麻栎种子和胚方脱水敏感性及其低温贮藏技术

doi:10.11707/j.1001-7488.LYKX20240144

摘要/Abstract

摘要： 目的研究麻栎种子和胚方的脱水敏感性特征与临界含水量，探索麻栎种子低温保存技术和胚方超低温保存技术，为麻栎等顽拗性种子的保存提供技术参考。方法以野生麻栎种子为试验材料，采用硅胶减重法快速脱水至目标含水量，测定不同含水量种子和胚方的萌发指标；利用差示扫描量热技术（DSC）测定不同含水量胚轴和子叶的热力学参数，根据含水量和对应热焓的关系测算胚方结晶临界含水量；应用低温密封保存法保存不同含水量的种子，运用玻璃化法在超低温环境中保存不同含水量的胚方。结果 1）麻栎不同含水量种子和胚方萌发特征显示，二者分别从初始含水量34.90%和44.14% 降至10.00%，萌发率从93.00%和90.00%降至5.00%和60.00%。2）不同含水量麻栎胚轴和子叶DSC结果显示，随着胚轴和子叶含水量降低，结晶起始温度、峰值温度和热焓均呈规律性下降趋势，与含水量显著正相关；不同含水量胚轴平均热焓高于子叶平均热焓，胚轴自由水含量高于子叶自由水含量，初步判定本批次麻栎胚方理论结晶临界含水量为11.72%。3）脱水对麻栎种子生理生化特性的影响结果显示，随着脱水程度加深，种子细胞超氧化物歧化酶（SOD）和过氧化物酶（POD）活性、脯氨酸（PRO）和丙二醛（MDA）含量均呈先上升后下降趋势。4）麻栎种子低温保存结果显示，初始含水量种子在4 ℃下保存21天，萌发活力没有变化，重度脱水（10.00%~20.00%）种子能够忍受一定程度的低温伤害；不同含水量胚方超低温保存结果显示，含水量对超低温保存结果有显著影响，PVS2预处理30 min时10.00%和15.00%含水量麻栎胚方成活率分别为5.00%和3.00%，不同含水量胚方经预处理和超低温保存后，热力学特征发生改变，15.00%含水量麻栎胚方重新获得自由水。结论麻栎种子和胚方萌发率随含水量下降而降低，具有脱水敏感性特征，脱水对麻栎种子生理生化特性有显著影响；麻栎胚方理论结晶临界含水量为11.72%；超低温保存麻栎胚方具有可行性，但仍需完善保存方法，提高胚方超低温保存后恢复萌发率。

关键词: 麻栎, 顽拗性种子, 脱水敏感性, 超低温保存, 差示扫描量热技术

Abstract: Objective This study aims to investigate the desiccation sensitivity as well as the critical moisture content of the seed and embryonic axis of Quercus acutissim and explore low-temperature preservation techniques for Q. acutissima seeds and ultra-low temperature preservation techniques for the embryonic axis, in order to provide technical guidance for the preservation of recalcitrant seeds such as Q. acutissima. Method Wild Q. acutissim seeds were used as experimental materials, the silica gel weight reduction method was used to rapidly dehydrate the seeds to the target moisture content, and then the germination indexes of seeds and embryonic axes with different moisture contents were determined. The differential scanning calorimetry (DSC) was employed to determine the thermodynamic parameters of embryonic axes and cotyledons with different moisture contents, and the critical moisture content of crystallization in the embryonic axis was evaluated according to the relationship between moisture content and corresponding enthalpy. The low-temperature sealed storage method was used to preserve seeds with different moisture contents, and the vitrification method was used for cryopreservation of the embryonic axis. Result 1) When the initial moisture content of the seeds and embryonic axes decreased from 34.90% and 44.14% to 10.00%, their germination percentage decreased from 93.00% and 90.00% to 5.00% and 52.00%, respectively. 2) The DSC results of Q. acutissim embryonic axis and cotyledon with different moisture contents showed that with the decrease of moisture content of embryonic axis and cotyledon, the crystallization initiation temperature, peak temperature and enthalpy value showed a regular downward trend, which were significantly positively correlated with moisture content. The average enthalpy value of the embryonic axis was higher than that of the cotyledon, and the free moisture content of the embryonic axis was higher than that of the cotyledon. It was preliminarily determined that the theoretical critical moisture content of crystallization in this batch of the embryonic axes was 11.72%. 3) The effects of dehydration on the physiological and biochemical characteristics of Q. acutissima seeds showed that as dehydration progressed, the activities of superoxide dismutase (SOD) and peroxidase (POD), as well as the contents of proline (PRO) and malondialdehyde (MDA) in seeds initially increased and then decreased. 4) Q. acutissim seeds with initial moisture content stored at 4 ℃ for 21 days showed no change in the germination vitality, and the seeds with severe dehydration (10%–20%) were still able to tolerate a certain degree of low temperature damage. The results of cryopreservation of embryonic axis with different moisture contents showed that moisture content had a significant impact on the results of cryopreservation. When pretreated with PVS2 for 30 min before cryopreservation, the embryonic axis with 10.00% and 15.00% moisture content had a 5.00% and 3.00% survival rate, respectively, and the thermodynamic characteristics of the embryonic axis with different moisture content changed. The embryonic axis with 15.00% moisture content regained free moisture. Conclusion The germination rate of Q. acutissim seeds and embryonic axes decreases with the decrease of moisture content, which is a typical characteristics of recalcitrant seeds with the dehydration sensitivity. Dehydration has a significant effect on the physiological and biochemical characteristics of the seeds. The theoretical critical moisture content of crystallization in Q. acutissim embryonic axis is 11.72%. Cryopreservation of Q. acutissimembryonic axis is possible, but the method still needs adjustment to increase the recovery of germination rate, and then is applied to the long-term preservation of the recalcitrant seed.

