Chapter I

General introduction

Chapter I

General Introduction

2

enetic improvement of a crop plant is a never ending process and every

improvement is aimed at particular requirement of time and situation like

higher productivity, disease and pest resistance, nutritive value, abiotic stress

etc. The present day fast changing world is characterized by two main

problems – the growing population with ever increasing demand for various

crop production and global climatic change and environmental detoriation.

The later has generated all pervasive abiotic stress which affects every crop

to varied extent with possibility of drastic reduction in growth and crop

productivity. Understanding of how plants respond to or cope with abiotic

stress like drought at physiological, biochemical and molecular level is of

paramount importance. Drought tolerance is governed by genetic factor and

known to be a multigenic trait. Understanding drought tolerance at molecular

level particularly identification of relevant genes is likely to pave the ways to

overcome drought stress of major crops.

1.1 Tea (Camellia sinensis L.)

Tea (Camellia sinensis) is the most widely consumed non-alcoholic

beverage in the world, and consequently the most important crop

species in the genus Camellia, which has over 325 species reported so far

(Mondal, 2002). Tea currently made in the world can be classified into five

main types namely- black, green, white, yellow and oolong tea. However, the

principal types of tea produced and consumed in the world are black and

green tea, with small amounts of other types (International Tea Committee,

2003). Tea is mainly produced in most of the tropical countries in Asia, South

America and South Africa (Figure 1.1). In Asia, India, Sri Lanka, China,

Vietnam, Japan and Indonesia are some of the leading countries that produce

tea (Figure 1.1). India, Sri Lanka and Kenya produce most of the black tea

while the other countries produce green tea and other varieties. India is the

second largest tea producing country in the world after China (FAO statistical

year book 2009).

According to the latest Tea Board data (www.teaboard.com), the volume and

value of tea from India is declining in International market and loosing the

GGGG

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General Introduction

Figure 1.1: Major tea producing countries of the world and Tea Exports (US$’000)

(FAO Statistical Year book 2009)

competition to Kenyan and Sri Lankan brews because of the high prices of

Indian tea compared to other countries.

Tea plantation and tea industry represents a classic example of ‘green

economy’ long before the present day concern for environmental damaging

industrial activity. Tea production of Assam and India not only cater to the

domestic market but also a major foreign exchange earner. Being manpower

orientated industry it has enormous employment generation capacity to semi-

skilled or even unskilled manpower. Like any other plantation crop, tea

plantation also faces various biotic and abiotic stresses. Particularly with

concern for global warming and climatic change, abiotic stresses such as

drought stress is likely to aggravate. One of the ways to counter this is to

intensify research and development works to gain insight about drought

tolerance and to breed clones for inherent tolerance to drought.

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General Introduction

4

In North-East India, loss in tea production due to drought has been reported in

Cachar, Golaghat, Nowgong and Mangaldai (Assam), Terai and Dooars

(W.B.) (Manivel and Handique, 1983). Among the three types of tea i.e.,

Assam, Cambod and China, Cambod is fairly drought tolerant followed by

China (Manivel and Handique, 1983). Singh and Handique (1993) reported

that about 30% of total tea growing areas in North-East India are affected by

reoccurring drought causing considerable loss. The dry spell has also affected

other major tea growing countries, thus causing tea prices to firm up in the

global markets (Anon, 2009).

1.2 Drought stress

Drought is one of the most wide spread environmental stresses reducing crop

yields by as much as 50% (Bray et al., 2000). According to the assessment

report of the Inter-governmental Panel on Climate Change (IPCC, 2007),

drought-affected areas are expected to increase with the potential for adverse

impacts on multiple sectors, e.g., agriculture, water supply, energy production

and health. Consequently, the development of drought-tolerant varieties will

become increasingly important and efforts are directed towards a better

understanding of plant responses to water deficit.

Plant adaptation to environmental stresses is controlled by cascades of

molecular networks resulting in a combination of metabolic, physiological and

morphological changes. Stress perception by osmosensors leads to signal

transduction via primary and secondary messengers (Figure 1.2). Secondary

signals can be phytohormones such as abscisic acid (ABA) and ethylene as

well as Ca2+, reactive oxygen species (ROS) and intracellular second

messengers such as phospholipids (Xiong and Zhu, 2002). Stress associated

genes encoding functional proteins are controlled by regulatory proteins such

as transcription factors, mitogen activated and calcium-dependent protein

kinases and phospholipases (Shinozaki and Dennis, 2003; Beck et al., 2007).

