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.

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

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含水量 Moisture content (%)	实际含水量范围 Actual moisture content range (%)	萌发率 Germination percentage (%)	萌发指数 Germination index	萌发时间 Germination time/d	根长 Length of root/mm	发霉数量 Mildew number
34.90	—	93.00±0.04c	4.36±0.04c	7.49±1.24a	6.59±0.43a	1.66±0.15a
30.00	29.14~30.89	82.00±0.02c	4.12±1.32c	7.81±0.80a	7.02±0.85a	1.66±0.15 a
25.00	24.69~26.05	85.00±0.09c	2.24±0.14b	12.05±0.32ab	6.98±1.63a	1.00±0.00a
20.00	18.32~21.35	42.00±0.02b	1.94±0.06ab	10.34±0.11a	4.32±0.66b	4.33±0.33c
15.00	13.98~15.87	19.00±0.10a	1.03±0.33a	15.92±0.32b	5.18±0.16ab	3.00±0.00b
10.00	8.33~12.50	5.00±0.01a	0.33±0.04a	15.45±1.79b	2.69±0.45b	4.33±0.33c

种子部位 Seed site	含水量 Moisture content (%)	冷却结晶阶段Cooling crystallisation			加热熔融阶段Warming melt
种子部位 Seed site	含水量 Moisture content (%)	起始温度 Onset temperature/℃	峰值温度 Peak temperature/℃	热焓 Enthalpy/(J·g^?1)	起始温度 Onset temperature/℃	峰值温度 Peak temperature/℃	热焓 Enthalpy/(J·g^?1)
胚轴 Embryonic axes	10.00	无结晶峰No peak
	15.00	?34.01±5.84a	?43.50±4.50a	81.60±13.6a	?80.71±67.97a	?10.07±4.30a	48.23±24.01a
	20.00	?32.40±2.51a	?38.70±5.90a	85.50±3.50a	?19.08±0.35a	?9.75±1.05a	49.00±1.00a
	25.00	?31.92±4.02b	?37.52±0.48a	101.37±6.93a	?17.10±0.39a	?8.14±0.44ab	60.11±7.88a
	30.00	?26.29±0.94b	?28.65±3.76ab	142.69±0.02b	?12.62±3.57a	?5.04±2.21ab	75.35±2.34a
	55.33	?18.01±1.73c	?16.30±2.72b	358.14±13.41c	?5.80±0.14a	?0.67±0.11b	143.40±6.28b
子叶 Cotyledon	10.00	无结晶峰No peak
	15.00	无结晶峰No peak
	20.00	?32.19±3.48a	?29.30±1.92a	92.30±5.84a	?15.16±1.31a	?7.13±0.55a	40.05±4.51a
	25.00	?26.70±2.20ab	?23.05±1.05ab	114.11±2.11a	?10.99±0.48b	?4.33±0.22b	42.31±6.68a
	30.00	?21.47±3.02ab	?21.31±2.79ab	129.11±12.89a	?10.06±0.44bc	?4.32±0.21b	42.01±6.38a
	44.25	?18.00±2.21b	?18.62±2.09b	220.06±81.64a	?6.69±1.18c	?2.19±1.01b	80.52±11.95b

含水量 Moisture content (%)	胚轴热焓 Embryonic axes enthalpy/(J·g^?1)			子叶热焓 Cotyledon enthalpy/(J·g^?1)			平均热焓 Average enthalpy/(J·g^?1)
含水量 Moisture content (%)	冷却结晶 Cooling crystallisation	加热熔融 Warming melt	平均热焓 Average enthalpy	冷却结晶 Cooling crystallisation	加热熔融 Warming melt	平均热焓 Average enthalpy	平均热焓 Average enthalpy/(J·g^?1)
10.00	—	—	—	—	—	—	—
15.00	81.60	48.23	65.42	—	—	—	—
20.00	85.50	49.00	71.75	92.30	40.05	71.18	88.90
25.00	101.37	60.11	80.91	114.11	42.31	78.21	107.74
30.00	142.69	75.35	109.02	129.11	42.01	85.56	135.90
34.94	358.14	143.40	250.77	220.06	80.52	150.29	289.10

温度 Temperature/℃	萌发率Germination rate (%)
温度 Temperature/℃	初始含水量Initial moisture content	30.00%	25.00%	20.00%	15.00%	10.00%
4	93.33±4.40a	68.00±2.77a	58.00±10.13b	55.00±10.40b	18.00±4.40b	15.00±2.88c
–20	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
–40	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
–80	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00

胚方大小 Embryonic axis size/g	胚方体积 Embryonic axis square volume/cm³	萌发率 Germination percentage（%）	染菌率 Contamination rate（%）
2.50~3.00	3.00~3.20	88.00±4.4a	48.33±8.33a
1.50~2.00	1.20~1.50	85.00±0.00a	41.66±7.26a
0.05~1.00	0.30~0.40	92.00±1.66a	3.44±1.66b