Biotechnological Approaches for Tea Improvement

BIOTECHNOLOGICAL APPROACHES FOR TEA IMPROVEMENT

4

H. Ranjit Singh*, Pranita Hazarika† ⁎

Cotton University, Guwahati, India, †Tocklai Tea Research Institute, Jorhat, India

4.1 Introduction The word “tea” is derived from “t’e,” the Chinese Fukien dialect. Tea plant was discovered by Robert Bruce during 1823 in Assam, India. Based on Wight’s nomenclature (Wight, 1959, 1962), tea may be of three races: (1) Camellia sinensis L. or the China tea plant, (2) Camellia assamica (Masters) or the Assam tea plant, and (3) Camellia assamica sub sp. lasiocalyx (Planch. MS), or the Cambodiensis or Southern form of tea plant. Cross pollination nature of tea plant make them genetically complex species. The genus Camellia belongs to family Theaceae accounting for >325 species (Mondal, 2002). About 600 varieties of tea are cultivated worldwide with unique traits such as high caffeine content, drought tolerance, blister blight disease tolerance, etc. (Mondal, 2004). Tea is the second most consumed beverage in the world, after ­water. Based on different processing of leaf, tea beverages may be of black (fermented), green (nonfermented), oolong (semifermented), white, and yellow teas. They differ in chemical constituent, appearance, and organoleptic taste. But black and green tea account for the major types of tea produced and consumed in the world. Indian tea industry plays a vital role in the economy of India. Tea business in India has accounted for an annual turnover of about US$660 million (Mondal, 2014). India, Sri Lanka, and Kenya produce most of the black tea while the other countries like China and Japan produce green tea. India is the second largest tea producing country in the world after China. Complete fermentation in black tea manufacturing process results in oxidation and polymerization of some vital secondary metabolites like polyphenols. This results in the formation of theaflavin Biotechnological Progress and Beverage Consumption. https://doi.org/10.1016/B978-0-12-816678-9.00004-7 © 2020 Elsevier Inc. All rights reserved.

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and ­thearubigin. These chemical constituents are responsible for the briskness, strength, color, taste, aroma, and pungency associated with black tea. Alternatively, in green tea manufacturing process fermentation does not take place. The leaves are collected and steamed immediately to inactivate the enzymes to check oxidation and polymerization of secondary metabolites like polyphenols. Thus, results in green color of made tea with the smell of fresh leaves. Chinese people initially used tea as a medicinal drink about 300 years ago, which later on has become a beverage and now considered as a potential drink with scope of attracting important industrial and pharmaceutical personals. There are many health benefits of drinking tea as supported by many scientific reports. Polyphenols were responsible for most of the beneficial effects of tea. A number of findings suggested that phenolic compounds may be responsible for reducing the prevalence of dreaded diseases like cancer and a­ rteriosclerosis. Other beneficial constituents include cinnamic acid derivatives and flavonoids. Since the natural polyphenols are mostly unaffected in green tea, it can be assumed that green tea is more ­beneficial than black tea. Other benefits of health include arthritis, cardiovascular diseases, diabetes, and obesity. With the advancement of many molecular techniques of biotechnology lots of information on different molecular approaches for plant improvement has been provided potentially uncovering the complexity of the demographic and adaptive processes underlying in the crop improvement of tea. The present chapter focusses on recent updates of some biotechnological techniques like micropropagation, somatic embryogenesis, genetic transformation, molecular markers, and functional genomics for tea improvement.

4.2  An Efficient Tool for Tea Propagation by Tissue Culture Technique Good planting material is the source of tea industry. The propagation of the plant plays a vital role for mass cultivation of tea garden. Although vegetative propagation was traditionally employed, but following limitations were encountered by tea planters (Mondal, 2014): (1) slow rate of propagation; (2) devoid of suitable planting material for selection due to some environmental factors like winter dormancy and drought in some tea plantation areas; (3) high mortality rate at nursery due to inefficient root development of some cultivars, and (4) seasonal rooting of the cuttings. The above limitations clearly demands for an efficient tea propagation technique. This led to the application of micropropagation

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t­ echnique to uplift conventional propagation method. Couple of merits of micropropagation are listed below: (1) rapid multiplication with huge number of plants, it is the requirement for a newly developed tea cultivar with high industrial demand to be supplied in large quantities within limited period of time and (2) important platform for successful transgenic technology, for the development of transgenic plant with desired quality micropropagation is indispensable for the transfer of the transgenic plants to the field within a limited period of time. A number of reports are available on micropropagation of tea (Kato, 1989; Das, 2001; Mondal, 2003). It is apparent from the publications that during 1980s micropropagation was highly reported. But the main determining factor about the issue of survivability of micropropagated tea in field condition was raised during 1990s. Report about the hardening and its field trial of micropropagated tea and its commercial application started during the onset of 2000s, which will be covered in our coming write-up. Many factors are involved for successful micropropagation protocols which are discussed below:

4.2.1 Selection of Explants For any in vitro culture the type, origin, and availability of explants is of utmost importance. Usually, shoot tips and nodal segments are mostly used as explants for tissue culture of tea (Vieitez et al., 1992). There are also report about using zygotic embryos and cotyledons for adventitious bud induction (Iddagoda et al., 1988; Jha and Sen, 1992). Other plant portion like flower stalks; stem pieces and leaf petioles have also been attempted for the same but with less efficiency of callus formation (Sarwar, 1985).

4.2.2 Standardization of Tissue Culture Media Standardization of tissue culture media is a crucial step for any tissue culture event like micropropagation. Commonly used basal medium remains either full- or half-strength of Murashige and Skoog (MS) salts (Murashige and Skoog, 1962). MS media was found to be better than B5 medium (Gamborg et al., 1968) and Nitsch and Nitsch (1969) for tea shoot multiplication (Nakamura, 1987a). Certain concentrations of thidiazuron (TDZ) were found to be useful in axillary shoot proliferation (Tahardi and Shu, 1992). Even though many workers have reported about full-strength MS media as a suitable basal medium, but it’s half-strength also worked very well for multiplication and shoot proliferation in tea (Phukan and Mitra, 1984; Banerjee and Agarwal, 1990; Agarwal et  al., 1992). Supplementation of halfstrength MS salts with vitamin also worked well to achieve initiation and multiplication of axillary shoots (Arulpragasam and Latiff, 1986).

