Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
2001-03-22
2003-07-29
Campell, Bruce R. (Department: 1661)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
Reexamination Certificate
active
06599743
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an efficient method for micropropagation of tea plants (
Camellia sinensis
) using explants excised from leaves.
BACKGROUND AND PRIOR ART REFERENCES
Tea is a popular caffeine containing beverage with anti-cancerous properties (Jankun et al. Why drinking green tea could prevent cancer, Nature 5:561; 1997). Although the genus Camellia has many species, only
C. sinensis
(L.) O. Kuntze or tea and its different cultivars are commercially important (Barua D. N. ed. Science and practice in tea culture, Tea Research Association Calcutta; 53-68; 1989).
Tea cultivation is not only an important employment generator but is also a major foreign exchange earner in all the tea growing areas of the world (Wilson, K. C. Botany and Plant Improvement in Wilson R. C., ed. Coffea Cocoa and Tea. CABI Publishing, Wallingford, UK: 167-173; 1999). However, the total production of tea is not sufficient enough to meet the demands of the domestic and the world markets (Kabra, G. D. Tea statistics for 1999 in Tea time, Vol VIII, No. 3 September-November 99, 30-31; 1999). The yield and quality of tea is further reduced by different biotic (fungi, pests and viruses) and abiotic (frost, hail, chilling, drought, nutritional deficiencies etc.) stresses (Wilson, K. C. Botany and Plant Improvement in Wilson R. C., ed. Coffea, Cocoa and Tea. CABI Publishing, Wallingford, UK: 167-173; 1999).
Tea actually being a woody tree species has a long life cycle coupled with a high degree of self incompatibility and inbreeding depression (Barua, D. N. The tea plant of commerce in Barua, D. N., ed. Science and practice in tea culture, Tea Research Association Calcutta; 53-68; 1989) that generally limit the production of high yielding but superior and stress resistant tea plants through conventional breeding methods. Therefore, application of biotechnological means would be an effective and alternative approach. However, an efficient as well as reproducible regeneration protocol is the most important pre-requisite for any biotechnological application.
The most severe problem in tea is the blister blight disease because it afflicts the young leaves and shoots that are used for making tea as a result of which 50% loss in yield is incurred. Therefore, resistance to blister blight is urgently required to compensate for this loss. Some clones have been identified which are high yielding as well as of high quality but these are susceptible to blister blight disease and hence require biotechnological improvement through homogenous tissues like leaf explants because heterogenous tissues like cotyledon explants would result in genetic segregation and loss of the desirable character of high yield and good quality. Therefore, the existing protocols involving heterogenous tissues like cotyledon explants is of no use with respect to the above objective and there is an urgent need to develop methods for micropropagation using homogenous tissues. Regeneration from leaf explants are maximally preferred because:
(i) leaf explants are homogenous.
(ii) leaves have chloroplast DNA that have extremely high copy number and thus the level of expression can be amplified by several folds if leaves are used during genetic manipulations like development of transgenics or somatic hybrids.
(iii) leaves offer larger surface area for application of any genetic manipulation techniques.
(iv) leaves are the major commercial source of made tea sold in the market.
(v) leaves provide an abundant supply of starting material
(vi) using leaves as explants will not hamper the general well being and growth of the plant.
Biotechnological crop improvement either through somatic hybridization or through transgenic technology generally requires regeneration via a callus phase provided there is no creation of somaclonal variants during the regeneration process. Since tea has a long life span, chances of chromosomal variability in the callus phase is low as compared to that of fast growing herbaceous plants. Therefore, an efficient indirect method for the micropropagation of tea plants using leaf explants via callus phase has been developed. The regeneration ability of woody plants is difficult and more so if either leaf explants are used or if the plants are very old trees of about 50 years or more.
Leaf explants have been used in other ornamental species of Camellia i. e.
C. japonica
and
C. reticulata
by (Sanjose and Vieitez, A. M. Adventitious shoot regeneration from in vitro leaves of adult
Camellia reticulata,
J.Hort.Sci. 67: 677-683; 1992; Sanjose, M. C. and Vieitz, A. M. Regeneration of Camellia plantlets from leaf explant cultures by embryogenesis and caulogenesis. Sci.Horti.54: 303-315; 1993; Pedroso, M. C. and Pais, M. S. Direct embryo formation in leaves of
C. japonica
L. plant Cell Rep. 12: 639-643; 1993) for generating plants via somatic embryogenesis or adventitious shoot bud formation via callus phase but either the conversion frequency was low (4-6%) or rooting was poor. Moreover, these are all ornamental species. However, there is no report on a method of plant regeneration from leaf explants for adventitious shoot bud formation through callus in
C. sinensis
i.e. the commercial Camellia or tea.
Attempts were first made in 1984 by Nakamura Y.(Effective methods of in vitro propagation of tea plant. Proc. Internat. Symp. On Recent Development in Tea Production, Taiwan Republic of China, 1984: 63-74 pp) for developing regeneration protocol from leaf explants wherein callus was obtained on Nitsch & Nitsch's medium (Nitsch, J. P. and Nitsch C., Haploid plants from pollen grains. Sci. (Washington), 163; 85-87; (1969) and Gamborg's medium (Gamborg, O. L., Miller, R. A. and Ojima, K. Nutrient requirements of suspension cultures of soyabean root cells. Experimental Cell Research 50: 151-58; 1968) supplemented with an auxin 2,4-Dichlorophenoxy acetic acid. The drawback of the protocol is that he failed to obtain morphogenesis or adventitious shoot bud formation. Again in 1985, Nakamura (Nakamura, Y. Effects of origin of explants on differentiation of root and its varietal difference in tissue culture of tea plants. Shizuoka Tea Experimental Station 62: 1-8; 1985) and Palni, Sood, Chand. Sharma, Rao and Jain (Palni, L. M. S. Sood, A., Chand, G., Sharma, M., Rao, D. V., Jain, N. K. Tissue culture studies in tea. Proc. International Sym. On Tea Science, Shizuoka, Japan, 395-399; 1991) attempted plant regeneration from leaf explants through callus phase wherein although he obtained rhizogenesis from the leaf callus but failed to regenerate plants from such rhizogenic calli. Thereafter, there was no report on plant regeneration from leaf explants until in 1996 wherein Kato,(Kato, M. Somatic embryogenesis from immature leaves of in vitro grown tea shoots. Plant Cell Rep. 15: 920-926; 1996) obtained a few plants from somatic embryos derived from leaf explants of in vitro grown plants on Murashige and Skoog medium (Murashige T. and Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497; 1962) medium supplemented with 0.5 mg/l 2,4-Dichlorophenoxy acetic acid in liquid and 5 mg/l in 0.8% agar solidified medium. Yet, the major drawbacks of Kato's protocol are as follows:
(i) The percent of explant response with respect to induction of somatic embryo is very low (6%).
(ii) The donor plants are seedlings leading to genetic variations in the progenies.
(iii) The frequency of somatic embryo conversion into plants is very poor i. e. 7.1%.
(iv) The embryos induced were confined to specific regions of the leaf and not from all over the leaf surface rendering them unsuitable for transgenic studies.
(v) Does not involve a system for culturing leaf explants from mature selected bushes with elite characters rather it involves development of embryogenic calli from leaf explants of seedlings.
(vi) Seedlings represent heterogenous population whereas, explants collected from selected mature trees represent elite characters because they are propagated
Ahuja P. S.
Bhattacharya Amita
Sandal Indra
Sharma Madhu
Campell Bruce R.
Council of Scientific & Industrial Research
McCormick Susan B.
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