Micropropagation of tea (Camellia sinensis (L.) O. Kuntze) using Thidiazuron

The effect of thidiazuron (TDZ) on the micropropagation of Camellia sinensis (China hybrid) was compared with that of benzylaminopurine (BAP) using nodal segments from in vitro raised seedlings. Extremely low concentrations of TDZ (1pM–100nM) alone were effective in inducing shoot bud proliferation and maintaining high rates of shoot multiplication on hormone-free media. On the other hand, higher concentrations of BAP (1–10M) and its continued presence were required to initiate and sustain shoot proliferation. While wider ranges of BAP combined favourably with auxins like NAA or IBA, only specific combinations of TDZ and NAA were effective for shoot proliferation. TDZ treated explants yielded healthy shoots, with sturdy leaves, even during the initial stages of growth, whereas, the effect of BAP was cumulative over subcultures in attaining a high proliferative rate.     

Clonal variation of tea [Camellia sinensis (L.) O. Kuntze] in countering water deficiency

Various clones of tea [Camellia sinensis (L.) O. Kuntze] such as TTL-1, TTL-2, TTL-4, TTL-5, TTL-6, UPASI-2 and UPASI-3 planted in the field were subjected to soil moisture stress conditions by withholding irrigation. A control set of the same clones were maintained by watering regularly. The soil water content of the irrigated and non irrigated plants was monitored through the soil moisture status. The extent of effect of drought on tea plants were monitored through various physiological parameters such as shoot weight, leaf water potential, chlorophyll and carotenoid content, chlorophyll fluorescence (Fv/Fm), net photosynthetic rate, transpiration rate, stomatal conductance and biochemical parameters such as extent of proline accumulation and free radical generation. These parameters were studied on the 30 d of non irrigation and on the 5 d during recovery from drought. The plants recovered when re-irrigated after 30 d of non-irrigation, which suggests that permanent wilting did not occur due to non-irrigation up to 30 d. On the 30 d of non-irrigation the clones TTL-1, TTL-6 and UPASI-2 showed lesser reduction of shoot weight, leaf water potential, chlorophyll fluorescence, photosynthetic rate, transpiration rate and stomatal conductance and increased proline and lesser lipid peroxidation as compared to the other clones. From these results it can be concluded that the clones TTL-1, TTL-6 and UPASI-2 are comparatively more drought tolerant than the clones TTL-2, TTL-4, TTL-5 and UPASI-3.     

In vitro clonal propagation of tea (Camellia sinensis (L.) O. Kuntze)

A system for in vitro clonal propagation has been developed in tea plants. Shoots obtained from primary explants were induced from terminal buds and axillary buds of mature field-grown plants. Cultures were initiated from both types of explants on Murashige and Skoog (MS) medium supplemented with 10% coconut milk (CM), 200 mg l^-1 of yeast extract (YE), 1.4 M indoleacetic acid (IAA) and 17.8 M benzyladenine (BA). The shoot tips were multiplied on 1/2 strength MS medium containing 10% CM, 2.9 M IAA and 17.8 M BA. The larger shoots were separated after multiplication and rooted on 1/2 MS medium supplemented with 11.4 M ascorbic acid and 34.5 M indolebutyric acid (IBA). A pretreatment of the plants with an aqueous solution of 493 M IBA greatly increased the frequency of rooting. More than 60% of the rooted plants have been transferred to soil successfully.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - YE yeast extract - CM coconut milk - MS Murashige and Skoog medium (1962)     

Molecular regulation of catechins biosynthesis in tea [Camellia sinensis (L.) O. Kuntze

Catechins are bioprospecting molecules present in tea and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. These are synthesized through the activities of phenylpropanoid and flavonoid pathways. Expression regulation of various genes of these pathways namely phenylalanine ammonia-lyase (CsPAL), cinnamate 4-hydroxylase (CsC4H), p-coumarate:CoA ligase (Cs4CL), flavanone 3-hydroxylase (CsF3H), dihydroflavonol 4-reductase (CsDFR) and anthocyanidin reductase (CsANR) was accomplished previously. In depth analyses of the remaining genes namely, chalcone synthase (CsCHS), chalcone isomerase (CsCHI), flavonoid 3'5'-hydroxylase (CsF3'5'H) and anthocyanidin synthase (CsANS) were lacking. The objective of the work was to clone and analyze these genes so as to generate a comprehensive knowledge on the critical genes of catechins biosynthesis pathway. Gene expression analysis was carried out in response to leaf age and external cues (drought stress, abscisic acid, gibberellic acid treatments and wounding). A holistic analysis suggested that CsCHI, CsF3H, CsDFR, CsANS and CsANR were amongst the critical regulatory genes in regulating catechins content.     

