Influence of genotypes, growth regulators and basal media on direct differentiation of shoot buds from leaf segments of marigold (Tagetes spp.)

Abstract

Marigold is a commercially important flower crop popular for its multipurpose use as loose flower, ornamental pot plant, pharmaceutical and industrial crop for lutein pigment extraction. The genus Tagetes is considered as a recalcitrant in terms of in vitro morphogenesis and plant regeneration. In the present study, we propose an efficient protocol for direct differentiation of shoot buds from immature leaf segments of African (Tagetes erecta L.) and French marigold (Tagetes patula L.) genotypes viz., Pusa Basanti Gainda and Pusa Arpita, respectively. We developed this protocol as follows. Leaf segments were collected from in vitro proliferated shoots established in aseptic culture from shoot tips of doubled haploids and dihaploids. Rapid and direct shoot bud induction was observed on enriched MS (EMS) medium supplemented with coconut water, AgNO3 , casein hydrolysate, polyvinylpyrrolidone and glutamine. Among the different treatments, the highest (89.7%) direct shoot bud induction, bud forming capacity index (BFC) (2.36) and rapid (14 days) organogenesis was observed from leaf segments of French marigold cv. Pusa Arpita cultured on EMS media supplemented with 2.0 mg L-1 6-benzylaminopurine (BAP) and 0.5 mg L-1 1-naphthalene acetic acid (NAA). African marigold cv. Pusa Basanti Gainda exhibited maximum regeneration (66.6%), BFC index (2.11) and early (20 days) shoot bud induction on EMS media supplemented with 0.5 mg L-1 BAP and 0.25 mg L-1 NAA. In order to improve the regeneration capacity, different maturity groups of distal and proximal leaf ends were cultured on genotype specific medium. Among the different explant groups, immature leaf basal segments exhibited highest BFC index of 4.2 and 2.6 in Pusa Arpita and Pusa Basanti Gainda respectively. All the regenerated shoots were excised, rooted in 0.5 mg L -1 IBA and transplanted under polyhouse conditions, where they exhibit normal morphological growth similar to mother plant.