Enzymes involved in stress avoidance maintain the osmotic pressure and

stabilisation of the quaternary structure of proteins as well as synthesize

proteins involved in damage repair.

Chapter I

General Introduction

Figure 1.2: Plant responses to drought stress (modified from Vinocur and Altman,

2005 and Beck et al., 2007).

1.2.1 Stress avoidance and damage repair

One prominent response to drought stress is the modulation of the osmotic

level of the plant cell’s cytosol and the vacuoles by accumulation of manifold

substances, with the aim to counteract the loss of turgor (Cushman, 2001).

These osmolytes do not interfere with normal cellular biochemical reactions,

but help to maintain an osmotic balance. Osmolytes include amino acids,

TRANSCRIPTIONAL

CONTROL

STRESS RESPONSIVE

MECHANISMS

DROUGHT

SIGNAL SENSING,

PERCEPTION AND

TRANSDUCTION.

Osmosensors (e.g. AtHK1, RPK1),

phospholipids, secondary messengers

(e.g. Ca2+, ROS, ABA, ethylene

e.g., Transcription factors (e.g. ABF, bZIP,

MYC/MYB, zinc finger proteins), MAP

kinases

Gene activation

REGULATORY PROTEINS

(e.g. ABA, MAP kinases,

transcription Factors)

Disruption of osmotic homeostasis, damage of

functional and structural proteins and membranes.

FUNCTIONAL PROTEINS - Water and ion movements e.g.

aquaporin

- Detoxification e.g. peroxidases

- Osmoprotection e.g. proline, polyols, carbohydrates

- Chaperone function e.g. HSP, LEA

Re-establishment of cellular homeostasis, functional

and structural protection of proteins and membranes.

STRESS TOLERANCE/ RESISTANCE

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General Introduction

6

such as proline and quaternary ammonium compounds (e.g., glycine betaine),

hydrophilic proteins (e.g., late embryogenesis abundant proteins),

carbohydrates (e.g., sucrose, trehalose, fructan), polyols (e.g., pinitol,

mannitol) and polyamines (Chaves et al., 2003; Liu et al., 2004).

Accumulation of proline and glycine betaine under water deficit and salt stress

has been demonstrated in many different plants (Delauney and Verma, 1993;

Rhodes and Hanson 1993). Interestingly, several plant species such as

Arabidopsis and Tobacco do not synthesise glycine betaine indicating that it is

not universal (Sakamoto and Murata, 2002). Increased levels of sucrose

and/or reducing sugars have been also frequently reported and are known to

contribute towards the maintenance of turgor (Hare et al., 1999). For example,

high concentrations of the monosaccharides - glucose and fructose were

recorded in maize and soybean leaves upon osmotic stress (Pelleschi et al.,

1997; Liu et al., 2003). Also sugar alcohols and cyclic polyols are known to

accumulate under drought stress. Mannitol, a six carbon polyol, is the most

widely distributed sugar alcohol in nature (Stoop et al., 1996) and has been

shown to accumulate under drought stress, for example, in Fraxinus

(Guicherd et al., 1997). Increased concentrations of the cyclic pinitol were

demonstrated in white clover (McManus et al., 2000) and soybean (Streeter et

al., 2001).

Beside their contribution to osmotic adjustment some solutes may also

function as osmoprotectants by way of stabilization of proteins, protein

complexes or membranes at osmotically insignificant concentrations (Bohnert

and Shen, 1999).

1.2.2 Stress Signaling

A generic signal transduction pathway starts with signal perception by, for

example, receptor-like kinases followed by the generation of second

messengers and activation of stress responsive genes, whereas second

messengers such as plant hormones and reactive oxygen species can initiate

another series of signaling events. The activation of stress responsive genes

is further modulated by phosphoprotein cascades and transcription factors

(Shinozaki and Dennis, 2003; Xiong et al., 2002). The manifold features of

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General Introduction

7

gene families encoding signaling molecules, protein kinases and transcription

factors provide both complexity and flexibility in plants responses to

environmental stresses.

