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Checkbiotech: Biotechnological improvements of tea
Posted by: DR. RAUPP & madora (IP Logged)
Date: August 10, 2004 07:35AM

www.czu.cz ; www.raupp.info

Tea (Camellia sinensis L.; family Theacea) is the oldest non-alcoholic
caffeine-containing beverage crop in the world, and India is currently the
foremost producer, consumer, and exporter of commercial tea. The plant is a
woody perennial, traditionally propagated through either seeds or stem
cuttings, with a life span of more than 100 years. Tea is classified
morphologically into two varieties: Assam and China. The young leaves are
processed into different types of tea, such as black, green, and oolong.
Health benefits attributed to tea consumption are well proven, August 2004
by Tapan Kumar Mondal .


Background
Conventional tea breeding is well established, though time-consuming and
labor intensive due to its perennial nature and long gestation period (4?5
years). Vegetative propagation is standard, yet limited by slow
multiplication rate, poor survivability of some clones, and need for copious
initial planting material. Seed-borne plants are heterogeneous due to their
highly allogamous nature; consequently, it is difficult to maintain their
superior character. Additionally, tea breeding has been slowed by lack of
reliable selection criteria. Although few morpho-chemical markers are
available for identification of superior cultivars, these markers are
greatly influenced by environmental factors and show a continuous variation
with a high degree of plasticity.

To overcome these problems, a limited number of isozyme markers have been
used, resulting in less polymorphism. With the advancements of molecular
biology, however, efforts have shifted to using various DNA markers.
Understanding genetic diversity at the molecular level of tea germplasm will
help to: (1) preserve the intellectual property rights of the tea breeder;
(2) identify individual tea cultivars through use of a molecular passport;
(3) prevent duplicate entry of different genotypes into the tea gene pool;
(4) increase efficient selection of varieties for hybridization, composite
plant production, etc.; (5) classify tea genotypes taxonomically using
molecular markers; and (6) improve tea varieties for agronomically important
characteristics through marker assisted selection. Consequently,
biotechnological tools appear to be the ideal choice to circumvent problems
of conventional tea breeding.

Micropropagation
The development of micropropagation, a rapid in vitro multiplication method,
of tea has passed through three phases. Until the 1980s, emphasis was on
standardizing parameters of the in vitro protocol, such as using a suitable
explant, overcoming microbial contamination, and optimizing media
composition combined with growth regulation for better proliferation. It is
now accepted that nodal segments (0.5-1 cm) cultured on MS medium with BAP
(1-6 mg/l) are best for multiplication of shoots, along with either a high
dose (500mg/l) pulse treatment or a low dose (1-2mg/l) long duration
treatment of auxin such as IBA for in vitro rooting. Until the1990s, efforts
turned toward hardening micro-shoots to achieve a higher survival
percentage. Accordingly, several nonconventional approaches, such as a
CO2-enriched hardening chamber, biological hardening, and micrografting,
were developed. Presently, attention is increasingly focused on evaluating
field performance of the micropropagated plant.

In our laboratory we developed a micropropagation protocol by using a novel
plant hormone, thidiazuron, which was commercialized at the Research and
Development Department of Tata Tea Ltd., India. This protocol provides much
faster proliferation rates.

Somatic embryogenesis
One prerequisite for genetic transformation of tea is an efficient system of
regenerating the complete plant from a single cell. Until today, somatic
embryogenesis in tea was considered the most efficient regeneration system.
Unlike micropropagation, tea somatic embryogenesis started in the late
1980s. Thus, emphasis was focused on standardizing parameters, such as
genotypes, seed maturity, media formulation, growth regulator, physical
condition, etc. We developed a complete pathway of tea somatic embryogenesis
in which somatic embryos were first induced within 6?8 weeks on the
cotyledon segments of mature tea seed, which were then further multiplied
synchronously. A germination medium was formulated that yielded a 70%
conversion rate. Following this protocol, we transferred 3,000 plants to the
field at the Research and Development department, Tata Tea Ltd, India (1).

Bioreactor technology for secondary embryogenesis
Applications of bioreactor technology further ensure the speedy, continuous,
and large-scale supply of propagule. A bioreactor system for repetitive
embryogenesis in tea has also been developed in Australia (2) in which
uniform sizes of globular somatic embryos were obtained for a bioreactor
technology called the temporary immersion system (TIS). By controlling
immersion cycles, synchronized multiplication (24 fold) and embryo
development were achieved with greater consistency and with a high rate of
plant recovery. Plantlets recovered through this method were hardy, with a
well-formed taproot. Therefore, this technique was the first significant
step for commercial application of bioreactor technology to produce
large-scale tea somatic embryos.

Field performance of micropropagated raised plants
The ultimate success of any in vitro protocol depends upon performance of
plants in the field compared to vegetative counterparts. For the last
several years, researchers at the Research and Development Department of
Tata Tea Ltd, India, have transferred more than 45,000 plants of eight tea
cultivars to the field, from which leaves are harvested regularly to
manufacture black tea.