Key words: Quercus acutissim, desiccation sensitivity, cryopreservation, recalcitrant seeds, differential scanning calorimetry (DSC).

中图分类号:

张铭佳, 仝伯强, 曲凯, 咸洋, 崔程程, 王永正, 臧一村, 韩彪. 麻栎种子和胚方脱水敏感性及其低温贮藏技术[J]. 林业科学, 2025, 61(4): 129-139.

Zhang Mingjia, Tong Boqiang, Qu Kai, Xian Yang, Cui Chengcheng, Wang Yongzheng, Zang Yicun, Han Biao. Desiccation Sensitivity and Low-Temperature Preservation Techniques of Quercus acutissim Seeds and Embryonic Axes[J]. Scientia Silvae Sinicae, 2025, 61(4): 129-139.

参考文献

冯　景, 沈永宝, 史锋厚. 2019. 银杏种子脱水敏感性的研究. 南京林业大学学报(自然科学版), 43(6): 193-200.
Feng J, Shen Y B, Shi F H. 2019. Study on desiccation sensitivity of Ginkgo biloba seeds. Journal of Nanjing Forestry University (Natural Sciences Edition), 43(6): 193-200. ［in Chinese］
韩　彪. 2020. 板栗种子顽拗性特征解析及贮藏研究. 北京: 北京林业大学.
Han B. 2020. Analysis on recalcitrant characteristics and preservation of Castanea mollissima Bl. seed. Beijing: Beijing Forestry University. ［in Chinese］
韩　彪, 李文清, 郭素娟, 等. 2020. 基于差示扫描量热技术的板栗胚轴低温保存技术及临界含水量. 林业科学, 56(3): 21-27.
Han B, Li W Q, Guo S J, et al. 2020. Cryopreservation and critical moisture content of embryo axis of Castanea mollissima based on differential scanning calorimetry. Scientia Silvae Sinicae, 56(3): 21-27. ［in Chinese］
何明高, 王瑞霞, 宋希强, 等. 2010. 束花石斛种子超低温保存的研究. 云南植物研究, 32(4): 334-338.
He M G, Wang R X, Song X Q, et al. 2010. Study on cryopreservation of Dendrobium chrysanthum (Orchidaceae) seeds. Acta Botanica Yunnanica, 32(4): 334-338.［in Chinese］
冀智清, 张存旭, 张昌胜, 等. 2012. 栓皮栎胚性组织低温保存技术研究. 西北林学院学报, 27(6): 88-92.
Ji Z Q, Zhang C X, Zhang C S, et al. 2012. Cryopreservation of embryogenic tissues in Quercus variabilis. Journal of Northwest University, 27(6): 88-92. ［in Chinese］
李东兴, 钱家连, 许慧慧, 等. 2022. 脱水对栓皮栎种子基因表达的影响. 东北林业大学学报, 50(3): 1-8.
Li D X, Qian J L, Xu H H, et al. 2022. Expression analysis of genes in response to desiccation of Quercus variabilis seeds. Journal of Northeast Forestry University, 50(3): 1-8. ［in Chinese］
李　磊, 孙雪婷, 张广辉, 等. 2014. 脱水速率对顽拗性三七种子脱水敏感性和抗氧化酶活性的影响. 种子, 33(12): 1-5.
Li L, Sun X T, Zhang G H, et al. 2014. Effect of drying rates on the desiccation sensitivity and antioxidant enzyme activities of recalcitrant Panax notoginseng seeds. Seed, 33(12): 1-5. ［in Chinese］
李艳林, 渠慎春, 栾雨婷, 等. 2019. 苹果茎尖超低温脱毒体系的建立. 分子植物育种, 17(9): 2982-2995.
Li Y L, Qu S C, Luan Y T, et al. 2019. Establishment of cryopreservation detoxification system of apple shoot-tips. Molecular Plant Breeding, 17(9): 2982-2995. ［in Chinese］
罗婷婷. 2017. 基于黄连种子后熟生理特性的种子保存技术的初步研究. 成都: 成都中医药大学.
Luo T T. 2017. A preliminary study on seed preservation technology based on post- ripening physiological characteristics of Coptis chinensis Franch. seeds. Chengdu: Chengdu University of TCM. ［in Chinese］
田新民, 李洪立, 何　云, 等. 2014. 热带作物顽拗性种子贮藏研究进展. 热带农业科学, 34(8): 52-58.
Tian X M, Li H L, He Y, et al. 2014. Research progress on recalcitrant seeds storage of tropical crops. Chinese Journal of Tropical Agriculture, 34(8): 52-58. ［in Chinese］
韦树根. 2017. 桑寄生顽拗性种子生物学特性及脱水敏感性机理研究. 北京: 北京协和医学院.
Wei S G. 2017. Study on the biological characteristics of recalcitrant seeds and mechanisms of desiccation sensitivity of Taxillus chinensis (DC.) Danser. Beijing: Peking Union Medical College. ［in Chinese］
文　彬. 2019. 漫话顽拗性种子. 生命世界, 11: 22-33.
Wen B. 2019. Rambling stubborn seed. Life World, 11: 22-33. ［in Chinese］
薛　鹏, 文　彬. 2015. 脱水速率对非洲柚种子脱水耐性的影响. 植物分类与资源学报, 37(3): 293-300.
Xue P, Wen B. 2015. Effects of drying rates on the desiccation tolerance of Citrus maxima ‘Feizhouyou’ seeds. Plant Diversity and Resources. 37(3): 293-300. ［in Chinese］
闫兴富, 杜　茜, 王建礼, 等. 2009. 脱水处理对水蜡树种子萌发的影响. 种子, 28(7): 93-96.
Yan X F, Du Q, Wang J L, et al. 2009. Efects of dehydrating treatments on seeds germination of Ligustrum obtusifolium. Seed, 28(7): 93-96. ［in Chinese］
