I hereby declare that the matter contained in this thesis is the result of investigations carried out by me in the Department of Biotechnology, Indian Institute of Technology Guwahati, India, under the direction of Dr. It is certified that the work described in this thesis, entitled "Genetic engineering of cowpea for storage pest resistance", done by Mr.
ABSTRACT
Nos poly-(A) Nopaline synthase Poly (adenylimes) nptII gene neomycin phosphotransferase II NPTII Neomycin Phosphotransferase II.
UNITS
LIST OF TABLES
INTRODUCTION
Introduction
Therefore, the bean αAI-1 gene has been considered to be a strong candidate to confer resistance to Callosobruchus spp. However, the lack of a robust plant regeneration system suitable for routine genetic transformation methods and the lack of an efficient selection scheme for recovering viable and fertile transgenic plants from transformed explants at a reasonably high frequency have resulted be the main obstacles in the adaptation of both. previously published protocols (Popelka et al. 2006; Chaudhury et al. 2007) for transfer of candidate genes to pea.
Objectives
REVIEW OF LITERATURE
Plant Genetic Engineering
- Plant regeneration
- Plant regeneration in Asiatic Vigna species
- Gene delivery systems
- Agrobacterium- mediated gene transfer
- Gene delivery by Biolistics
- In planta transformation
- Alternative methods of transformation
- Marker genes for selection of transgenic plants
- Selectable marker genes
- Reporter genes
- Genetic transformation of Asiatic Vigna species
Subsequent pollination with bombarded pollen led to the recovery of transgenic plants (Touraev et al. 1997). Grass gene (bialaphos resistance) which confers resistance to BASTA® herbicide (Murakami et al., 1986;.
Regeneration and genetic transformation of cowpea
- In vitro plant regeneration in cowpea
- Genetic transformation of cowpea
Fertile cowpea plants have been successfully regenerated using nodal thin cell layer (TCL) explants (Le et al. 2002). The progeny (first and second generations) of all self-fertilized transgenic lines revealed the presence of the transgenes (gus and ahas) co-segregated in a Mendelian fashion (Ivo et al. 2008).
Storage pest resistance
- Seed defence
- Proteinase inhibitors
- α-Amylase inhibitors
- Lectins
- Conventional breeding for resistance to insect pests in legumes
- Transgenic grain legumes for storage pest resistance
- Genetic improvement of cowpea for storage pest resistance
Arcelins work by binding to the peritrophic membrane of the insect gut and interfering with nutrient absorption and causing the rupture of intestinal membranes (Paes et al. 2000). Uptake and expression of aAI-1 in azuki bean resulted in complete resistance to the azuki bean beetle (Ishimoto et al. 1996).
DEVELOPMENT OF AN EFFICIENT PLANT
MATERIALS AND METHODS .1 Plant materials
- Explants preparation and plant regeneration
- Transplantation
Explants were grown on MSB medium supplemented with different cytokinins [benzylaminopurine (BAP), kinetin and thidiazuron (TDZ)] individually at different concentrations and 7.5 µM) in culture tubes (25 × 100 mm) for multi-shoot induction. Therefore, the cultures were induced on MSB medium containing 5 µM BAP, after 4 weeks transferred to basal media supplemented with kinetin and gibberellic acid (GA3) separately at different concentrations (0.1, 0.5 and 1.0 µM) for 2 weeks for shoot extension.
RESULTS AND DISCUSSION .1 Multiple shoot induction
- Shoot elongation
- Cyclic organogenesis
- Rooting and acclimatization
Changing the physiological state of the initial explants is one approach that can alter the regenerative capacity in vitro (Malik and Saxena, 1991; Santalla et al. Among the eight commercially important cultivars compared for their shoot regeneration potential, Pusa Komal produced the most shoots (9 .1 shoots/explant) in 94% of cultures.
ESTABLISHMENT OF EFFICIENT SELECTION SYSTEM
MATERIALS AND METHODS
- Effect of kanamycin, paromomycin and geneticin on shoot regeneration
Control experiments were carried out by cultivating explants in shoot induction medium in the absence of any antibiotics. The selection scheme, i.e., the choice of the aminoglycoside antibiotic and its optimal concentration, for the selection of transformed shoots was based on those that allowed the initial proliferation of shoot buds followed by their bleaching without necrotic effect on the explants.
RESULTS AND DISCUSSION
- Effect of kanamycin, geneticin, paromomycin on shoot regeneration
Furthermore, early necrosis was observed in explants grown in higher concentrations of paromomycin, which interfered with the regeneration process to varying degrees. These results supported the observation that early necrosis may be due to substances leached from the dying cells, which probably had a negative influence on regeneration. Therefore, low concentration of geneticin (45 mg/l) was chosen for selection and maintenance of putative transformed shoots in our study.