An universal secondary messenger responding to stress is calcium. Changes

in cytosolic Ca2+ levels can be sensed by calcium-dependent protein kinases

(CDPKs), which modify the phosphorylation status of substrate protein (Knight

and Knight, 2001). Several genes encoding CDPKs of broad bean (Vicia faba)

and wheat (Triticum aestivum) have been shown to be induced under drought

stress (Liu et al., 2006; Li et al., 2008). Besides CDPKs, mitogen activated

protein kinases (MAPKs) play an important role in cell signaling. MAPK

cascades amplify and transmit signals through a series of phosphorylation

events from MAPKKK to MAPKK to MAPK (Cardinale et al., 2002). Targets of

MAPKs can be again protein kinases as well as various transcription factors.

It has further been shown, that MAPK signaling pathways can regulate ROS

production (Pitzschke and Hirt, 2009). Beside their negative effect on cell

damage, ROS can also act as signalling molecules as they are small and able

to diffuse over a short distance. Among different ROS, only H2O2 can cross

plant membranes and therefore can directly function in cell-to-cell signaling

(Pitzschke and Hirt, 2006). Furthermore, H2O2 has been shown to change the

intercellular calcium levels to mediate stomatal closure (McAinsh et al., 1996).

Downstream signaling events associated with ROS sensing involve Ca2+ and

Ca2+ binding proteins, the accumulation of phospholipid signaling and

mitogen-activated protein kinase (MAPK) cascades (Mittler et al., 2004). In

Arabidopsis, H2O2 has been shown to activate the MAPKKK ANP1, which

further initiates phosphorylation cascade involving two stress MAPKs,

AtMPK3 and AtMPK6 (Kovtun et al., 2000).

After signal sensing, perception and transduction, gene expression is

controlled by transcription factors. Among stress inducible transcription

factors, members of the dehydration-responsive element-binding (DREB)

protein family, the ethylene-responsive element binding factor (ERF) family,

the zinc-finger family, the WRKY and MYB family, the basic helix-loop-helix

family, the basic-domain leucin zipper (bZIP) family, the NAC family and the

homeodomain transcription factor family have been identified (Shinozaki et

Chapter I

General Introduction

8

al., 2003). Transcription factors of the bZIP family are known to recognize the

core sequence of the ABA-responsive element (ABRE) (Hattori et al., 1995;

Choi et al., 2000; Uno et al., 2000). This element has been identified from the

promoter analysis of ABA-regulated genes and is described in several plant

species. Like ABRE-binding proteins, the MYC and MYB transcription factors

AtMYC2 and AtMYB2 from Arabidopsis are known to bind cis-elements and

suggested to be also involved in the ABA-dependent signaling pathway

(Shinozaki et al., 2003). In addition, an ABA-independent signalling pathway

is known via the dehydration-responsive element (DRE)/C-repeat (CRT).

Transcription factors belonging to the ERF/APETALA2 (AP2) family that bind

to DRE/CRT have been isolated from Arabidopsis thaliana (Stockinger et al.,

1997; Liu et al., 1998). NAC transcription factors are suggested to be involved

in both the ABA-dependent and ABA-independent pathway (Shinozaki and

Yamaguchi-Shinozaki, 2007).

1.2.3 Gene expression

Plants have the ability to dramatically alter their gene expression patterns to

cope with a variety of environmental stresses. These transcriptional changes

are sometimes successful adaptations, leading to tolerance while in other

instances the plant ultimately fails to adapt to the new environment showing

susceptibility or intolerance (Hazen et al., 2005). However, ability to improve

plant tolerance to environmental stresses has remained limited due to lack of

insight and understanding of the inherent complexity of stress signaling and

adaptation processes (Cushman and Bohnert, 2000). Breeding for increased

abiotic stress tolerance has been found to be difficult partly due to

multigenicity of abiotic stress tolerance (Bohnert et al., 2001). Recent insights

into the molecular basis of stress tolerance have begun to suggest new

strategies of crop improvement. The first step of this approach is to learn

more about gene regulation and signal transduction pathways involved in

stress tolerance.

