TRANSGENIC TEA

Individual putative transformants of tea (cv Kangra jat) for osmotin and chitinase genes produced by transforming their somatic embryos via biolistic mediated transformation were confirmed by PCR and used for grafting. The shoots were grafted on root stocks of Kangra jat and shifted to containment facility. About 65% of the transgenic grafts survived. Performance of transgenics were analysed (Table 4.1).

Variation in leaf size was observed in transgenic plants as compared to control (un-transformed). Stable integration and expression of osmotin gene in the plants was further confirmed by Southern and Northern hybridization, (Fig.)


Fig. Southern hybridization of transgenic tea harboring osmotin gene showing expected hybridization signals. T= transgenic, C= control;	Fig. a RNA gel of osmotin transformants for Northern Blot. b. Northern blot of osmotin transformants showing very high expression

Tea leaves of several cvs are recalcitrant to Agrobacterium mediated genetic transformation, due to high contents of bactericidal poly-phenols. Catchin content at 400 µg/ml was found to inhibit Agrobacterium growth both in liquid and solid medium. It was found that different compositions of co-cultivation medium supplemented with L-glutamine induced infection at lower temperature i.e., 18ºC. High Agrobacterium infection of Kangra jat leaves was observed when L-glutamine was added to the co-cultivation medium prior to autoclaving (Table 4.2, Fig.). This was employed as standard medium for transformation of tea cv Kangra jat.

Fig. Agrobacterium infection of Kangra jat leaves irrespective of pH or co-cultivation medium

CHARACTERIZATION AND IMPROVEMENT OF TEA THROUGH BIOTECHNOLOGICAL TOOLS (Funded by Department of Biotechnology, New Delhi)

The aim of the large network programme is to fingerprint all the tea germplasm of the country so that redundancy is revoked and better management and utilization of the germplasm is possible. Concomitantly, work is in progress on micro satellite markers for their application in improvement programme.

Genomic DNA was isolated from 300 tea clones of which 200 belonging to TRA-Tocklai, Jorhat and 100 belonging to IHBT. Pre-amplified DNA of 462 tea clones taken from TERI and AFLP profiles were generated with specific primer combinations. A representative photograph with E-ACA + M-CACG primers is depicted in fig.

Fig: AFLP profile of 96 tea clones generated with the primer pair E-ACA+ M-CACG

Earlier standardized protocol for transgenic production was further extended to genes of interest such as osmotin, chitinase and SOD. Several independent lines of putative transformants were maintained in vitro. The putative transformants harbouring the chitinase and osmotin genes were morphologically distinct (Fig.). Continuing studies on host range specificity of Agrobacterium, the leaf surface characteristics of five different cultivars of tea and an unrelated species (Artemisia parviflora) were assessed with respect to the ability of being infected by Agrobacterium. Degree of Agrobacterium infection varied with leaf wetness, micromorphology and surface chemistry. While wettable leaf surfaces of TV1, UPASI-9 and Kangra jat showed higher rate (75%) of Agrobacterium infection as compared to U-10 and ST-449, non-wettable

leaves of A. parviflora showed minimum(25%) infection. Thus leaves withglabrous surface, lower q (larger surfacearea covered by water droplet), higherphenol and wax content were moresuitable for Agrobacterium infection (Fig.). Though wax generally preventsinfection, tea wax contains caffeine thatpromotes Agrobacterium infection.While wax free of caffeine inhibited bothAgrobacterium growth and infection, supplementation of caffeine to leaves poor in wax (U-10) could bring aboutsignificant infection (Fig.) The studyprovided a base for screening aclone/cultivar of particular species most suitable for Agrobacterium infection, first step in Agrobacterium mediated genetic transformation.

Fig. Effect of tea wax, caffeine free wax and caffeine fraction on Agrobacterium infection of tea leaves with and without cuticle. Vertical bars ± SE

A major problem for in vitro propagation of tea (Camellia sinensis) was poor conversion of somatic embryos into plantlets as they failed to complete normal stages of embryogeny, generally common to zygotic embryos. While endogenous level of free ABA was highest during maturation stage of zygotic embryos, treatment of somatic embryos, with exogenous ABA (5.0 mg/l for 14 days) could alleviate the problem of lack of reserve accumulation during maturation of somatic embryos, the major cause of poor and abnormal somatic embryo germination. Further, starch and protein contents were negligible in untreated control embryos that increased several fold with a simultaneous increase in oil globules (Fig.) when ABA was added at heart or early maturation stage. Germination of somatic embryos also improved. More than 50 % converted into normal plants when their starch and total soluble sugar content increased to levels equivalent to those found in zygotic embryos at late maturation stage (Table 4.1).

