6

2. REVIEW OF LITERATURE

2.1. ORIGIN, TAXONOMY AND IMPORTANCE

Groundnut, Arachis hypogaea L., is native to South America,

originated probably from a region including central Brazil and

Paraguay (Gregory et al., 1980). It is a member of the genus Arachis

in the subtribe Stylosanthinae of tribe Aeschynomeneae of the family

Leguminosae. The cultivated groundnut, (Arachis hypogaea L.) (2n =

40) is a self-pollinated, allotetraploid species and is thought to be of

monophyletic origin, harbouring relatively little genetic diversity

(Pattee and Young, 1982). There are two subspecies of A. hypogaea,

distinguished primarily on branching pattern and distribution of

vegetative and reproductive axis. Subspecies hypogaea has two

varieties (hypogaea and hirsuta), whereas ssp. fastigiata has four

varieties (fastigiata, vulgaris, peruviana and aequatoriana). The only

species A. hypogaea L. in the genus of Arachis has significant

economic importance which is cultivated in over 100 countries

across Asia, Africa and America in around 24.5 m ha, generating an

annual production of nearly 38.2 m t (FAOSTAT, 2008). Of the 38.2

m t of groundnut produced worldwide, China produce 14 m t, India

produce 7.3 m t, Nigeria produce 3.9 m t, United States of America

produce 2.3 m t and rest mostly produced in other countries of Asia

and Africa (FAOSTAT, 2008).

7

2.2. BIOTIC AND ABIOTIC STRESSES

The important widespread biotic constraints that limit

groundnut productivity are late leaf spot (Phaeoisariopsis personata

(Berk. and Curtis) Deighton), early leaf spot (Cercospora arachidicola

Hori.), rust (Puccinia arachidis Speg.) (Pretorius, 2005), aflatoxin

contamination caused by Aspergillus flavus (Kennan and Savage,

1994), web blotch caused by Didymella arachidicola (Chock.) Taber,

Pettit and Philley (Subrahmanyam et al., 1994), stem or root rot

caused by soilborne fungi, such as Sclerotium rolfsii Sacc. (Mayee

and Datar, 1988), pod rot caused by Fusarium spp. (Mehan et al.,

1981), bacterial wilt caused by Ralstonia solanacearum (E.F. Smith))

(Tomlinson and Mogistein, 1989), groundnut rosette virus (Gibbons,

1977), peanut clump virus (Reddy et al., 1983), peanut bud necrosis

(Reddy et al., 1995), peanut stem necrosis disease (Reddy et al.,

2002), peanut stunt virus (Blount et al., 2002), peanut stripe virus

(Demski et al., 1984), nematodes (Starr et al., 1990), leaf miner, and

spodoptera spp. (Wightman and Amin, 1988). Based on the estimate

made by the International Crops Research Institute for the Semi-arid

Tropics (ICRISAT), late leaf spot, drought, and rust are the most

important constraints in terms of economic losses globally (Dwivedi

et al., 2003).

2.3. FOLIAR FUNGAL DISEASES

Several fungal diseases damage the groundnut crop at different

stages of growth. Among the foliar fungal diseases, late leaf spot

8

(LLS) and rust are distributed worldwide and cause significant pod

and haulm yield loss besides adversely affecting their quality. In

most areas, both diseases occur together, but the incidence and

severity of each disease vary with environment, location, and cultivar

(Mehan et al., 1996).

2.3.1. Late leaf spot (LLS)

LLS is caused by Phaeoisariopsis personata ((Berk. and M.A.

Curtis) Arx) = Cercosporidium personatum ((Berk. and M.A. Curtis)

Deighton). Groundnut is the only known natural host of P. personata

(Mc Donald et al., 1985). This pathogen generally occurs mainly at

reproductive stage and is often seen as a complex with other leaf

spots. Infection of leaflets by C. personata develops with small

cholorotic spots, which enlarge and become brown and black,

subcircular, 1 to 10 mm or more in diameter. The characteristic

features of these lesions are darker brown without a definite chlorotic

halo surrounding each lesion (Jenkins, 1938 and Woodruff, 1933).

Horne et al. (1976) reported that the LLS fungus produced haustoria

that penetrate individual plant cells and that leaves infected with the

fungus showed a marked increase in respiration.

