Mandal B B, Ahuja-Ghosh Sangeeta
Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, India.
Cryo Letters. 2007 Sep-Oct;28(5):329-36.
The encapsulation-dehydration protocol for the cryopreservation of in vitro shoot tips of Dioscorea floribunda was optimized. Maximum survival of 87% was obtained when overnight pretreatment with 0.3 M sucrose was followed by encapsulation, preculture in 0.75 M sucrose for 4 d, dehydration in a laminar air flow for 5.5 h, quenching in liquid nitrogen and thawing at 40 degrees C. During recovery growth, 29% shoot formation was obtained when cryopreserved shoot tips were initially cultured for 25 d on a medium with 1.5 mg per liter (-1) BAP, 0.2 mg per liter(-1) NAA and 0.2 mg per liter(-1) GA3 followed by culturing for 15 d on a medium with reduced BAP (1 mg per liter(-1)) but increased NAA (0.5 mg per liter(-1)) and GA3 (0.3 mg per liter(-1)). Finally, transfer on to a medium with further reduced doses of BAP (0.05 mg per liter(-1)) and NAA (0.15 mg per liter(-1)) but without GA3 stimulated production of fully grown plantlets. All plants regenerated without callus formation. Modification of post-thaw culture media with plant growth regulators was essential for regrowth of shoot tips to plantlets.
对薯蓣离体茎尖冷冻保存的包埋脱水法进行了优化。当用0.3M蔗糖过夜预处理，然后进行包埋，在0.75M蔗糖中预培养4天，在层流空气中脱水5.5小时，投入液氮并在40℃解冻时，获得了87%的最大存活率。在恢复生长期间，当将冷冻保存的茎尖最初在含有1.5毫克/升（-1）BAP、0.2毫克/升（-1）NAA和0.2毫克/升（-1）GA3的培养基上培养25天，然后在含有降低的BAP（1毫克/升（-1））但增加的NAA（0.5毫克/升（-1））和GA3（0.3毫克/升（-1））的培养基上培养15天时，获得了29%的芽形成。最后，转移到含有进一步降低剂量的BAP（0.05毫克/升（-1））和NAA（0.15毫克/升（-1））但不含GA3的培养基上，刺激了完全长成的小植株的产生。所有再生植株均未形成愈伤组织。用植物生长调节剂对解冻后培养基进行改良对于茎尖再生为小植株至关重要。
