In-vitro Production of Cabbage and Cauliflower

Background: An efficient method of artificial mass propagation was optimized for two very nutritious vegetables of Pakistan, the cabbage and cauliflower. Being an agrarian economy, Pakistan’s more than half of the population depends directly or indirectly on agricultural products.Methodology: Hypocotyls of germinating of seeds (5-7 days old seedlings) were used as explants. Murashige and Skoog basal medium was supplemented with different concentrations of auxin (2,4 Dichlorophenoxy acetic  acid) and cytokinin (benzyl amino purine) in combination to study the callus forming tendency of cabbage and cauliflower; and found better if used in ratio of 2:1 (at least) respectively.Results: For evaluation of regeneration potential Kinetin, Zeatin, Gibrellic acid-3, and Indole acetic acid were found good in combination with benzyl amino purine. Higher (than auxin) concentration of cytokinin was found essential to obtain good regeneration response of callus.Conclusion:  Effectively concluded that these techniques can be used to raise the disease free stock of cabbage and cauliflower for genetic improvement of in-vitro bulk of varieties on commercial scale in a very brief time span

Molecular characterization, cloning and sequencing of coat protein gene of a Pakistani potato leaf roll virus isolate and its phylogenetic analysis

Total RNA was extracted from potato leaf roll virus (PLRV) positive potato plants and complementary DNA were synthesized. Reverse transcriptase polymerase chain reaction (RT-PCR) based detection conditions were optimized by using coat protein (CP) gene specific primers. A 346 bp amplicon of PLRV- coat protein (CP) gene was amplified. Amplified CP-gene of PLRV was cloned in TA cloning vector, PCR ® 2.1. The clone was confirmed subsequently through restriction digestion analysis. PCR amplification used cDNA clone as a template and nucleotide sequencing. Expected nucleotide sequence of amplified PLRV-CP gene show homology of 94 to 97% when compared to the sequences already reported in GenBank database. This explored novel PLRV-CP gene was submitted at NCBI GenBank for characterization of PLRV Pakistani isolate (Accession No. JN039286). Phylogenetic analysis was also carried out and tree was made by using MEGA 4.0.Key words: Potato, potato leaf roll virus (PLRV), coat protein (CP) gene, reverse transcriptase polymerase chain reaction (RT-PCR), PLRV-CP Pakistani isolate

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 (R2≥0.05); 7.1% of the SSR marker pairs showed significant pairwise LD in 90 accessions of G. hirsutum. Also we observed a significant (R2) 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 (R2) 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

Tolerance of Roselle (Hibiscus Sabdariffa L.) Genotypes to Drought Stress at Vegetative Stage

Background: Hibiscus sabdariffa L. is an important medicinal and fiber plant in Sudan. Among other stresses, drought extremely limits the growth, quality and net yield of the crop. The drought effects the crop plants by imposing certain morphological, physiological and biochemical changes at different periods of growth.Methods: Current study was carried out in greenhouse settings at Center of Excellence in Molecular Biology (CEMB) to investigate the effects of drought stress. Five (5) different genotypes of Hibiscus Sabdariffa L., namely Baladimostadir (H1), Um shiak (H2), Abu shankal (H3), Rahad mix (H4) and Abu Najma (H5) were studied. Thirty (30) days old Roselle seedlings were drought stressed for 10 days and its implications on plant growth, gas exchange, water relation, chlorophyll content and proline accumulation were estimated. Substantial genotypic differences in their adaptive response to drought were observed.Results: Drought stress significantly affected the plant height; lowered the relative gas exchange efficiency and altered the physiological and biochemical responses.  In comparison with others, H2 and H4 genotypes tolerated the osmotic stress well with lower osmotic potential and higher osmotic adjustment, better water content, higher stomatal conductance, photosynthetic efficiency and chlorophyll content. Accumulation of osmoprotectant and gas exchange indicators clearly distinguished the responses of different genotypes towards water stress.Conclusion: Our results can be used for evaluation, screening, and manipulations of Hibiscus Sabdariffa L. genotypes for improvement of drought tolerance through conventional breeding or drought responsive gene isolation

Punjabi population data for seven X-chromosome short tandem repeat (X-STR) loci using a new miniplex system

Short tandem repeat (STR) markers are extensively being used for human identification as well as paternity and forensic case work. X Chromosome STR (X-STR) markers are a powerful complementary system especially in deficiency paternity testing. This study presents development of a new Xchromosomal STR multiplex kit providing short-amplicon (<200 bp) fragments. About 200 samples from Punjabi population were typed for seven X-STR markers: DXS101, DXS6789, DXS7132, DXS7423, DXS8378, GATA172D05 and GATA31E08. A total of 51 alleles were detected in the range of five to 10 alleles for each marker. The data can be used as reference database for Punjabi population along with the current battery of autosomal STR for forensic case work to increase the discrimination capacity and strengthen the existing system.Keywords: X-chromosome short tandem repeats (X-STRs), mini-STRs, Punjabi populatio