Advances and technical developments in genomics, bioinformatics and

‘functional genomics’ made in the recent years have offered the opportunity to

gain a more complete understanding of how many genes become integrated

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General Introduction

9

to effect abiotic stress tolerance. Thus, it is now possible to address the

complexity of stress response on a large scale through genome wide

‘expression profiling’ (Reymond et al., 2000; Richmond and Somerville, 2000).

Scientists are now equipped to perform gene expression analysis to

characterize and define the functional roles of all genes - essential, important,

and ancillary to the stress response of tolerant genotypes. Novel genes and

regulators identified by gene expression profiling can be explored further for

their specific role(s) in the tolerance or susceptibility to the stress in question.

The new and novel genes can also serve as genetic markers for diversity

analysis in native, improved and exotic germplasm. The results obtained

through genomic approaches can also be helpful in the development of gene

constructs, which could be used to genetically modify crop plants for elevated

stress tolerance.

1.2.4 Transcriptome and techniques for transcriptome analysis

Transcriptome is the set of all RNA molecules including mRNA, rRNA, tRNA

and other non-coding RNA produced in one or a population of cells under

particular physiological condition. It is a well established fact that gene

expression varies from tissue to tissue and for the same tissue from one

physiological condition to another as well as age of the organisms. As a

consequence transcriptome profile is highly variable for the same plant and

hence lot of precaution is to be exercised for transcriptome study.

Suppression subtractive hybridization (SSH) is powerful method for isolating

differentially expressed transcripts, which can be used for the generation of

subtracted cDNA or genomic DNA libraries. This technique can be used to

compare mRNA or genomic DNA populations and to obtain cDNAs of genes

exclusively expressed or over-expressed in one population. SSH is based on

hybridization and suppression PCR and combines normalization and

subtraction in a single procedure. The normalization step equalizes the

abundance of DNA fragments within the target population. In the subtraction

step, sequences that are common to both populations are excluded. During

this step, the cDNA containing the transcripts of interest and the reference

cDNA are hybridized and the hybrid sequences are removed. Thus, the

Chapter I

General Introduction

10

probability of obtaining low-abundance differentially expressed cDNA or

genomic DNA fragments is clearly increased (Diatchenko et al., 1996;

Rebrikov et al., 2004).

The cDNA in which specific transcripts are to be found is referred to as tester

and the reference cDNA as driver. The SSH technique involves the following

steps:

1. cDNA synthesis : Tester and driver cDNA are prepared from the two

mRNA samples under comparison.

2. Rsa I digestion: Tester and driver cDNA are separately digested to

obtain shorter, blunt-ended molecules.

3. Adaptor ligation: Two tester polpulations are created with different

adaptors. Driver cDNA has no adaptors.

4. First Hybridization: Differentially expressed sequences are equalized

and enriched.

5. Second Hybridization: Templates for PCR amplification are

generated from differentially expressed sequences.

6. First PCR amplification: Only differentially expressed sequences are

exponentially amplified by suppression PCR.

7. Second PCR amplification: Background is reduced and differentially

expressed sequences are further enriched.

Another technique called cDNA AFLP is an electrophoretic, gel-based

transcript profiling method to display differentially expressed genes for any

organism on a genome-wide scale. It has been reported as one of the most

robust, sensitive and attractive technologies for gene discovery on the basis

of fragment detection (Cappelli et al., 2005; Gabriels et al., 2006; Guo et al.,

2006; Hmida-Sayari et al., 2005; Ling et al., 2000). It has also been applied

for temporal quantitative gene expression analysis (Breyne et al., 2002; De

Paepe et al., 2004; Goossens et al., 2003; Mao et al., 2004; Vandeput et al.,

2005); for generating quantitative gene expression phenotypes; for expression

quantitative trait loci mapping (Vuylsteke et al., 2006; Brugmans et al., 2002);

for construction of transcriptome map (Brugmans et al., 2002; Ritter et al.,

Chapter I

General Introduction

11

2008) and for particularly in organisms that lack the gen(om)e sequences

necessary for development of transcript profiling DNA chips or microarray.

