Methods in Molecular Biology

Not AvailableParticle bombardment/biolistic delivery is a very popular method of genetic transformation of diverse targets including cells and intact tissues. Delivery of DNA through particle bombardment is genotype and species independent, nevertheless, an ef fi cient protocol for large-scale generation of transgenic plants through embryogenic tissues with a high ( ³ 80%) shoot regeneration ef fi ciency is a prerequisite. Young embryogenic tissues or multiple shoot buds in early stages of induction are the most suited target tissues for recovery of transgenic plants. We describe the protocol for delivery of foreign genes using particle delivery system (Biorad gene gun, PDS-1000/He) in to the meristematic tissues of embryonic axes derived from mature seeds of castor. With the optimized physical and biological parameters, putative transformants were obtained at a frequency of 1.4% through particle gun bombardment of castor embryo axes. Also, transformation of embryogenic calli of sorghum using particle in fl ow gun (PIG) is described.Not Availabl

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Cytogenetics of Sorghum

Not AvailableSorghum is an interesting genus having a large number of well-recognized species taxonomically classified into five subgenera. Cytogenetic analysis led to the understanding of the nature of chromosomal variations, origins, and probable relationships based on chromosome morphology. Progress in the science of conventional and molecular cytogenetics, and genomic research provide a detailed insight into the genome organization of an individual or species, leading to enhanced utilization of genetic and physical information towards improvement of the crop. The integration of genetic, physical, and cytomolecular maps of the Sorghum genus is useful to scientists working on genomics of grass species. Large-scale molecular karyotyping of grass genomes would facilitate alignment of related chromosomal regions among different grass species and also facilitate genetic and cytogenetic studies of chromosome organization and evolution. As compared to other crop species little is known about the karyomorphology in sorghum mainly due to the small size of its chromosomes. In this chapter efforts have been made to collate the scattered information on karyotype studies, cytotaxonomy, phylogenic relation, numerical and structural variations, genome architecture, and wide introgression in sorghum. Implications of the information on sorghum improvement are discussed.Not Availabl

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Not AvailableTo select agronomically useful transgenic plants, a large number of transgenic events are initially produced, gene transfer confirmed, and advanced to obtain homozygous lines for testing in field trials. Direct in planta assays for identifying the transgene carriers in the segregating progeny are based on the activity of selectable marker gene and are easy, simple and inexpensive. For this purpose, expression of bar gene as measured by tolerance to damage by glufosinate ammonium, the active ingredient in the herbicide BASTA, was investigated. Dose damage curves were generated by leaf paint tests with BASTA on four genotypes of sorghum. Transgenic plants were characterized in terms of sensitivity to the concentration of glufosinate ammonium. In transgenics, symptoms of BASTA swab tests at different growth stages and PCR analysis for cry1B were carried out and correlated. Germination tests could not be employed for large scale evaluation of transgenic progeny because of mortality of tolerant seedlings after transplantation to soil. Based on the above findings, a simple, inexpensive, time-saving, twostep scheme for effective evaluation of transgenics and their progeny containing bar gene as selection marker using BASTA swab tests is described.Not Availabl

Transgenic Bt sorghum

Not AvailableTransgenic sorghum plants were produced through particle bombardment and Agrobacterium methods in two elite, but recalcitrant genotypes of Sorghum bicolor L. Moench. Use of target cells from developing tissues (immature embryos and multiple shoot buds), pre-culture of target tissue, small size of target tissue (2–3 mm), and regular subculture improved the selection and regeneration efficiencies. Addition of amino acid l-cysteine during co-cultivation and blotting sheet interface was helpful for complete decontamination of Agrobacterium tumefaciens and regeneration of transgenic plants. We demonstrated production of transgenic sorghum plants expressing a Bacillus thuringiensis lepidopteran toxin, through tailored in vitro protocols. Our results showed that decontamination of agrobacteria employing subtle treatments aided recovery of transgenic plants in recalcitrant genotypes. We generated 14 independent transgenic lines carrying different classes of B. thuringiensis toxin genes, cry1Aa and cry1B. Many single copy events were generated in two elite parental lines, CS3541 and 296B. Accumulation of the B. thuringiensis protein in leaves during the susceptible period of plant growth ranged from 35 to 500 ng/g fresh leaf tissue. Comprehensive insect bioassays for tolerance to spotted stem borer (Chilo partellus) were conducted through leaf disk and whole plant assays. Transgenic progeny plants showed 20–30% of damage as compared to 70–80% in non-transformed controls

Biolistic DNA delivery and its applications in Sorghum bicolor

Biolistic DNA delivery has been considered a universal tool for genetic manipulation to transfer exotic genes to cells or tissues due to its simplicity, versatility, and high efficiency. It has been a preferred method for investigating plant gene function in most monocot crops. The first transgenic sorghum plants were successfully regenerated through biolistic DNA delivery in 1993, with a relatively low transformation efficiency of 0.3%. Since then, tremendous progress has been made in recent years where the highest transformation efficiency was reported at 46.6%. Overall, the successful biolistic DNA delivery system is credited to three fundamental cornerstones: robust tissue culture system, effective gene expression in sorghum, and optimal parameters of DNA delivery. In this chapter, the history, application, and current development of biolistic DNA delivery in sorghum are reviewed, and the prospect of sorghum genetic engineering is discussed