2.3.2. Rust

Rust on groundnut is caused by the fungus Puccinia arachidis

Speg. (Spegazinni, 1884). Rust pustules are orange colored (uredia)

and appear on all aerial parts except on flowers. In leaflets, they

9

initially appear on the lower surface and spreads to adaxial surfaces

later in susceptible varieties. Pustules may also form on shells of

developing pods (Van Wyk and Cilliers, 2000). On rupturing, they

release masses of reddish-brown spores. Pustules are circular and

range from 0.5 to 1.4 mm in diameter. On highly susceptible

cultivars, secondary pustules may develop around the primary

pustules.

2.3.3. Disease management for LLS and rust

Several management options such as agronomic practices,

chemical and biological methods are available to control these foliar

diseases in groundnut. However, the excessive usage of fungicides is

no longer a sustainable approach due to safety issues such as

environmental concerns, consumer health as well as the increasing

incidence of pathogen resistance to fungicides. The biological control

of fungal plant pathogens is also not completely successful in all

cases because the functional activation of these agents differs with

different geological and environmental conditions (Gohel et al., 2006).

2.3.4. Breeding for disease resistance

As the chemical and biological control methods are ineffective,

the alternative way is to identify groundnut germplasm resistant to

these diseases and incorporate the resistance into adapted cultivars.

Sources of resistance to these foliar diseases have been identified in

cultivated A. hypogaea and also in wild species (Chiteka et al.,

10

1988a, 1988b; Anderson et al., 1993; Waliyar et al., 1993;

Subrahmanyam et al., 1995; Mehan et al., 1996; Singh et al., 1997;

Pande and Rao, 2001; Liao, 2003; Igze et al., 2007 and Hossain et

al., 2007). These resistant sources belong to var. fastigiata and var.

peruviana having poor agronomic traits, including low shelling

outturns, thick pod shells, heavy pod reticulation, and unacceptable

seed coat colors. This undesirable genetic linkage between disease

resistance and the poor pod characters impeded the progress of

breeding. For example, among 49 resistant lines used as rust

resistance donors in breeding at ICRISAT, only ICG 1697 and ICG

4747 have led to the release of rust-resistant cultivars such as ICGV

86590, ICGV (FDRS) 4, and ICGV (FDRS) 10 in India and West Africa

(Waliyar et al., 1993; Singh et al., 1997), but still with poor pod

traits. Hence, high degree of resistance could not be transferred to

the high yielding background mainly because of the complexity of

inheritance of resistance and undesirable linkages (Miller et al.,

1990). However, much of these efforts are being redirected from

developing cultivars with high yields to conferring resistance to

disease that are only adaptable to certain locations.

2.4. ROLE OF GENETIC ENGINEERING TO ENHANCE FUNGAL

DISEASE RESISTANCE

Recent application of techniques in plant molecular biology

and biotechnology to study host-pathogen interactions have resulted

in the identification and cloning of numerous genes involved in

11

defense response of plants following the pathogen infection.

Introduction and expression of these endogenous genes as well as

some antimicrobial genes from non-plant sources may enhance

plants resistance to fungal diseases (Punja, 2001). Various resistant

genes have been transformed into A. hypogaea to control fungal

diseases (Bent and Yu, 1999; Melchers and Stuiver, 2000; Rohini

and Rao, 2000). At present, no transgenic groundnut cultivars

transformed with resistant genes have been released for commercial

production. In the long term, the transformation technology will

become increasingly important for groundnut breeding, as more

genes with disease resistance and increased performance of

agronomic potential are isolated.

2.5. STRATEGIES FOR THE DEVELOPMENT OF FUNGUS

RESISTANT TRANSGENIC PLANTS

With the beginning of the molecular era of plant biology in the

early 1980’s, identifying, cloning and characterizing plant disease

resistance genes has become a major research area (Punja, 2001;

Crute and Pink, 1996). Over the past 10 years, numerous genes

involved in several mechanisms of plant response to pathogen

infection have been identified (Nicholson and Hammerschmidt, 1992;

Crute and Pink, 1996; Donofrio and Delaney, 2001). The evaluation

of the specific roles participated by these genes in disease response

pathways, summarized to categorize them in five general categories

12

(Punja, 2001) in which their protein products can show the following

response:

1) They are directly toxic to pathogens or reduce their growth,

e.g., pathogenesis-related proteins (PR proteins) such as

hydrolytic enzymes (chitinases, glucanases), antifungal

proteins (osmotin, thaumatin-like), antimicrobial peptides

(thionins, defensins, and lectin), ribosome inactivating

proteins, and phytoalexins.