Interestingly, the developmental stage of initial explants and their subsequent regeneration was found to coincide with geneticin activity, while kanamycin and paromomycin exerted a faster inhibitory activity, thus targeting early necrosis. Attempts to perform selection with organized tissue have not been very successful with the notable exception of shoot apical meristem remodeling in lupine, cotton and soybean (Pigeaire et al. 1997; Zapata et al. 1999; Aragao et al. 2000). The main problem with organized tissue is that transformed cells allow the proliferation of non-transformed tissue in their vicinity by effectively detoxifying the selective agent.
The judicious choice of selection levels for organized tissues is critical to facilitate the growth and division of transformed cells, as higher levels would be detrimental even to transformed cells in the initial stages of screening (Sahoo et al. 2001 ).
AGROBACTERIUM -MEDIATED GENETIC TRANSFORMATION OF
MATERIALS AND METHODS
- Plant material and explant preparation
- Agrobacterium strains and transformation vector
- Triparental mating
- Bacterial inoculation
- Cocultivation
- Selection and regeneration of transformants
- Transient and stable gus expression analysis
- Genomic DNA isolation and PCR analysis
- Southern hybridization analysis
- RNA isolation and RT-PCR
- Analysis of transgene inheritance
Pusa Komal was surface sterilized and cultured on MSB medium [MS salts (Murashige and Skoog, 1962) + B5 vitamins (Gamborg et al. 1968)]. Four Agrobacterium tumefaciens strains, characterized by different chromosomal backgrounds and their respective tumor-inducing plasmids [LBA4404 (Hoekema et al. 1983), GV2260 (Deblaere et al. 1985), AGL1 (Lazo et al. 1991) and EHA1 et al. . 1993)], all with a binary vector pCAMBIA2301 (http://www.cambia.org) were compared for their efficacy through early detection of transient transformation events in infected explants. The vector pCAMBIA2301 is a pPZP-based small binary vector (Hajdukiewicz et al. 1994) and the T-DNA of pCAMBIA2301 includes neomycin.
Plasmid SB1 lacks T-DNA but has all the virulence genes present in pTOK233 (Hiei et al. 1994) and contains a tetracycline resistance gene as a selection marker. The donor strain DH5apCAMBIA2301 and the helper strain DH5apRK2013 were plated on LB medium (Sambrook et al. 1989) containing kanamycin (50 mg/l) and incubated at 370°C one day before triparental mating. Individual colonies of the bacterial strains were transferred to 2 ml of liquid AB minimal medium (Chilton et al. 1974) with appropriate antibiotics and grown overnight at 28°C.
Amplified DNA fragments were analyzed by 1% agarose gel electrophoresis, visualized by ethidium bromide staining (Sambrook et al. 1989), and photographed under ultraviolet light in the gel documentation system.
RESULTS AND DISCUSSION .1 Effect of Agrobacterium strains
- Effect of pH
- Effect of acetosyringone
- Effect of cocultivation period and temperature
- Effect of additional virulent genes
- Selection and regeneration of transgenic plants
- Stable GUS expression analysis
- Molecular analysis of transgenics
- Transgene expression and segregation in the T 1 generation
The presence of nptII and gus transcripts was determined by reverse transcription polymerase chain reaction. A previous evaluation of different Agrobacterium strains (AGL0, AGL1 and LBA4404) in pea transformation found no difference in their transient transformation efficiency (Popelka et al. 2006). Constitutive expression of the vir genes in the resident vector pSB1 dramatically increased the frequency of transient transformation to 100% compared to only 80% in the absence of additional copies of the vir genes (Chaudhury et al. 2007).
GUS expression in T1 germinated seedlings clearly demonstrated the inheritance and expression of the transgene in the progeny (Fig. 5i). The result verified the functional expression of nptII and gus genes in the transgenic plants. Strong GUS activity in T1 germinated seedlings indicated inheritance and expression of the gus gene in the progeny.
PCR analysis of the progeny of each selected transgenic line showed the inheritance of the nptII and gus genes in a Mendelian manner (Fig. 6e, f).
MATERIALS AND METHODS
- Agrobacterium strain and mobilization
- Plant material and explant preparation
- Transformation, selection and plant regeneration
- GUS assay
- Molecular analysis of putative transformants
- Polymerase chain reaction analysis
- Southern hybridization analysis
- Segregation analysis
- Extraction of seed protein
- α-amylase inhibitory activity
- Insect bioassays
The standard binary vector pCAMBIA2301 (CAMBIA, Australia) was used to subclone α-amylase inhibitor 1 gene expression cassette between the T-DNA borders of the binary vector. The optimized Agrobacterium-mediated genetic transformation protocol was used for the overexpression of common bean α-amylase inhibitor 1 gene in cowpea. Histochemical GUS assays (Jefferson, 1987) were used to assess transient and stable expression of the gus gene.