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Addition of plant growth regulators (PGR) like 6-benzyladenine (BAP, 1–6 mg/L) and indole-3-butyric acid (IBA, 0.01–2.0 mg/L) in the culture medium was appropriate for both shoot initiation and multiplication (Mondal, 2014). The PGR, like 2,4-dichlorophenoxyacetic acid (2,4-D) and α-naphthaleneacetic acid (NAA) were successfully used for callus induction. But these PGRs were unable to initiate the growth and development of tea shoots (Nakamura, 1988). NAA was very effective in callus induction and shoot development when used in combination with BAP (Phukan and Mitra, 1984; Bag et al., 1997). Another PGR 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was reported for the successful shoot elongation (Jain et  al., 1991). Many workers gave an emphasis on the use of indole-3-acetic acid (IAA, 0.1–2.0 mg/L) and kinetin (Kn, 0.21–8.0 mg/L) for the induction and multiplication of axillary shoots in tea (Das and Barman, 1988). Mondal et al. (1998) highlighted a very interesting fact on the effect of TDZ on micropropagation of tea by showing that very low concentrations of TDZ (0.02 mg/L) alone was sufficient to induce shoot bud proliferation along with enhanced rates of shoot multiplication on a media devoid of any hormone. And higher concentrations of BAP (0.22–2.2 mg/L) showed good effect for shoot proliferation. In most of the cases higher concentration of TDZ (1.1, 2.2, and 3.3 mg/L) i­ nduces callusing of explants when used in combination with either 2,4-D, NAA or IBA at concentrations ranging from 1 to 3 mg/L. Higher efficiency (98%) of shoot proliferation was noticed with a mixture of TDZ (1.1 mg/L) and NAA (2 mg/L). TDZ was found to be more effective than BAP in shoot formation, but multiplication rates were almost similar in both the cases. Thus, TDZ was found to be an effective cytokininlike growth factor for tea micropropagation. Liquid culture medium was also well established for tea shoot culture (Sandal et al., 2001). Addition of TDZ (0.55–1.1 mg/L) in MS was again proved to be good growth promotion for tea shoot development in liquid culture. Liquid culture volume of 20 mL in 250 mL Erlenmeyer flasks was the most acceptable compared to other tested volumes in terms of result and cost effectiveness. Liquid medium performed better than solid medium (Carlisi and Torres, 1986). In addition to above requirements for micropropagation of tea, some important growth adjuvant have also been highlighted by some reports that include coconut milk (Nakamura and Shibita, 1990; Agarwal et al., 1992), yeast extract (Phukan and Mitra, 1984; Banerjee and Agarwal, 1990), casein acid hydrolysate (Chen and Liao, 1983; Jha and Sen, 1992), and serine and glutamine as nitrogen sources, etc. Sucrose (3%–6%) was the best carbon source (Nakamura, 1990). It was very interesting to find that caffeine, major constituents of tealeaves, was found to inhibit growth/development of tea shoots, stem, and roots in vitro (Owuor et al., 2007).

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4.2.3 Rooting Efficiency The transfer of in vitro-raised microshoots to field through hardening phase depends on the effectiveness of root formation and establishment. Literatures are available where hardening of microshoots is done after in vitro and ex vitro rooting in tea. In vitro rooting was found to depend on auxin treatment, strength of MS salt, and physical status of the cultures. Ex vitro rooting depends on pH of the hardening substance and relative humidity of the hardening room (Mondal, 2014). Besides root induction in tea, root elongation was also supported by reducing the strength of MS to half (Kato, 1985; Banerjee and Agarwal, 1990). In most cases, IBA (0.5–8 mg/L) was found to be more effective than NAA in root induction (Gunasekare and Evans, 2000; Bidarigh and Azarpour, 2011; Bidarigh et al., 2012). Liquid medium was also found to work well for rooting in tea. Ex vitro rooting media performed better than in vitro rooting (Kato, 1985; Nakamura, 1987b; Jain et  al., 1993) Pretreatment of cut ends of tea shoot by dipping in IBA (50 mg/L) solution for 2 h before hardening phase showed 97% rooting efficiency. Low light and low pH (4.5–4.6) also showed supporting result for root induction (Nakamura, 1987a; Banerjee and Agarwal, 1990). Genotypic character of cultivars also play a critical role in root induction in addition to above mentioned factors Murali et al. (1996).

4.2.4 Hardening Phase Any true tea plant, whether raised through in  vitro rooting or ex vitro rooting, is not worthy unless it is successfully transferred to soil or field condition. This transfer phase is called hardening phase and it is the determining stage of micropropagation. Generally, in vitro plantlets are first acclimatized under a controlled environment in greenhouse before transferring to open field. In greenhouse condition, the plantlets are taught how they can adapt in open environment condition. There are different approaches of hardening which are discussed below.

4.2.4.1  Conventional Hardening Conventional practice of hardening is done by planting in  vitroraised tea microshoots on soil mixture containing different ratios of cow dung, soil rite, etc. and kept in a polytunnel for about 6 months (Mondal, 2014). There is successful story about transferring 5–8-cm rooted plantlets to small plastic pots containing fumigated soil followed by its storage in humid chamber for 10 days before transferring to open field (Arulpragasam et al., 1988). Das and Barman (1988) reported about achieving better result by preconditioning plants at low

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temperature (22°C) and low light intensity (250 μmol /m2/s) after transferring to soil. Most of the standard protocols used soil mixture containing peat and soil (1:1) under high humid condition (Banerjee and Agarwal, 1990; Agarwal et al., 1992), but vermiculite and soil (1:1) also worked well (Kato, 1985). An exclusive survey was also done ­relating to time of microshoot harvesting, shoot size, soil pH, PGRs, CO2 enrichment, and light condition (Sharma et  al., 1999). Even though the protocol developed by some workers like Rajasekaran and Mohankumar (1992) and Mondal et  al. (1998) are little bit complex but the Research and Development Department of Tata Tea Ltd., India was able to transfer >45,000 tea plants to the field (Mondal et al., 2004). No significant difference was noticed between normal field-grown plants and tissue culture raised plants based on their physiological and biochemical data (Marimuthu and Raj Kumar, 2001).

4.2.4.2  Biological Hardening This is an alternative method of hardening specially to counter high mortality rate during laboratory-to-land transfer. The main reason of high mortality rate is that “aseptically” raised tissue culture plants get shock due to their sudden exposure to soil microflora. Initially the plants are unable to counter the microbial attack. Trichoderma, vesicular arbuscular mycorrhiza, Piriformospora indica, Bacillus subtilis, and Pseudomonas corrugata, are mostly used as biocontrol agents by their application to micropropagated tea plants before their transfer to soil (Singh et al., 2000; Pandey et al., 2000). Thus, biological hardening can be quite promising method of hardening.

4.2.4.3 Micrografting Micrografting is a widely used hardening method in citrus, cherry, kiwifruit, pistachio, stone fruits, apple, and grapes. Here the in vitroraised (scions) are grafted either onto in  vitro-raised rootstocks under sterile conditions or in vivo-raised stocks (Banerjee et al., 2000). Micrografting has been reported in tea (Prakash et al., 1999). Usually, conventional hardening method takes 12–18 months time whereas the same cultivar of micrografted one requires only 6–8 months time. This plays a vital role in tea breeding method. Micrografting not only reduces the hardening period of the tissue culture plant but also produces better root system in the plant to resist the subsequent drought periods in the field. Some important factors responsible for successful micrografting are: (1) effect of PGRs, (2) assessment of compatibility, (3) effect of the age of rootstock, and (4) season (Prakash et al., 1999; Mondal et al., 2005).

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4.2.5 Field Assessment of Micropropagated Plants Limited literatures are available on field assessment of micropropagated tea plants. There was no sharp difference in 17-month-old micropropagated and vegetative propagated (VP) tea plants of cultivar Banuari-96 at nursery level except root induction time, which was earlier by 1 month in case of micropropagated shoots (Sharma et al., 1999). Average height and stem thickness at collar region were higher in VP plants, leaf number was twice (16 leaves/plant) in micropropagated plants. Mondal et al. (2004) found comparable performance between field-grown micropropagated and VP tea cultivars, namely UPASI-9 and TTL-1 in terms of yield, biochemical analysis, and physiological parameters. However, two morphological variations were marked: (1) lateral shoot number was sufficiently higher in micropropagatedraised plants compared to VP plants and (2) root volumes were higher in micropropagated plants than VP plants.

4.2.6 Troubles Encountered During Micropropagation Two very common troubles encountered in micropropagation of woody perennial plant like tea are phenolic exudation from explants and microbial contamination in tissue culture medium, which are discussed below.

