Optimization of different factors for an Agrobacterium-mediated genetic transformation system using embryo axis explants of chickpea (Cicer arietinum L.) (original) (raw)
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ASSESSMENT THE RESPONSE OF CHICKPEA GENOTYPES TO AGROBACTERIUM -MEDIATED TRANSFORMATION SYSTEM
Transformation protocol based on the inoculation of chickpea mature embryos with Agrobacterium suspension was carried out. Four chickpea lines and one Iraqi local variety were used as recipient to the foreign gene of Agrobacterium tumefaciens strain (AgL1). Three plasmids were already inserted in the bacteria cells. The first plasmid carries the bar gene coding for phosphinothricin acetyle transferase (PAT), which confers resistance to the herbicide phosphinothricin or glofosinate ammonium(PPT) and uidA (gusA) gene coding for β-glucuronidase (GUS). The other two plasmids carried the LeEREBP gene which confers drought resistance and bar gene coding for phosphinothricin acetyle transferase (PAT). Successfully regenerated explants were subjected to selection pressure on 10 mg /l of phosphinothricin PPT and the putative transgenic explants were rooted on root induction medium consisting of MS basal medium with B5 medium vitamins supplemented with 2.5 ml of 1mg /ml IBA in addition to grafting on 7 days old non-transformed rootstock. PCR approved transgenic chickpea. 600 mg/l of PPT was used by painting the leaves of surviving plants to detect the expression of bar gene which encodes for phosphinothricin acetyle transferase and confirmed herbicide resistance in transgenic plants.
Agrobacterium- mediated transformation of chickpea using mature embryos
Chickpea is an important food legume, valued for its seeds with high protein content. Its production is bedeviled by major biotic and abiotic stresses. Plant transformation is a relatively new tool developed with potentially huge benefits for plant improvement. In this study, a transformation protocol based on the inoculation of decapitated mature embryos with the Agrobacterium tumefaciens strain AgL0 was used. Two chickpea lines, ILC482 and ICC12004 were transformed by the binary vector pCGP1258 which contains the bar and the gusA genes. Regenerated explants were selected on 10 mg/l of phosphinothricin. The putative transgenic explants were subjected to hormone-free media for rooting or were grafted on 5-day old non-transgenic rootstock. Transgene to chickpea was confirmed by the polymerase chain reaction (PCR). The expression of the bar gene encoding for the enzyme phosphinothricin acetyl transferase and confering resistance to the herbicide phosphinothricin (PPT)was detected by surviving plants after painting the leaves with 600 mg/l PPT. The activity of gus gene encoding for β-glucuronidase was confirmed by histochemical which revealed GUS-positive expression in T 0 and T 1 plants.
Agrobacterium‐mediated Genetic Transformation of Local Cultivars of Chickpea (Cicer arietinum L.)
Agrobacterium‐mediated genetic transformation protocol was established for two chickpea (Cicer arietinum L.) varieties, namely Barichhola‐4 (Bch‐4) and Barichhola‐5 (Bch‐5). Transformation ability of different explants such as decapitated embryo with single cotyledon disc (DEC), decapitated embryo (DE) and slice embryo decapitated at shoot end with single cotyledon disc (SEC) were tested using Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI121, containing the GUS and nptII genes. Maximum transformation ability was exhibited by explants of decapitated embryo (DE) from Barichhola‐5 (Bch‐5). The optimum regeneration from the transformed tissue was achieved on MS supplemented with 0.5 mg/l BAP, 0.5 mg/l Kn and 0.2 mg/l NAA along with double the amount of CaCl2 and KNO3. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin to 150 mg/l. Stable expression of the GUS gene was detected in various parts of the transformed shoots through GUS histochemical assay. Stable integration of nptII gene within the genomic DNA from these transformed shoots was confirmed through PCR analysis.
