Alternative Plant Vitrification Solution A3-80% and Initial Ammonium-Free Regrowth Medium Enable Cryobanking of Chrysanthemum Germplasm

Abstract

Cryopreservation, storing biological material in liquid nitrogen (LN, −196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocol is hampered by a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing a droplet-vitrification cryopreservation procedure for chrysanthemum shoot tips. The standard procedure includes two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 40 min, cryoprotection with alternative plant vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with 1 mg L−1 gibberellic acid (GA3) and 1 mg L−1 benzyl adenine (BA) followed by an ammonium-containing medium with and without growth regulators was essential for the development of normal plantlets from cryopreserved shoot tips. A pilot cryobanking of 154 accessions of chrysanthemum germplasm initiated with post-cryopreservation regeneration of 74.8%. This approach will facilitate the cryobanking of the largest Asteraceae family germplasm as a complementary long-term conservation method.

Keywords

• C4-35%
• droplet-vitrification
• liquid overlay
• shoot tips
• two-step preculture
• three-step regrowth