Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
2001-03-20
2003-04-15
Campbell, 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
C800S295000
Reexamination Certificate
active
06548300
ABSTRACT:
FIELD
The present invention relates to a novel one step method for micro-shoot production from tea leaves.
BACKGROUND
Tea is a popular caffeine containing beverage with anti-cancerous properties (Jankun, J., Selman, S. H., Swiercz, R. 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 like the Chinary, Assamica and Cambod are commercially important (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).
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 Sep-Nov 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. 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 50% loss in tea 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 improvements through homogenous tissues like leaf explants because heterogeneous tissues like cotyledon explants would result in genetic segregation and loss of the desirable characters of high yield and good quality. Therefore, the existing protocols involving heterogeneous tissues like cotyledon explants is of no use with respect to the above objective and also where the true to type character of an elite plant needs to be maintained. Therefore, 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) regeneration from leaf explants would always result in the development of plants true to type to the original selected elite
(iii) 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 while doing any genetic manipulations like the development of transgenics or somatic hybrids.
(iv) leaves offer larger surface area for application of any genetic manipulation techniques.
(v) leaves are the major commercial source of made tea sold in the market.
(vi) leaves provide an abundant supply of starting material.
(vii) 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 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, the applicants report an efficient one step method for the micropropagation of tea plants using leaf explants via callus phase. The regeneration ability of the 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
(Sanjose, M. C. and Vieitez, A. M. Adventitious shoot regeneration from in vitro leaves of adult
Camnellia reticlilata
. J.Hort.Sci. 67: 677-683; 1992; Sanjose, M. C. and Vieitez, 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. There is no report of plant regeneration from leaf explants for adventitious shoot bud formation through callus in
C. sinensis
i.e. the commercial Camellia or tea. Also there is no report till date on one step method of shoot regeneration from leaf explants in either tea or any of the other ornamental species.
Attempts were first made in 1984 by Nakamura, (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: 63-74pp) for developing regeneration protocol from leaf explants wherein callus was obtained on Nitsch and 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 method is that he failed to obtain morphogenesis or adventitious shoot bud formation despite using different media in the different steps of morphogenesis.
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 to regenerate plants from leaf explants through callus wherein rhizogenesis or root formation was obtained from the leaf callus however, the drawback was that such rhizogenic calli failed to produce shoots and two different media were used. 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) supplemented with 0.5 mg/l 2,4-Dichlorophenoxy acetic acid in liquid and 5 mg/l 2,4-Dichlorophenoxy acetic acid in 0.8% agar solidified medium.
The major drawbacks of Kato's protocols are enumerated 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
Ahuja Paramvir Singh
Bhattacharya Amita
Sandal Indra
Sharma Madhu
Campbell Bruce R.
Council of Scientific & Industrial Research
McCormick Susan B.
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