Development of an intragenic gene transfer and selection protocol for sugarcane resulting in resistance to acetolactate synthase-inhibiting herbicide (original) (raw)
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In Vitro Cellular & Developmental Biology - Plant, 2013
Selection genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells. In order to find alternatives to the use of antibiotics as selection agents, we followed a targeted approach utilizing a plant gene, encoding a mutant form of the enzyme acetolactate synthase, to convey resistance to herbicides. The sensitivity of sugarcane callus (Saccharum spp. hybrids, cv. NCo310) to a number of herbicides from the sulfonylurea and imidazolinone classes was tested. Callus growth was most affected by sulfonylurea herbicides, particularly 3.6 μg/l chlorsulfuron. Herbicide-resistant transgenic sugarcane plants containing mutant forms of a tobacco acetolactate synthase (als) gene were obtained following biolistic transformation. Post-bombardment, putative transgenic callus was selectively proliferated on MS medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose, 0.5 g/l casein, and 3.6 μg/l chlorsulfuron. Plant regeneration and rooting was done on MS medium lacking 2,4-D under similar selection conditions. Thirty vigorously growing putative transgenic plants were successfully ex vitro-acclimatized and established under glasshouse conditions. Glasshouse spraying of putative transgenic plants with 100 mg/l chlorsulfuron dramatically decreased the amount of non-transgenic plants that had escaped the in vitro selection regime. PCR analysis showed that six surviving plants were als-positive and that five of these expressed the mutant als gene. This report is the first to describe a selection system for sugarcane transformation that uses a selectable marker gene of plant origin targeted by a sulfonylurea herbicide.
Establishment of alternative selection systems for transgenic sugarcane callus
Selection-marker genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells after transformation. In order to find alternatives to the use of antibiotics as selection agents we focused on so-called positive selection systems. Four alternative systems were investigated for their potential as selection agents in sugarcane genetic transformation. The systems included i) galactose, ii) 2-deoxyglucose iii) NaCl and iv) gluconic acid as selective agents in combination with the galactose--phoshate uridyl-transferase (galT), 2-deoxyglucose-6-phosphate-phosphatase (2Dog6PP), "cytosolic" betaine aldehyde dehydrogenase (BADH), and gluconokinase selection genes, respectively. Control sugarcane callus sensitivity towards these selection agents were determined and resulted in gluconic acid being eliminated as a possible selection agent. Transgenic calli were produced for the remaining three selection systems and results monitored. Galactose and 2-deoxyglucose showed the most promise as alternative selection agents in the production of transgenic sugarcane callus.
Sugarcane commercial variety RA 87-3 was transformed with a genetic construct harboring the epsps gene from Agrobacterium strain CP4 conferring tolerance to glyphosate and nptII gene for kanamycin selection. Transformed lines were multiplied in greenhouse, and herbicide tolerance was evaluated using different concentrations (3, 4, 8 and 16 l/ha) of glyphosate (Helm 48 % p/v). All herbicide-tolerant (HT) lines were field tested to confirm glyphosate tolerance and perform preliminary evaluations of phenotypic resemblance to parental cultivar. All transformed lines maintained herbicide tolerance, but many showed phenotypic changes and/or growth aberrations. Ten HT lines, showing close growth resemblance to RA 87-3, were analyzed using nine compulsory morphologic markers proposed by the International Union for the Protection of New Varieties of Plants (UPOV) and 339 molecular markers. Out of the ten HT lines tested, six showed minor morphologic and genetic variations and were selected for field testing over two vegetative crop cycles (plant cane and first ratoon) at two production areas in Argentina. The six field-tested HT lines were found to be almost indistinguishable when comparing agro-nomic and industrial characteristics and chemical composition. Stable heritance of the CP4 epsps gene and glyphosate tolerance throughout different clonal generations were confirmed by RT-qPCR and Southern blot. Taking into account all results, two out of the six lines tested were selected for a possible commercial release. Our study confirms the utility of genetic transformation as a complementary tool to classical breeding procedures and highlights the usefulness of UPOV traits together with molecular markers for early selections of transgenic events that closely resemble their parental genotype.
BIOTECHNOLOGICAL APPROACHES FOR HIGH SUGARCANE YIELD
2011
In sugarcane, biotechnological applications are yielding encouraging results in the areas of genome characterization, mapping for specific traits, molecular variability of pathogens, marker aided selection for insect/disease resistance, transformation, precise detection of plant pathogens etc. Increased sensitivity has generally made the assays for pathogens more reliable and contributes in making the assays more rapid and economical for routine detection and identification. Tissue culture is playing an important role in improving the plant quality and producing pathogen free plants. This can be used to produce clones of phytoplasma infected plants that are healthy. For virus elimination the micropropagation using the shoot apical meristem as the explants is the best way to produce virus free plants. Research efforts need to be made to minimize somaclonal variation for use in clonal propagation and transgenic research. New information on the molecular biology and genetics of selected tropical crop plants need to be generated. Genomics structure should be worked out to identify markers linked to important agronomics traits. Transgene constructs that mitigate potential environmental and biosafety risks of transgenic plants need to be developed so that it can be managed. Regulatory controls over metabolisms need to be identified and characterized to improve yield and quality of sugarcane.