2) May destroy or neutralize a component of the pathogen infection

e.g., polygalacturonase, oxalic acid, and lipase.

3) May potentially enhance the structural defenses (elevated levels

of peroxidase causing lignification) in the plant.

4) Would release signals that can regulate plant defenses. e.g.,

production of specific elicitors, hydrogen peroxide, salicylic

acid, and ethylene.

5) Get involved in the hypersensitive response (HR) when

interacted with avirulence factors.

2.5.1. Pathogenesis-related (PR) proteins

PRs are usually defined as host-specific proteins that are

induced in several, if not all, plant species during pathological and

wounding situations (van Loon et al., 2006). Since the discovery of

PR proteins, they have been classified into 17 families based on

primary structures, serological relatedness, enzymatic and biological

13

activities (van Loon et al., 2006). The function and source of these

families are summarized in Table 2.1. In recent years, the expression

of PR proteins in transgenic plants has become a useful technology

to obtain disease resistance. The genes belonging to 3 PR-protein

groups viz., PR-2 (ß-1,3-glucanases), PR-3 (chitinases) and PR-5

(thaumatin-like proteins, TLPs) have been used successfully to

enhance plant resistance to fungal pathogens (Yun et al., 1997).

Among the PR-proteins, chitinases belonging to the PR-3 group

appear to be potential candidates for management of fungal diseases.

Table 2.1. Recognized families of pathogenesis-related proteins

(modified from van Loon et al., 2006).

Family Type member Properties

PR-1 Tobacco PR-1a Unknown

PR-2 Tobacco PR- 2 ß-1,3-glucanase

PR-3 Tobacco P,Q Chitinase type I, II,

IV-VII

PR-4 Tobacco R Chitinase type I, II

PR-5 Tobacco S Thaumatin-like

PR-6 Tomato inhibitor I Proteinase-inhibitor

PR-7 Tomato P6g Endoproteinase

PR-8 Cucumber chitinase Chitinase type III

PR-9 Tobacco lignin-forming peroxidase Peroxidase

PR-10 Parsley “PR-1” Ribonuclease-like

PR-11 Tobacco “class V” chitinase Chitinase type I

PR-12 Radish Rs-AFP3 Defensin

PR-13 Arabidopsis THI2.1 Thionin

PR-14 Barley LTP4 Lipid-transfer

protein

PR-15 Barley OxOa (germin) Oxalate oxidase

PR-16 Barley OxOLP Oxalate-oxidase-like

PR-17 Tobacco PRp27 Unknown

14

2.5.2. Chitinase and mode of action

Chitinases (E.C.3.2.1.14) are poly(1,4-(N-acetyl-ß-D-

glucosaminide))-glycanohydrolases. They are widely distributed in

nature, occurring in bacteria, fungi, animals, and plants. During

pathogen invasion, the chitinases inhibit the fungal growth both

directly and indirectly. They directly hydrolyze fungal cell walls,

which contain chitin, the substrate for the enzyme, and by this

action, fungal hyphal lysis and inhibition of fungal growth occurs

(Roberts and Selitrennikoff, 1988; Schlumbaum et al., 1986). The

chitinases can also release elicitors from the fungal cell walls by their

enzymatic action and these elicitors induce various defense

responses indirectly in plants (Ren and West, 1992). The activity of

elicitor-inducible chitinases was reported to be more useful for

disease resistance as their activity will increase several-fold upon a

pathogen’s invasion. Some of the chitinases are not elicitor-inducible.

In barley, the 34 kDa chitinase is not induced in aleurone

protoplasts upon treatment with elicitor (Sheba et al., 1994). In

cucumber, among the three acidic class III chitinase genes, Chi 1 and

Chi 3 genes are not induced by the elicitors whereas the Chi 2 gene is

induced by pathogens as well as by abiotic elicitors (Lawton et al.,

1994). Similarly, in rice suspension-cultured cells, basic chitinase

transcripts were induced upon elicitor treatment, whereas acidic

chitinase genes showed very weak induction (Xu et al., 1996). Proper

selection of chitinase genes is very important for the development of

transgenic plants with enhanced disease resistance.