Betaine a novel candidate for rapid induction of somatic embryogenesis in tea (Camellia sinensis (L.) O. Kuntze)

Addition of betaine to the inductionmedium significantly enhanced the rapid formation ofsomatic embryos directly without callusing from maturefresh seeds of tea within two weeks of cultureinitiation. The induction response was furtherenhanced when ABA (7.5 mgl^–1) was co-supplementedwith betaine in the induction medium. The rate ofinduction of somatic embryogenesis increased linearlywith external betaine concentration. Globular somaticembryo-like structures (embryoids) were observed in 4-week old cultures when inoculated on the inductionmedium without ABA and betaine. The positive effectof ABA on the induction process was found to bedependent on the presence of betaine in the medium. ABA alone in the medium could not bring the inductionstimulus in the explants; on the contrary, it provedinhibitory. The optimum response of ABA was observedwhen the medium was supplemented with 500 to1000 mgl^–1 of betaine. Primary somatic embryosobtained in the presence of ABA and betaine were ableto produce secondary embryos. A conversion rate of15–20% was achieved upon transfer of somatic embryosof size 3–5 mm in diameter to the basal medium consistof half strength of macro nutrients, full strength ofmicro nutrients and vitamins of MS. Medium wasfurther supplemented with 100 mg l^–1 each ofadenine hemisulfate sulphate and L-glutamine, 30 gl^–1 sucrose, gelled with 7 gl^–1 bitek agar. The plantlets regenerated by this procedure did notshow any visible abnormalities. This report for thefirst time details the potential use of betaine inplant tissue culture.     

Boron re-translocation in tea (Camellia sinensis (L.) O. Kuntze) plants

Boron (B) re-translocation is an important factor determining tolerance to B deficiency in plants. In this work growth, B content of leaves with different ages, B partitioning between soluble and cell wall (CW) fractions, and B re-translocation were investigated in tea (Camellia sinensis (L.) O. Kuntze) plants grown hydroponically without (<2.5 μM) and with adequate (46 μM) B supply. Under B deficiency, the proportion of CW bound B increased in the old leaves but decreased in roots. Contrastingly, the proportion of CW bound B was not influenced by B supply in the young leaves. A continuous reduction of B content was observed in all fully expanded leaves as well as in roots of low B plants. Taken together, these results revealed considerable re-translocation of B from mature to growing leaves. Leaf extract and phloem exudate samples were analyzed and sucrose, glucose, and fructose were detected while xylitol, sorbitol, mannitol, maltose, galactose, cellobiose or rafinose were not found in these samples. In the leaf extracts, concentration of sucrose increased under B deficiency conditions, concentration of glucose decreased, while that of fructose remained unchanged. Our results provide circumstantial evidence for a considerable re-translocation of B in tea plants despite lacking polyol compounds.     

Differential display-mediated identification of three drought-responsive expressed sequence tags in tea [Camellia sinensis (L.) O. Kuntze]

There is no information on drought-modulated gene(s) in tea [Camellia sinensis (L.) O. Kuntze], a woody and perennial plant of commercial importance. Using differential display of mRNA, three drought-modulated expressed sequence tags (ESTs) were identified. Northern and BLAST analysis revealed that clonedr1 (droughtresponsive), induced only by drought but not by ABA, showed significant scores with PR-5 (pathogenesis related) family of PR-protein gene. Another clonedr2, repressed by drought but not by ABA, had nucleotide repeats for polyasparate that are also present in chicken calsequestrin-like mRNA. Clonedr3, responded similarly to clonedr2 but did not show significant homology with the reported genes, hence appears to be novel. Identification of these ESTs is an initial step to clone the full length genes and their promoters     

Potentiality of actinobacteria to combat against biotic and abiotic stresses in tea [Camellia sinensis (L) O. Kuntze]

Tea (Camellia sinensis (L) O. Kuntze) is a long-duration monoculture crop prone to several biotic (fungal diseases and insect pest) and abiotic (nutrient deficiency, drought and salinity) stress that eventually result in extensive annual crop loss. The specific climatic conditions and the perennial nature of the tea crop favour growth limiting abiotic factors, numerous plant pathogenic fungi (PPF) and insect pests. The review focuses on the susceptibility of tea crops to PPF/pests, drought, salinity and nutrient constraints and the potential role of beneficial actinobacteria in promoting tea crop health. The review also focuses on some of the major PPF associated with tea, such as Exobasidium vexans, Pestalotiopsis theae, Colletotrichum acutatum, and pests (Helopeltis theivora). The phylum actinobacteria own a remarkable place in agriculture due to the biosynthesis of bioactive metabolites that assist plant growth by direct nutrient assimilation, phytohormone production, and by indirect aid in plant defence against PPF and pests. The chemical diversity and bioactive significance of actinobacterial metabolites (antibiotics, siderophore, volatile organic compounds, phytohormones) are valuable in the agro-economy. This review explores the recent history of investigations in the role of actinobacteria and its secondary metabolites as a biocontrol agent and proposes a commercial application in tea cultivation.     

Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos

A protocol for the production of transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] was developed via Agrobacterium-mediated genetic transformation of somatic embryos. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the nptII gene and gus-intron were evaluated as vector systems. A number of parameters were tested with respect to maximizing transformation efficiency. While pre-culture, wounding and acetosyringone treatment were inhibitory, the bacterial growth phase (optical density; OD₆₀₀ = 0.6), cell density (10⁹/ml), co-cultivation period (5 days) and pH of the co-cultivation medium (5.6) had positive effects on transformation. Following co-cultivation, globular somatic embryos were placed on multiplication medium and stressed with kanamycin (50 µg/ml). Further selection occurred in the maturation and germination medium at an elevated kanamycin level (75 µg/ml). An average of 40% transient expression was evident based on the GUS histochemical assay. Kanamycin-resistant, GUS-positive embryos were germinated, and the resulting microshoots were multiplied in vitro. Integration of the transgenes into the tea nuclear genome was confirmed by PCR analysis using nptII- and gus-specific primers and by Southern hybridization using an nptII-specific probe. The transgenic shoots were micrografted onto seed-grown rootstocks of cv. Kangra Jat and eventually hardened in a walk-in polyhouse. This is the first report on the production of transgenic tea.     

Diversity and genetic differentiation among populations of Indian and Kenyan tea (Camellia sinensis (L.) O. Kuntze) revealed by AFLP markers

AFLP markers were successfully employed to detect diversity and genetic differentiation among Indian and Kenyan populations of tea (Camellia sinensis (L.) O. Kuntze). Shannon's index of diversity was used to partition the total phenotypic variation into between and within population components. On average, most of the diversity was detected within populations, with 79% of the variation being within and 21% being between populations of Indian and Kenyan tea. A dendrogram constructed on the basis of band sharing distinctly separated the three populations of tea into China type (sinensis), Assam type (assamica) and Cambod type (assamica ssp. lasiocalyx) in a manner consistent with the present taxonomy of tea, the known pedigree of some of the genotypes and their geographical origin. Principal coordinate (PCO) analysis grouped Assam genotypes both from India and Kenya supporting the suggestion that the Kenyan clones have been derived from collections made in this region. The China types were more dispersed on the PCO plot which is a reflection of wider genetic variation. As would be expected, clones collected from the same region exhibited less overall genetic variation. AFLP analysis discriminated all of the tested genotypes from India and Kenya, even those which cannot be distinguished on the basis of morphological and phenotypic traits. Received: 2 May 1996 / Accepted: 14 June 1996     

Green tea [Camellia sinensis (L.) O. Kuntze] extract reverses the despair behaviour in reserpinised and diabetic mice

Green tea (C. sinensis) extract (GTE) dose dependently produced reversal of despair in normal, reserpinised and diabetic mice, thereby demonstrating an antidepressant effect. Although the exact mechanism is yet to be explored, the possible inhibition of catechol-o-methyl transferase and monoamine oxidase enzymes may be responsible for antidepressant activity of GTE.     

Characterization of fluoride uptake by roots of tea plants (Camellia sinensis (L.) O. Kuntze)

Backgrounds and aims

Tea plants (Camellia sinensis (L.) O. Kuntze) accumulate high fluoride in the leaves whereas the mechanism on its uptake is poorly understood. The measured F^? uptake was compared to calculated uptake from transpiration rates assumuing no discrimination between F^? and water to characterize the property of F^? absorption by tea plant roots.

Methods

The F^? uptake was examined by depletion method under variable external F^? concentrations, pH, temperature, relative air humidity, anion channel blockers and metabolism inhibitors in solution experiments.

Results

Measured F^? uptake rates were significantly larger than those calculated from transpiration rates regardless of external F^? concentrations, uptake durations, relative humidity, and solution pH. The measured and net F^? uptake (subtracting that calculated from transpiration rate from the measured uptake) were reduced by low temperature and inhibited by anion channel and metabolism inhibitors anthracene-9-carboxylic acid (A-9-C), niflumic acid (NFA), and carbonylcyanide m-chlorophenylhydrazone (CCCP) but not by dihydro-4, 4′ diisothiocyanostilbene-2, 2′-disulphonic acid (DIDS). The F^? uptake showed biphasic response patterns, following saturable Michaelis–Menten kinetics in the range of low external F^? (below 100 μmol?L^?1) while increased linearly with external supply in the range of high concentrations.

