以新鲜葡萄柚(Citrus paradise)种子为材料，使用3种脱水速率处理至约20%含水量，然后用饱和盐溶液平衡至10种不同的含水量，再进行简化的二步法超低温保存；同时，以经过15 ℃、50%相对湿度(RH)条件脱水至含水量约12%的葡萄柚种子为材料，使用程序降温仪进行5个降温速率和3个预冷温度的传统二步法超低温保存。结果表明，葡萄柚种子对脱水敏感，种子含水量越低，成苗率越低。而且，脱水速率越快，对种子的损伤越大，成苗率呈现75% RH>50% RH>15% RH；冷冻处理对种子有进一步的伤害，含水量6%～8%的种子超低温保存效果最佳，但脱水速率对超低温保存的种子成苗率的影响不明显。用传统二步法超低温保存葡萄柚种子，与预冷对照相比，经液氮冷冻的种子成苗率都有明显下降。在预冷对照组中，-2.5 ℃·min-1降温速率的种子成苗率最高，在冷冻保存中-0.5 ℃·min-1和-0.25 ℃·min-1的降温速率最有利于超低温保存。预冷温度从-40 ℃降至-60 ℃，未超低温处理种子的发芽率降低，但超低温处理种子的发芽率提高。
Fresh grapefruit seeds were firstly desiccated at 3 different dehydration rates to about 20% moisture content (MC), and equilibrated to 10 different MC by saturated salt solutions, then cryopreserved using a simplified two-step protocol; Meanwhile, another fresh grapefruit seeds was firstly dehydrated to about 12% MC under 15 ℃ and 50% relative humidity (RH) conditions, and then cool down at 5 cooling rates and 3 pre-cooling temperatures using a programmable cooler before frozen in liquid nitrogen. It was found that grapefruit seeds were sensitive to dehydration, and the emergence percentage fell with the decrease of MC. The damage to seeds increase with the acceleration of dehydration rate under slow dehydration. The germination percentage was expressed as 75% RH > 50% RH > 15% RH; Freezing treatment brought further damage to seeds, but there were no significant difference among germination percentage of frozen seeds dried at the three dehydration rates, and the best post thaw germination found in seeds at 6%—8% MC. Compared with the pre-cooling control, freezing in liquid nitrogen significantly reduced seed emergence. The seeds pre-cooled at rates of -0.5 ℃·min-1 and -0.25 ℃·min-1 had the best cryopreservation results, while the pre-cooling rate of -2.5 ℃·min-1 was optimal for the pre-cooling control seed. The decrease in pre-cooling temperature from -40 ℃ to -60 ℃ reduced the emergence of non-cryopreservation seeds, but increased the emergence of cryopreserved seeds.
[1] 叶荫民. 柚Citrus grandis (L.) Osbeck种质多样化中心的探讨[J]. 中国南方果树, 1997(1): 3–5.
[2] Wen B, Cai C, Wang R, Tan Y, Lan O. Critical moisture content windows differ for the cryopreservation of pomelo (Citrus grandis) seeds and embryonic axes[J]. Cryo Letters, 2010,31(1): 29–30.
[3] Ellis R H, Hong T D, Roberts E H. An intermediate category of seed storage behaviour? I. COFFEE [J]. Journal of Experimental Botany, 1990,41(9): 1167–1174.
[4] 文彬. 试论种子顽拗性的复合数量性状特征[J]. 云南植物研究, 2008(1): 76–88.
[5] 宋松泉,龙春林,殷寿华,兰芹英. 种子的脱水行为及其分子机制[J]. 云南植物研究, 2003(4): 465–479.
[6] Zhang N, Wen B, Ji M Y, Yan Q. Low-temperature storage and cryopreservation of grapefruit (Citrus paradisi Macfad.) seeds[J]. Cryo Letters, 2014,35(5): 418–26.
[7] 文彬. 植物种质资源超低温保存概述[J]. 植物分类与资源学报, 2011,33(3): 311–329.
[8] Engelmann F, Takai H. Cryopreservation of tropical plant germplasm. Current research progress and application[C] // Joint International Workshop. JIRCAS & IPGRI, 2000.
[9] Engelmann F, Arnao M-T G, Wu Y, Escobar R. Development of encapsulation dehydration[M] // Reed B M. Plant Cryopreservation: A Practical Guide, New York: Springer New York, 2008: 59–75.
[10] 陈晓玲,张金梅,辛霞,黄斌,卢新雄. 植物种质资源超低温保存现状及其研究进展[J]. 植物遗传资源学报, 2013,14(3): 414–427.
[11] Pérez R M, Navarro L, Duran-Vila N. Cryopreservation and storage of embryogenic callus cultures of several Citrus species and cultivars[J]. Plant Cell Reports, 1997,17(1): 44–49.
[12] González-Arnao M T, Engelmann F, Urra C, Morenza M, Rios A. Cryopreservation of citrus apices using the encapsulation- dehydration technique[C] // Cryopreservation of tropical plant germplasm: current research progress application. Cuba: 13th International Congress on Plant Biotechnology and Agriculture BIOVEG 2021, 2000: 177–182.
[13] Al-Ababneh S S, Karam N S, Shibli R A. Cryopreservation of sour orange (Citrus aurantium L.) shoot tips[J]. Vitro Cellular Developmental Biology Plant, 2002,38(6): 602–607.
[14] González-Arnao M T, Juárez J, Ortega C, Navarro L, Duran-Vila N. Cryopreservation of ovules and somatic embryos of citrus using the encapsulation-dehydration technique[J]. Cryo Letters, 2003,24(2): 85–94.
[15] 中国科学院中国植物志编辑委员会. 中国植物志(第43卷第2分册)[M]. 北京: 科学出版社, 1997.
[16] 中国科学院昆明植物研究所. 云南植物志(第6卷)[M]. 北京: 科学出版社, 1995.
[17] ISTA. International Rules for Seed Testing[M]. Basserdorf: International Seed Testing Association, 2004.
[18] Hor Y L, Kim Y J, Ugap A, Chabrillange N,?Sinniah U R,?Engelmann F,?Dussert S. Optimal Hydration Status for Cryopreservation of Intermediate Oily Seeds: Citrus as a Case Study[J]. Annals of Botany, 2005,95(7): 1153–1161.
[19] Samarah N H, Mullen R E, Alqudah A M. An index to quantify seed moisture loss rate in relationship with seed desiccation tolerance in common vetch[J]. Seed Science and Technology, 2009,37(2): 413–422.
[20] Huang H, Song S Q, Wu X J. Response of Chinese wampee axes and maize embryos to dehydration at different rates[J]. Journal of Integrative Plant Biology, 2009,51(1): 67–74.
[21] Liang Y. Desiccation tolerance of recalcitrant Theobroma cacao embryonic axes: the optimal drying rate and its physiological basis[J]. Journal of Experimental Botany, 2000,51(352): 1911–1919.
[22] 郭延平,李嘉瑞. 超低温保存技术在果树种质保存中的应用[J]. 果树学报, 1993(s1): 1–5.
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