15

Generally, the chitinases are constitutively expressed at low

levels in the plants. However, various abiotic agents (ethylene,

salicylic acid, salt, ozone, UV radiations), biotic factors (infection by

fungi, bacteria, viruses, and viroids), fungal cell wall components (N-

acetyl-β-D-glucosaminide) and oligosaccharides induces

overproduction of this enzyme (Roby et al., 1987; Nasser et al., 1988;

Broglie et al., 1989; Tuzun et al., 1989; Irving and Kuc, 1990; Ward

et al., 1991 and Jacobsen et al., 1992). It has been demonstrated

that high-level expression of chitinases in transgenic plants can

enhance resistance to a variety of pathogens (Broglie et al., 1991; Lin

et al., 1995; Marchant et al., 1998; Tabei et al., 1998). However, the

sensitivity of different pathogens to different chitinases may vary

widely (Rokem et al., 1986). Chitinases purified from thorn-apple,

tobacco and wheat inhibited growth of saprophytic fungi but did not

inhibit growth of Botrytis cinerea (Broekaert et al., 1988). Similarly,

the purified Arabidopsis chitinase inhibited growth of Trichoderma

reesei, but growth of several pathogenic fungi including Alternaria

solani, Fusarium oxysporum, Sclerotinia sclerotiorum,

Gaeumannomyces graminis and Phytophthora megasperma was not

inhibited (Verburg and Huynh, 1991).

Some chitinases do not exhibit antifungal action. Tobacco

chitinase expression in carrot failed to reduce the infection caused by

Theilaviopsis basicola and Alternaria radicina (Punja and Raharjo,

1996). No activity of this enzyme was also found against Cercospora

nicotianae in tobacco (Neuhaus et al., 1991). Sugarbeet chitinase in

16

tobacco did not lead to resistance against Cercospora nicotianae

(Nielsen et al., 1993).

2.5.2.1. Classification of chitinases

On the basis of the amino acid sequence homology, the

glycosyl hydrolases were classified into 58 families (Henrissat, 1991;

Henrissat and Bairoch 1993). According to this classification,

chitinases form families 18 and 19 with endo and exo-chitinases.

They hydrolyze ß-1,4-glycosidic linkage between N-acetylglucosamine

(GlcNAc) residues. The endochitinases, cleave randomly in the chitin

chain, and exochitinases which cleaves off chitobiose (GlcNAc)2

(”chitobiosidase”; exo-N,N’-diacetylchitobiohydrolase) or chitotriose

(GlcNAc)3 (”chitotriosidase”; exo-N,N’,N’’-triacetylchitotriohydrolase)

from the reducing or the nonreducing end of the chitin chain

(Monreal and Reese, 1969; De la Cruz et al., 1992; Tronsmo and

Harman, 1993; Suzuki et al., 1999). These chitinases are widely

exploited for the control of insect and fungal pathogens of plants

(Kramer and Muthukrishnan 1997; Roberts and Selitrennikoff, 1988;

Herrera-Estrella and Chet, 1999). Several chitinases from bacteria,

fungus, plants, virus and some insects have been isolated, cloned

and characterized (Table 2.2, 2.3, 2.4).

17

Table 2.2. Examples of cloned and characterized bacterial chitinases.

Organism/plant Enzyme (MW) kDa Gene Target References

Streptomyces olivaceoviridis ATCC11238

Chit30 (30) Chit30 Pea Hassan, F. Ph. D.

Dissert. (2006)

Bacillus cereus 28-9 ChiCW (70)

ChiCH (37)

ChiCW,

ChiCH

E. coli Huang et al. (2005)

Streptomyces species Bacillus chitinolyticus

Chitinase (32) chIS Hoster et al. (2005)

Streptomyces sp. J-13-3

Chitinase (32) E. coli Yamashita and

Okazaki (2004)

Streptomyces griseus HUT6037

ChiC (33) ChiC Rice Itoh et al. (2003)

Bacillus subtilis

BC121 Chitinase enzyme of

25 kDa

Basha and

Ulaganathan (2002)

Streptomyces olivaceoviridis ATCC

11238

Chi30 (30), Chi92 (92) Chi30,chi92 Li (2000)

Streptomyces thermoviolaceus OPC-

520

Chi40 (40) Chi40 Tsujibo et al. (2000)

Stenotrophomonas maltophilia strain C3

Chitinase (32) Zhang and Yuen

(2000)

Serratia sp. Chitinase (22-54) Woytowich et al.