The cDNA-AFLP method involves reverse transcription of mRNA into double-

stranded cDNA, followed by restriction digestion, ligation of specific adapters

and fractionation of this mixture of cDNA fragments into smaller subsets by

selective PCR amplification. The resulting cDNA-AFLP fragments are

separated on high-resolution gels, and visualization of cDNA-AFLP

fingerprints is described using either a conventional autoradiography platform

or an automated LI-COR system. Observed differences in band intensities

between samples provide a good measure of the relative differences in the

gene expression levels. Identification of differentially expressed genes can be

accomplished by purifying cDNA-AFLP fragments from sequence gels and

subsequent sequencing. This method has found widespread use as an

attractive technology for gene discovery on the basis of fragment detection

and for temporal quantitative gene expression analysis.

1.3 Tea breeding

Tea is known to be self incompatible and hence cross pollination is the rule

(Banerjee, 1992). However, for commercial cultivation it is normally

vegetatively propagated. The vegetative propagation ensures that genetic

purity remain intact and so also the quality of tea. Known as a freely cross-

pollinated plant, with many overlapping morphological, biochemical and

physiological attributes (Purseglove, 1968; Wickremasinghe, 1979; Banerjee,

1988), tea cannot be separated into discrete groups to identify various taxa

(Wickramaratne, 1981). Tea taxonomy is still a challenge today, but did not

receive the attention it deserved possibly because of the complexities

involved (Banerjee, 1992). From the very early days of tea growing, it was

recognized that breeding of tea creates problems that are somewhat unique

to the plant. This is so because, firstly, unlike other woody perennials, in tea

only a part of the total biomass constitutes the harvest, and secondly the plant

is highly heterogeneous and self-incompatible (Banerjee, 1992).

Selection is the most popular, long standing practice in tea breeding. Since

commercial tea plantations earlier were established with seedlings, hence lots

Chapter I

General Introduction

12

of variability exists among them. The majority of the tea clones have been

developed through selection. However, pedigrees of such clones remain

unknown. Earlier tea plant selection was mainly based on the morphological

characteristics for yield and quality rather than biotic and abiotic stress

resistance. Until now tea plantation is developed largely from the selected

genotypes based on the performance of yield and quality amongst the

previously existing planting materials. As a consequence of wide-spread

cultivation of clones, the genetic diversity of tea has diminished. In tea, clonal

identification has traditionally been based on morphological descriptors such

as plant shape, leaf shape and young leaf type. Vegetative propagation

(clones) began to replace seed propagation in the 1960s and reduced genetic

diversity within tea cultivation (Visser, 1969). Vegetatively propagation is good

from commercial view point but it limits genetic variability and evolutionary

development process is also disrupted.

Conventional tea breeding is well established and contributed much to tea

improvement over the past several decades, but the process is slow due to

some bottlenecks: tea is perennial nature, long gestation periods, high

inbreeding depression, self-incompatibility, unavailability of mutants with

tolerance to different biotic and abiotic stress, lack of clear selection criteria

(Kulasegaram, 1980), low success rate of hand pollination, short flowering

time (2-3 months), long duration for seed maturation (12-18 months), clonal

differences of flowering time and fruit bearing capability of some clones

(Mondal et al., 2004). A new technology for varietal improvement of tea is

genetic transformation. However, central to any successful transgenic

technology for a vegetatively propagated plant like tea is an efficient in vitro

regeneration protocol. While an efficient regeneration protocol is essential for

introduction of the foreign gene into plant tissues, micropropagation is

important for the transfer of large number of genetically modified plants to the

field within a short span of time (Mondal et al., 2004).

Seed propagated trees show a high degree of variability, therefore, the

alternative choice is through vegetative propagation from the cuttings.

Recently, grafting as an alternative propagation technique has gained

considerable popularity. In such case, both root-stock (commonly a drought

Chapter I

General Introduction

13

tolerant cultivar) and scion (often either good quality or high yielding cultivar)

which is generally fresh single leaf internode cuttings. Upon grafting, the scion

and stock influence each other for the characteristics and thus composite

plants combine both high yield and good quality characteristics. For further

improvement, the tender shoots were grafted on young seedlings; hence an

additional advantage of grafted tea seedling is the presence of tap root

system.