4.2.6.1  Phenolic Exudation Even though tea is valued for its high phenolic contents, yet its adverse effect occurs in tissue culture. Phenols exude from the cut ends of explants and allow enzymatic oxidation to form some toxic compounds in tissue culture medium making browning of in  vitro cultures. This problem has been overcome by putting chemicals like ascorbic acid, catechol, l-cysteine, phloroglucinol, phenyl-thiourea, PVP (polyvinyl pyrrolidone)-10, sodium diethyl dithiocarbonate, sodium fluoride, and thiourea along with highly reducing strength of MS salts (Iddagoda et al., 1988; Murali et al., 1996).

4.2.6.2  Microbial Contamination Contamination of tea plant by epiphytic and endophytic organisms has been reported (Debergh and Vanderschaeghe, 1988). So any part of the contaminated plant in field if taken as an explant, then it will contaminate any in vitro cultures. Therefore, prescreening of explants is of utmost importance. Several strategies were tried by many workers to overcome the problem: surface sterilization of stem explant with 70% ethanol followed by 7% sodium hypochloride solution,

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7% calcium hypochloride solution (Kato, 1985), 10%–15% Clorox solution (Arulpragasam and Latiff, 1986), benomyl (1, 2, or 4 g/L), and rifampicin at 10, 25, or 50 mg/L (Haldeman et al., 1987), streptomycin sulfate (1%) (Das and Barman, 1988), mercuric chloride solution (0.05%–1%) (Rajasekaran and Mohankumar, 1992; Jha and Sen, 1992), reducing the size of the explant to <0.5 mm (Kuranuki and Shibata, 1993).

4.3  Biotechnological Approaches for Selecting Good Planting Material for Helping Tea-Breeding Program Good planting material forms the backbone of tea industry. One of the major requirements of tea-breeding program is reliable selection criteria for good planting material (Kulasegaram, 1980). Tea scientists have tried with many morpho-biochemical markers for selection (Wachira, 1990; Singh, 1999; Ghosh-Hazra, 2001), but it is evident that they are unable to come up with an acceptable marker which could help the much needed tea industry for mass selection of planting materials with desired agronomic traits. Their inability may be due to the fact that most of the morphological markers reported so far were influenced greatly by the environmental features and hence showed a high degree of flexibility of the marker developed. As a result, these markers could not be formed as a dependable tool for identification (Wickramaratna, 1981). In the last couple of decades great efforts had been put forward to incorporate some modern techniques such as molecular markers to overcome some of the problems of conventional breeding. With the advancement of molecular biology and with the development of efficient DNA sequencing platforms DNA-based markers for crop improvement of tea was focused by tea scientists. There are several merits of molecular markers over the process of conventional selection. The utmost advantages of the technology are: (1) the markers are independent of the influences from the environmental factors and (2) the markers can be identified at an early stage of plant growth. The present discussion takes care about the molecular markers developed so far for tea improvement.

4.3.1 Random Amplified Polymorphic DNA (RAPD) Random amplified polymorphic DNA (RAPD) is one of the oldest types of molecular marker developed. RAPDs are DNA fragments amplified by PCR using single short synthetic primers (generally 10 bp). Initially, RAPD was widely used in the study of population genetics.

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It was the most preferred form DNA marker due to the fact that it is very rapid, user friendly, and it does not demand for radioactive materials (Williams et al., 1990). Good number of reports can be found regarding the use of this marker in tea which is summarized below.

4.3.1.1  Germplasm Characterization of Tea at the Molecular Level In the present practice, tea plantation is done mostly from the selected genotypes with desirable traits like crop yield, quality, and tolerance to biotic and abiotic stresses. As a result of which, the genetic diversity among the tea germplasms of a particular plantation area is diminishing. This demands for germplasm characterization at the molecular level of tea to help the tea industry: (1) in varietal development of tea with agronomically desired traits, (2) to protect the intellectual property right of tea breeders, (3) to identify a particular tea cultivar by developing a specific molecular signature, (4) to check the entry of exogenous genotypes in a particular tea gene pool, (5) to aid the selection of varieties for hybridization breeding, grafting, etc., and (6) in taxonomic classification of tea genotypes based on molecular markers. The first report on the use of RAPD marker came from Wachira et  al. (1995) to characterize 38 different cultivars of Kenyan tea in classifying into Assam, Cambodia, and China tea. Another group Tanaka et al. (1995) was able to detect the variation among Korean, Japanese, Chinese, Indian, and Vietnamese tea in the same year. And Japanese tea showed a closer relationship with Chinese and Indian counterparts, indicating that tea in Japan might have been introduced from China and India. Mondal (2000) successfully characterized 25 Indian tea cultivars and two ornamental species. These reports suggested that RAPD markers could be used for characterizing tea germplasm.

4.3.1.2  Detection of Genetic Stability Among the Micropropagated Plants The main objective of any micropropagation program is to produce large number of plantlets which are phenotypically stable and genetically similar with their mother/donor plant. The approaches like phenotypic variation (Vuylsteke et al., 1988), karyotypic analysis of metaphase chromosomes (Jha and Sen, 1992), and biochemical analysis (Damasco et al., 1996) were used to identify variants among micropropagated plants. But these techniques cannot produce adequate number of useful markers and are depended on environmental conditions (Rani et al., 1995). Due to these limitations, RAPD was used to study the genetic variability among micropropagated plants (Rani et  al., 1995; Damasco et  al., 1996). Mondal and Chand (2002) used RAPD marker to study genetic diversity of tissue culture tea plants of

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cultivar T-78. Other workers also used this technique to study the generic diversity in tissue culture products (Hammerschlag, 1992; Smith, 1998).

4.3.1.3  Identification of Pure-Line of Tea Cultivar RAPD markers can also be used to identify the pure-line of progenies of a particular cultivar. This is very important for successful in tea-breeding program. It helps to identify the true-crossing progenies and relating them with their exact parents which will finally help to indentify a particular tea cultivar. Two Japanese tea cultivars, Yutakamidori and Meiryoku, were successfully identified with the help of this marker (Tanaka and Yamaguchi, 1996). Wright et al. (1996) also used the same marker to screen five different South African tea cultivars, namely SFS 150, SFS 204, PC1, PC81, and MFS87. Singh et al. (1999) were able to demonstrate the originality of commercial tea which can be used by a tea manufacturer for identifying a particular cultivar for a particular tea brand. They have done this by isolating DNA from processed and dried tea samples and subjecting to polymerase chain reaction (PCR) amplification by taking RAPD primers. Tanaka et al. (2001) were able to identify the pollen parent of popular Japanese green tea cultivar “Sayamakaori” by using RAPDs. RAPD markers were used to classify different Camellia species in Chang’s manual of taxonomic classification (Prince and Parks, 2000; Tiao and Parks, 2003; Yoshikawa and Parks, 2001; George and Adam, 2006; Orel et al., 2007). But RAPD markers are dominant markers with very little reproducibility, which demand for alternative advance markers.

4.3.2 Inter-Simple Sequence Repeat (ISSR) In many plant species inter-simple sequence repeat (ISSR) markers had been used for studying genetic diversity (Tsumura et al., 1996). The main advantage of using ISSR marker is their large length of primer due to which genome mapping of closely related genotypes will be more stringent (Zietkiewicz et  al., 1994). Using ISSR marker (Mondal, 2002) was able to classify 25 diverse teas into three distinct clusters of Cambodia, Assam, and China type.