Molecular characterization was done with the help of high throughput AFLP marker for 150 tea clones growing at IHBT, TRA and UPASI. Due to large genome size, one of the selective primers was modified with an additional nucleotide at 3’ end. A representative figure of AFLP profile generated with primer pair E-ACA+M-CTGC is depicted in Fig.

Fig. AFLP profile of 150 tea clones belonging to IHBT, TRA-Tocklai and UPASI generated with the primer pair E-ACA+M-CTGC.

A novel inducer for rapid shoot growth and multiplication was identified for in vitro propagation (Fig.) The growth response was concentration specific for each of the cvs. UPASI 9, UPASI 10, TV1 and Kangra jat. Experiments with field grown shoots dipped in liquid media containing the novel inducer and a red coloured dye showed that the inducer stimulated rapid absorption and translocation of nutrients. Further biochemical assays revealed a concomitant fast rate of nutrient assimilation. A positive correlation between Relative Growth Rate (RGR) and the compound was observed in all the cvs. tested. The positive effect of this compound in micro-propagation was equally effective in both liquid and solid media. While liquid medium yielded higher number of plants, an early response was observed when solid medium was used. However, the compound was ineffective in inducing growth and multiplication in the field grown plants.

Previously standardized protocol for transgenic production was further extended to genes of interest. When DNA of genes like osmotin and SOD were used to bombard recurrent somatic embryos of Kangra jat (Fig.), putatively transformed plants could be regenerated (Fig.) in good frequencies. Genomic DNA obtained from these putative transformants gave the expected PCR amplification product of 750 bp fragment when primers were designed from within internal sequence of the osmotin gene. While the amplification product of 750 bp matched with that of plasmid DNA, no amplification was obtained in the control.

Fig. Genetic transformation (A) Somatic embryos (B) Putative transformants growing on selection medium (C) PCR amplification of 750 bp internal fragment of osmotin gene (C= untransformed somatic embryos; T=putatively transformed somatic embryos; M=molecular marker)

Somatic embryos of tea do not mature normally and germination is either poor or precocious. Studies were, therefore, conducted to identify whether the (i) cause of poor or abnormal germination was due to inadequate reserve mobilization or (ii) lack of reserve accumulation and desiccation sensitivity during the embryo maturation phase. Different factors affecting (a) reserve mobilization such as chilling, desiccation or GA₃ and (b) the maturation process and reserve accumulation such as ABA were tested. The somatic embryos were sensitive to desiccation like their zygotic counterparts. Normal development and germination could not also be evoked by external agents like chilling and GA₃. However, supplementation of nutrient precursors and readily available forms of carbohydrates like sucrose or maltose together with trans-cinnamic acid improved somatic embryo germination significantly to the extent of 70% (Fig.). While maltose was slowly broken down to provide a steady supply of a readily metabolizable carbon source (glucose), trans-cinnamic acid served as the precursor of malonyl-CoA, an important compound in the fatty acid biosynthesis pathway.

Fig. Effect of maltose and trans-cinnamic acid on germination of somatic embryos

In an earlier study, the vital role of ABA in the tea seeds during the actual phase of embryo maturation was ascertained. However, since seed development also requires an understanding of the changes in each of the other major types of plant growth regulators, the role of endogenous auxins (IAA) and associated biochemical constituents like proteins and RNA were examined in the developing seeds. The levels of endogenous free IAA (Fig.) proteins and total RNA (Fig.) remained high at all stages of development. The moisture content, which was high at early stages of development, declined progressively with seed maturity. The persistence of appreciable contents of IAA, RNA and proteins even at full seed maturity indicated a state of continuum and a lack of clear end point to development thereby further confirming the ‘recalcitrant nature’ of the tea seeds.


Fig. Changes in (A) protein and RNA (B) IAA in the developing seeds of tea

		Fig. Putative transformants harbouring (a)osmotin & (b) chitinase genes
	Fig. Leaf surface characteristics (A) phenol, (B) wax, (C) contact angle (theta), (D) trichome density and (E) stomatal density of tea and A.parviflora. The line above the histogram showed the percent Agrobacterium infection in these species: Different letters above bars of histogram indicate that the means are significantly different at P<0.01. Vertical bars ± SE.