Vegetative propagation is an effective method of tea propagation, yet it is

limited by several factors such as: slower rates of propagation, unavailability

of suitable planting material due to winter dormancy, drought in some tea

growing area, poor survival rate at nursery due to poor root formation of some

clones and season dependent rooting ability of the cuttings (Mondal et al.,

2004). Though several genetic transformation techniques are available (Klee

et al., 1987; Kuhlemeiere et al., 1987; Hooykaas and Schilperoort, 1992;

Smith and Hood, 1995), yet few have been employed to produce transgenic

tea and the production of transgenic tea remains difficult mainly due to low

transformation efficiency as well as its difficult regeneration system (Mondal et

al., 2004).

Micropropagation technique appears to be an ideal choice for circumvention

of the problems related to conventional propagation. Forrest (1969) was

pioneer for initiating the work on the tissue culture of tea; then Kato (1985) did

a systematic study on micropropagation of tea, but studies on field

performance of micro-propagated tea and commercial exploitation only

started at the beginning of the 21st century (Mondal et al., 2004). The biggest

difficulty in micro-propagation of tea is to regenerate the adventitious shoots

from explants. Like other woody perennials, major problems encountered in

tea micropropagation are phenolic exudation from explants and microbial

contamination in tissue culture medium (Mondal et al., 2004).

1.4 Drought stress tolerance in tea and molecular breeding

Tea is one of the widely cultivated and main net foreign exchange earner

plants in India. Thirty to forty percent of tea growing areas of north-east India

Chapter I

General Introduction

14

are affected by drought (Singh and Handique, 1993). Jain (1999) reported

40% loss of total yield in tea due to drought.

Historically, improvement of tolerance to abiotic stresses has been a major

target of plant-breeding programs globally. The major challenge, however,

results from the complex nature of abiotic-stress-tolerance traits and the

difficulty in dissecting them into manageable genetic components amenable to

molecular breeding. In crop breeding, advances in molecular biology and

genomics have had a large impact on the speed of identification and

characterization of genes and genetic regions associated with quantitative

and qualitative traits. Marker-assisted selection through the use of high-

throughput marker systems is currently being used extensively in breeding

programs to improve selection efficiency, accuracy and to direct focus

towards traits of importance. As key genes are identified, efficiency increases

and opportunities for genetic engineering are realized. An underlying factor

important for gene discovery in relation to traits of interest is naturally

occurring genetic diversity. This is a fundamental aspect of research into

abiotic-stress tolerance, and discoveries of abiotic-stress-tolerance genes

revealing novel mechanisms of adaptation in cultivated plants and their wild

relatives. Genetic-diversity screening is a starting point for many functional

genomics projects relating to gene discovery.

In the face of global warming, tolerance to abiotic stresses such as drought,

salinity and low- or high-temperature has become very urgent breeding

objectives to maintain sustainability of crop performance. Advances in plant

genomics have offered an opportunity to identify regulatory genes and

networks that control important traits. Recent genetic analyses have become

able to contribute our understanding of phenotypic variation involving

tolerance to abiotic stresses and allowed the dissection of genes for

quantitative traits in major crops. Plants have adapted to respond to drought

stress at the molecular level by inducing the expression of genes, which

enables them to survive (Ingram and Bartels, 1996; Shinozaki and

Yamaguchi-Shinozaki, 2007). The products of stress-induced genes not only

function in stress tolerance (functional or single action genes), but also in

Chapter I

General Introduction

Figure 1.3: Molecular Breeding strategies for improving drought stress tolerance

in Tea.

Drought tolerance

Genetic engineering Natural genetic variation for Potential trait

Identification and characterization of target genes

Expression of genes

Osmolytes

Transcriptionfactors

Transgenic plants

Evaluation of drought resistance

Field performance

Functional genomics

QTL ; eQTL

Development of markers for MAS

Breeding using resistancelines

Cloning of target genes

signal amplification (regulatory genes) of the stress response (Shinozaki and

Yamaguchi-Shinozaki, 2007).

Drought tolerance in tea is a difficult trait to define as it encompasses a wide

range of characteristics involving multiple genetic, physiological, cellular and

biochemical strategies. Dissecting drought tolerance to the level of a single

gene or group of genes amenable to genetic engineering will be difficult. A

major challenge in the use of functional genomics to enhance the

development of drought tolerance is to define the system and focus on key

traits of interest. So, the first approach ideally will be a forward genetic

approach aimed at defining the genetic basis for differences in tolerance in

adapted germplasm. The second step will aim to build a database of

transcript, protein and metabolite induced when tea plant is exposed to

drought stress which can later be used to support candidate-gene discovery.

