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2. REVIEW OF LITERATURE - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/4511/12/12... ·...
Transcript of 2. REVIEW OF LITERATURE - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/4511/12/12... ·...
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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).
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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
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(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
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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.,
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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
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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
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(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
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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
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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.
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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
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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).
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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)
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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)
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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)
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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;
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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
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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).
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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)
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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)
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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)
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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
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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
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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
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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
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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.