Asif Ali Khan

Assessment of salinity tolerance based upon seedling root growth response functions in maize (Zea mays L.)

Root growth response of 10-days-oldseedlings of 100 maize accessions at, 0 mM, 60 mM, 80 mM and 150 mM NaCl concentration was assessed in solution culture. The non-linear least square method was used to quantify the salt tolerance of maize accessions. The estimated salinity threshold, Ct, the NaCl concentration at which root growth starts to decrease, C0, and C50, the concentrations at which roots stop growing and 50% of its control value revealed considerable differences between the accessions. No general consistency for tolerance was, however, found between the estimates of Ct and C50. Different genetic systems appeared to be involved in controlling the inheritance of Ct and C50.Both Ct and C50 appeared to quantify accession tolerance, and the expression of root growth as a function of NaCl concentrations provides a useful guideline for salt tolerance. Estimates of broad sense heritability for relative root length were moderate in size (0.62 to 0.82), suggesting the scope for enhancing salt tolerance in maize through selection and breeding.

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.

Uncovering the salt response of soybean by unraveling its wild and cultivated functional genomes using tag sequencing.

Soil salinity has very adverse effects on growth and yield of crop plants. Several salt tolerant wild accessions and cultivars are reported in soybean. Functional genomes of salt tolerant Glycine soja and a salt sensitive genotype of Glycine max were investigated to understand the mechanism of salt tolerance in soybean. For this purpose, four libraries were constructed for Tag sequencing on Illumina platform. We identify around 490 salt responsive genes which included a number of transcription factors, signaling proteins, translation factors and structural genes like transporters, multidrug resistance proteins, antiporters, chaperons, aquaporins etc. The gene expression levels and ratio of up/down-regulated genes was greater in tolerant plants. Translation related genes remained stable or showed slightly higher expression in tolerant plants under salinity stress. Further analyses of sequenced data and the annotations for gene ontology and pathways indicated that soybean adapts to salt stress through ABA biosynthesis and regulation of translation and signal transduction of structural genes. Manipulation of these pathways may mitigate the effect of salt stress thus enhancing salt tolerance.

Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses

The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV) and cotton leaf curl Kokhran virus (CLCuKoV), led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP) gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4) and CLCuKoV-CP gene (2.706X10-4), whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent.

Molecular Markers and Cotton Genetic Improvement: Current Status and Future Prospects

Narrow genetic base and complex allotetraploid genome of cotton (Gossypium hirsutum L.) is stimulating efforts to avail required polymorphism for marker based breeding. The availability of draft genome sequence of G. raimondii and G. arboreum and next generation sequencing (NGS) technologies facilitated the development of high-throughput marker technologies in cotton. The concepts of genetic diversity, QTL mapping, and marker assisted selection (MAS) are evolving into more efficient concepts of linkage disequilibrium, association mapping, and genomic selection, respectively. The objective of the current review is to analyze the pace of evolution in the molecular marker technologies in cotton during the last ten years into the following four areas: (i) comparative analysis of low- and high-throughput marker technologies available in cotton, (ii) genetic diversity in the available wild and improved gene pools of cotton, (iii) identification of the genomic regions within cotton genome underlying economic traits, and (iv) marker based selection methodologies. Moreover, the applications of marker technologies to enhance the breeding efficiency in cotton are also summarized. Aforementioned genomic technologies and the integration of several other omics resources are expected to enhance the cotton productivity and meet the global fiber quantity and quality demands.

Effects of Salinity on Growth, Ionic Relations and Solute Content of Sorghum Bicolor (L.) Monench

ABSTRACT The effects of increasing levels of calcium (Ca2+), potassium (K+), and sodium chloride (NaCl) salinity on the growth and ionic relations of Sorghum bicolor (L.) Monench, cv. ‘ICI-5521’ seedlings are reported. Increasing levels of Ca2+ in the solution culture enhanced growth, lowered sodium (Na+) uptake, and increased K+:Na+ ratio significantly. Elevated K+ level also improved growth significantly, but had no significant effect on Na+, Ca2+, or magnesium (Mg2+) accumulation of either roots or shoots. Accumulation of K+ and consequently K+:Na+ ratio increased with external K+ concentration. The effects of sodium chloride (NaCl) versus potassium chloride (KCl) salinity on the growth and ionic relations of sorghum (‘ICI-5521’) were also examined. Growth inhibition was greatest when KCl alone (160 mM) was used, but was enhanced when a mixture of NaCl and KCl (15:1) was used. The influence of salinity on the accumulation of proline, amino acids and carbohydrates and total osmolality is also discussed.

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.

Comprehensive screening and selection of okra (Abelmoschus esculentus) germplasm for salinity tolerance at the seedling stage and during plant ontogeny

The okra germplasm was screened for salinity tolerance at the seedling stage and during plant ontogeny. Substantial variation existed in okra for salinity tolerance at the seedling stage. An 80 mmol/L NaCl concentration was suitable for discriminating tolerant and non-tolerant okra genotypes. The pooled ranking of the genotypes, based on individual rankings for each trait (root and shoot length, germination percentage, and relative Na+ and K+) in individual NaCl concentrations, was effective for selecting tolerant genotypes. Genotypes selected at the seedling stage maintained their tolerance to NaCl during plant ontogeny, suggesting that screening of the germplasm entries and advanced breeding materials for salt tolerance at the seedling stage is effective. Among 39 okra genotypes, five were identified as the most tolerant genotypes and showed potential for use in breeding programs that focus on the development of salt-tolerant, high-yield okra cultivars.

Microsatellite Analysis of Rosa damascena from Pakistan and Iran

For the perfume industry, Rosa damascena is the most important species used in the production of rose attar which is made by distilling volatile oils from the petals of flowers. It is also used widely in the manufacture of rose water, as a flavoring agent. Other species like Rosa gallica L., Rosa centifolia L., Rosa × borboniana Desp. and the rose cultivar Gruss an Teplitz also exhibit the fragrance that is sought by perfumeries in the world. The main rose oil producers in the world are Turkey and Bulgaria, and they are obtaining the rose oil almost exclusively from the single clone of R. damascena. In the recent studies a wide genetic diversity for R. damascena has been revealed in Iran, but there was a need to explore and evaluate the R. damascena in the neighboring areas of Iran. Thus the new research project was planned with the objective to evaluate the genetic diversity amongst R. damascena landraces grown in Pakistan and the subsequent comparison with tested germplasm grown in Iran. Further, the study is based on the collection of Damask and selected garden roses from Pakistan, Iran and USA, determining the characterization through microsatellite markers. The SSR markers confirmed the high level of diversity of the Rosa damascena germplasm within Iran and showed that the Pakistani genotypes were similar to those from the Iranian provinces of Isfahan, Kerman and Fars. The garden rose cultivars were distantly related.