4.3.3 Restriction Fragment Length Polymorphism (RFLP) The first report on RFLP marker in tea was given by Matsumoto et  al. (1994) where they were able to classify Japanese green tea cultivars into five different groups of origin. The use of the same marker was also reported in another report (Matsumoto et al., 2002). In Japan, tea produced from the cultivar “Yabukita” had high demand in mar-

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ket, and therefore, Tea Company used to adulterate low-grade tea with Yabukita, which was not easy to be detected through visually or organoleptic performance of made tea. But this problem was solved by Kaundun and Matsumoto (2003b) with the help of sequence tag site (STS)-RFLP marker using the sequence information of three genes, namely, phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and dihydroflavonol 4-reductase (DFR). The restriction fragments thus produced after performing PCR was able to authenticate 46 tea samples.

4.3.4 Simple Sequence Repeat (SSRs) Simple sequence repeats (SSRs) are tandem repeats of DNA sequences of about 2–5 bp long which are reported to be highly polymorphic in plant genomes (Wu and Tanksley, 1993). This marker is also known as microsatellites. SSR marker is considered to be the most dependable genetic markers due to their high reproducibility and high scorable polymorphism. Couple of reports on the use of SSRs markers in Camellia japonica is available (Ueno et al., 2000). In silico data mining has also been done and validated for SSRs on different species of Camellia (Yang et al., 2009).

4.3.5 Amplified Fragment Length Polymorphism (AFLP) Amplified fragment length polymorphism (AFLP) markers are also another dependable DNA marker (Vos et  al., 1995). AFLP markers can detect higher polymorphism than RFLPs or RAPDs making them an accurate option for genetic studies in closely related population (Meksen et al., 1995). AFLP marker was first used in tea by Paul et al. (1997) where they were able to differentiate 32 tea clones comprising Indian and Kenyan origin into three known types, that is, Assam, China, and Cambodia. Rajasekaran (1997) also did AFLP analysis for 42 tea clones (23 UPASI, 17 popular South Indian estate clones, and 2 Kenyan tea clones). They reported that 90% of the UPASI clones were inbred and thus inappropriate for commercial exploitation. They were also able to identify the clones with blister blight disease resistance and susceptibility. The marker was also successfully used to identify drought tolerant tea clone (Mishra and Sen-Mandi, 2004).

4.3.6 Single Nucleotide Polymorphism (SNP) Compared to other available molecular markers, SNPs are highly available and stable during inheritance due to which they form a reliable tool for marker-assisted selection. In tea, Huang et  al. (2007)

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reported about the SNPs found in the coding sequence of polyphenol oxidase gene.

4.3.7 Sequence-Tagged Microsatellite Site (STMS) In nuclear DNA and organellar DNA there are polymorphic loci which are made of repeating units of 1–10 base pairs in length called SSRs, sequence-tagged microsatellite sites (STMS). STMS marker has been reported by Caser et al. (2010) in their genetic diversity study taking 132 accessions of tea germplasm, which particularly highlighted genetic overlap among Camellia sasanqua cultivars and those belonging to Camellia vernalis, Camellia hiemalis, and Camellia hybrida. Similar type of report about STMS markers was also given by Matteo et al. (2010).

4.3.8 Single-Strand Conformation Polymorphism (SSCP) Single-strand conformational polymorphism (SSCP) analysis is a simple and reliable technique to study the changes in conformation of single-stranded DNA due to mutation. Wachira et al. (1997) were able to show species introgression into cultivated gene pool of tea with the help of SSCP marker taking cpDNA-specific primers. This study indicated that Camellia furfuracea, Camellia assimilis, Camellia nokoensis, and Camellia tsaii belong to a common haplotype, which in turn indicated a possible hybridization between the species considered.

4.3.9 Cleaved Amplified Polymorphic Sequence (CAPS) Cleaved amplified polymorphic sequence (CAPS) marker is the combination of PCR and RFLP techniques where a very small amount of DNA is required for PCR analysis to show polymorphisms. CAPS markers were successfully employed in many perennial plants due to the availability of their nucleotide sequence in public domain. CAPS marker was used to study the genetic diversity of tea based on PAL, chalcone synthase 2 (CHS2), and DFR genes (Kaundun and Matsumoto, 2003a). These genes function for catechin and tannin biosynthesis, which are responsible for tea taste and aroma. In their study, it was found that China type was genetically distant from Assam type. In another work, Ujihara et al. (2011) reported about development of EST-based CAPS markers to screen different tea cultivars.

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4.3.10 Construction of Linkage Map In order to understand the genomic structure of an organism construction of genetic linkage map is important. The linkage map will be very useful for molecular plant breeding program. But for construction of linkage map, the initial requirement is a mapping population to understand the segregation pattern of a marker. This is difficult in a perennial woody species like tea due to its complex genetic makeup. The first linkage map for tea plant was constructed with RAPD markers along with phenotypic characters such as theanine content, date of bud sprouting, resistance to anthracnose, and tolerance to cold (Tanaka, 1996). RAPD and AFLP markers developed from two known noninbred parents were also scored to construct a linkage map (Hackett et  al., 2000). There is also a report about an AFLP linkage map c­ onstruction for tea plant in China (Huang et al., 2005). In another work, “Fuding Dabaicha” × “Yunnna Dayecha” was allowed for open ­ pollination. Backcross between F1 generation “Zhenong 129” and one of their parent “Fuding Dabaicha” was done. A partial genetic map of this ­backcross was constructed using RAPD and ISSR markers (Huang et  al., 2006). A reference map was constructed by merging the linkage maps from the data of markers like 441 SSR, 7 CAPS, 2 STS, and 674 RAPD, which can be used as a basic reference linkage map of tea (Taniguchi et  al., 2007, 2012). Most recently, there is another report about integrated genetic map of tea developed from the F1 generation derived from a cross between two commercial cultivars (“TTES 19” and “TTES 8”). This newly constructed integrated map comprised of 367 linked markers, including 36 SSR, 3 CAPS, 1 STS, 250 AFLP, 13 ISSR, and 64 RAPD markers. This integrated genetic map having the highest genetic coverage so far, could be very useful for comparative mapping, QTL mapping, and marker assisted selection of tea in the future (Hu et al., 2013).

4.4  An “Omic” Approach for Tea Improvement In the era of “omics,” functional genomics studies are very important to understand the genomic nature for any particular trait or character. The branch of molecular biology that deals with the functions and interactions of genes and proteins by using the genomics and proteomics data available in the public domain is called functional genomics. The role of functional genomics in plant breeding is of immense potential (Mondal, 2013; Mukhopadhaya et al., 2013). Functional genomics study in tea was started with the characterization of CHS gene from “Yabukita,” a Japanese green tea cultivar (Takeuchi et al., 1994).

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A good number of researches have been on functional genomics in tea by characterizing its trait-specific genes. Functional genomics studies target (Mondal, 2014): (i) cloning and c­ haracterization of trait-specific genes and (ii) differential gene profiling for identification of gene(s) responsible for a desired trait.

4.4.1 Classification of Genes of Interest Most of the tea growing states focus on two parameters, quality and yield, for commercial tea cultivation. Therefore, the tea biotechnologists are interested in those genes which are responsible for the quality and yield of the plant. In order to understand the complex molecular mechanisms and to improve the quality and yield of the plant, a number of functional genomics studies have been done worldwide. Tea genome has been successfully sequenced a few months back. Majority of the quality and stress-related genes have been sequenced and studied.

