Abstrcat of Ph.D. Thesis; Biosciences

Genetic engineering of vegetable Brassicas for resistance to Diamondback moth (DBM) is of paramount importance. Plant transformation technology offers unique opportunities to transfer a wide spectrum of functionally relevant genes from organisms that are known for producing toxic substances specific for pests of crop plants. Over the past four years, the success in producing insect-resistant Bt-transgenic plants through gene transfer has been impressive. The global adoption rates for transgenic crops are increasing and 19% of transgenic crops grown are Bt crops.

Among the vegetable Brassicas in India, cabbage is grown on large scale accounting to 10% of the world production. One of the major problems in cabbage cultivation is insect infestation, especially by DBM. DBM has become the most destructive insect pest of Cruciferous plants throughout the world and has become resistant to synthetic chemicals used against it as well as to environmentally benign Bt sprays in the field.

Due to the simplicity and cost effectiveness besides the ease with which Agrobacterium tumefaciens can infect Brassica sp, during the course of research carried out for this thesis, efforts were made to introduce the cry1B-cry1Ab fusion gene through A. tumefaciens-mediated transformation. In the present study, a synthetic fusion gene was constructed in a continuous reading frame. This fusion gene encodes for Cry1B and Cry1Ab δ-endotoxins as translational fusion protein. A. tumefaciens-mediated transformation method using hypervirulent helper strain, EHA105 was successfully used to transform a tropical cabbage breeding line, DTC 507 with cry1B-cry1Ab fusion gene driven by a constitutive cauliflower mosaic virus (CaMV) 35S promoter to confer resistance to DBM. This tropical cabbage breeding line is otherwise susceptible to DBM. The important feature of this breeding line is that it can set viable seeds without vernalization under the subtropical conditions prevailing in North Indian plains. The present study provides a reliable transformation and regeneration system for development of Bt-transgenic cabbage.

It was observed that MS medium supplemented with 10 µM BAP, 1 µM NAA and 10 µM silver nitrate was efficient in regenerating transformed cells into shoots from hypocotyl explants of 5 d old seedlings. For selection of transformed shoots 20 mg/l kanamycin was used in the selection medium and 20 putative transformants were selected. Regeneration of transformed cells into shoots took place four to six weeks after incubation in the selection medium. Application of selection pressure 3 d after cocultivation minimized the regeneration of untransformed shoots. After infection with Agrobacterium, shoot induction occurred from any one cut ends of the hypocotyl explants.

PCR analysis with specific primers for npt II, cry1B and cry1Ab genes was used for initial screening of putative transformants selected in the selection medium. It was noted that six putative transformants exhibited PCR positive signal for npt II, cry1B and cry1Ab genes. Southern hybridization confirmed the integration of cry1B-cry1Ab fusion gene in one of the six putative transformants. Southern hybridization with cry1B-cry1Ab gene as probe showed the presence of three distinct bands that got hybridized with the probe. It was also noted that one of the hybridized band was a truncated portion of the T-DNA because the molecular weight of the hybridized band (4.2 kb) was less than the molecular weight of T-DNA (6.1 kb). The variation seen in the position of bands on autoradiogram must be due to random integration of T-DNA in the genome. Inheritance of the transgene was investigated in the T₁ generation (obtained from bud pollination of the T₀ transgenic plants) by PCR analysis for the presence of npt II, cry1B and cry1Ab fusion gene. In order to ensure that all three genes were inherited in the T₁generation, specific primers for all three genes were used in PCR analysis. PCR analysis of 100 individuals of T₁ population confirmed the presence of all three genes in 47 individuals and rest of the individuals did not exhibit PCR signals for any of the three primers used. This data suggest that the transgenes were closely linked wherein unlinked loci or if integrated in three different chromosomes would give 63:1 ratio. Southern hybridization of 8 plants (that produced large size head) of T₁generation confirmed the stable inheritance of the fusion gene. Among the eight Southern positive plants five transgenic plants showed hybridization of cry1B-cry1Ab gene at three loci as observed in autoradiogram of T₀ transgenic plant. This suggests that the transgenes were closely linked. However, two T₁ plants (BtC-3 and BtC-4) showed hybridization of cry1B-cry1Ab fusion gene at a single locus. This again suggests that this particular locus was not closely linked to two other loci or must be present on different chromosome. Hence during meiosis, this particular locus was segregated from two other loci where the T-DNA got integrated. This Bt transgenic cabbage plant that showed hybridization of the fusion gene at a single locus was segregated into 3:1 ratio (monohybrid) for fusion gene in T₂ generation.

Double antibody sandwich ELISA analysis was performed to quantify the levels of the fusion protein expression in the transgenic plants. Expression of the fusion protein in T₀ transgenic plant was 0.1% (in mature leaves) to 0.15% (in young leaves) of buffer extractable protein. In T₁transgenic plants, expression of the fusion protein levels ranged from 0.04% (in mature leaves) to 0.16% (in young leaves) of buffer extractable protein.

Insect bioassays were conducted with all four larval stages of DBM. In all three (leaf disc, detached leaf and whole plant) types of bioassays, transgenic plants exhibited high level of resistance to infestation by DBM larvae. Even, mature leaves with the lowest level of expression, 0.04% of buffer extractable protein caused 100% mortality to all larval stages of DBM. This clearly demonstrates the lethal effect of Cry1B-Cry1Ab fusion protein expressed in the transgenic tropical cabbage plants against DBM larvae.

