Silver nitrate promotes high-frequency multiple shoot regeneration in cotton (Gossypium hirsutum L.) by inhibiting ethylene production and phenolic secretion

Abstract

The regeneration frequency of cotton (Gossypium spp.) is greatly influenced by its genetic makeup and recalcitrant nature. In particular, phenolic secretion is a major problem in cotton tissue culture. The present study was carried out to develop a rapid and efficient in vitro regeneration method, without phenolic secretion, from cotyledonary node explants of cotton. Multiple shoot induction was achieved after 3 wk of culture on MSB5 medium supplemented with N6-benzyl adenine (BA), kinetin (KN), thidiazuron (TDZ) (each tested at 0.5–3.0 mg L−1), Pluronic F-68 (0.1–0.6% [w/v]), or silver nitrate (AgNO3; 0.5–3.0 mg L−1). AgNO3 at 2 mg L−1 produced the greatest number of shoots (a mean of 22.2 ± 0.21 shoots per cotyledonary node explant), with no phenolic secretion. The induced shoots were healthy with intense green color and multiplied rapidly on the same medium. BA at 2.5 mg L−1 produced 10.3 ± 0.37 shoots/explant with moderate phenolic secretion. Pluronic F-68 at 0.3% produced 7.6 ± 0.28 shoots/explant along with high phenolic secretion. Individual shoots were elongated on MSB5 medium + gibberellic acid (GA3; 0.2 mg L−1; shoot length 7.8 ± 0.31 cm) and rooted on MSB5 medium + α-naphthalene acetic acid (NAA; 0.3 mg L−1; 8.3 ± 0.32 roots/shoot). Rooted plantlets were acclimatized in paper cups inside a plant growth chamber at 25°C and 70% relative humidity with a 70% survival rate. From the present study, AgNO3 was found to be suitable for obtaining a high frequency of multiple shoot production from cotyledonary node explants of cotton without phenolic secretion. The current protocol can be adopted for developing transgenic plants of commercial cotton cultivars through Agrobacterium-mediated genetic transformation.