4.4.1.1  Genes Responsible for Intrinsic Tea Quality Drinking tea has become an indispensable habit of mankind and we value tea for its intrinsic quality. Therefore, research works are more directed toward the improvement of quality and commercial value of the crop plant. Similarly, in the field of functional genomics, several genes which are responsible for various biochemical pathways are studied in detail, which is discussed below: Genes Involved in Flavonoid Biosynthesis Tea is a desired drink for its distinctive flavor which is due to the presence of flavonoids in tea. Flavonoids are a type of polyphenols composed of 15%–35% of the total dry weight of tea plant. Several genes of flavonoid biosynthesis pathway have been cloned and characterized from tea plants. These genes include PAL, CHS, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3′H), flavanone-3-hydroxylase (F3′5′H), leucoanthocyantin reductase (LAR), DFR (Matsumoto et al., 1994a, b; Ma et  al., 2010; Singh et  al., 2008, 2009a, b, c). Polyphenol oxidase plays an important role during processing of black tea. This gene was also cloned and characterized from young tealeaf by many workers (Zhao et al., 2001; Wu et al., 2010). The vital enzymes involved in polyphenolic proanthocyanidins pathway (PA) of tea was also characterized by Pang et al. (2013). Genes Involved in Purine Biosynthesis In tea, purine alkaloids consists of caffeine, theobromine, and theophylline. The gene expression patterns of caffeine biosynthetic

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­ athway are studied (Suzuki, 1972; Ashihara et  al., 1997; Kato et  al., p 2000; Feng and Liang, 2001; Deng et al., 2008; Mohanpuria et al., 2009). Genes Responsible for Aroma Aroma is an important index to understand the quality of black tea. The formation of aroma in tea is done by enzymes like β-glucosidases and β-primeverosides (Takeo, 1981). Therefore, if we want to enhance the quality of made tea then it is important to increase the aromaforming compounds. Gene expression pattern of β-glucosidases and β-primeverosides are studied (Mizutani et  al., 2002; Li et  al., 2004; Zhao et al., 2006a). Genes Involved in Theanine Biosynthesis Theanine is a particular type of amino acid found in tea. It is found to be a glutamate derivative and mostly present in tender leaves (Sugiyama and Sadzuka, 2004). Theanine is responsible for the briskness of tea, which is directly related to the quality of black tea. Theanine constitutes the half of the total amino acids found in tea. It is synthesized in the roots and stored in the tender parts of the plant. Under sunlight, it is converted to polyphenols, another major secondary metabolite of the plant which gives the taste of tea and some medicinal values. In theanine biosynthesis pathway assimilation of ammonia generated by various biochemical processes is done by glutamine synthase. The glutamine synthase gene has been cloned from tea (Rana et  al., 2008a, b) and its expression pattern is also studied (Edwards et al., 1990).

4.4.1.2  Genes Responsible for Biotic Stress Biotic stresses the harmful agents that infect tea bushes and decreases the productivity of tea. This basically includes pests and pathogens. The cystain gene was cloned by Wang et al. (2005). Yoshida and Homma (2005) were also able to clone eight wound/ pathogen inducible cDNAs and the performed in silico analysis (Yoshida and Homma, 2005).

4.4.1.3  Genes Responsible for Abiotic Stress Abiotic stresses of tea cultivation are the stresses due to nonliving things like light, heat, cold, drought, and food, which hamper the productivity of tea significantly. The corelation between light or dark conditions on the utilization of nitrate as well as ammonia has been studied from the differential expression of cytosolic glutamine synthetase gene (CsGS) from tea (Rana et al., 2010). Ammonium transporter (AMT) gene, involves in utilization of nitrogenous fertilizer, has also been cloned from tea root (Taniguchi and Tanaka, 2004). CsCOR1 is

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one type of cold responsive gene cloned recently from tealeaves. The first enzyme of sulfate assimilation pathway, ATP sulfurylase (APS1 and APS2) was cloned from tea (Zhu et  al., 2008). Selenocysteine methyltransferase (SMT) gene, responsible for selenium metabolism, was cloned from tea (Zhu et al., 2008).

4.4.1.4  Genes Responsible for Photosynthesis Plants are green due to the presence of chlorophyll which helps them to prepare their food (cabohydrate) by photosynthesis in the presence of solar energy and release of oxygen. There exist two ­photosynthesis-related genes in tea plants, photosystem II protein D1 and violaxanthin de-epoxidase (VDE) out of which the later has been studied extensively. Two photosynthesis-related genes had been cloned, which were photosystem II protein D1 and VDE genes. Among them, VDE had been studied extensively (Wei et  al., 2003, 2004). Ribulose 1,5-bisphosphate carboxylase (RUBISCO) is the most abundant protein in any organism. RUBISCO is a key enzyme for Calvin cycle pathway of photosynthesis. In energy metabolism of the Calvin cycle pathway of photosynthesis. The full length of RUBISCO small subunit (RbcS) has been cloned from tea and studied (Ye et al., 2009).

4.4.1.5  Genes Responsible for Cellular Structural Elements Microtubules are the major constituent eukaryotic cytoskeleton. Microtubules are of two types, α-tubulin and β-tubulin dimmers, which acts as an important structural element involved in mitosis, cytokinesis, and vesicular transport. The β-tubulin gene has been cloned from tea (Takeuchi et  al., 1994). The eukaryotic nucleosome composed of four histones subunits, H3, H2A, H2B, and H4. Out of them H3.1 cDNA (CsH3) was cloned from tealeaves and its expression profile had been studied (Singh et al., 2009d). Cloning and expression analysis of another important gene, lipoxygenase (LOXs) has been done in tea plant (Fang et al., 2006; Liu and Han, 2010).

4.4.2 Gene Expression Profiling Understanding the changes at the transcript level will give a clear picture about the molecular mechanisms taking place inside the plant under stress condition. Therefore, differential gene expression studies have a role to go for transcriptomic study. There are reports about the use of different techniques to study transcriptome (Mondal and Sutoh, 2013; Das et al., 2013) but suppression subtractive hybridization (SSH), cDNA AFLP is mostly preferred to study the differential gene expression in tea. SSH was used to study the genetic regulation of secondary metabolism pathways in the young leaves (Park et al., 2004; Chen et  al., 2005), to isolate the cold-related genes (Wang et  al., 2009), to

