జర్నల్ ఆఫ్ ప్లాంట్ పాథాలజీ & మైక్రోబయాలజీ

నైరూప్య

Gene Transformation of Alfalfa Plants (Medicago varia L.) Domestic Cultivar ‘Burgaltai’ for Increasing Leaf Size

Uuganzaya Myagmarjav*, Enkhchimeg Vanjildorj, Altantsetseg Khajidsuren

‘Burgaltai’ cultivar of alfalfa is the most important forage in Mongolia and its yield had decreasing last decades due to cross pollination. This study was conducted for increasing leaf size of ‘Burgaltai’ cultivar through Agrobacterium mediated genetic transformation transferring growth regulating factor AtGRF2 gene of Arabidopsis thaliana. In vitro regeneration was derived from seven day old hypocotyl and cotyledon explants for callus induction. Induced calli were used for shoot induction and proliferation. Proliferated shoots carried to root induction medium. The best calli induction medium was MSB5 (Murashige Skoog’s medium with Gamborg’s vitamin) medium supplemented with 2 mg/L 2,4-D, 0.2 mg/L BAP and 1 mg/L NAA, shoot induction and proliferation media from calli were MSB5 medium supplemented with 1mg/L BAP, 0.1 mg/L NAA, proliferated shoots were rooted on half strength MSB5 medium supplemented with 0.5 mg/L NAA. For the genetic transformation, hypocotyl explants used as an initial source. Totally 235 explants were tested to transformation and 30 explants (12.7%) were resistant to hygromycin antibiotics and regenerate plantlets. Plantlets were transferred to the soil and fresh young leaf used for GUS histochemical assay and RT-PCR for confirmation. Among them fully regenerated seven independent transgenic plantlets (T6, 8, 11, 12, 13, 15, 22) were confirmed. Transgenic ‘Burgaltai’ cv. of alfalfa leaf size was increased by 0.7 cm² comparing to the wild type. T6 and 11 lines showed 3:1 segregation ratio in T1 progenies (X2=0.04-0.42, p ≤ 0.52-0.84).