Development of a Phosphomannose Isomerase-Based Agrobacterium-Mediated Transformation System for Chickpea (Cicer Arietinum L.) (original) (raw)
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Journal of Plant Biotechnology, 2022
In this study, we developed a reliable and efficient Agrobacterium-mediated genetic transformation system by applying sonication and vacuum infiltration to six chickpea cultivars (ICCV2, ICCV10, ICCV92944, ICCV37, JAKI9218, and JG11) using embryo axis explants. Wounded explants were precultured for 3 days in shoot induction medium (SIM) before sonication and vacuum infiltration with an Agrobacterium suspension and co-cultivated for 3 days in co-cultivation medium containing 100 µM/l of acetosyringone and 200 mg/l of L-cysteine. Responsive explants with putatively transformed shoots were selected using a gradual increase in kanamycin from 25 mg/l to 100 mg/l in selection medium to eliminate escapes. Results showed optimal transformation efficiency at a bacterial density of 1.0, an optical density at 600 nm wavelength (OD 600), and an infection duration of 30 min. The presence and stable integration of the β-glucuronidase (gusA) gene into the chickpea genome were confirmed using GUS histochemical assay and polymerase chain reaction. A high transformation efficiency was achieved among the different factors tested using embryo axis explants of cv. JAKI 9218. Of the six chickpea cultivars tested, JAKI9218 showed the highest transformation efficiency of 8.6%, followed by JG11 (7.2%), ICCV92944 (6.8%), ICCV37 (5.4%), ICCV2 (4.8%), and ICCV10 (4.6%). These findings showed that the Agrobacterium-mediated genetic transformation system will help transfer novel candidate genes into chickpea.
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 (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 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 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 transformation of chickpea using shoot meristem
2009
Agrobacterium-mediated gene transfer to pre-organized meristematic tissue combined with axillary regeneration was standardized for transformation and regeneration of chickpea, which otherwise was difficult to achieve from other explants. Different Agrobacterium strains harbouring binary vectors pCGP1258, containing the GUS as a reporter and bar [gene for resistance to phosphinothricin (PPT)-the active ingredient of the herbicide Basta] as the selectable marker, were used for the transformation experiments. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops (2 mm) were thoroughly wetted with PPT solution (2 mg/mL). The multiple axillary shoots developing from the shoot apices were excised and placed onto a medium containing 10 mg/L PPT. The surviving shoots were subcultured every 2 nd wk onto fresh medium containing 20 mg/L PPT. After each subculture, the number of surviving shoots decreased until it stabilized. Some of the chimeric shoots surviving the PPT selection eventually developed new healthier axillary shoots, which could be rooted or grafted on in vitro grown seedling. This whole process took 6-9 months. Average transformation frequency was found between 1.29-3.33%. Transmission of the transgenes into progeny was also studied following the inheritance of uid A gene in T 1 and T 2 progenies. The overall segregation ratio among progenies of plants derived from T 0 plants appeared to be close to 3:1 Mendelian ratio, indicating integration of the transgene at single locus.
Different selectable marker genes are used in the genetic transformation of plants, but the efficiency of these genes in the selection of transgenic plants is different. In this study, we have used two different selectable marker gene systems in an established chickpea transformation protocol, and have compared the effectiveness of each selection system based on transformation efficiency. Using the herbicide resistance bar gene, together with a low concentration of the selective agent phosphinothricin, results in greater transformation efficiency than using the antibiotic resistance nptII gene with a high concentration of the selective agent kanamycin. In addition, modification of the rooting media and the use of a grafting method further improved the transformation efficiency of both selection systems. The transformation efficiency using the nptII and bar genes as selectable markers was 0.37% and 4.3%, respectively.
A reproducible and efficient transformation method was demonstrated for two Indian desi chickpea (Cicer arietinum L.) cultivars using bar as a selectable marker gene. In order to select the transformants in tissue culture medium, 2.5 mg/l of phosphinothricin was optimized to use as a selective agent in regeneration and selection medium. Cotyledonary explants containing half embryonic axes were infected and co-cultivated with Agrobacterium strain harboring binary plasmid (pBK16.2) containing a chimeric cre (Cyclization recombination) gene of bacteriophage P1 and a bar gene as selectable marker gene. The frequencies of putative transformed plants were 1.23% in cv. 'ICCV 89314' and 1.12% in cv. 'Vijay'. Explant to plant duration was 61-71 days. Putative transformed plants/shoots that were selected on regeneration and selection medium were confirmed by PCR analysis for the presence of transgenes. A " leaf painting assay " using 0.6 mg/ml of phosphinothricin confirmed expression of the bar gene in the putative transformed plants. This protocol generated a slightly higher frequency of putative transformed plants in our laboratory when compared to generation of transformed chickpea plants using nptII as the selection marker gene.