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clone genes associated with dormancy (Wang et al., 2010; Krishnaraj et al., 2011; Paul and Kumar, 2011; Yang et al., 2012; Phukon et al., 2012; Thirugnanasambantham et  al., 2013) and to screen drought-related genes (Sharma and Kumar, 2005; Das et al., 2012; Gupta et al., 2012; Muoki et al., 2012; Gupta et al., 2013). There are reports about the construction of drought-induced SSH library (Das et al., 2012; Taniguchi et al., 2012); light-induced library (Wang and Ruan, 2012; Wang et al., 2012); aroma-related library (Yang et  al., 2011). Differential expression of PAL1, CHS, DFR, leucoan-thocyanidin reductase, and flavanone 3-hydroxy-lase genes were also studied (Eungwanichayapant and Popluechai, 2009); Ectropic obliqua infections induced defense-­ related genes (Qiao et al., 2011). The genes responsible for flavored tea popularly known as ‘oriental beauty’ in Taiwan was also analyzed by gene expression profiling and biochemical study with the help of DNA microbead array and fluorescence labeled-based technique (Choi et al., 2007). Darjeeling tea is world famous for its flavor, aroma, and quality. Besides its complex genetic makeup, the inherent quality of the plant is found to be triggered by insect infestation, particularly jassids and thrips. Several genes and transcription factors were involved in the aroma and flavor formation of the plant (Gohain et al., 2012). Expression of genes encoding enzymes involved in flavan-3-ol biosynthesis pathway such as, CHS, CHI, F3H, F3′-5′H, DFR, ANS, ANR, and LAR was investigated (Takechi and Matsumoto, 2003; Ashihara et al., 2010; Zhao et al., 2012). It was noticed that shade had influential effects on flavonoids, proanthocyanins, and lignin biosynthesis, but hardly any on anthocyanin accumulation (Wei et al., 2013). On the other hand, it was proposed by some workers that phenolic acids compete with lignin and flavonoid biosynthesis in tealeaves under different conditions of lighting (Wang et al., 2012). Gene expressions related to the variations in morphology of callus are also studied by Yang et al. (2012). The first report on cDNA microarray of tea plant was developed with 1680 gene tags developed from the SSH library of gray blight disease infection in tolerant cultivar UPASI-10 Senthilkumar et al. (2012). This shows that cDNA microarray can be used for high-throughput detection in the gene expression profiling in tea (Zhao et al., 2006b). Attempts were also made to understand the gene expression of tea by cross species hybridization (Murayama et al., 2007; Venkatesh et al., 2006). The genes responsible for albino phenotype of “Anji Baicha” tea cultivar widely cultivated in China have been analyzed using microarray technology (Ma et al., 2012).

4.4.3 In Silico Analysis The application of computer science has played a tremendous role in biological sciences. Nowadays bioinformatics and biostatics has become an integral part of the functional genomics. Large n ­ umber of

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data are generated from research works which demands for a specific system from computer science to store and to analyze them. This led to an in silico analysis which is very important during pre- and postlaboratory work. The availability of large number of expressed sequence tags (ESTs) in the public domain has provided the researchers with alternative process of SSR development without employing the conventional methods. Submission of EST sequences of tea to public domain is increasing day by day which can be used for studying functional genomics across species (Sahu et al., 2012). The EST sequences submitted can also be used for studying miRNAs of tea (Das and Mondal, 2010; Prabu and Mandal, 2010). In silico analysis of a CsGS from tea was conducted. This was done by comparing its structural aspects with other known structures of CsGS available in public domain. The 3-D structure of tea CsGS protein showed high degree of similarity with maize GS (Yadav, 2009). Similarly, other proteins in tea like ICE1 (Shi et  al., 2012), stearoyl-acyl carrier protein desaturase (SAD) was also tried (Pan et al., 2013) with great success.

4.4.4 Next-Generation Sequencing Platform Different platforms of DNA sequencing are available nowadays. The next-generation sequencing technology, particularly RNAseq is commonly used for studying differentially expressed genes (Mondal and Rana, 2013). The first transcriptome data set was generated by RNA-Seq from the young leaf of tea plants, which can be used for gene expression, genomics, and functional genomics studies (Shi et  al., 2011). In the same year, Jiang et  al. (2011) reported about deep transcriptome sequencing of Camellia oleifera, Camellia chekiangoleosa, and Camellia brevistyla using 454 GS FLX platform. The genes involved in secondary metabolism were identified with the help of high-throughput sequencing of tealeaf transcriptomes (Wu et al., 2013). RNA-SEq was also used to reveal the molecular mechanisms of cold induction in tea plants by genome wide expression profiling. The study indicated that “carbohydrate metabolism pathway” and “calcium signaling pathway” might play a critical role during cold induction in tea plants (Wang et al., 2013).

4.5  In Vitro Plant Regeneration System of Tea By Somatic Embryogenesis The manipulation of cell in laboratory condition (in vitro) can be an additional tool to counter some of the problems of traditional tea breeding. The development of somatic embryos from ant somatic cell

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is called somatic embyrogenesis. The most crucial step of somatic embyrogenesis is the development of an efficient in vitro plant regeneration system (Jain and Newton, 1990). The effectiveness of somatic embyrogenesis depends on the rate of multiplication and regeneration of somatic embryos. The main advantage of somatic embryogenesis is the growth and development of embryos from explant tissue without going for callus phase which helps in maintaining genetic integrity (Bano et al., 1991). Thus, it will highly applicable for clonal propagation (Mondal et  al., 2001a) and genetic transformation of tea (Mondal et al., 1999, 2001b). With the help of this technology artificial seed production (Mondal et  al., 2000a, b; Mondal, 2002) and some interspecific hybrid crosses of Camellia (Nadamitsu et al., 1986) are now possible. Somatic embryogenesis can also be used for producing disease free plants. In recalcitrant tea, somatic embryogenesis can be alternative means to conventional micropropagation. Following factors are responsible for achieving successful somatic embryogenesis.

4.5.1 Induction of Primary Somatic Embryogenesis Primary somatic embryogenesis can be induced by several factors discussed below.

4.5.1.1 Explants Reports are available about the use of explants like immature cotyledons (Abraham and Raman, 1986; Nakamura, 1988a; Bano et  al., 1991), de-cotylenated embryos (Nakamura, 1985; Paratasilpin, 1990; Mondal et al., 2000a, 2000b), deembryonated cotyledons (Ponsamuel et  al., 1996), nodal cuttings (Akula and Akula, 1999), juvenile leaves (Sarathchandra et al., 1988), and leaf stalk (Hua et al., 1999).

4.5.1.2  Season of Seed Collection The optimum time for cotyledon culture varies with season and the type of cultivar. The preferable timing for Camellia sinensis was late September to mid-October (Paratasilpin, 1990; Nakamura, 1988a). Mondal et al. (2000a, b) found that UPASI-9 was favorable during JulyAugust; Tuckdah-78 during September-October and Kangra Jat was during November-December. Induction of somatic embryogenesis in Camellia japonica was maximum with seeds collected during July, September, and October (Vieitez and Barciela, 1990). Therefore, it can be said that somatic embryogenesis is also controlled by some genetic factors in Camellia (Nakamura, 1988a).

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4.5.1.3  Media Composition and Growth Regulators Media plays a very crucial role in any tissue culture work. Lots of work has been done on the type, concentration, and time of application of different growth regulators in culture media. In addition to commonly used MS media, WPM (Woody Plant Medium, Lloyd and McCown, 1980) and Nitsch and Nitsch (1969) media were tried with good result. From full to one-third strength modified-MS was tried. This was supplemented with sucrose (20 g/L) or d-glucose (25 g/L) (Pedroso-Ubach, 1991). 6-Benzylaminopurine (BAP, 0–10 mg/L) was used as a growth regulator by many workers (Bennett and Scheibert, 1982; Beretta et  al., 1987; Barciela and Vieitez, 1993; Kato, 1986b; Zhuang et al., 1988; Zhuang and Liang, 1985a, b; Vieitez et al., 1991). The workers also tried with other elicitors like NAA (Paratasilpin, 1990; Bag et al., 1997; Balasubramanian et al., 2000), 2,4-Dichlorophenoxyacetic acid (2,4-D) (Das and Barman, 1988; Bano et al., 1991), IAA (Wu et al., 1981; Sood et  al., 1993) and abscisic acid (ABA) (Akula et  al., 2000). Reports are available on somatic embryogenesis using MS media containing a cytokinin (frequently BAP) with or without auxin (frequently NAA). But these reports were unable to determine optimum conditions for somatic embryogenesis suitable for all the cultivars of Camellia (Mondal, 2014).

