植物学报 ›› 2021, Vol. 56 ›› Issue (5): 573-583.DOI: 10.11983/CBB21096
刘柯1, 刘彬1, 袁璐1, 帅辉1, 杨洋1, 王挺进1, 陈德良2, 陈小荣2, 杨锴斌2, 金孝锋3, 陈利萍1,*()
收稿日期:
2021-06-14
接受日期:
2021-09-30
出版日期:
2021-09-01
发布日期:
2021-10-12
通讯作者:
陈利萍
作者简介:
* E-mail: chenliping@zju.edu.cn基金资助:
Ke Liu1, Bin Liu1, Lu Yuan1, Hui Shuai1, Yang Yang1, Tingjin Wang1, Deliang Chen2, Xiaorong Chen2, Kaibin Yang2, Xiaofeng Jin3, Liping Chen1,*()
Received:
2021-06-14
Accepted:
2021-09-30
Online:
2021-09-01
Published:
2021-10-12
Contact:
Liping Chen
摘要: 百山祖冷杉(Abies beshanzuensis)种子萌发率低, 发育特性尚不明确, 极大地限制了种群在原生境的自然更新。该研究以百山祖冷杉不同发育时期的雌球果为材料, 通过研究球果中种子的胚与胚乳数量、重量和显微结构特性, 及测定种子发育关键时期胚乳的初生代谢物, 探究种子发育特征及影响种子发育的主要因素。结果表明, 7月是百山祖冷杉种子发育的关键时期, 此期间胚乳重量快速增加, 胚乳中碳水化合物代谢活动旺盛。解剖学和形态学分析显示, 7月下旬球果中大量种子发育异常。进一步对发育关键时期胚乳中初生代谢物进行分析表明, 海藻糖-6-磷酸含量显著降低。推测在种子发育的关键时期胚乳中海藻糖-6-磷酸降低可能是种子发育异常的重要原因之一。基于种子发育特征, 该研究建立了百山祖冷杉未成熟胚培养技术体系, 并成功获得试管苗。
刘柯, 刘彬, 袁璐, 帅辉, 杨洋, 王挺进, 陈德良, 陈小荣, 杨锴斌, 金孝锋, 陈利萍. 濒危植物百山祖冷杉种子发育特征及其胚培养. 植物学报, 2021, 56(5): 573-583.
Ke Liu, Bin Liu, Lu Yuan, Hui Shuai, Yang Yang, Tingjin Wang, Deliang Chen, Xiaorong Chen, Kaibin Yang, Xiaofeng Jin, Liping Chen. Seed Development and Embryo Culture of Endangered Abies beshanzuensis. Chinese Bulletin of Botany, 2021, 56(5): 573-583.
图2 百山祖冷杉传粉阶段的珠孔形态变化和花粉转移 (A) 珠被发育形成漏斗状珠孔(箭头); (B) 花粉(箭头)附着于珠孔边缘; (C) 珠被向内折叠, 珠孔闭合(箭头); (D) 花粉(箭头)进入储粉室。MF: 漏斗状珠孔; Po: 花粉; Poc: 储粉室。(A)-(C) Bars=100 μm; (D) Bar=500 μm
Figure 2 Morphological changes of the micropylar funnel and pollen transfer during the pollination stage of Abies beshanzuensis (A) The integument develops a stigmatic micropylar funnel (arrow); (B) The pollen (arrow) attaches to the edge of micropylar funnel; (C) The integument folds inward and the micropylar funnel is closed (arrow); (D) The pollen (arrow) enters the pollen chamber. MF: Micropylar funnel; Po: Pollen; Poc: Pollen chamber. (A)-(C) Bars=100 μm; (D) Bar=500 μm
图3 2019年和2020年百山祖冷杉不同发育时期球果含胚乳率和含胚率变化 (A) 百山祖冷杉球果含胚乳率随时间的变化; (B) 百山祖冷杉球果含胚率随时间的变化。不同小写字母表示在0.05水平差异显著。
Figure 3 The endosperm rate and embryo rate of Abies beshanzuensis cones at different developmental stages in 2019 and 2020 (A) Changes of endosperm rate of A. beshanzuensis seeds with time; (B) Changes of embryo rate of A. beshanzuensis seeds with time. Different lowercase letters indicate significant differences at 0.05 level.
图4 2019年与2020年百山祖冷杉不同发育时期种子及其胚和胚乳重量变化 (A) 2019年与2020年种子重量随时间的变化; (B) 2019年胚与胚乳重量随时间的变化; (C) 2020年胚与胚乳重量随时间的变化。不同小写字母表示在0.05水平差异显著。
Figure 4 Changes in seed, embryo and endosperm weight of Abies beshanzuensis seeds at different developmental stages in 2019 and 2020 (A) Changes in the weight of seed with time in 2019 and 2020; (B) Changes in the weight of embryo and endosperm with time in 2019; (C) Changes in the weight of embryo and endosperm with time in 2020. Different lowercase letters indicate significant differences at 0.05 level.