Conclusion

The uptake of F^? by roots of accumulator tea plants was likely an active process and energy-dependent. This helps to explain why tea plants are able to accumulate considerably high F^?.     

Shoot epicatechin and epigallocatechin contents respond to water stress in tea [Camellia sinensis (L.) O. Kuntze

An experiment was conducted to determine the association of tea catechins to water stress in tea, with the objective of determining their suitability as indicators for predicting drought tolerance in tea (Camellia sinensis). The study consisted of six tea clones (BBK 35, TRFK 6/8, TRFK 76/1, TRFK 395/2, TRFK 31/30, and TRFK 311/287) and four levels of soil water content (38, 30, 22, and 14% v/v), which were arranged in a complete randomized design and replicated 3 times. The treatments were maintained for a period of 12 weeks. Tea shoots were sampled for catechin analysis during the 6th week of water treatment, in which fresh shoots with two leaves and a bud were plucked and steamed for 2 min, and dried at 70 degrees C to constant weight. Subsequently, the samples were ground and analyzed for catechins using an HPLC system. The total catechins showed significant correlation with shoot growth (r=0.65, P=0.006), soil water content (r=0.54, P=0.0066), and water stress index (r=0.67, P=0.0004). The epicatechin (EC) correlated with shoot growth (r=0.58, P=0.0032), soil water content (r=0.62, P=0.0014), and water stress index (r=0.63, P=0.0010). Similarly, epigallocatechin (EGC) correlated with shoot growth (r=0.65, P=0.0006), soil water content (r=0.50, P=0.0133), and water stress index (r=0.60, P=0.0021). However, epigallocatechin gallate (EGCg) and epicatechin gallate (ECG) showed no significant response to changes in soil water content. The shoot contents of EC and EGC in the six clones showed varied responses, with a distinct pattern in the water-stress tolerant clones (TRFK 6/8 and TRFK 31/30). The results suggest a potential use for EC and EGC as indicators in predicting drought tolerance in tea.     

Agrobacterium tumefaciens-mediated transformation of embryogenic tissues of tea (Camellia sinensis (L.) O. Kuntze)

Embryogenic tissues of tea were cocultivated withAgrobacterium tumefaciens LBA4404. The plasmid pBi121, which contains the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the β-glucuronidase (uidA) reporter gene, was used as binary vector. The highest transformation frequency (12 transformants/g fresh weight [FW] of treated embryogenic tissue) was obtained with 5-day-old tissues grown in liquid medium and cocultivated withAgrobacterium for 2 d in the same medium but containing 50 μM acetosyringone. There was improvement in the recovery of kanamycin-resistant tissues when tissues were first grown for 10 d on a medium containing 350 mg/L Timentin to prevent bacterial overgrowth, before application of the selection pressure. Resistant tissues obtained after 6 wk on kanamycin-selection medium showed stableuidA expression. Polymerase chain reaction demonstrated the presence of the transgenes, while Southern hybridization confirmed their integration into the genome. Transgenic plants were regenerated from transformed tissues within 4 mo after coculture.     

Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves

The anatomical localization of caffeine within young Camellia sinensis leaves was investigated using immunohistochemical methods and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in resin was deemed the best protocol as it retained most of the caffeine and allowed for the samples to be sectioned with ease. Immunohistochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localized and accumulated within vascular bundles, mainly the precursor phloem. With the use of a pressure bomb, xylem sap was collected using a micro syringe. The xylem sap was analyzed by thin-layer chromatography and the presence of caffeine was determined. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defense against various pathogens and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine’s localization.     

Changes in endogenous auxins during winter dormancy in tea (Camellia sinensis L.) O. Kuntze

Changes in endogenous auxin (IAA) were determined in tea shoots with the onset of winter dormancy (30 October) and subsequent to dormancy release (15 April). Very low levels of free IAA were detected in dormant shoots when they were still in deep dormancy. The levels increased after 30 January and reached highest value after dormancy release. Conjugated IAA levels increased with onset of dormancy reaching its maximum value when free IAA levels were lowest. With increase in free IAA levels, the conjugated IAA levels decreased in shoots prior to dormancy release suggesting of metabolic interconversion of IAA during these periods. The possible roles of free and conjugated IAA in dormancy and following its release are discussed in relation to winter dormancy in tea shoots.