Abstract：
Petunia × Calibrachoa \'Light Yellow\' (× Petchoa \'Light Yellow\') is a kind of perennial herbaceous flower obtained through intergeneric hybridization of Petunia and Calibrachoa with high ornamental value and wide application, facing challenges in seed acquisition. Expanding propagation through tissue culture is an economically efficient means. Hence, establishing an effective procedure for the storage of callus is essential for × Petchoa \'Light Yellow\'. Cryopreservation is an effective method for the in vitro propagation and long-term preservation of × Petchoa \'Light Yellow\' germplasms. For formulating the optimization of the vitrification procedure, first, an orthogonal experimental design was employed to pinpoint critical steps in the vitrification protocol (pre-culture, osmoprotection, dehydration, and dilution) for Petunia × Calibrachoa callus tissues and then five additional factors (pre-culture, osmoprotection I and II, dehydration, and dilution) were optimized to further reduce the sample water content and enhance cell viability levels. The vitrification procedure was described as follows: callus tissues were precultured in MS solid medium with 0.3 M sucrose for 5 d, incubated with osmoprotection solution I and II for 15 min at 25 °C, respectively, cryoprotected with PVS2 for 30 min at 0 °C, and rapidly immersed in liquid nitrogen. Cryopreserved callus tissues were then diluted in MS liquid medium with 1.2 M sucrose for 20 min at 25 °C and recovered on MS solid medium with 0.5 mg/L 6-BA and 0.1 mg/L NAA, and sucrose. The cell viability measured by TTC staining was approximately 16 %-18 % after 72 h-recovery. Following 45 days, the relative survival of callus reached up to 49.48 %. Furthermore, EST-SSR analysis showed no significant difference in the genetic stability of cryopreserved callus compared to the control. Based on the cryopreservation of × Petchoa \'Light Yellow\' callus, we further evaluated the response of callus water contents to the osmotic stress in the optimized and original protocols (CK) for a higher cryopreservation survival. A comparative analysis of water content demonstrated that the procedure of gradual and gentle dehydration significantly improved water content and cell survival. Ultrastructural changes between cryopreserved and non-cryopreserved callus were examined and high vacuolation emerged as a key determinant, indicating its substantial impact on the low survival of cryopreserved cells, which should help us to understand the effectiveness of osmotic protectants in dehydration.
摘要：
矮牵牛×Calibrachoa\'LightYellow\'(×Petchoa\'LightYellow\')是通过矮牵牛与Calibrachoa属间杂交获得的一种多年生草本花卉,具有很高的观赏价值和广泛的应用,在种子获取方面面临挑战。通过组织培养扩大繁殖是一种经济有效的手段。因此，建立储存愈伤组织的有效程序对于×Petchoa\“浅黄色\”至关重要。低温保存是一种有效的离体繁殖和长期保存的方法。为了制定玻璃化程序的优化，首先,采用正交实验设计来确定玻璃化方案中的关键步骤(预培养，渗透保护,脱水,和稀释）用于矮牵牛×Calibrachoa愈伤组织，然后是五个其他因子（预培养，渗透保护I和II，脱水,和稀释）进行了优化，以进一步降低样品水含量并提高细胞活力水平。玻璃化过程描述如下:愈伤组织在含有0.3M蔗糖的MS固体培养基中预培养5d，与渗透保护溶液I和II在25°C下孵育15分钟，分别,在0°C下用PVS2冷冻保护30分钟，并迅速浸入液氮中。然后将冷冻保存的愈伤组织组织在25°C下在具有1.2M蔗糖的MS液体培养基中稀释20分钟，并在具有0.5mg/L6-BA和0.1mg/LNAA的MS固体培养基上回收。和蔗糖。在72h恢复后,通过TTC染色测量的细胞活力为约16%-18%。45天后,愈伤组织的相对存活率高达49.48%。此外，EST-SSR分析表明，与对照相比，冷冻保存的愈伤组织的遗传稳定性没有显着差异。基于×Petchoa\'浅黄色\'愈伤组织的冷冻保存，我们在优化和原始方案（CK）中进一步评估了愈伤组织含水量对渗透胁迫的响应，以获得更高的冷冻保存存活率。对水分含量的比较分析表明，逐渐和温和脱水的过程显着改善了水分含量和细胞存活率。检查了冷冻保存和非冷冻保存的愈伤组织之间的超微结构变化，并且高空泡化成为关键决定因素，表明它对低温保存细胞的低存活率有重大影响，这应该有助于我们了解渗透保护剂在脱水中的有效性。