The third involves specific targeting of genes and screening the cultivated and

wild germplasm (Figure 1.3).

Chapter I

General Introduction

16

Numerous studies to evaluate tea diversity have been conducted by using

morphological characteristics (Guohua et al., 1995; Chen et al., 2005),

biochemistry components (Magoma et al., 2000; Chen et al., 2005), isoozyme

(Yee et al., 1996; Chen et al., 2005) and genetic markers, e.g., CAPs

(Kaundun and Matsumoto, 2003), cpDNA (Katoh et al., 2003), RFLPs

(Matsumoto et al., 1994; Devarumath et al., 2002; Matsumoto et al., 2002),

RAPDs (Wachira et al., 1995, 1997; Chen et al., 1998; Kaundun et al., 2000;

Kaundun and Park, 2002; Park et al., 2002), AFLPs (Paul et al., 1997;

Balasaravanan et al., 2003) and ISSRs/microsatellites (Ueno et al., 1999; Lai

et al., 2001; Mondal, 2002). However, information about molecular basis of

tolerance level towards biotic and abiotic stress, particularly drought tolerance

is very scanty.

Nagaraja and Ratnasurya (1981) studied relation between differences in

rooting depth, root weight and vertical distribution of roots in the soil with

different clones with varied degree of drought resistance. The results showed

that among these factors, only rooting depth influenced drought resistance.

Shallow rooted clones were drought susceptible and deep rooted clones

drought resistant. In shallow rooted clones drought resistance increased with

rooting depth. However, in deep rooted clones drought resistance was not

related to rooting depth. Mishra and Swati Sen-Mandi (2004) reported RAPD

analysis of DNA of ten short-listed (on the basis of field performance for

drought tolerance) clones. Upadhaya and Panda (2004) studied the effects of

dehydration on tea seedlings. They found that the contents of proline, H2O2

and superoxide anions increased and so also lipid peroxidation, catalase and

superoxide dismutase activities increased after five days of drought induction.

Sharma and Kumar (2005) studied drought responsive genes in tea and

reported three drought responsive ESTs using differential display method. But

for a polygenic trait like drought, three ESTs is not adequate. Wachira et al.,

2007 from Kenya tried to determine the association of tea polyphenols with

drought stress and their suitability as indicators of drought tolerance. The

result indicated that the declining soil water content (SWC) reduced both

growth and content of polyphenols in tea. They suggested that polyphenols

can be used as indicators for selection of drought tolerant tea cultivars.

Chapter I

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17

Cheruiyot et al. (2007) reported that the polyphenol content of tea plant has a

strong correlation with drought tolerance. They found that clones having high

polyphenol content are relatively more tolerant than clone with less

polyphenol content. They also emphasized that polyphenol content can be

used as a potential marker for selection of drought tolerant tea cultivars.

Upadhyay et al. (2008) studied the physiological and anti-oxidative responses

of tea plant under water stress and after rehydration. They found that water

stress decreased non-enzymatic antioxidants like ascorbate and glutathione

contents with differential responses of enzymatic antioxidants in selected

clones of Camellia sinensis. Similar studies were reported by Jayaramraja et

al. (2005). Thus, the molecular biology of tea with respect to abiotic stress

tolerance is still in a preliminary stage in comparison to other crop plants.

Understanding the drought signal perception mechanism and succession of

signal transduction events leading to switch on of molecular, cellular and

whole plant adaptive processes will help to improve drought tolerance in tea.

Identification of novel abiotic stress regulatory genes, key pathways that are

up regulated or down regulated in response to water stress and functional

characterization of genes involved is a must to understand water stress

tolerance mechanisms in tea. There are several example of studies to

compare expression profiling of stressed versus non-stress plants resulting

into the identification of several abiotic stress related genes (Reddy et al.,

2002; Rabbani et al., 2003; Kushwaha et al., 2009).

