular markers and gene conservation 1. RFLP 2. RAPD 3. AFLP 4. Microsatellite system 5. SSR 6. ISSR 7. i. e. (PCR based markers) Field Gene bank. This is the traditional method of conservation to keep the germplasm in plantations as mature trees. It provides mature material for vegetative propagation, hyrbidization, and characterization. This method is cost intensive. Plants are very susceptible to the spread of diseases. However, this method requires a minimum of special techniques. Seed storage. Seed storage avoids cost complications, the risk involved in growing plants at different intervals and there is minimum genetic alteration. This method is difficult for citrus because of the recalcitrant nature of citrus seeds. Pollen storage. This is also one of the potential ways of conserving the plant genetic resources, but the practical period of storage appears to be only a few years. In Vitro Methods. To overcome the difficulty encountered in the conventional storage methods, in vitro methods are being adopted for germplasm conservation in addition to uses for propagation and genetic manipulation. The main advantages for long term storage in this method are as follows: 1) Plantlets or tissue cultures are small sized with very slow development at lower temperatures. 2) High speed of multiplication at optimum temperature. 3) They can be kept free from virus, fungus and bacteria in protected growth chambers.

5 Phenotypic Characterization Use of Molecular markers. DNA technology can be used to avoid duplication in the conserved germplasm. Various DNA markers can be used for this purpose (Rao and Riley, 1994). 1) RFLP (Restricted Fragments Length Polymorphism) 2) RAPD (Random Amplified Polymorfic DNA) 3) AFLP (Amplified Fragment Length Polymorphisim) 4) Microsatellite System 5) SSR (Simple Sequence Repeat) 6) Isozymes 7) ISSR 8) Other PCR Based Markers Conclusions 1) There is urgent need for conservation of the rapidly depleting genetic resources of citrus for present and future use in citrus improvement programmes. 2) The germplasm may be stored in the form of field gene banks at various research stations. 3) The wild spp. or genera which do not grow very well outside their natural habitat must be conserved in situ. 4) More research on seeds and pollen storage is needed. 5) Possibility of storing vegetative material viz. cuttings or other tissues of citrus germplasm at low temperature should be further explored. 6) Germplasm at various places should be properly evaluated and there should be free exchange of germplasm between the countries for future utilization. 7) Potential use of molecular markers techniques should be explored for conservation. Literature Cited Cooper, C. and H. Chapot Fruit production with special emphasis on fruit processing. Citrus Science and Technology. Vol. II : Rai, M., K. P. S. Chandel and P. N. Gupta Occurrence, distribution, and diversity in the genus citrus in the Indian Gene Centre. Proc. Int. Citriculture Congress. 1996; Vol. 2: Rao, V. R. and K. W. Riley The use of biotechnology for conservation and utilization of plant genetic resources. Plant Genetic Resources Newsletter No. 97 :3-19. Swingle, W. T The botany of citrus and its wild relatives of the orange, sub-family. Citrus Industry Vol. I : Swingle, W. T. and P. C. Reece The botany of citrus and its relatives. Citrus Industry Vol. I :