Agrobacterium – mediated transformation of chickpea (Cicer arietinum L.) mature embryos
Chickpea is an important food legume, valued for its seeds with high protein content. Its production is bedeviled by major biotic and abiotic stresses. Plant transformation is a relatively new tool developed with potentially huge benefits for plant improvement. In this study, a transformation protocol based on the inoculation of decapitated mature embryos with the Agrobacterium tumefaciens strain AgL0 was used. Two chickpea lines, ILC482 and ICC12004 were transformed by the binary vector pCGP1258 which contains the bar and the gusA genes. Regenerated explants were selected on 10 mg/l of phosphinothricin. The putative transgenic explants were subjected to hormone-free media for rooting or were grafted on 5-day old non-transgenic rootstock. Transgene to chickpea was confirmed by the polymerase chain reaction (PCR). The expression of the bar gene encoding for the enzyme phosphinothricin acetyl transferase and confering resistance to the herbicide phosphinothricin (PPT)was detected by surviving plants after painting the leaves with 600 mg/l PPT. The activity of gus gene encoding for β-glucuronidase was confirmed by histochemical which revealed GUS-positive expression in T 0 and T 1 plants.
Agrobacterium mediated transformation of chickpea ( Cicer arietinum L.) embryo axes
Plant Cell Reports, 2000
Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction.
GM Crops & Food, 2019
An efficient genetic transformation system is a prerequisite for studying gene functions, molecular breeding program, and introducing new traits. Agrobacterium tumefaciens-mediated genetic transformation is a widely preferred and accepted method for many plants, including pigeon pea. However, the efficiency of transformation of pigeon pea using the existing protocols is low and time-consuming. In the present study, we developed a rapid and highly efficient transformation system of pigeon pea, using embryonic axis-attached cotyledons as explants. We systematically investigated the influence of varying optical densities of Agrobacterium suspension, duration of incubation, and co-cultivation on the transformation efficiency. In our system, a transformation efficiency of approximately 83% was achieved using Agrobacterium cells at an optical density (OD 600) of 0.25, infection time of 15 min, and co-culturing with explants for 72 h in the light with 100µM acetosyringone. The entire procedure, starting from seed to establishment of transformed plants in soil, was achieved in 35-40 days. This is a rapid and highly efficient protocol for
An effective method of sonication-assisted Agrobacterium-mediated transformation of chickpeas
Plant Cell, Tissue and Organ Culture, 2008
An efficient and reproducible transformation method of sonication-assisted Agrobacteriummediated transformation (SAAT) was developed for chickpea (Cicer arietinum L.). Agrobacterium tumefaciens (LBA4404) harboring pCAMBIA1305.2 was used to transform decapitated embryo explants of two cultivars of chickpeas. By using a series of co-cultivation, callus induction, shoot initiation and root inducing media, a large number of transgenic plants were recovered. Transient expressions of GUS gene were detected by X-Gluc histochemical assay in transformed tissues. DNA analysis of T0 and T1 plants by PCR and Southern hybridization confirmed the integration of transgenes in initial and next generation transformants in different transgenic lines. The transformation efficiency was more than two times higher in SAAT treatment than simple Agrobacterium without sonication.
Journal of Animal & Plant Sciences, 2017
The yield of chickpea in Pakistan is unexpectedly low and unstable that may be due to biotic and abiotic stresses. To combat with these stresses, genetic make of chickpea should be improved incorporating important genes. For successful gene incorporation, an efficient tissue culture system may be established. Therefore, this study was conducted in two local chickpea genotypes namely Bittle-98 and Dasht-2000 for the establishment of an efficient tissue culture system. Among various concentrations of BAP, MS medium fortified with 3.0 mg/l BAP showed the highest multiple shoot formation (69.2 and 70.66%) in cvs Dasht-2000 and Bittle-98, respectively. Similarly, the highest elongation of regenerated shoots (58.33 and 91.66%) was recorded in Bittle-98 and Dasht-2000, respectively on MS media supplemented with 0.25 mg/l IAA. The highest rooting formation (60 and 80%) was recorded in Dasht-2000 and Bittle-98, respectively on MS media having 1.0 mg/l IBA. Half Embryo with single cotyledon was found to be the best explant for genetic transformation. During this study, hygromycin (75 mg/l), acetosyringone (100 µM) and cefotaxime sodium (500 mg/l) were found to be optimum for Agrobacterium-mediated transformation in chickpea. The putative transgenic plants were subjected to GUS and microscopic observations that confirmed the presence of uidA gene in both genotypes. The optimized protocols will provide a base line to improve transformation system and incorporate important genes in chickpea in future research programs.