4.5.1.4  Growth Adjuvants Growth adjuvants are required in low concentration but their presence in tissue culture media is indispensable. Even though it was reported that tea does not require any growth adjuvant, yet some ­researchers got good result of tea tissue culture practice after using different types of adjuvants like yeast extract (Yamaguchi et al., 1987; Arulpragasam et  al., 1988), coconut milk (Sarathchandra et al., 1988), adenine sulfate (Jha et al., 1992), betain (Akula et al., 2000), etc. These adjuvants also showed positive result of yeast extract had also been found in some Camellia hybrids (Yamaguchi et al., 1987).

4.5.2 Secondary Embryogenesis Secondary embryogenesis is very important for transgenics work if primary somatic embryos are used as explants for Agrobacterium infection Mondal et al. (2001a). Different workers tried several types of media for secondary somatic embryogenesis which composed of stages like induction, germination, and maintenance of somatic embryos. Some used single media for all the stages (Abraham and Raman, 1986) and others used different media compositions for different stages (Kato, 1986a; Jha et al., 1992; Balasubramanian et al., 2000).

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4.5.3 Maturation and Germination of Tea Somatic Embryos Some of the factors responsible for efficient somatic embryogenesis are elevated germination rate and successive plant regeneration (Webster et  al., 1990). In comparison to other woody plants tea cotyledon was found to be easier in induction of somatic embryogenesis (Vieitez, 1994). Two common difficulties frequently encountered during tea somatic embryogenesis were precocious and abnormal germination. To overcome such problems the following factors need to be addressed.

4.5.3.1  Carbon Source Besides acting as a carbon source, sugars are the inducer of desiccation tolerance during maturation and germination of somatic embryos (Lecouteux et al., 1993; Mondal et al., 2002) by providing an osmotic medium (Tremblay and Tremblay, 1995).

4.5.3.2 Desiccation The capacity of somatic embryogenesis can be promoted by desiccation treatment (Roberts et  al., 1990). Tea somatic embryos were found to be sensitive to desiccation. This may be due to its recalcitrant nature. Impact of high relative humidity (60%–90%) on tea somatic embryos resulted in poor germination. Therefore, it was suggested to use osmotic protectant in culture media to avoid the tea somatic embryo from desiccation (Mondal et al., 2002).

4.5.3.3  Plant Growth Regulators Some reports (Roberts et  al., 1990; Avgioglu and Knox, 1989) indicated that ABA promotes germination in woody plants but Mondal et al. (2002) did not find the same effect in tea somatic embryos. Same thing happened in case of gibberellic acid (GA3), which promoted germination in several woody species but not significantly in tea. BAP, IBA, and IAA were also found to promote rooting and shooting based on different combinations (Kato, 1986a; Vieitez and Barciela, 1990; Vieitez et  al., 1991; Pedroso and Pais, 1993; Plata and Vieitez, 1990; Plata et al., 1991; Jha et al., 1992).

4.5.4 Hardening of Tea Somatic Embryo Derived Plants Limited literatures are available on hardening of tea somatic embryo derived plants. The first report about the transfer of such plants to field by using soil mixture came from report of Wu et  al. (1981).

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In another report (Kato, 1989) a mixture of vermiculite and soil (1:1) was used to perform hardening of tea somatic seedlings under natural conditions. Many other soil mixtures like peat: soil (1:1) (Jha et al., 1992), autoclaved mixture of sand: peat (3:1) (Wachira and Ogado, 1995); sand and cowdung (1:1) (Akula and Akula, 1999) were also used for hardening. Mondal et al. (2002) established a complex technique of hardening by the application of which 3000 somatic seedlings of tea were reported to be transferred to the field by Tata Tea Ltd., Kerala, India (Mondal et al., 2004). In spite of the availability of above successful reports on hardening of tea somatic embryo derived plants, none of them has reported about the commercial exploitation of the protocol by any tea industry of the world.

4.5.5 Variations in Somaclones and Gametoclones Usually in breeding program of perennial crops demands for stable somaclones are there but no attempt has been made to produce useful somaclones in tea. This may be due to two reasons (1) since tea exhibit open cross-pollinated somaclones already exist naturally and most of the breeders are interested in the superior variants and (2) since hardening is not well established in tea, therefore, sizable amount of hardened tea somatic embryo derived plants may not be present to study the somaclonal variants. Rajkumar et al. (2001) studied somaclonal variation of tea plants raised from somatic embryos of clone UPASI-10. They reported five somaclonal variants in the field based on morphological, physiological, and biochemical parameters. Das (1992) was able to identity a wide range of chromosomal variations in callus derived from in vitro leaf and cotyledon explants of tea. In another experiment with UPASI-10, 15 somaclonal variants were derived in field which were taken for characterization by morphological, physiological, and biochemical characters. The result showed that a very few variants possessed unique “Chinary” characters while others exhibited “Assam” characters.

4.6  Transgenic Tea for Crop Improvement The conventional tea breeding method is unable to provide good planting material to tea breeders which can resist the biotic and abiotic stresses prevalent in tea plantation areas. The stresses can be controlled with the help of chemicals. But the adverse side effects of such chemicals on nature make them undesirable for application. Every year human population is increasing due to which the demand for food items is also increasing simultaneously. Geographical area is fixed but the yield must be increased to cope with the increasing demand. Therefore, transgenic approach can be an option to overcome the problems relating to biotic, abiotic, quality, and yield of tea.

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The initial requirement was to develop a protocol for gene transfer. Transformation of tea can be achieved by any of the following means:

4.6.1  Agrobacterium tumefaciens-Mediated Transformation of Tea The first report about producing of transgenic tea, cv. Kangra jat, was given by Mondal et  al. (1999). Transformation was done via Agrobacterium-mediated genetic transformation taking Agrobacterium tumefaciens strains like EHA 105 and LBA 4404. Many attempts were made to maximize the transformation efficiency. Acetosyringone (AS) treatment acts as an inducer of the virulence genes. After cocultivation, the somatic embryos were inoculated on a multiplication medium and selected with kanamycin (50 mg/L). GUS histological assay shows the insertion of gene of interest into plant genome. Kanamycin-resistant, GUS-positive embryos were selected and germinated. Later, the transgenic plants developed thereafter were micrografted onto seed-grown rootstocks (Mondal et al., 2005). Then the transgenic plants were taken for hardening. Integration of transgene was tested by PCR and southern hybridization. Agrobacterium tumefaciens-mediated transformation of tea was done by many workers (Mondal et al., 2005; Mondal et al., 2001b; Matsumoto and Fukui, 1998, 1999; Wu et al., 2003; Liang et al., 2000).

4.6.2  Agrobacterium rhizogenes-Mediated Transformation of Tea Agrobacterium rhizogenes is also successfully used to transform tea and reported by some workers (Zehra et al., 1996; John et al., 2009). They used Agrobacterium rhizogenes strains like A4, MTCC 532, etc. to transform tealeaves. Transformation frequency (70%) obtained was very encouraging. AS of concentration 60 mg/L was effective for the transformation work. The tissue culture activities were done in MS media supplemented with maltose (30 mg/L) and IAA (5 mg/L) which was well enough to produce callus and hairy root culture. The hairy roots were found to grow rapidly against geotropism along with the formation of dense white fibrils and strong diversification capacity. The capacity of callus formation was found to be different in different cultivars (Peng et al., 2004).

