H. M. N. Cheema

Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L.

Good quality deoxyribonucleic acid (DNA) is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction (PCR) applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950–1050 μg of good quality DNA, free of protein and RNA. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method. The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat (SSR) primers. This modified protocol can also be used to extract DNA from other woody plants having similar problems.

Comparative analysis of resistance gene analogues encoding NBS‐LRR domains in cotton

BACKGROUND Plant production is severely affected by biotic and abiotic stresses R-genes exhibit resistance against a range of diseases and pathogens in plants. The nucleotide binding site and leucine rich repeat (NBS-LRR) class of R-genes is the most comprehensively studied in terms of sequence evolution and genome distribution. The differential response for resistance against biotic and abiotic stress has been observed in cultivated and wild relatives of the genus Gossypium. RESULTS Efforts have been made to address the recent evolution of NBS-LRR sequences within Gossypium hirsutum and resistance gene analogue (RGA) sequences derived from G. arboreum and G. raimondii. The % identity and phylogenetic analysis of NBS-LRR-encoded RGAs from tetraploid New World cotton and its diploid ancestors G. raimondii and G. arboreum suggest that the evolution of NBS-LRR-encoding sequences in G. hirsutum occurred by gradual accumulation of mutants that led to positive selection and a slow rate of divergence within distinct R-gene families. CONCLUSION The allotetraploid genome of cotton, after separating from its diploid parents, experienced polyploidisation, natural and artificial selection, hybrid necrosis, duplication and recombination which became the reason to shed off and evolve new genes for its survival. These driving forces influenced the development of genomic architecture that make it susceptible to diseases and pathogens as compared to donor parents.

Assessment of Bt cotton genotypes for the Cry1Ac transgene and its expression

Genetically modified (GM) plants expressing Bt toxin provide protection against lepidopteran pests. The only GM crop in Pakistan is Bt cotton, which was illegally imported and adopted rapidly by cotton producers. Farmers gained access to the seed of many unapproved Bt genotypes before the matter was picked up and formal approval granted by the relevant governmental agencies. The present study was conducted to evaluate the samples of Bt cotton, collected from farmers and seed dealer, for transgene integration and expression. Seeds of 52 cotton genotypes, labelled as Bt, were collected from various farmers and seed dealers. An immunoblot strip test was carried out, which showed that only 0·86 of the samples collected were synthesizing Cry1Ac toxin. According to multiplexed polymerase chain reaction (PCR) results, 0·86 of the genotypes tested were positive for the Mon531 event (an ‘event’ is a specific genetic modification in a specific species) and 0·14 were negative for any transgene. Transcript analysis of transgenes in positive genotypes by real-time Rt-PCR confirmed the synthesis of mRNA in all genotypes but with significant variation. The concentration of Bt toxin revealed by enzyme linked immunosorbent assay (ELISA) showed that only 0·02 genotypes had the reported optimum level. The real-time PCR and ELISA results further confirmed the attenuation of transgene expression at transcriptional and translational level by various internal and external factors. The same type of event was found in all genotypes, with significant variation in toxin level, revealing the impact of genetic background on transgene expression. The findings support the recommendation to improve the existing quality criteria for transgenic cotton variety approval and certification in Pakistan, with the inclusion of toxin concentration in the list of parameters to be considered.

Identification and characterization of plasma membrane aquaporins isolated from fiber cells of Calotropis procera

Calotropis procera, commonly known as “milkweed”, possesses long seed trichomes for seed dispersal and has the ability to survive under harsh conditions such as drought and salinity. Aquaporins are water channel proteins expressed in all land plants, divided into five subfamilies plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like proteins (NIPs), small basic intrinsic proteins (SIPs), and the unfamiliar X intrinsic proteins (XIPs). PIPs constitute the largest group of water channel proteins that are involved in different developmental and regulatory mechanisms including water permeability, cell elongation, and stomata opening. Aquaporins are also involved in abiotic stress tolerance and cell expansion mechanisms, but their role in seed trichomes (fiber cells) has never been investigated. A large number of clones isolated from C. procera fiber cDNA library showed sequence homology to PIPs. Both expressed sequence tags (ESTs) and real-time polymerase chain reaction (PCR) studies revealed that the transcript abundance of this gene family in fiber cells of C. procera is greater than that of cotton. Full-length cDNAs of CpPIP1 and CpPIP2 were isolated from C. procera fiber cDNA library and used for constructing plant expression vectors under constitutive (2×35S) and trichome-specific (GhLTP3) promoters. Transgenic tobacco plants were developed via Agrobacterium-mediated transformation. The phenotypic characteristics of the plants were observed after confirming the integration of transgene in plants. It was observed that CpPIP2 expression cassette under 2×35S and GhLTP3 promoter enhanced the numbers of stem and leave trichomes. However, 2×35S::CpPIP2 has a more amplified effect on trichome density and length than GhLTP3::CpPIP2 and other PIP constructs. These findings imply the role of C. procera PIP aquaporins in fiber cell elongation. The PIPs-derived cell expansion mechanism may be exploited through transgenic approaches for improvement of fiber staple length in cotton and boosting of defense against sucking insects by enhancing plant pubescence.