(2000)

Serratia marcescens

2170

Chitinases C1 and C2

(36)

chic Suzuki et al. (1999)

18

Organism/plant Enzyme (MW) kDa Gene Target References

Clostridium paraputrificum

ChiB (87) chiB Morimoto et al. (1997)

Enterobacter agglomerans IC 1270

Chia-Entag (59) ChiA (60)

chiA Chernin et al. (1995), Park et al. (1997)

Serratia marcescens 2170

Chitinase A (57), B

(52)

chiA, chiB Watanabe et al. (1997)

Serratia marcescens KCTC2172

54 and 22 chitinases Not designated

Gal et al. (1997)

Streptomyces lividans

66

Chitinase A (36), C

(65), D (41), B (46)

chiA, chic, chiB

Miyashita et al.

(1991 ; 1997)

Aeromonas sp. No10s-

24

Chitinase II (53) Chitinase III (55)

Chit II, III ORF-1-4

Ueda et al. (1994); Shiro et al. (1996)

Streptomyces griseus

HUT6037

ChiC (33), C-1, C-2

(27)

Chic Ohno et al. (1996);

Mitsutomi et al.

(1995)

Serratia marcescens BJL200

Chit A (62), Chit B (55)

chiA, chiB Brurberg et al. (1994) and (1995)

19

Table 2.3. Examples of fungal chitinases successfully expressed in

different crops.

Table 2.4. Examples of chitinase genes that have been cloned

from other organisms.

Target crop Chitinase gene source References

Pea Mycorrhiza (Glomus mosseae)

Slezack et al. (2001)

Tobacco (N. tabacum) Yeast (Sacchromyces cerevisiae)

Carstens et al. (2003)

Tobacco (N. tabacum) Hornworm (Manduca Sexta)

Ding et al. (1998)

Tobacco (N. tabacum) Baculovirus chitinase Shi et al. (2000)

Source Gene Target References

Trichoderma harzianum

Endochitinase Apple Wong et al. (1999) and Bolar et al.

(2000)

Potato(Solanum tuberosum), Broccoli

Mora and Earle

(2001)

Tobacco (N. tabacum)

Lorito et al. (1998)

Grape (Vitis vinifera L.)

Kikkert et al.

(2000)

Black spruce and hybrid poplar

Noel et al. (2005)

Endochitinase

(CHIT42)

Tobacco (N. tabacum) and

Potato (Solanum tuberosum)

Lorito et al. (1998)

Rhizopus oligosporus

Chitinase Tobacco (N. tabacum)

Terakawa et al.

(1997)

Rhizopus sp. Chitinase Yanai et al. (1992)

20

2.5.2.2. Plant chitinases

Studies conducted in plants like Phaseolus vulgaris (Broglie et al.,

1989), Petunia (Linthorst et al., 1990), Arachis hypogaea (Herget et al.,

1990), Populus (Davis et al., 1991), tobacco (Fukuda et al., 1991) and rice

(Zhu and Lamb 1991) showed that chitinases are coded by multigene

family. Plant chitinase have been classified into more than four classes

(Class I, II, III and IV) (Collinge et al., 1993). In general, class I chitinases

have the highest antifungal activity, perhaps due to the presence of a

chitin-binding domain (Sela-Buurlage et al., 1993). They also have higher

specific activities compared to other classes of chitinases. While a chitin

binding domain (CBD) is not required for chitinolytic or antifungal

activities, it increases both, perhaps by anchoring to the substrate and

increasing its effective concentration for hydrolysis (Iseli et al., 1993).

Another explanation is that CBD might have antifungal activity of its

own, acting on another substrate. All other chitinase classes have lower

to no antifungal activity as compared to class I chitinases.

These classes of enzymes are developmentally regulated in several

tissues including flower, roots and lower leaves (Mauch et al., 1988 a,b;

Lotan et al., 1989; Neale et al., 1990; Jacobsen et al., 1990; Samac and

Shah 1991; Neuhaus et al., 1991) exhibiting their major role in plant

defense mechanism. Chitinase along with other PR-proteins also plays a

role in somatic embryogenesis (de Jong, 1992; van Hengel et al., 2001;

21

Domon et al., 2000; Gerhardt et al., 1997; Helleboid et al., 2000), freeze

tolerance (Yeh et al., 2000) and in nodule development (Goormachtig et

al., 2001; Xie et al., 1999). Evidence for other physiological functions of

chitinases in flowering, reproduction, germination and plant growth are

also beginning to emerge.