图5 百山祖冷杉种子及其胚与胚乳发育过程的形态结构 (A) 5月25日的胚珠; (B) 6月25日类型I种子的胚与胚乳; (C) 6月25日类型II种子的胚乳; (D) 5月25日胚珠的石蜡切片, 红色方框内为花粉(上)与颈卵器(下); (E) 6月25日类型I种子的石蜡切片, 红色方框内为胚; (F) 6月25日类型II种子的石蜡切片; (G) 7月25日类型I种子的胚与胚乳; (H) 7月25日类型II种子的胚乳; (I) 7月25日类型III种子内皱缩的胚乳或/和胚; (J) 7月25日类型I种子的石蜡切片, 红色方框内为胚; (K) 7月25日类型II种子的石蜡切片; (L) 7月25日类型III种子的石蜡切片; (M) 8月25日类型I种子的胚与胚乳; (N) 8月25日类型III种子; (O) 9月25日类型I种子的胚与胚乳。Bars=1 000 μm。MF: 珠孔; In: 珠被; Em: 胚; Es: 胚乳; Po: 花粉; FG: 雌配子体; Re: 树脂囊; Ext: 外种皮; Ent: 内种皮; Cy: 子叶; Hy: 下胚轴; Su: 胚柄系统; ST: 皱缩组织; ER: 胚根; CC: 腐蚀腔
Figure 5 Morphology of Abies beshanzuensis seed and its embryo and endosperm during development (A) The ovule on May 25th; (B) The embryo and endosperm of type I seeds on June 25th; (C) The endosperm of type II seeds on June 25th; (D) The paraffin section of the ovule on May 25th, the pollen (top) and archegonium (bottom) are in the red box; (E) The paraffin section of type I seeds on June 25th, the embryo in the red box; (F) The paraffin section of type II seeds on June 25th; (G) The embryo and endosperm of type I seeds on July 25th; (H) The endosperm of type II seeds on July 25th; (I) The shriveled tissue of endosperm or/and embryo within type III seeds on July 25th; (J) The paraffin section of type I seeds on July 25th, the embryo in the red box; (K) The paraffin section of type II seeds on July 25th; (L) The paraffin section of type III seeds on July 25th; (M) The embryo and endosperm of type I seeds on August 25th; (N) Type III seeds on August 25th; (O) The embryo and endosperm of type I seeds on September 25th. Bars=1 000 μm. MF: Micropylar funnel; In: Integument; Em: Embryo; Es: Endosperm; Po: Pollen; FG: Female gametophyte; Re: Resinocyst; Ext: Exotesta; Ent: Endotesta; Cy: Cotyledon; Hy: Hypocotyl; Su: Suspensor system; ST: Shriveled tissue; ER: Embryo root; CC: Corrosion cavity
图6 6月和7月百山祖冷杉胚乳的初生代谢物变化 (A) 6月和7月发育正常胚乳的差异代谢物; (B) 第1、4、5类模式中关键差异代谢物数量占每类模式全部代谢物总数的比例与占所有被测样品中同类代谢物总数的比例; (C) 6月和7月百山祖冷杉种子胚乳中的蜜二糖、海藻糖-6-磷酸和甲基马来酸相对含量。6_NE: 6月发育正常的胚乳; 7_NE: 7月发育正常的胚乳; 7_AE: 7月发育异常的胚乳。不同小写字母表示在0.05水平差异显著。FC: 差异倍数值; LPC: 溶血磷脂酰胆碱; LPE: 溶血磷脂酰乙醇胺; PC: 磷脂酰胆碱
Figure 6 Changes of primary metabolites in endosperm of Abies beshanzuensis in June and July (A) Differential metabolites of normal endosperm in June and July; (B) The proportion of key difference metabolites in the first, fourth, and fifth models in the same change pattern in all metabolites and the proportion of the same metabolites tested in all samples; (C) Relative content of melibiose, trehalose-6-phosphate and methylmaleic acid in endosperm of A. beshanzuensis seeds in June and July. 6_NE: Normal endosperm in June; 7_NE: Normal endosperm in July; 7_AE: Abnormal endosperm in July. Different lowercase letters indicate significant differences at 0.05 level. FC: Fold change; LPC: Lysophosphatidylcholine; LPE: Lysopnosphatidylethanolamine; PC: Phosphatidylcholine
图7 百山祖冷杉不同种子发育时期的胚培养萌发率变化 (A) 百山祖冷杉胚萌发(bar=200 μm); (B) 胚培养获得的试管苗(bar=2 cm); (C) 不同百山祖冷杉种子发育时期的胚培养萌发率变化。不同小写字母表示在0.05水平差异显著。
Figure 7 Changes in germination rate of embryo of Abies beshanzuensis seeds at different developmental stages (A) Embryo germination (bar=200 μm); (B) Seedlings obtained by embryo culture (bar=2 cm); (C) Changes in germination rate of embryo of A. beshanzuensis seeds at different developmental stages. Different lowercase letters indicate significant differences at 0.05 level.
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