S. Riazuddin

The myth of plant transformation.

Technology development is innovative to many aspects of basic and applied plant transgenic science. Plant genetic engineering has opened new avenues to modify crops, and provided new solutions to solve specific needs. Development of procedures in cell biology to regenerate plants from single cells or organized tissue, and the discovery of novel techniques to transfer genes to plant cells provided the prerequisite for the practical use of genetic engineering in crop modification and improvement. Plant transformation technology has become an adaptable platform for cultivar improvement as well as for studying gene function in plants. This success represents the climax of years of efforts in tissue culture improvement, in transformation techniques and in genetic engineering. Plant transformation vectors and methodologies have been improved to increase the efficiency of transformation and to achieve stable expression of transgenes in plants. This review provides a comprehensive discussion of important issues related to plant transformation as well as advances made in transformation techniques during three decades.

Mini-scale Genomic DNA Extraction from Cotton

Large amounts of polyphenolics in cotton leaves make it difficult to obtain high-quality genomic DNA during extraction. A procedure to isolate nuclear DNA from local cotton leaves (gossypium hirsutum, MNH93, CIM443, FH672) was therefore developed. It consists of rapid isolation of stable nuclei, which hinders covalent interactions with phenolics, followed by DNA extraction. The yield and quality of the resulting DNA is satisfactory and the protocol can be scaled up or down according to sample size. It is suitable for PCR and the restriction enzyme digestion needed for Southern and RFLP analysis.

Gossypium arboreum GHSP26 enhances drought tolerance in Gossypium hirsutum

Heat‐shock proteins (HSP) are molecular chaperones for protein molecules. These proteins play an important role in protein–protein interactions such as, folding and assisting in the establishment of proper protein conformation and prevention of unwanted protein aggregation. A small HSP gene GHSP26 present in Gossypium arboreum responds to dehydration. In the present study, an attempt was made to overcome the problem of drought stress in cotton. A cDNA of GHSP26 was isolated from G. arboreum, cloned in plant expression vector, pCAMBIA‐1301 driven by the cauliflower mosaic virus 35S promoter and introduced into Gossypium hirsutum. The integration and expression studies of putative transgenic plants were performed through GUS assay; PCR from genomic DNA, and quantitative real‐time PCR analysis. Transgenic cotton plants showed an enhanced drought tolerance, suggesting that GHSP26 may play a role in plant responsiveness to drought.

Variability in expression of insecticidal Cry1Ab gene in Indica Basmati rice

The expression of an insecticidal gene cry1Ab, under three differentpromoters was studied in leaves, stem and panicles to determineorgan-specificity in Basmati rice. Enhanced resistance against twoLepidopteran insects, stem bore (Scripophaga incertulas) and leaffolder (Cnaphalocrocis medinalis) was observed. The result of westernhybridization and insect bioassays demonstrated that all these promotersexpress the cry1Ab gene at similar levels in leaves and panicles. Thecry1Ab gene was expressed in stems at 0.05% of the total protein underthe control of the PEPC promoter alone or in combination with thepollen-specific promoter. On the other hand it was expressed at 0.15%under the control of the ubiquitin promoter. Southern blot hybridization ofthese plants indicated integration of the complete plant transcriptional unitat multiple insertion sites. These results demonstrated that a specificpromoter could be used to limit the expression of cry1Ab gene in thedesired parts of Basmati rice plants.

Cotton somatic embryo morphology affects its conversion to plant

Somatic embryos differentiated from hypocotyl explant in cotton (Gossypium hirsutum L.) exhibited very divergent morphologies. Six different types of somatic embryos based on cotyledon development were observed. The growth hormones (2,4-dichlorophenoxyacetic acid and kinetin) used in induction and maintenance media did not affect embryo rooting and germination. The 95 % conversion of normal embryos (with two cotyledons) was achieved, while an overall conversion was only 38 %. Horn shaped embryos failed to exhibit shoot growth. Poorly developed apical meristems were responsible for lower conversion percentages in some of embryo classes. However, regenerated plants phenotypically resembled to seed grown control plants regardless of somatic embryo morphology.

Identification and expression of six drought-responsive transcripts through differential display in desi cotton (Gossypium arboreum)

There is not enough information available on drought-modulated gene(s) in Gossypium arboreum, which can be a valuable gene pool for improving modern cotton cultivars. In the present work, differential display reverse transcriptase PCR (DDRT) was used to compare overall differences in gene expression between water-stressed and control plants. By screening 93 primer-pair combinations, the DDRT technique resulted in upregulation of 30 cDNA transcripts. Through reamplification and quality control assay, ten cDNA transcripts appeared false positive. The remaining 20-cDNA transcripts were extracted from the gel, reamplified, cloned and sequenced. Homology search revealed that six transcripts showed significant homology with known genes. Real-time RT-PCR showed that, among six transcripts, five showed significant overexpression in water-stressed leaves as compared to control. This is an important finding since there are only few reports of universal stress protein, and transposable elements are available in plants but none in cotton under drought condition.

Isolation and functional analysis of cotton universal stress protein promoter in response to phytohormones and abiotic stresses.

The 949 bp promoter fragment upstream from the translation initiation site of the GUSP gene encoding a universal stress protein was isolated from the genomic DNA of Gossypium arboreum. Some putative cis-acting elements involved in stress responses including E-box, ABRE, DPBF-box, and MYB-core elements were found in the promoter region. In an Agrobacterium-mediated transient expression assay, strong activation of the GUSP full promoter region occurred in tobacco leaves following dehydration, abscisic acid, salt, heavy metal, gibberellic acid and dark treatments. Deletion analysis of the promoter revealed that the dehydration, abscisic acid and salt responses were affected by the deletion between −208 and −949 bp and showed 2–4-fold induction. However, in response to dark, gibberellic acid and heavy metals the induction was only 2-fold. These findings further our understanding of the regulation of GUSP expression. This is an important study as no report of this universal stress protein promoter is available in literature.