4.6.3 Transformation of Tea by Biolistic Gene Gun For transient exogenous gene expression biolistic-mediated transformation is employed with the help of gene gun. This type of work was first done by Akula and Akula (1999). Gold particles (1.5–3 μm

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diameters) coated with plasmid DNA (p2k7) were used to bombard somatic embryos. The plasmid DNA (p2k7) was prepared by using binary vector pBI221 with the incorporation of nptII gene and gus gene driven by a constitutive promoter 35S from cauliflower mosaic virus. The coating of gold particles with DNA (1 μg/μL) is done by precipitating with CaCl2 (111 mg/mL) and spermidine (14.52 mg/mL). The overall performance of the action is determined by various factors: (1) the distance between the site of delivery of the microprojectile and the target tissue, (2) helium pressure, and (3) the condition of target tissue considered to obtain transient expression. The transformants were analyzed with the help of GUS assay after 30–40 h of bombardment. With the help of biolistic-mediated transformation maximum transformation efficiency of about 1085 blue spots/shot were obtained by taking somatic embryos. Helium pressure of 550 kPa was used for the target tissue placed at 9.5 cm away from the DNA delivery position. Initial treatment with mannitol was not effective for the transient expression level. The gfp gene guided by organelle specific signals was incorporated with the help of particle bombardment or co-transformation with Agrobacterium tumefaciens taking embryogenic tea callus. Putatively transformed embryos were selected on culture media mixed with kanamycin. The incorporation of gfp gene into tea genome was checked by PCR analysis and its preliminary gene expression system was studied with the help of a spectral imaging system and later confirmed by performing real-time PCR (Kato et al., 2004).

4.6.4 Applications of Transformation Technologies The application of transformation technology in tea science was started with the silencing of gene. Glutathione synthetase (gs) gene responsible for restoring cellular redox balance was silenced which produced transgenic tea plant with reduced glutathione content (Mohanpuria et al., 2008). DNA fragment of size 457 bp of gs gene was cloned to RNAi construct and used to transform tea somatic embryo with the help of Agrobacterium tumefaciens. In order to overcome the problems pertaining to transgenic tea development, Mohanpuria et al. (2010) established a rapid Agrobacterium-mediated transformation in tea root system. The protocol was used to establish caffeine-free transgenic tea plants. They successfully constructed RNAi construct (pFGC1008-CS) with 376 bp of caffeine synthase (cs) cDNA fragment, then used Agrobacterium-mediated transformation technique to infected tea root system. The transgenic tea plant showed reduced expression of cs gene and a significant decrease in caffeine and theobromine contents. Reconstructed RNAi construct (pF-GC1008-CS) with 376 bp ofcs gene fragment was also used to produce transgenic tea with reduced caffeine content with the help of gene gun (Mohanpuria et al., 2011).

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In case of overexpression of exogenous gene transgenic tea plants were produced by overexpression of osmotin gene with the help of gene gun. The recombinant tea plants were reported to have ­improved stress tolerance. Transgenic tea plants with blister blight resistance were also reported with the overexpression of exogenous class I chitinase (Singh et al., 2015) and glucanase gene (Singh et al., 2018).

4.7 Conclusion Tea being the most economical plantation crop, special emphasis must be given for its crop improvement since its marketing is counted as one of the major revenue sources in tea growing countries. Till today sufficient reports are available relating to micropropagation of tea. Different media compositions have been reported so far with different physiological stages of explants. Although MS is the widely used basal medium, BAP is the most preferred PGR. It is important to mention here that micropropagation of tea plants have not been successfully established for commercial benefit. Hardly any commercial tea garden has used micropropagated plants for plantation. The major drawbacks are the absence of juvenility of in vitro cultures making them incompatible for long-term production, due to its inability to establish taproot system micropropagated tea cannot withstand drought and protocols established are not cost effective. For a cultivar of superior quality with high demand for planting materials, including transgenic tea to supply within a short period of time, micropropagation is the best option for commercial exploitation. Traditionally, genetic improvement of tea was done by conventional breeding practice but with the development of modern techniques such as molecular markers during the last two decades conventional method can be replaced with modern molecular breeding technique. The idea is nice since it is more concrete and accurate but it is easier said than done. Different workers have claimed about the development of molecular markers for screening desirable tea planting material but hardly any marker has been used for commercial exploitation by tea planters. This implies that the biotechnologists need to come up with more convincing and reproducible molecular marker. Nowadays, due to the advancement of modern molecular techniques like DNA sequencing “omics” approaches are mostly pronounced. This led to the development of functional and structural genomics. Such studies can help to establish whole genome sequencing, to develop sequence-based association mapping, gene introgression study, linkage map constructions, small RNAs, allele-mining

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which are very important for sequence-based conservation and utilization of tea genetic resources. Even though somatic embryogenesis is an intrinsic nature of tea cotyledon but it is performed excellently in tissue culture by induction, maturation, germination, and multiplication of tea. Somatic embryogenesis is comparatively preferred than conventional micropropagation due to the presence of taproots in somatic embryo-derived seedlings, they can overcome drought. Therefore, efforts should be taken for commercial exploitation of somatic embryogenesis. There are recent reports about the successful transformation of tea with some beneficial genes. Even though it is well known that the transgenic technology can play a tremendous role for tea improvement but no transgenic tea plant has been released commercially so far. The reason may be due to some ethical issues or government policies.

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Further Reading Chen, Q., Yang, L., Ahmad, P., Wan, X., Hu, X., 2011. Proteomic profiling and redox status alteration of recalcitrant tea (Camellia sinensis) seed in response to desiccation. Planta 233, 583–592. Ishida, M., Kitao, N., Mizuno, K., Tanikawa, N., Kato, M., 2009. Occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants. Planta 229, 559–568. Ku, K.M., Choi, J.N., Kim, J., Kim, J.K., Yoo, L.G., Lee, S.J., Hong, Y.S., Lee, C.H., 2010. Metabolomics analysis reveals the compositional differences of shade grown tea (Camellia sinensis L.). J. Agric. Food Chem. 58, 418–426. Lee, J.E., Lee, B.J., Chung, J.O., Hwang, J.A., Lee, S.J., Lee, C.H., Hong, Y.S., 2010. Geographical and climatic dependencies of green tea (Camellia sinensis) metabolites: a 1H NMR-based metabolomics study. J. Agric. Food Chem. 58, 10582–10589. Lee, J.E., Lee, B.J., Hwang, J.A., Ko, K.S., Chung, J.O., Kim, E.H., Lee, S.J., Hong, Y.S., 2011. Metabolic dependence of green tea on plucking positions revisited: a metabolomic study. J. Agric. Food Chem. 59, 10579–10585. Li, J., Chen, J., Zhang, Z., Pan, Y., 2008. Proteome analysis of tea pollen (Camellia sinensis) under different storage conditions. J. Agric. Food Chem. 56, 7535–7544. Li, Q., Huang, J., Liu, S., Li, J., Yang, X., Liu, Y., Liu, Z., 2011. Proteomic analysis of young leaves at three developmental stages in an albino tea cultivar. Proteome Sci. 9, 44–56. Mondal, T.K., Bhattacharya, A., Ahuja, P.S., Chand, P.K., 2001c. Factor effecting Agrobacterium tumefaciens mediated transformation of tea (Camellia sinensis (L). O. Kuntze). Plant Cell Rep. 20, 712–720. Wang, X., Yang, Y., Ma, C., Jin, J., Ma, J., Cao, H., 2011. Cloning and expression analysis of cyclin gene (CsCYC1) of tea plant. Acta Botan. Boreali-Occiden. Sin. 31, 2365–2372.