袁　鸣, 朱铭玮, 侯　静, 等. 2022. 利用低场核磁共振技术检测刺槐种子吸水过程水分的变化. 南京林业大学学报(自然科学版), 46(2): 135-142.
Yuan M, Zhu M W, Hou J, et al. 2022. Changes of water content in Robinia pseudoacacia seeds during imbibition by a low nuclear magnetic resonance. Journal of Nanjing Forestry University (Natural Sciences Edition), 46(2): 135-142. ［in Chinese］
张铭佳, 韩　彪, 朱　强, 等. 2023. 渗透处理对麻栎和栓皮栎种子生物学特性的影响. 基因组学与应用生物学, 42(9): 927-940.
Zhang M J, Han B, Zhu Q, et al. 2023. Effects of osmotic treatment on the biological properties of Quercus acutissima and Quercus variabilis’ seeds. Genomics and Applied Biology, 42(9): 927-940.［in Chinese］
郑郁善, 陈礼光, 李庆荣, 等. 2002. 板栗种子超低温保存研究. 林业科学, 38(6): 146-149.
Zheng Y S, Chen L G, Li Q R, et al. 2002. Study of cryopreservation on Castanea mollissima seeds, Scientia Silvae Sinica, 38(6), 146-149. ［in Chinese］
Al Zoubi O M, Normah M N. 2015. Critical moisture content for successful cryopreservation of embryonic axes of Fortunella polyandra determined by differential scanning calorimetry (DSC). Acta Physiologiae Plantarum, 37(1): 1727.
Araldi C G, Coelho C M M, Gaziola S A, et al. 2016. Storage elicits a fast antioxidant enzyme activity in Araucaria angustifolia embryos. Acta Physiologiae Plantarum, 38(8): 201.
Berjak P, Vertucci C W, Pammenter N W. 1993. Effects of developmental status and dehydration rate on characteristics of water and desiccation-sensitivity in recalcitrant seeds of Camellia sinensis. Seed Science Research, 3(3): 155-166.
Ellis R H, Hong T D, Roberts E H. 1991. An intermediate category of seed storage behaviour? II. effects of provenance, immaturity, and imbibition on desiccation-tolerance in coffee. Journal of Experimental Botany, 42(5): 653-657.
Engelmann F. 2011. Cryopreservation of embryos: an overview. Methods in Molecular Biology, 710: 155-184.
Fu J R, Xia Q H, Tan L F. 1993. Effects of desiccation on excised embryonic axes of three recalcitrant seeds and studies on cryoproservation. Seed Science and Technology, 21: 85-95.
[24] ISTA. 2021. Chapter 9: determination of moisture content//International rules for seed testing, Bassersdorf. Switzerland: International Seed Testing Association.
Kenzo T, Ichie T, Ninomiya I, et al. 2003. Photosynthetic activity in seed wings of Dipterocarpaceae in a masting year: does wing photosynthesis contribute to reproduction? Photosynthetica, 41(4): 551-557.
King M W, Roberts E H. 1979. The storage of recalcitrant seeds: achievements and possible approaches. Roma: IBPGR.
Lin L, Xu Q, Ma J C, et al. 2014. Cryopreservation of protocorm-like body-derived shoot tips of Calanthe davidii by droplet-vitrification. Cryo Letters, 35(2): 129-137.
Pammenter N W, Berjak P, Walters C. 2000. The effect of drying rate on recalcitrant seeds: lethal water contents, causes of damage, and quantification of recalcitrance. UK: CABI Publishing, 215-221.
Pammenter N W, Vertucci C W, Berjak P. 1991. Homeohydrous (recalcitrant) seeds: dehydration, the state of water and viability characteristics in Landolphia kirkij. Plant Physiology, 96(4): 1093-1098.
Pence V C. 1990. Cryostorage of embryo axes of several large-seeded temperate tree species. Cryobiology, 27(2): 212-218.
Roberts E H. 1973. Predicting the storage life of seeds. Seed Science and Technology, 1: 499-514.
Spitz D R, Oberley L W. 1989. An assay for superoxide dismutase activity in mammalian tissue homogenates. Analytical Biochemistry, 179(1): 8-18.
Xia K, Daws M I, Hay F R. et al. 2012. A comparative study of desiccation responses of seeds of Asian evergreen oaks, Quercus subgenus Cyclobalanopsis and Quercus subgenus Quercus. South African Journal of Botany, 78: 47-54.

