Genetic transformation of Lentil (Lens culinaris M.) and production of transgenic fertile plants

Introduction Lentil is an important pulse crop in Iran. Lentil is a seed propagating, self-pollinating crop originating from Near East. It is containing 27.5% -31.75% protein and it has high level of iron, calcium, phosphor, thiamin, niacin, and some unusual amino acid as hydroxyarnitin, hydroxyarginin and homo arginin. FAOSTAT reported that the world production of lentils was 4,975,621 metric tons in 2013. Major producing countries are India, Australia, Canada and Turkey. According to Iranian agricultural statistic (2014), area of pulses cultivation estimated 770000 hectares and its 20.2% belong to Lentil. Important abiotic stresses that affect lentil are cold, freezing, drought, heat and salinity. Highland and cold temperatures areas are the main producer in Iran and production of lentil in these areasis limited. So we need tolerant cultivars against cold and freezing stresses for sowing during autumn.Glycine betaine (GB) accumulates in some plants under abiotic stress such as cold. GB stabilizes the structures and activities of enzymes and protein complexes and maintains the integrity of membranes against the damaging effects of excessive salt, cold, heat and freezing. The codA gene from Arthrobacter globiformis bacteria encodes choline oxidase enzyme that catalyzes synthesis of glycine betaine from choline. The efficiency of biotechnology techniques, especially tissue culture and gene transformation is noticeable in this aspect. The objective of this research is gene transformation of lentil using codA gene to enhance its tolerance against abiotic stress especially cold tolerance.
Materials and MethodsSeeds of lentil (Lens culinaris Medik) variety of Gachsaran (ILL6212) collected from Shiravan Agriculture Research center in Iran were used in this investigation.
Gene transformation of lentil was done using pChlCOD plasmid. This plasmid is containing codA gene.Gene transformation was done by Agrobacterium tumefaciens using Cotyledon with slight part of Embryo Axes explant after shooting for cu-cultivation. Co-cultivation and following selection and regeneration was done according to Zaker Tavallaieet al, (2011) protocol. The plasmid was containing one intron of rice that it eliminated before cu-cultivation. Three weeks after transferring seedling to glasshouse DNA was extracted from leaves samples using modified CTAB protocol. Existence of codA gene was investigated by PCR reaction using codA-4 and codA-5 primers. Putative transgenic plants were verified by dot blotting also. To determine integration of the gene into transgenic lentil southern blotting also was done. RT-PCR after RNA extraction from leaves sample from mature plants was done to investigate transcription of codA gene. We collected some seeds from putative transgenic plants and after cultivation in glasshouse T1 plants were obtained. PCR, RT-PCR experiments on investigate existence of codA gene and dot blotting was done on T1 transgenic plant.
Results and DiscussionThe existence of codA gene into T0 putative transgenic plants was confirmed by PCR and dot blotting.Integration of codA gene into genome was revealed by Southern blotting and transcription of the gene confirmed by RT-PCR assay, respectively. The existence of gene into T1 plants and its transcription was also confirmed using PCR and RT-PCR. Considering the availability transgenic seeds, it is possible to perform further bioassay experiments regarding to enhancement of tolerance of transgenic plants against abiotic stress especially cold stress.
ConclusionGene transformation of lentil by Agrobacterium using codA gene was achieved successfully. After co-cultivation, regeneration of transgenic shoots including shoot induction, shoot elongation, root induction, hardening and growth in glasshouse to rich to mature plants was done successfully according to Zaker Tavallaie et al, (2011) protocol. Hygromycine was used as selection agent in selection media. T0 transgenic plants verified using PCR, Dot blotting, Southern blotting and RT-PCR. T1 transgenic plants also confirmed by PCR and RT-PCR. It is necessary to achieve all bio assays about tolerance of transgenic plants against abiotic stress. Also we will investigate inheritance of treat in following generation of T1 plants. Also we will do other necessary investigation such as biosafety experiments. We hope the transgenic plants will be tolerant against abiotic stresses such as cold and freezing.