Photosynthesis is the most important metabolic event that regulates the plant growth and development and finally the seed production. The measurement of primary photochemical reactions mediated by PS II and PS I showed that there was no significant difference in PSII and PS I activities in Bt transgenic (T₀and T₁) cabbage plants and its wild type counter part. So, the integration of synthetic Bt fusion gene, cry1B-cry1Ab in the cabbage nuclear genome did not alter photosynthetic activity of the transgenic plant. Both T₀ and T₁ Bt transgenic plants flowered normally and set seeds normally like its wild type counterpart.

Insect resistance to Bt-transgenic plants has arisen, especially in lepidoptera. Although there are no reports of insects directly developing resistance to Bt-transgenic plants, survival to maturity has been reported for resistant strains of DBM, tobacco budworm and pink bollworm on Bt-transgenic plants expressing single toxin in broccoli and cotton (Zhao et al., 2000). In all these reports the resistance exhibited by the insect strains did not develop directly from selection on Bt-transgenic crops. However, Bt-transgenic cabbage plants developed in the present study carry two different cry genes with binding affinity to different receptors in the midgut epithelial membranes of DBM. Therefore, there is little chance for DBM to develop resistance to such toxic combinations expressed in transgenic plants. Hybrids of Cry1Ac and Cry1Fa have a wider target spectrum than either of the parental toxins from which they were derived (Malvar and Gilmer, 1998). Theoretical model also suggests that pyramiding two dissimilar toxin genes in the same plant has the potential to delay the onset of resistance much more effectively than single toxin plants released spatially or temporally (Roush, 1994; Gould, 1998). In a recent study, Moellenback et al. (2001) found that co-expression of two new δ endotoxins with molecular masses of 14 kD and 44 kD (expressed during sporulation growth phase of Bacillus thuringiensis) caused mortality to corn root worm (Diabrotica virgifora).

Because of its specificity and mode of action, Bt has historically been considered a safe option for pest control and has often been the preferred insect control method in insect pest management programme. Biotechnological development of Bt transgenic crop plants is thus a potentially valuable for the agricultural economy and promises to increase yield and reduce pesticide inputs.

Following are the major conclusions that can be derived from the present studies:

1	A fusion gene comprising of synthetic cry1B and cry1Ab genes encoding a translational fusion product of the N-terminal halves of the respective proteins was constructed and cloned into binary vector plasmid pBinAR. This binary vector carrying cry1B-cry1Ab fusion was designated as pBinBt6 and mobilized into Agrobacterium tumefaciens strain EHA105.
2	Hypocotyl explants excised from 5 d old seedlings were the most suitable explant for caulogenesis and shoot induction.
3	Agrobacterium tumefaciens mediated transformation method was successfully used for insertion of synthetic cry1B-cry1Ab fusion gene into the cells of hypocotyl explants.
4	MS medium supplemented with 10 µM BAP, 1µM NAA, 10 µM silver nitrate and 20 mg/l kanamycin was efficient in regeneration of transformed cells into shoots from 5 d old seedlings.
5	Half-strength MS medium was efficient in inducing profuse rooting at the cut end of shoots of putative transformants that were selected in the selection medium.
6	PCR amplification with specific primers for npt II, cry1B, cry1Ab genes was useful in screening putative transformants as well as useful in identifying transgenic plants of T₁ populations.
7	Southern hybridization with the probe, cry1B-cry1Ab gene confirmed the integration of cry1B-cry1Ab gene in the genome of tropical cabbage.
8	Two of T₁ Bt-transgenic cabbage plants that showed hybridization signal at single locus were segregated into 3:1 ratio for the fusion gene in T₂ generation.
9	The expression of the integrated cry1B-cry1Ab fusion gene in the Bt -transgenic cabbage plants was quantified by double antibody sandwich ELISA analysis. ELISA analysis confirmed the expression of the fusion protein in Bt -transgenic plants and not in wild type.
10	Expression of the fusion protein in T₀ transgenic plant was ranged from 0.1% (in mature leaves) to 0.15% (in young leaves) of buffer extractable protein. In T₁ plants, expression of fusion protein levels ranged from 0.04% (in mature leaves) to 0.16% (in young leaves) of buffer extractable protein.
11	Insect bioassays with all four larval stages of DBM confirmed the efficacy of the fusion gene expressed in Bt-transgenic cabbage plants. Bt-transgenic plants that expressed the fusion protein were highly resistant to DBM and caused 100% mortality of all larval stages of DBM.
12	Mortality was observed in neonate stage of DBM within 24 h on the Bt-transgenic cabbage plants. Also, second instar to fourth instar could survive on the Bt transgenic cabbage plants for not more than 48 h.
13	In the insect bioassay conducted with detached-mature leaf of a transgenic line expressing least amount of fusion protein, 0.04% of buffer extractable protein also caused 100% mortality to all larval stages of DBM. This clearly demonstrates the lethal effect of Cry1B-Cry1Ab fusion protein that expressed in Bt transgenic cabbage plant to all larval stages of DBM.
14	The primary photochemical reactions mediated by PSII and PSI showed that in both T₀ and T₁Bt-transgenic cabbage as well as wild type plants, PS II and PSI activities did not differ significantly. So, integration of synthetic fusion gene, cry1B-cry1Ab in the nuclear genome of the tropical cabbage did not alter photosynthetic efficiency of the transgenic plants. Both T₀ and T₁ Bt transgenic plants flowered and set seeds normally like its wild type counterpart.


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Author	Supervisor
Anderson Paul Dept. of Biosciences Jamia Millia Islamia	Dr. P. Pardha Saradhi Dept. of Biosciences Jamia Millia Islamia