Genes encoding for the various isoforms of chitinases have been

cloned from many plants including Arabidopsis (Samac et al., 1990),

barley (Swegle et al., 1989; Leah et al., 1991), bean (Broglie et al., 1989;

Hedrick et al., 1988; Margis-Pinherio et al., 1991), cucumber (Metraux et

al., 1989), maize (Wu et al., 1994); pea (Vad et al., 1991), groundnut

(Herget et al., 1990), Petunia (Linthorst et al., 1990), poplar (Parsons et

al., 1989; Davis et al., 1991), potato (Gaynor, 1988; La flamme and

Roxby, 1989), rice (Huang et al., 1991; Nishizawa and Hibi, 1991; Zu and

Lamb, 1991), sugarbeet (Nielsen et al., 1993, 1994) and tobacco (Shinshi

et al., 1990; Payne et al., 1990; Fukuda et al., 1991; Van Buuren et al.,

1992; Lawton et al., 1992).

2.6. TRANSGENIC APPROACHES FOR DEVELOPING FUNGAL

DISEASE RESISTANT PLANTS

2.6.1. Overexpression of chitinases in plants

To date, several plant chitinase genes have been cloned and

characterized (Collinge et al., 1993). Several transgenic plants (e.g., rice,

tobacco, canola, tomato, and cucumber) overexpressing chitinases have

22

been produced to increase resistance to fungal pathogens, with varying

levels of protection (Broglie et al., 1991; Grison et al. 1996; Marchant et

al. 1998; Zhu et al., 1994; Lin et al., 1995; Terakawa et al., 1997; Datta

et al., 2000, 2001; Nandakumar et al., 2007) (Table 2.5).

23

Table 2.5. Plant chitinases successfully expressed in different crops.

Target crop Gene Source Fungus tested References

Tobacco (N. tabacum L.)

Bean chitinase

CH5B

Bean

(Phaseolus vulgaris)

Rhizoctonia solani

Broglie et al. (1991)

Canola (Brassica napus L.)

Bean chitinase

CH5B

Bean

(Phaseolus vulgaris)

Rhizoctonia solani

Broglie et al. (1993)

Tobacco (N. tabacum L.)

RCH10 Rice Cercospora nicotianae

Zhu et al. (1994)

Canola (Brassica napus var. oleifera)

Hybrid

endochitinase gene

Tobacco-

Tomato (chimeric)

Cylindrosporium concentricum and

Sclerotinia sclerotiorum

Grison et al. (1996)

Cotton (Gossypium hirsutum)

Heterologus bean

chitinase gene

Bean Verticillium dahliae Tohidfar et al.

(2005)

Tobacco (N. sylvestris L.)

Tab Tobacco (N. tabacum L.)

Cercospora nicotianae

Vierheilig et al. (1993)

Cucumber (Cucumis

sativus) L.)

Chitinase (RCC2) Rice Botrytis cinerea Tabei et al. (1998)

Cucumber (Cucumis sativus L.)

Class I chitinase

CR32

Rice Phytophthora nicotianae var. parasitica

Kishimoto et al.

(2002)

Groundnut (Arachis hypogaea L.)

Class I ChiC Tobacco Cercospora arachidicola

Rohini and Rao

(2001)

24

Target crop Gene Source Fungus tested References

Carrot (Dacus carota) Class I ChiC Tobacco Botrytis cinerea, Rhizoctonia solani, and Sclerotium rolfsii

Punja and Raharjo

(1996)

Carrot (Dacus carota) Class I ChiC Tomato Cylindrosporium concentricum Sclerotinia sclerotiorum

Punja and Raharjo

(1996)

Potato (Solanum

tuberosum)

BjCHI1 Brassica

juncea

R. solani Chye et al. (2005)

Italian ryegrass (Lolium multiflorum)

RCC2 Rice (Oryza sativa)

Puccinia coronata Takahashi et al.

(2005)

Chrysanthemum (Dendranthema grandiflorum (Ramat.)

Chitinase (chi11) Rice (Oryza sativa)

Botrytis cinerea Takatsu et al. (1999)

Grape (Vitis vinifera L.) RCC2 Rice (Oryza sativa)

Uncinula necator and Elisinoe ampelina

Yamamoto et al.

(2000)

Rose (Rosa hybrida L.) Chitinase (chi11) Rice (Oryza sativa)

Diplocarpon rosae Marchant et al.

(1998)

Strawberry (Fragaria sp.

L.)

Chitinase

RCC2 Rice (Oryza sativa)

Sphaerotheca humuli Asao et al. (1997)

Indica Rice Class I chitinase (chi11)

Rice (Oryza sativa)

Rhizoctonia solani

Lin et al. (1995)

Indica Rice Class I chitinase (chi11)

Rice (Oryza sativa)

Rhizoctonia solani

Datta et al. (2000)

Japonica rice Class I chitinase

(cht-2 and cht-3)

Rice (Oryza sativa)

Magnoporthe grisea

Nishizawa et al.

