Cryopreservation of asian Dioscorea bulbifera l. and D. alata l. by vitrification: importance of plant growth regulators.
The aim of this study was to develop cryopreservation protocols for Asian races of Dioscorea bulbifera and D. alata with high survival and plant regeneration after cryopreservation. Using a vitrification procedure, survival of shoot tips postcryopreservation of up to 89% in D. bulbifera and up to 82% in D. alata were recorded when excised shoot tips were pretreated overnight with 0.3 M sucrose in MS medium, followed by loading with 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degrees C, exposure to PVS2 solution for 90 min at 0 degrees C, immersion in liquid nitrogen for 1 h, rewarming at 40 degrees C for 2 min, unloading in medium with 1.2 M sucrose for 20 min and culturing on growth recovery medium. During growth recovery, 58% shoot regeneration was obtained in D. bulbifera when cryopreserved shoot tips were initially cultured for 40 days on MS medium with 1.5 mg/L BAP, 0.15 mg/L NAA and 0.2 mg/L GA3 followed by culturing on a medium with 0.05 mg/L BAP and 0.15 mg/ L NAA. However, a maximum of 39% shoot regeneration was recorded in D. alata when cryopreserved shoot tips were initially cultured for 40 days on medium M2 (MS containing 1/5 NH4NO3 and 40 g/L sucrose) supplemented with 1.0 mg/L BAP, 1.0 mg/L zeatin, 0.15 mg/L IAA and 0.2 mg/L GA3. Subsequently, the regenerating shoots were cultured for 30 days on medium M2 with 1.0 mg/L BAP, 0.3 mg/L zeatin, 0.02 mg/L NAA and 0.2 mg/L GA3 followed by culturing for another 30 days on medium with 0.5 mg/L BAP, 0.02 mg/L NAA and 0.2 mg/L GA3. Finally, transfer onto medium with 0.05 mg/L BAP and 0.15 mg/L NAA stimulated production of fully grown plantlets. Alteration of post-thaw culture media with plant growth regulators and their application at various stages of growth recovery was crucial for regeneration of shoot tips and formation of plantlets in D. alata.
Mukherjee
Papiya
P
Tissue Culture and Cryopreservation Unit, NBPGR, Pusa Campus, New Delhi, India. Agharkar Research Institute, Maharastra, India.
Mandal
B B
BB
Bhat
K V
KV
Biswas
A K
AK
D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D027742:Dioscorea / Q000187:drug effects* / Q000254:growth & development*; D009566:Nitrates / Q000494:pharmacology; D010937:Plant Growth Regulators / Q000494:pharmacology*; D018520:Plant Shoots / Q000187:drug effects* / Q000254:growth & development*; D014022:Tissue Survival / Q000187:drug effects
D003451:Cryoprotective Agents; D009566:Nitrates; D010937:Plant Growth Regulators; C006568:ammonium nitrate