A systematic study at 1.7, 4, and 8 year-old field-grown micropropagated and
vegetatively propagated tea plants in our laboratory and elsewhere in India
demonstrated that overall yields and quality were comparable. Although
different physiological parameters such as photosynthetic rate, chlorophyll
content, etc. remained the same, two morphological variations were noticed.
First, the number of lateral shoots produced after `centering' were
significantly greater in micropropagated-raised plants compared to
vegetatively propagated plants. This is perhaps due to effects of various
growth regulator treatments applied under in vitro conditions. Second, root
volumes of tissue culture plants were also greater than in
vegetatively-propagated plants. Micropropagated shoots were treated with IBA
to induce rooting, which may be responsible for better root development in
the field. Therefore, we concluded that the micropropagation protocol should
be used only when required to produce a large number of plantings from a
limited source.

Other tissue culture techniques
Other techniques have been applied in tea with specific objectives. Efforts
to improve these techniques are ongoing at laboratories worldwide.

Genetic transformation
Transgenic technology has immense potential for genetic improvement of tea;
however, until 2000 there were no reports on tea transgenesis. The initial
challenge was to develop a protocol for gene transfer. Recently, we reported
the optimization of transformation conditions and production of transgenic
tea via Agrobacterium tumefaciens (3). In this study, we produced transgenic
tea using GUS reporter and NPT-II marker genes under control of strong
monocot gene promoters, and stepwise antibiotic selection. Using this
protocol, further experiments are underway to transfer the chitinase gene
for production of a fungus-resistant tea plant.

Biolistic-mediated genetic transformation is another effective method to
produce transgenics in a wide variety of plant species. Although no
transgenic tea plants have been grown in the field using this technique,
experimental conditions have been standardized by Australian and Chinese
scientists.

Genetic characterization
Morphological markers such as leaf pose, dry matter production,
partitioning, flesh evenness, etc. and biochemical markers such as total
catechin/polyphenol content, caffeine, etc. are used to identify the
superior tea plant. However, tea breeders are often unable to use markers
effectively because they are greatly influenced by environmental factors and
show a continuous variation with a high degree of plasticity. Hence, to
overcome these problems, research has shifted to using more sensitive DNA
markers.

Work on molecular markers in tea began in our laboratory in 1994 and a
significant amount of work is continuing worldwide (discussed below).

Research in India:
We have used the random amplified polymorphic DNA (RAPD) assay to
characterize 25 important Indian tea cultivars and two ornamental species.

In a separate study, twenty-five diverse tea cultivars were analyzed using
the simple sequence repeat anchored polymerase chain reaction (SSR-anchored
PCR) or Inter SSR-PCR (ISSR). In both cases, cultivars were analyzed using
Shannon's diversity index, which revealed that the China type tea group is
more diverse than the Assam group. Additionally, we noted that molecular
classification matches conventional classification of tea. A
species-specific primer was also developed for distinguishing between the
Assam and China type tea cultivars.

Amplified fragment length polymorphism (AFLP) markers were also studied in
depth to detect diversity and genetic differentiation of several important
tea clones, including the famous `Darjeeling tea', mainly to protect
cultivars for intellectual property rights purposes. Interestingly, the RFLP
technique was also used to detect adulteration with cashew husk in 10
different tea samples (4).

Research abroad:
Researchers in different countries have made fingerprints of tea cultivars
in their countries of origin. In Kenya, fingerprints of popular tea
cultivars were made through RAPD and AFLP analysis at the Tea Research
Institute, Kenya. The same group also initiated a genetic linkage map of
tea. Work is ongoing to develop a complete tea database with chemical as
well as molecular data, which will assist with easy identification of the
different cultivars.

In Japan, a wide range of markers has been used with various applications.
The markers used for genetic characterization of different green tea
cultivars are RAPD, AFLP, SSR, CAPS, and RFLP. Importantly, the RFLP
technique was also applied in Japan to prevent adulteration of higher grade
with lower grade tea. Several other minor tea-producing countries have used
different molecular markers to characterize the tea gene pool of introduced
tea cultivars available to that country. Such efforts were made using RAPD
in Portugal, ISSR in Taiwan, and RAPD in South Africa. All work focused on
the genetic characterization and molecular taxonomy of the introduced
variety available in the respective countries. Similarly, South Korea and
China tea cultivars were characterized through RAPD or AFLP, and RAPD,
respectively.

Simple sequence repeats (SSR) were derived from C. japonica, a closely
related species of tea in Japan. Using these primer pairs, 53 C. japonica
ecotypes were genotyped and population genetic parameters calculated. Later,
the same group investigated the spatial genetic structure of C. japonica
using four of these microsatellite primers. Spatial distribution of
individuals was also assessed to obtain an insight into spatial
relationships between individuals and alleles.

Gene cloning and expression
Japanese researchers have isolated the cDNA chalcone synthase (CHS) gene as
well as -tubulin gene from the Japanese green tea cultivar `Yabukuta'. More
recently, a few important genes such as phenyl ammonia lyase (PAL), caffeine
synthetase, and primeverosidase have been isolated.

References

(1) Mondal TK et al. (2004) Plant Cell Tissue Org Cult, Netherlands 76,
195-254.

(2) Akula A, Akula C (1999) In: Jain SM, Gupta PK, & Newton RJ (eds.),
Somatic embryogenesis in Woody plants, Vol. 5, pp 239-259. Kluwer Academic
Publishers: The Netherlands.

(3) Mondal TK et al. (2001) Plant Cell Reports 20,712-720.

(4) Dhiman & Singh (2003) Planta Med 69(9), 882-4.

Tapan Kumar Mondal
Centre for Advance Study in Tea Science and Technology
Uttar Banga Krishi Viswavidalaya, India

[www.isb.vt.edu]

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