Bahaeldeen Babiker Mohamed

Chloroplast-targeted expression of recombinant crystal-protein gene in cotton: An unconventional combat with resistant pests

Plants transformed with single Bt gene are liable to develop insect resistance and this has already been reported in a number of studies carried out around the world where Bt cotton was cultivated on commercial scale. Later, it was envisaged to transform plants with more than one Bt genes in order to combat with resistant larvae. This approach seems valid as various Bt genes possess different binding domains which could delay the likely hazards of insect resistance against a particular Bt toxin. But it is difficult under field conditions to develop homozygous plants expressing all Bt genes equally after many generations without undergoing recombination effects. A number of researches claiming to transform plants from three to seven transgenes in a single plant were reported during the last decade but none has yet applied for patent of homozygous transgenic lines. A better strategy might be to use hybrid-Bt gene(s) modified for improved lectin-binding domains to boost Bt receptor sites in insect midgut. These recombinant-Bt gene(s) would express different lectin domains in a single polypeptide and it is relatively easy to develop homozygous transgenic lines under field conditions. Enhanced chloroplast-localized expression of hybrid-Bt gene would leave no room for insects to develop resistance. We devised and successfully applied this strategy in cotton (Gossypium hirsutum) and data up to T3 generation showed that our transgenic cotton plants were displaying enhanced chloroplast-targeted Cry1Ac-RB expression. Laboratory and field bioassays gave promising results against American bollworm (Heliothis armigera), pink bollworm (Pictinophora scutigera) and fall armyworm (Spodoptera frugiperda) that otherwise, were reported to have evolved resistance against Cry1Ac toxin. Elevated levels of hybrid-Bt toxin were confirmed by ELISA of chloroplast-enriched protein samples extracted from leaves of transgenic cotton lines. While, localization of recombinant Cry1Ac-RB protein in chloroplast was established through confocal laser scanning microscopy.

Linkage disequilibrium and association mapping of drought tolerance in cotton (Gossypium hirsutum L.) germplasm population from diverse regions of Pakistan

Drought stress is a major abiotic stress that limits crop production. Molecular association mapping techniques through linkage disequilibrium (LD) can be effectively used to tag genomic regions involved in drought stress tolerance. With the association mapping approach, 90 genotypes of cotton Gossypium hirsutum , from diverse regions of Pakistan were used. The morpho-physiological traits of all genotypes were evaluated in greenhouse under well-watered and drought stress conditions. Mean squares from analysis of variance for all morpho-physiological traits revealed highly significant variations (P≤0.05) between water levels and genotypes. Cotton varieties were screened for polymorphism with 180 simple sequence repeat (SSR) markers. Out of these 180 SSR markers, 95 were polymorphic. Genotyping of the selected 95 SSR primer pairs generated 57.5% polymorphism, and the number of polymorphic alleles per primer was 2.10. Population structure, linkage disequilibrium, and association mapping between pairs of SSR marker loci were studied. The significance of pairwise LD (P≤0.005) among all possible SSR loci was evaluated at significant threshold values (R 2 ≥0.05); 7.1% of the SSR marker pairs showed significant pairwise LD in 90 accessions of G. hirsutum . Also we observed a significant (R 2 ) LD between 13 pairs of SSR loci; each pair within the same chromosome in a range of 180 cM between NAU1230 and NAU3095 loci in chromosome (D5) and 1.612 cM between NAU462 and NAU3414 in chromosome A9. This indicates tight linkage between two alleles on the same chromosome. Markers, NAU3414, NAU2691, NAU1141 and NAU1190 were associated with more than single traits under drought treatments. Highest phenotypic variance explaining (R 2 ) was ascribed to NAU3011 chromosome D13 significantly (p ≤ 0.001) associated with root length under drought treatment. Keywords: Cotton Gossypium hirsutum , drought stress, association mapping, linkage disequilibrium (LD), simple sequence repeats (SSRs)

Identifying salt stress-responsive transcripts from Roselle ( Hibiscus sabdariffa L.) roots by differential display

No previous study has been reported on the salt-modulated gene(s) of roselle ( Hibiscus sabdariffa L.). Identifying the potentially novel transcripts responsible for salt stress tolerance in roselle will increase knowledge of the molecular mechanism underlying salt stress responses. In this study, differential display reverse transcriptase PCR (DDRT-PCR) was used to compare the overall differences in gene expression between salt-stressed and control plants. A total of 81 primer combinations were used and false positive clones were rejected during a screening and quality control assay. The remaining nine cDNA transcript fragments were extracted from the gel, reamplified, cloned and sequenced. A homology search revealed that four transcripts showed significant homology with known genes. Out of five transcripts, real-time PCR demonstrated that four exhibited high expression in salt-stressed root tissues relative to the control and one transcript was down-regulated. These transcripts may be useful for improving tolerance in salt stress-sensitive plants. Keywords: Roselle, Hibiscus Sabdariffa L., differential display, salt-stress, differentially expressed transcripts, signal transduction. African Journal of Biotechnology , Vol 13(53) 4775-4781