Panicum hallii: Tissue Culture & Transformation

Panicum hallii (PAH) is a small perennial C4 grass plant (family Poaceae), and a potential model plant for genetic studies of polyploid panicoid species. PAH’s status as a model plant is largely dependent on tissue culture characteristics (e.g. starting explant material, callus type, ease of callus establishment and maintenance, callus longevity, transformation efficiency, plant regeneration efficiency). Previous studies have indicated that callus can be induced from seeds using an MS-based medium, and the genome of P. hallii filipes has been fully sequenced, although no studies have developed a method for transformation of PAH. Tissue culture of PAH was initiated using root tips, tillers, and seeds as starting explant materials for callus induction. Callus longevity was determined by maintenance on the initial medium with sub-culture at two week intervals. Callus transformation was monitored using two pANIC vectors (pANIC-10A & pANIC-12A) containing a hygromycin resistance gene and a fluorescent reporter gene. Transformation efficiency was determined as the number of hygromycin resistant calli expressing the fluorescent reporter per 100 pieces of callus transformed. Additionally, the regeneration efficiency of calli (number of shoot- and root-developing callus pieces per 100 pieces of callus regenerated) was calculated for wild-type and transformed PAH. Callus induction was most efficient on MS-medium containing L-Proline with 5 µM 2,4-D and 5 µM BAP. We present a comparison of tissue culture and transformation techniques for PAH. Our results demonstrate a method in which >90% explants are able to induce callus and ~50% of the calli can be regenerated into whole plants.