[1]	曾素平,厉月桥,刘儒,王佳,赵梅芳,张华聪. 脱水方式和贮藏时间对苦槠种子发芽的影响[J]. 林业科学, 2023, 59(3): 84-93.
[2]	袁鸣,朱铭玮,解志军,康真,张中会,李淑娴. 核磁共振技术在沼生栎种子失水过程中水分相态变化[J]. 林业科学, 2023, 59(11): 42-48.
[3]	张维伟,赵忠,刘金良,邓平. 桥山林区3种麻栎群落类型的种群动态与幼苗特征[J]. 林业科学, 2021, 57(7): 1-10.
[4]	陈媛,晏婷婷,杨昇,付宗营,李改云,任海青. 栓皮栎和麻栎抽提物的化学成分分析[J]. 林业科学, 2021, 57(4): 191-198.
[5]	韩彪,李文清,郭素娟,陆璐,解孝满. 基于差示扫描量热技术的板栗胚轴低温保存技术及临界含水量[J]. 林业科学, 2020, 56(3): 21-27.
[6]	赵文瑞, 刘鑫, 张金池, 王玲, 谢德晋, 袁颖丹, 王金平, 王鹰翔. 添加酸雨酸度和硫氮比对麻栎林细根生长的影响[J]. 林业科学, 2017, 53(4): 158-165.
[7]	赵文瑞, 刘鑫, 张金池, 王鹰翔, 王金平, 庄家尧. 南京城郊典型树种光合蒸腾、固碳释氧及降温增湿能力[J]. 林业科学, 2016, 52(9): 31-38.
[8]	王君, 严小莉, 李凌. 不同种源麻栎幼苗对Cd²⁺-Pb²⁺复合污染的吸收累积特性[J]. 林业科学, 2014, 50(7): 23-30.
[9]	刘艳;李庆梅;刘广全;;刘勇;侯龙鱼;李国雷. 麻栎种子萌发的抑制机制[J]. 林业科学, 2012, 48(9): 164-170.
[10]	翟晓巧;程斐朱延林. 二乔刺槐愈伤组织超低温保存及适宜降温方法*[J]. 林业科学, 2009, 12(10): 49-54.
[11]	赵文飞;王迎王华田亓立云董玉峰. 不同季节麻栎树干贮水量的动态变化[J]. 林业科学, 2007, 43(4): 115-120.
[12]	辛霞;景新明孙红梅;于文文;. 富含多酚的麻栎种子中蛋白质提取的探讨[J]. 林业科学, 2007, 43(2): 26-30.
[13]	陈礼光郑郁善李庆荣张祝金. 肉桂离体胚超低温保存研究[J]. 林业科学, 2005, 41(5): 38-44.
[14]	陶月良朱诚. 板栗种子成熟前后脱水敏感性与蛋白质、可溶性糖的关系[J]. 林业科学, 2004, 40(2): 45-50.
[15]	崔建国崔文山白瑞兴李德民兰显臻. 辽西半干旱地区栎树人工造林技术的研究[J]. 林业科学, 2003, 39(6): 68-76.