Subsequently, 1/10 volume of the original cDNA was used to amplify the αAI-1 gene using the same primer sets as used in the genomic PCR. These templates were used to amplify αAI-1 gene transcripts using the same primer sets as those used in genomic PCR. α-Amylase activity was measured using a modification of the Bernfeld method (Bernfeld, 1955) as previously described by Morton et al.
The weights of the newly hatched adults were measured and the lifespan of the adults was also recorded.
RESULTS AND DISCUSSION
- GUS assay
- Molecular analysis of transgenics
- Expression of αAI-1 gene in transgenic plants
- Inheritance of αAI-1 gene in transgenic plants
- α-amylase inhibitory activity in transgenic seeds
- Insect bioassays
Gus expression in germinated T1 seedlings clearly demonstrated the inheritance and expression of the transgene in the progeny (Fig. 9i,j). Molecular analysis of the transgenic plants was performed to detect the presence, integration and expression of the αAI-1 gene. Progeny plants of 4 independent T0 transgenic lines were examined by Southern analysis to confirm the integration of the αAI-1 gene into the genome of the transgenic plants (Fig. 10c).
No hybridization signal was detected in the untransformed plant used as a negative control (Fig. 10c, lane C), indicating the verification of the results. These results were further verified with the functional expression of the αAI-1 gene in the transgenic seeds. Detection of strong GUS activity in germinated T1 seedlings indicated the inheritance and stable expression of the gus gene in the progeny.
There were clear reductions in the emergence of adult insects from the seeds of both transgenic lines compared to the untransformed control in all insect bioassays.
CONCLUSIONS
SIGNIFICANCE AND SALIENT FEATURES OF THE PRESENT STUDY
Black peas make an enormous contribution to human nutrition, as the seeds and fresh peas are a rich source of protein, some minerals and vitamins (Sahoo et al. 2003). Sustained and adequate levels of bruchid resistance are lacking in the primary gene pool, but are available in distant wild species, which present barriers to gene transfer through conventional breeding techniques (Singh et al. 2000). Furthermore, limited genetic diversity in cowpea breeding programs is of particular concern because black bean appears to have lower inherent genetic diversity than other cultivated plants due to a presumed single domestication event (Fang et al. 2007).
There have been three reports published on the generation of stable transgenic plants in cowpea, by Agrobacterium-mediated transformation (Muthukumar et al. We used an improved Agrobacterium-mediated cowpea transformation method (Solleti et al. 2008) for the introduction of the bean ( Phaseolus vulgaris) α-amylase inhibitor-1 (αAI-1) gene in a commercially important Indian cowpea cultivar, Pusa Komal and generated fertile transgenic plants. The preliminary studies on synergistic effect of the two cytokinins, BAP and kinetin on shoot proliferation showed that the time of exposure to the combination of these two cytokinins was critical for accelerated regeneration response from cotyledon nodule explants of cowpea.
The presence, integration and expression of the α-amylase inhibitor gene in the transgenic lines was confirmed by PCR, Southern hybridization and RT-PCR.
FUTURE PROSPECTS
Collen A M C and Jarl C I (1999) Comparison of different methods for regeneration of the legume Galega orientalis Lam. Hansen G, Das A and Chilton M D (1994) Constitutive expression of virulence genes improves efficiency of plant transformation by Agrobacterium. Ishimoto M and Chrispeels M J (1996) Defense mechanism of Mexican bean downy mildew against high levels of alpha-amylase inhibitor in common beans.
Orczyk AN ken Orczyk W (2000) Panagadal kadagiti banag a mangimpluensia ti Agrobacterium-a naipamaysa a panagbalbaliw ti gisantes (Pisum sativum L.). Orlikowska TK, Cranston HJ ken Dyer WE (1995) Dagiti banag a mangimpluensia ti Agrobacterium tumefaciens-a naipamaysa a panagbalbaliw ken panagpabaro ti kultibar a safflower. Popelka JC, Gollasch S, Moore A, Molvig L ken Higgins TJV (2006 ) Genetiko a panagbalbaliw ti gisantes (Vigna unguiculata L.) ken natalinaay a pannakayakar dagiti transgenes kadagiti kaputotan.
Suzuki K and Ishimoto M (1999) Characterization of the third alpha-amylase inhibitor alpha AI-3, in the common bean (Phaseolus vulgaris L.). DNA from untransformed plant (negative control); Lanes 1-7: DNA from T1 transgenic plants. f) PCR amplification of the 240-bp fragment of the gus gene from T1 plants. Molecular weight marker; Lane P: pSiva plasmid DNA (positive control); Lane C: DNA from untransformed plant (negative control); Lanes 1-8: DNA from T1 transgenic plants. e) RT-PCR analysis of the aAI-1 gene mRNA transcript level in the T0 transgenic plants.
LIST OF PUBLICATIONS 1. In Refereed journals
In Conferences