Viability assessment of in vitro produced synthetic seeds of cucumber

Friable, embryogenic calli of F1 cucumber (Cucumis sativus) cultivar, Royal, were induced from the hypocotyl pieces cultured on solidified MS-basal media supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyl amino purine (BAP). Embryogenic calli were transferred to liquid Murashige and Skoog (MS)-basal  media supplemented with 5 ƒÊM naphthaleneacetic acid (NAA) and 1 µM BAP. The mature somatic embryos  were encapsulated in sodium alginate mixture in synthetic seeds. The encapsulation mixture containing 3%  sodium alginate, 100 mM calcium chloride and one-fourth volume of the cell suspension nutrient mixture  containing 5x10-4 somatic embryos per ml was found the best. Synthetic seeds remain viable up to 14 weeks  when stored at 4°C. Germination efficiency of synthetic seeds was decreased to 57% after 10 weeks of  storage followed by rapid decrease in survival rate to 0% after 15 weeks. Genetic diversity between mother  plants and in vitro produced synthetic seeds showed resemblance as assessed by amplified fragment length polymorphism (AFLP) markers.Key words: Artificial seed, Cucumis sativus, encapsulation, somatic embryogenesis, sodium-calcium alginate

Evaluation of two cotton varieties CRSP1 and CRSP2 for genetic transformation efficiency, expression of transgenes Cry1Ac+Cry2A, GT gene and insect mortality

AbstractExpression of the transgene with a desirable character in crop plant is the ultimate goal of transgenic research. Transformation of two Bt genes namely Cry1Ac and Cry2A cloned as separate cassette under 35S promoter in pKHG4 plant expression vector was done by using shoot apex cut method of Agrobacterium. Molecular confirmation of putative transgenic cotton plants for Cry1Ac, Cry2A and GT gene was done through PCR and ELISA. Transformation efficiency of CRSP-1 and CRSP-2 was calculated to be 1.2 and 0.8% for Cry1Ac while 0.9 and 0.6% for Cry2A and 1.5 and 0.7% for GTG respectively. CRSP-1 was found to adopt natural environment (acclimatized) earlier than CRSP-2 when exposed to sunlight for one month. Expression of Cry1Ac, Cry2A and GTG was found to be 1.2, 1 and 1.3ng/μl respectively for CRSP-1 as compared to CRSP-2 where expression was recorded to be 0.9, 0.5 and 0.9ng/μl respectively. FISH analysis of the transgenic CRSP-1 and CRSP-2 demonstrated the presence of one and two copy numbers respectively. Similarly, the response of CRSP-1 against Glyphosate @1900ml/acre was far better with almost negligible necrotic spot and efficient growth after spray as compared to CRSP-2 where some plants were found to have necrosis and negative control where the complete decay of plant was observed after seven days of spray assay. Similarly, almost 100% mortality of 2nd instar larvae of Heliothis armigera was recorded after three days in CRSP-1 as compared CRSP-2 where insect mortality was found to be less than 90%. Quantitatively speaking non transgenic plants were found with 23–90% leaf damage by insect, while CRSP-1 was with less than 5% and CRSP-2 with 17%. Taken together CRSP1 was found to have better insect control and weedicide resistance along with its natural ability of genetic modification and can be employed by the valuable farmers for better insect control and simultaneously for better production

Genetic effects of Calotropis procera CpTIP1 gene on fiber quality in cotton (Gossypium hirsutum)

Background: The importance of cotton crop (Gossypium hirsutum) in textile industry is based on its fiber quality. A number of fiber-specific genes play important role in the development of cotton fiber. Previous studies have demonstrated the importance of genes that are responsible for metabolic functions and their involvement in cotton fiber development.Methods: This study was focused at successful Agrobacterium mediated stable transformation of the fiber gene CpTIP1, isolated from the wild plant Calotropis procera, into cotton variety NIAB-846 for one generation. Results: Transformation efficiency was calculated to be 1.01% for the target gene. Different molecular techniques such as PCR were used for confirmation and Real-Time PCR was used to check the level of quantitative expression of fiber expansin gene in putative transgenic cotton plants. On the base of molecular analysis, results showed higher expression level of fiber gene (CpTIP1) in transgenic plants as compared to the control plants.Conclusion: The results of this study support the idea of improved cotton fiber through genetic modification especially the cotton fiber strength

Defense strategies of cotton against whitefly transmitted CLCuV and Begomoviruses

Cotton leaf curl virus (CLCuV) is economically important monopartite Geminivirus which is transmitted by whitefly in persistent circulative manner. In Pakistan, CLCuV causes severe damage to Gossypium hirsutum whereas G. arboreum is resistant to this virus. A total of 86 articles were included in this study that were searched through web of knowledge, web of science and google scholar by using the keywords of cotton, CLCuV, waxes, Begomoviruses and transgenic techniques used against Begomoviruses in plants. Various transgenic strategies i.e., pathogen derived resistance (with and without protein expression) and non - pathogen derived resistance have been adopted to control this virus or its vector. Beside these, some natural defense mechanisms of plant also protect it against the vector. The cuticular waxes make the insects’ attachment difficult to plant surfaces and act as a physical barrier. The cuticular waxes in G. arboreum act as first line of defense against whiteflies and thus CLCuV. Some other defense strategies may involve hindering the insect movements or depriving it from food due to thick waxy layer. Biotechnological strategies against various Begomoviruses including CLCuV were found successful in some crops except cotton. Whereas, the natural defense strategies in G. arboreum i.e., long trichome or presence of inorganic salts with increased concentration of waxes, provide good defense strategy against whiteflies, CLCuV and other pathogens