Final Bt Poster

Genetic engineering approach to develop male sterility in Jute (Corchorus spp) plant

Results-2. Isolation, cloning, partial Tirthartha Chattopadhyay,Materials ALPGE and and methods Mrinal K. Maiti, Department of Biotechnology, IIT, Kharagpur characterization and preparation of Male sterility (inability to produce Isolation of genomic DNA, RNA and cDNA siRNA mediated gene silencing construct functional male gamet) phenomenon in synthesis: of 3 potential candidate genes from Jute plant system is crucial for hybrid seed The plant genomic DNA isolation was carried out seedling cDNA production. Limitations of naturally as described by Doyle and Doyle, 1990. Total Potential candidate genes are selected on occurring male-sterility demand RNA from jute seedlings was isolated by Trizol the basis of the following strategies: application of genetic engineering method (Simms et al., 1993). cDNA was prepared A. Deregulating the energy metabolism of technique to develop transgenic male by using anchored oligo-dT primer with the help the cells in the anther tissue (for this sterility in important crop plants. of Transcriptor First Strand cDNA Synthesis Kit purpose partial CDS of the gene encoding (Roche). Several genetic beta subunit of F1 ATPase is cloned) B. Standard molecular biological techniques: engineering Deregulation of lipid metabolism in anther Carried out as described by Sambrook et al., strategies have tissue (for this purpose partial CDS of the 1989. been devised in gene encoding Stearoyl ACP Desaturase is Agrobacterium mediated Plant transformation: different crops but cloned) C. Cell ablation mediated by cytoSuitable explants (leaf disc for tobacco and nothing is available skeletal damage (for this purpose partial embryonic meristem with attached petiole for for developing a CDS of the Actin gene is cloned) jute) were surface sterilized and taken for male sterility transformation by Agrobacterium LBA4404 system in Jute strain bearing the concerned genetically (Corchorus spp.) , engineered plasmid. After transformation and 3 an important fibre days of co-cultivation, transformed explants were crop. Here, a placed on agar solidified MS basal media Figure 1: Jute plants, strategy of fibre and some of the (containing 8mg/l IAA, 2.5mg/l kinetin, 50mg/l developing jute-products hygromycin and 250mg/l cefatoxin for tobacco genetically engineered male-sterile Jute whereas 0.5mg/l IBA, 0.5mg/l BAP, 15mg/l plant is elucidated and progress made hygromycin and 250mg/l cefatoxin for jute). After in this regard is documented. 8 weeks of subculture, individual plantlets were B transferred to glass bottles containing sand for A C D Figure 4: Amino acid sequence alignment of the 3 cloned part Introduction hardening and root development. After 2 weeks genes (A), partial characterization of them by Southern A male sterile plant lacks functional hybridization (B), respective schematic diagram of the siRNA of hardening, individual plants were transferred mediated gene silencing construct s (C), and restriction pollen grains (male gamets) but has to field. analysis of the concerned constructs. functional female part. Therefore, all of Histochemical GUS assay: the genetic engineering strategies to As described by Jefferson, 1987. Results-3. Standardization of a protocol develop male-sterile plants intend to for plantlet regeneration and genetic interfere precisely with the anther or transformation in jute plant Results- 1. Anther specific novel promoter isolation Best organogenic media for multiple pollen development (Mariani et al.,1990, Spena et al., 1992, Poovaiah et al., 2002). On the basis of the bioinformatics analysis with shooting was found to be MS basal media However, transgenic male-sterile plants the Pea END1 anther specific protein (Gomez et supplemented with 0.5mg/l IBA and produce 50% male fertile plants due to al., 2004), a putative anther specific promoter 0.5mg/l BAP (taking embryonic meristem segregation in the filial progeny. In a (OsASP) was isolated from rice genome by PCR with attached petioles as the explant). hybrid seed production programme, mediated cloning (Figure 2). OsASP-GUS-NOS Transient GUS expression was found in these fertile plants are considered highly transformed transgenic tobacco plants showed the explants infected with Agrobacterium undesirable. So, any strategy to develop anther-predominant GUS expression in T1 and LBA4404 strain harboring pCAMBIA1300 transgenic male-sterile plants should T2 generations. Strong GUS expression was 2X35S-GUS-NOS plasmid. However, no include a plan for selective elimination of observed in the anthers of 13.5mm to 45mm long plantlets could be recovered through these fertile plants. flower buds of transgenic tobacco plants (Figure hygromycin selection. 3). Abstract

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Figure 3: A. PCR screening of transgenic lines B. Southern hybridization of PCR positive plants C. Comparative histochemical GUS assay of transgenic lines D. GUS assay of transgenic tobacco anther at different developmental stages

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On the basis of this background information, the following objectives are fixed – 1.Isolation and charaterization of a novel anther specific promoter 2.Isolation of a few candidate genes from jute plant or other sources 3.Preparation of a suitable gene construct capable of causing male sterility 4.Standardization of jute plant transformation protocol 5.Preparation of a linked herbicide resistance gene construct to facilitate chemical rouging of fertile plants

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Figure 2: A. Restriction analysis of OsASP PCR product B. Schematic diagram of pCAMBIAOsASP-GUS construct, C. Restriction analysis of this construct and D. OsASP-GUS transformmed putative transgenic tobacco plants

Figure 5: Composition of different multiple shooting media for jute, their relative performance and transient histochemical GUS assay of transformed jute explants References: 1. Mariani C., De Beuckeleer M., Truettner J., Leemans J. and Goldberg R.B. (1990): Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature. 347: 737-741. 2. Spena A., Estruch J.J., Prensen E., Nacken W., Van Onckeler H. and Sommer H. (1992): Anther specific expression of the rolB gene of Agrobacterium rhizogenes increases IAA content in anthers and alters anther development and whole flower growth. Theor. Appl. Genet. 84: 520-527. 3. Poovaiah B.W., Liu Z., Patil S. and Takezawa D. (2002): Compositions and methods for production of male sterile plants. Patent No. US6.362.395. 4. Gomez, M.D., Beltran J.P. and Canas L.A. (2004): The pea END1 promoter drives anther specific gene expression in different plant species. Planta. 219: 967-981. 5. Doyle J. and Doyle J. (1990): Isolation of plant DNA from fresh tissue. BRL FOCUS. 12: 13-15. 6. Sambrook, J., Fritisch, E.F. and Maniatis, T. (1989): Molecular cloning: a Laboratory Manual, 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. 7. Jefferson R A (1987): Assaying Chimeric Genes in Plants: The GUS Gene Fusion System. Plant Molecular Biology Reporter. 5: 387-405.