Breeding for drought tolerance in tea is a challenging task because of the

complexity of drought responses, environmental factors, and their

interactions. Conventional breeding approaches have been partly successful,

but progress has been slow. Recent advances in functional genomics

technology provide new powerful tools for the genetic dissection of drought

tolerance components. It is anticipated that molecular genetics research will

provide high-throughput DNA marker systems for marker-assisted selection

that will be more efficient and effective in combining out favorable drought

tolerance traits in breeding programs. It will also lead to a better

understanding of the molecular basis of the genes underlying drought

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18

tolerance, which can be used in a genetic engineering program for genetic

improvement of drought tolerance.

1.5 Research objectives and work plan of thesis

Over the years, many high yielding cultivars are being evolved and released

by Tocklai Experimental Station for cultivation in North-East India. These tea

cultivars are found to vary in their tolerance level towards drought. As a

contribution to the development and implementation of marker assisted

selection (MAS) approach in tea, the objective of this research was to develop

functional markers and gene targeted markers which will facilitate MAS for

drought tolerance in tea. Marker assisted selection or identification can be

used to pyramid the major genes including tolerance genes, with the ultimate

goal of producing tea varieties with more desirable characters. The concept

here is to study whole genome transcriptome polymorphisms between

drought tolerant and drought susceptible genotypes and use transcript derived

fragments and candidate genes identified from differential gene expression

study to assess stress tolerance mechanisms in the land race or wild lines

that show elevated tolerance. Where variation is identified in candidates,

either in the gene structure or at expression levels, the locus can be

transferred and tested in non-adapted or cultivated germplasm for

assessment. In this way, the variation will not only be used to validate

candidate genes for stress tolerance but also to provide a tool for allele

discovery. Desirable alleles can be transferred by molecular breeding

(transgenic approach) as well as conventional breeding and selection.

The present work commence with an overview and assessment of available

literature on basic aspects of tea and particularly the impact of drought stress

as well as how tea plant respond to drought stress from morphological to

molecular level (Chapter I). It was considered pertinent to study some of the

physiological parameters associated with drought stress since the impact of

drought stress always manifest in some physiological change or disturbance.

The Chapter II is devoted to this in which an induced drought experiment was

performed under control condition taking a drought tolerant (TV23) and a

drought susceptible cultivar (S3.A/3). The major focus of the present work

Chapter I

General Introduction

19

was to understand the molecular mechanisms at the level of mRNA during

drought stress. Therefore, one of the work component was transcriptome

analysis and the assumption was that up or down regulation of certain genes

could help to explain tea plant response to drought stress. With the idea of

identification of drought stress tolerance genes/transcripts based on their

stress induced-expression, two different strategies were pursued. In a first,

cDNA AFLP approach, it was hypothesized that transcripts which are

differentially expressed in the tolerant cultivar must have some role in

elevated tolerance against drought (Chapter III). So, the concept here was to

study whole genome transcriptome polymorphisms between drought tolerant

and drought susceptible cultivar and develop ESTs from polymorphic

transcript derived fragments (TDFs), so that it can be used as molecular

markers to select tolerant genotypes at early stage.

In a second, SSH approach (Chapter IV), work was designed to find out the

genes which are involved in tea defense against drought. As drought

response is multigenic in nature and the genes would vary with the type of

samples considered. Leaves are one of the most important sites where water

loss can be minimized and as a result synthesizes chemical signals for rapid

response of the plant to drought stress. The present work focuses on the

genes differentially expressed during ‘before wilting’ and ‘wilting’ stage of

drought in 3rd and 4th leaf of the plant. From the accumulated data, a workable

selection of interesting candidate genes was sorted out to further validate it as

gene marker for marker assisted breeding. But the EST data generation was

based on controlled condition of pot experiment which may be very much

different from what actually occurs in field. The Genotype x Environment (G x

E) interaction in controlled condition may not be the same at field drought

condition. With this idea another experiment (rain-out shelter) was done using

TS-463 progenies and expression analysis of genes was done using Real

Time PCR (Chapter V).

This work is one of the components for a long term objective for better

understanding of molecular mechanisms involved in drought tolerance with

the aim of genetic improvement in tea to combat drought stress. Accordingly

the work components are summarized below:

Chapter I

General Introduction

Drought stress (Overview and

assessment of literature)

Physiological analysis

Molecular analysis

Validation of candidate genes under field condition by Real Time PCR

Identification of candidate

gene by SSH approach

Development of EST from

transcript derived fragments

Transcriptome analysis by cDNA AFLP