Molecular Plant Breeding, 2012
Success rate in the selection of putative transformants is mainly dependent upon the dose rate of selective agent. Selection pressure determines the survival frequency of transformant and non transformant cells. Dose of selective agent, which is lethal for the non transformants, would be the optimum for the transgenic selection. We investigated the optimum dose of basta (selective agent), in two sugarcane genotypes viz., S-2003-us-359 and S-2003-us-127, in which we have already established a proficient in vitro regeneration system . Different dosages of basta viz., 1 mg/L, 3 mg/L, 5 mg/L, 7 mg/L and 10 mg/L were studied for regeneration media (RM) of both genotypes. For determining the optimal inhibitory dose of basta on regeneration, 21 days old calli of S-2003-us-359 were induced on CIM1, 28 days old calli of S-2003-us-127 were induced on CIM2 and were shifted to RSM1 and RSM2 respectively. In genotype S-2003-us-359, early callus death was observed on RSM having 10 mg/L basta, and RSM having 5 mg/L, 7 mg/L, 10 mg/L basta proved to be lethal for S-2003-us-127 with early death of calli. For genotype S-2003-us-127, among 5 mg/L, 7 mg/L and 10 mg/L basta levels, 5 mg/L basta was selected for regeneration media as optimal dose for the selection of transgenics of this genotype. For transgenic selection in both sugarcane genotypes, the affectivity of selected optimal doses of basta was checked. Twenty one (21) days old calli of S-2003-us-359 and 28 days old calli of S-2003-us-127 were bombarded with bar gene and plant were selected at 10 mg/L and 5 mg/L of basta respectively. Putative transgenic plants with integrated copy of bar gene were confirmed with PCR analysis.
Biotechnological Developments in Sugarcane Improvement: An Overview
Sugarcane (Saccharum officinarum L.) is one of the most important field crops grown in the tropics and sub-tropics. More than half of the world's sugar is derived from sugar cane. Conventional methods have greatly contributed to crop improvement; however limitations such as complex genome, narrow genetic base, poor fertility, susceptibility to biotic and abiotic stresses and long duration to breed elite cultivars still impose a challenge. Sugarcane, thus, is a suitable candidate for application of biotechnology and genetic engineering tools. In this direction, in vitro culture systems and related biotechnologies have been developed as novel strategies for sugarcane improvement. Studies have been conducted towards employing in vitro culture combined with radiation/chemical induced mutagenesis for mutant isolation. Advancements in genomics tools have paved the way for a detailed understanding of the mechanism underlying biotic and abiotic stress responses. The potential of the current genomics programs, aimed at elucidating the structure, function, and interactions of the sugarcane genes, will revolutionize the application of biotechnology to crop improvement. Genetically modified sugarcane with increased resistance to agronomic traits including biotic and abiotic stresses, yield and juice could become useful in breeding for better varieties. This review outlines some of the biotechnological developments that are in place and tailored to address important issues related to sugarcane improvement.
Cytology and Genetics, 2017
⎯The aim of this study was to ensure the systematic protein expression of two genes (GTG and Cry1Ac) under the influence of two different constitutive promoters i.e. Ubiquitin-1 and CaMV 35S promoters in different sugarcane lines. PCR amplification of GTG and Cry1Ac was achieved from putative transgenic plants through gene specific primers. Qualitative comparisons of GTG and Cry1Ac genes expression under two different promoters were obtained through protein dot blot and dipstick assay. The appearance of comparatively dark color dots in dot blot and dark color bands on dipstick with Ubiquitin as compared to light color bands with CaMV35S promoter, qualitatively confirmed high protein expression of two genes under Ubiquitin promoter. In quantitative gene expression comparisons maximum optical density (OD) at 450 nm of UV-light was obtained for GTG (3.7 OD) and Cry1Ac (3 OD) under Ubiquitin promoter, while for GTG (1.6 OD) and Cry1Ac (2.5 OD) with CaMV 35S promoter. The results indicated higher expression of two genes under Ubiquitin-1 promoter in sugarcane was found as compared to CaMV 35S promoter. This study provides a guide for stable and high expression of transgenes with reference to Ubiquitin-1 promoter which can be utilize in sugarcane as well as in other monocots.
⎯The aim of this study was to ensure the systematic protein expression of two genes (GTG and Cry1Ac) under the influence of two different constitutive promoters i.e. Ubiquitin-1 and CaMV 35S promoters in different sugarcane lines. PCR amplification of GTG and Cry1Ac was achieved from putative transgenic plants through gene specific primers. Qualitative comparisons of GTG and Cry1Ac genes expression under two different promoters were obtained through protein dot blot and dipstick assay. The appearance of comparatively dark color dots in dot blot and dark color bands on dipstick with Ubiquitin as compared to light color bands with CaMV35S promoter, qualitatively confirmed high protein expression of two genes under Ubiquitin promoter. In quantitative gene expression comparisons maximum optical density (OD) at 450 nm of UV-light was obtained for GTG (3.7 OD) and Cry1Ac (3 OD) under Ubiquitin promoter, while for GTG (1.6 OD) and Cry1Ac (2.5 OD) with CaMV 35S promoter. The results indicated higher expression of two genes under Ubiquitin-1 promoter in sugarcane was found as compared to CaMV 35S promoter. This study provides a guide for stable and high expression of transgenes with reference to Ubiquitin-1 promoter which can be utilize in sugarcane as well as in other monocots.