(1999)

25

Target crop Gene Source Fungus tested References

Wheat (Triticum aestivum

L.)

Chitinase (chi11) Rice (Oryza sativa)

Fusarium graminearum

Chen et al. (1998

and 1999)

Silver birches (Betula pendula R.)

Chitinase 4 gene Sugar beet Melamsporidium betulinum

Pasonen et al. (2004)

Silver birches (Betula pendula R.)

Chitinase 4 gene Sugar beet Pyrenopeziza betulicola

Pappinen et al.

(2002)

Tobacco (Nicotiana

tabacum)

Chitinase 4 gene Sugar beet Cercospora

nicotianae

Pasonen et al.

(2004)

Wheat (Triticum aestivum L.)

Class II chitinase Barley Blumeria graminis f. sp. Tritici Erysiphe graminis

Bliffeld et al. (1999)

Wheat (Triticum aestivum L.)

Class II chitinase Barley

Erysiphe graminis and Puccinia recondita

Oldach et al. (2001)

Tomato (Lycopersicon esculentum Mill.)

pcht28 Wild tomato

(Lycopersicon chilense)

Verticillium dahliae Tabaeizadeh et al.

(1999)

Tomato (Lycopersicon esculentum Mill.)

Chi-I Tobacco (N. tabacum L.)

Fusarium oxysporum Melchers et al. (1994)

Rice variety, Pusa

Basmati 1

Chitinase (chi11) Rice (Oryza

sativa)

Rhizoctonia solani Sridevi et al. (2003)

Rice (Indica genotypes) RC7 Rice (Oryza sativa)

Rhizoctonia solani Nandakumar et al. (2007)

Sorghum bicolor (L.)

Moench

Chitinase (chi11) Rice (Oryza sativa)

Fusarium thapsinum Zhu et al. (1998);

Krishnaveni et al.

(2001)

26

2.6.2. Co-expression of ß-1,3-glucanase and thaumatin-like proteins

with chitinases in plants

Expressing the chitinases with other pathogenesis-related proteins

such as thaumatin-like protein (TLPs) and ß-1,3-glucanases show

elevated plant defense response which are coregulated/coexpressed

developmentally (Peumans et al., 2002) when challenged by pathogens

(Jacobs et al., 1999). The synergistic effect of chitinases with glucanases

and other PR proteins have been widely studied in vivo and in vitro

(Mauch et al., 1988 a,b; Vogeli-Lange et al., 1988; Vad et al., 1991;

Herget et al., 1990; Kragh et al., 1990; Lawton et al., 1992; Zhu et al.,

1994; Jach et al., 1995; Jongedijk et al., 1995; Melchers and Stuiver

2000; Chenault et al., 2002; Longemann et al., 1992). Co-expression of

chitinase and glucanase synergistically showed enhanced fungal

resistance (Jongedijk et al., 1995, Jach et al., 1995; Zhu et al., 1994;

Bliffeld et al., 1999 and Sreeramanan et al., 2006) against a wide

pathogen range. Transgenic tomato plants expressing only a chitinase or

a ß-1,3-glucanase transgene were susceptible to Fusarium oxysporum,

but plants expressing both genes had significantly higher resistance

(Jongedijk et al., 1995). Expression of barely class II chitinase and class

II ß-1,3-glucanase, in tobacco produced significantly enhanced

protection against R. solani (Jach et al., 1995) and in wheat showed

enhanced resistance to Erysiphe graminis (Bliffeld et al., 1999). The

expression of rice class I chitinase gene and the alfalfa class II glucanase

27

gene by constitutive co-expression in transgenic tobacco resulted in

substantially greater protection against Cercospora nicotianae, causal

agent of frogeye leafspot, than either transgene alone (Zhu et al., 1994).

Delayed head blight development caused by Fusarium

graminearum was observed in wheat plants co-expressing chi11 and

glucanase or tlp (thaumatin-like proteins) genes (Anand et al., 2003),

demonstrating the combinatorial effects of PR-proteins as effective means

for incorporating durable protection against pathogens. Co-expression of

rice chitinase gene (chi11) and thaumatin like protein (tlp) in the

progenies of a rice transgenic Pusa Basmati1 line revealed an enhanced

resistance to the sheath blight pathogen, Rhizoctonia solani, as compared

to that in the lines expressing the individual genes (Maruthasalam et al.,

2007). Higher levels of sheath blight resistance was observed in elite

indica rice line, when co-transformed with rice chitinase and thaumatin-

like protein (TLP) than the chitinase or TLP transformants alone (Kalpana

et al., 2006).

