enhanced-photosynthesis-water-use-efficiency-transgenic-sugarcane

Enhanced Photosynthesis and Water-Use Efficiency in Transgenic Sugarcane Expressing Arabidopsis <em>DREB1A</em> Under Drought Stress

Drought stress imposes a critical limitation on sugarcane productivity, thereby necessitating the development of stress-resilient cultivars for ...

Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)

Rapid growth in the genetic transformation of plants is the outcome of versatile transformation methods, explant nature, and media regimes. Modern biotechnologists have now a toolkit embraced with different plant transformation methods to generate specific and targeted genetic variation for performance improvement of crop plants. Genetic information are created by proper custom synthesis/amplification of DNA sequences from natural sources, modification during gene cloning, and choice of regulatory sequences and delivered to plants via different plant transformation techniques. Cotton is known by different names like king of fiber crops, white gold, etc., due to its socioeconomic involvement in society livelihood. So cotton is the host of several transgenes delivered for the purpose of trait development of improvement outcompeting its wild counterparts. At present most of the cotton adopted by farmers is biotech and contributes significantly in meeting farmers and industry demands. It is the versatile nature of cotton that it has been subjected to different genetic transformation methods to provide the breeders with an opportunity to develop alien traits or improve the endogenous gene performance that are very difficult or impossible to develop through conventional breeding methods. Landmark achievements were achieved by expanding explant choice such as calli as explants as it reduces the extent of labor, time, and effort and thereby becoming cost-effective cotton transformation. Cotton calli becomes differentiated into embryogenic and non-embryogenic which requires regular screening, keeping in view texture, color, and growth behavior. Here we describe the calli features which are peculiar in nature when used as explants in a novel physical way of cotton transformation with different genes by using embryogenic calli as continuous source of explants. The dawn of genome editing has opened another horizon in transformation research and development enhancing the scope of cotton transformation.

Embryogenic Calli Explants and Silicon Carbide Whisker-Mediated Transformation of Cotton (Gossypium hirsutum L.).

Biosafety is a multidisciplinary approach that encompasses social, societal, ethical issues and policies for the regulations of genetically modified (GM) organisms. The potential health risks associated with GM sugarcane containing AVP1 gene confers resistance against drought and salinity were evaluated by animal feeding studies and some genotoxicity assays. Acute and sub-chronic toxicity examinations were carried out via oral dose administration of GM sugarcane juice supplemented with the normal diet (modified from certified rodent standard diet) on Wistar rats. AVP1 protein concentration in sugarcane juice was 1mg/1 mL. Biochemical, haematological blood analyses were performed and the results revealed that there were non-significant differences among all the treatment groups; GM sugarcane juice, non-GM sugarcane juice and the control group (normal diet and water). Genotoxicity assessment based on the comet assay and the micronucleus assay data exhibited that AVP1 GM sugarcane was not genotoxic or cytotoxic in rat's peripheral blood. These research findings supported the conclusion that GM AVP1 sugarcane was non-toxic in experimental animals. Therefore, data generated through this research work would be helpful for the commercial release of GM AVP1 sugarcane.

Risk assessment of genetically modified sugarcane expressing AVP1 gene.

Cotton is an international agricultural commodity and the main cash crop of Pakistan of which quality and quantity are subject to various whims of nature. Climate change, insect pest complex, and weeds are reducing its productivity. Here, we have developed triple gene cotton containing EPSPS gene along with two Bt toxin genes Cry1Ac and Cry2Ab using a strategy where all three genes are cloned in the same T-DNA, followed by successful cotton transformation via Agrobacterium-mediated transformation. This strategy has been developed to help cotton breeders in developing new cultivars by incorporating these genes into the non-transgenic or single Bt (Cry1Ac) gene cotton background where all three genes will inherit together. The expression of all three proteins was confirmed through immunostrips and was quantified through enzyme-linked immunosorbent assay (ELISA). The spatio-temporal expression of Bt protein in different parts of triple gene NIBGE cotton plants was determined. Maximum expression was found in leaves followed by seeds and boll rinds. Insect bioassays with cotton bollworms (Helicoverpa armigera), armyworms (Spodoptera litura), and pink bollworms (Pectinophora gossypiella) showed more than 90% mortality. The best performing line (NIBGE-E2) on the basis of spatiotemporal expression, glyphosate assays, and insect mortality data, was used for event characterization by using the genome sequencing approach. The event was successfully characterized and named NIBGE 20-01. A diagnostics test based on event-specific PCR was developed and its ability to distinguish NIBGE 20-01 event from other commercial transgenic cotton events was confirmed. To confirm stable expression of all three proteins in the field conditions, homozygous transgenic lines were grown in the field and the expression was confirmed through immunostrip assays. It was found that all three genes are expressed under field conditions. To show that all three genes are inherited together upon crossing with local elite cotton lines, the F generation was grown under glasshouse and field conditions. The expression of all three genes was confirmed under field conditions. Our results showed that transgenic cotton with three genes cloned in the same T-DNA can express all genes and can be conveniently transferred into elite cotton lines through a single cross.

Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance.

Cotton (Gossypium hirsutum L.) is of great economic importance as a cultivated crop in many parts of the world. In addition to being a pillar of the textile industry, cotton and its byproducts are used for livestock feed, seed oil, and other products. Bacillus thuringiensis crystal toxin (Bt) expression in cotton provides effective protection against chewing insects but does not defend plants from piercing/sucking insect pests. With the aim to create transgenic plants with resistance against piercing/sucking pests, we used Agrobacterium-mediated genetic transformation of cotton cultivar Coker 312 to express the Allium sativum leaf agglutinin (ASLA) gene from the phloem-specific rolC promoter. The ASLA transgene was stably inherited and showed Mendelian segregation in the T1 generation. Transgenic lines, expressing the ASLA gene, showed explicit resistance against major sap-sucking pests. Green peach aphid (Myzus persicae Sulzer) choice assays showed that 75% of aphids preferred untransformed cotton plants relative to those expressing the ASLA gene. In detached leaf bioassays, plants expressing ASLA caused 82% aphid mortality and 44-53% reduction in fecundity. Clip cage bioassays with whiteflies (Bemisia tabaci Gennadius) showed 74-82% mortality and 44-60% decrease in fecundity due to ASLA gene expression. In whole plant bioassays, whiteflies showed 77% mortality and a 54% decrease in fecundity on ASLA transgenics. Importantly, we did not observe a negative effect of the ASLA gene on ladybugs (Coccinella septempunctata) that consumed these whiteflies. Together, our findings demonstrate the potential of ASLA-transgenic cotton for providing protection against two devastating insect pests, whiteflies and aphids. The ASLA-transgenic cotton appears promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.

Article	Year
Embryogenic Calli Explants and Silicon Carbide Whisker-Mediated Transformation of Cotton (Gossypium hirsutum L.). Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30543063	2019
Risk assessment of genetically modified sugarcane expressing AVP1 gene. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association\| PubMed ID: 31132391	2019
Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance. Scientific reports\| PubMed ID: 36319662	2022
Transgenic cotton expressing Allium sativum leaf agglutinin exhibits resistance to whiteflies and aphids without negative effects on ladybugs. Gene\| PubMed ID: 39255858	2025

Shaheen Asad

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