Establishment of High Frequency Callus induction and Genetic Transformation in Neglected Leafy Vegetable Amaranthus trisis

In the present study, an efficient in vitro callus induction and genetic transformation protocol was optimized for neglected leafy vegetable Amaranthus tristis. In vitro grown plant leaves were used as explants for callus initiation. Six different hormone combinations were tested for callus induction. Of the different hormone combination tested, Murashige and Skoog (MS) medium supplemented with 2, 4-dichlorophenoxyacetic acid and 6-benzylaminopurine (0.5mg/l) showed effective callus proliferation (87%). The frequency of callus induction ranged from 30.3% to 87%. Even though different hormone combinations were tried, the proliferated callus failed to differentiate into shoots. After 5th subculture, the callus was turned brown and dried. For genetic transformation, the plasmid pCAMBIA1301 harboring the GUS gene was transformed into the leaf explants by Agrobacterium mediated method. The presence of GUS gene in the putative transformed callus was confirmed by Polymerase Chain Reaction (PCR) and GUS histochemical staining. In PCR, an expected band of 1.2kb size was observed in all transformed calli confirming the presence of GUS gene. In histochemical staining, the transformed callus develops blue color in the presence of X- Gluc (5- Bromo-4- Chloro-3- Indolyl- Beta-D- Glucuronidase) reagent. Although shoot regeneration was unsuccessful, the present study might helps to produce secondary metabolites from callus suspension cultures in Amaranthus tristis. This study can also prelude for metabolic engineering studies in this neglected vegetable to meet our future nutritional requirements.