2.6.3. Expressing bacterial and fungal chitinases in plants

More recently, chitinase encoding transgenes have been isolated

from bacteria and fungi. These genes on transformation into plants

highly improved the plant defense response against broad range of fungal

pathogens. Several plant species expressing the Trichoderma

endochitinase encoded by ech42 gene, which inhibits the fungal spore

28

germination and hyphal elongation, have been generated. This fungal

chitinase appeared to be more effective in controlling fungal pathogens

than the plant chitinases. The ech42 when transformed into grapevine

exhibited reduced hyphal growth of Botrytis cinerea (Kikkert et al., 2000).

Similarly, potato and tobacco expressing this gene exhibited a high level

and a broad range of resistance against foliar and soilborne fungal

pathogens (Lorito et al., 1998). Transgenic broccoli expressing this gene

showed limited resistance to Alternaria brassicola and

Sclerotinia sclerotiorum (Mora and Earle, 2001). Toyoda et al. (1991)

microinjected chitinase derived from Streptomyces griseus into barley

coleoptile epidermal cells infected with powdery mildew pathogen,

Erysiphe graminis and found that the enzyme was effective in completely

digesting haustoria at the stage of primordium formation and in

suppressing the subsequent formation of secondary hyphae. Chitinase C

(ChiC) from Streptomyces griseus HUT 6037 transformed in rice and

potato showed enhanced resistance to blast disease caused by

Magnaporthe grisea and early blight caused by Alternaria solani

respectively (Itoh et al., 2003; Khan et al., 2008).

2.6.4. Chitinolytic enzyme encoding genes from other organisms

Chitinases isolated from insects are introduced in plant genomes

for enhancement of crop resistance against phytophagous insects and

other pests (Kramer and Muthukrishnan, 1997). Antifungal activities

29

have been reported in transgenic plants expressing recombinant

bacterial (Carstens et al., 2003; Itoh et al., 2003) and fungal chitinases

(Terakawa et al., 1997; Emani et al., 2003) and a chitinolytic hen egg

white lysozyme (Trudel et al., 1995).

2.7. SIGNIFICANCE OF PRODUCING MARKER-FREE TRANSGENICS

Plant transformation using Agrobacterium has been a low

frequency method. Subsequently, the usage of selectable marker genes

so far have been pivotal in these studies because, they facilitate in

identifying the rare cells that are expressing the cloned DNA, thus

monitoring and selecting the transformed progeny. They usually no

longer serve a vital purpose once the transgenic plant has been

generated and characterized. Additionally, the consumers and the

environmental groups over the past several years have expressed

concerns about the use of the marker genes from an ecological and food

safety perspective. The European Union also recommends evading the

use of antibiotic resistance and other selectable markers and the

ultimate goal will be to introduce as few foreign sequences in excess of

the gene of interest, as possible (Bukovinszki et al., 2007). Consequently,

generating marker-free plants would certainly demarcate these issues,

thus contributing to the public acceptance of transgenic crops.

Several transformation strategies have been used for the

elimination of selectable markers including cotransformation, multi auto

30

transformation system (MAT), site specific recombination system,

transposon based marker methods, intrachromosomal recombination

system and transplastomics (De Block and Debrouwer 1991; Russell et

al., 1992; Xing et al., 2000; Hohn et al., 2001; McCormac et al., 2001;

Hoa et al., 2002; Hare and Chua 2002; Puchta 2003; Jeongmoo et al.,

2004; Miki and McHugh 2004; Darbani et al., 2007). An efficient plant

transformation vector without harbouring any marker genes for

producing transgenics (de Vetten et al., 2003; Popelka et al., 2003;

Rosellini et al., 2007; Weeks et al., 2008; Malnoy et al., 2007; Doshi et

al., 2007), are efficient, rapid and does not require genetic segregation

step to remove marker genes. Further this approach will eliminate the

risk of introducing unintended effect in the transgenics that arises due to

position and pleiotropic effects of selectable marker gene.

With this background, the present study was planned to employ a

novel, antibiotic-free transformation strategy to result in rapid and

efficient expression of rice chitinase protein in groundnut plants,

performing defense response against fungal pathogens.