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紫斑牡丹Paeonia rockii为芍药科Paeoniaceae芍药属Paeonia牡丹组Sect.Moutan小灌木[1], 是野生牡丹受威胁程度最高的种类之一, 被列为国家三级保护植物[2]。其种子需沙藏催根, 生根后种子的上胚轴仍处于休眠, 如休眠不解除, 则种子成苗率极低, 这是导致野生紫斑牡丹濒危的重要原因[3]。KENTZER[4]和AMEN[5]认为, 抑制物质的存在是植物种子休眠的主要原因之一。周理平等[6]发现, 紫斑牡丹的种皮、胚乳中含有抑制油菜Brassica campestris种子萌发及幼苗生长的物质, 认为种子内的抑制物质是紫斑牡丹种子休眠的重要原因。周仁超等[7]通过紫斑牡丹种皮和胚乳水提液对白菜Brassica pekinensis种子生长影响的研究, 认为其内的抑制物质不是种子萌发的决定因素。上述研究仅测定未生根种子中的抑制物质, 并未动态观察生根及发芽的种子, 因此, 单纯靠生根前种子各部位浸提液得出种子中抑制物质是否为制约紫斑牡丹种子萌发的结论值得商榷。本研究选用与紫斑牡丹种子结构类似, 具明显上下胚轴、同时又可迅速生根、发芽的豌豆Pisum sativum种子为材料,观察紫斑牡丹种子生根及发芽前后的种皮、胚、胚乳浸提液对豌豆种子萌发的影响,测试紫斑牡丹各提取物的抑制强度,并结合种皮透性及种子离体培养探究紫斑牡丹种子休眠原因,为紫斑牡丹的繁育和保护提供理论支持。
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由图 1可见:去皮种子吸水率在前18 h内增长非常快, 至24 h时, 吸水率已接近饱和, 此后一直维持在54%左右; 完整种子吸水率增长较慢, 浸种24 h时仅约28%, 72 h后才近饱和。可见, 种皮在浸种前72 h内对种子吸水率的影响较大。
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从图 2可以看出:浸水前完整种子与去皮种子的呼吸速率均很低, 去皮种子的呼吸速率在浸种24 h内迅速增大, 到24 h时达最大值, 约24 μmol·g-1·s-1, 72 h后, 其呼吸速率又迅速下降, 解剖发现其种胚变黄软化; 完整种子的呼吸速率增长速度慢, 浸种72 h后才达最大值, 约19 μmol·g-1·s-1, 此后保持不变。完整种子所能达到的最大呼吸速率值明显低于去皮种子。可见, 种皮阻碍了种子的呼吸。
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从图 3和图 4可见:在生根前紫斑牡丹种子胚乳、胚、种皮浸提液中豌豆的生根率、根长依次升高, 但都小于对照, 说明紫斑牡丹种子胚乳、胚、种皮都会抑制豌豆生根及根生长, 但抑制物质强度依次降低。在生根后紫斑牡丹种子的胚乳、种皮浸提液中豌豆的生根率及根长均显著大于在生根前紫斑牡丹种子浸提液中的, 而在发芽后的紫斑牡丹种子的胚乳、种皮浸提液中豌豆的生根率、根长虽有所增加, 但与生根后紫斑牡丹种子浸提液中的并无显著差异。说明紫斑牡丹种子胚乳和种皮中抑制物质对豌豆生根抑制强度随生根进程而降低, 但生根后到发芽过程中对豌豆生根的抑制强度并无显著变化。与胚乳、种皮不同, 在生根前、生根后及发芽后紫斑牡丹种子胚浸提液中豌豆的生根率依次降低, 根长无明显变化。
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由图 5和图 6可知:在生根前紫斑牡丹种子种皮、胚、胚乳浸提液中豌豆的上胚轴萌发率均为0, 而对照可达87.78%, 可见生根前紫斑牡丹种子种皮、胚、胚乳完全抑制了豌豆上胚轴的生长; 在生根后和发芽后的的紫斑牡丹种子种皮及胚乳浸提液中的豌豆均有部分上胚轴萌发, 且两者无显著差异, 说明生根后的紫斑牡丹种子种皮及胚乳浸提液的抑制物质已经降低到可以使豌豆上胚轴萌发的强度, 且生根后到发芽过程中紫斑牡丹种子种皮及胚乳浸提液对豌豆上胚轴生长具有抑制作用的物质不再发生显著变化。在生根前、生根后及发芽后的紫斑牡丹种子胚浸提液中豌豆的发芽率均为0。
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从图 7和图 8可见:处理1在60 d内紫斑牡丹种子无生根, 首粒生根时间明显晚于其他处理; 处理2可以消除种皮的机械障碍及透水、透气性影响, 保留种皮中可以影响胚生长的物质, 与处理1相比, 紫斑牡丹种子生根快, 但60 d内仅6%的种子生根; 而处理3比处理2早生根约14 d, 60 d时生根率约38%, 说明去除种皮有利于紫斑牡丹种子生根而种皮中确实有抑制下胚轴萌发的物质; 与处理3相比, 处理4和处理5提早约26 d生根, 两者接种12 d后, 下胚轴即开始伸长、生根, 60 d时生根率约90%, 在5个处理中生根最快, 生根率最高, 且两者无显著差异, 可见子叶对胚的生根并无影响。胚乳也会影响胚根生长, 裸胚(处理4和处理5)较有胚乳的胚(处理3)生根更快更好, 去除种皮及胚乳可以促进下胚轴快速伸长, 使其迅速生根。
从表 1可见:未经4 ℃低温处理的生根胚及生根去子叶胚的上胚轴萌发率均为0, 而低温处理后两者的上胚轴萌发率均达73%以上, 上胚轴长达2.7 cm, 且两者无显著差异。可见仅去除子叶而不经过低温不能打破紫斑牡丹种子上胚轴休眠。这与JING等[9]的研究结果不同, 他认为子叶是控制牡丹Paeonia种子上胚轴休眠的关键部位, 而周仁超等[7]研究表明, 子叶对上胚轴休眠无影响, 这与本试验结论类似。
表 1 不同处理紫斑牡丹种胚的上胚轴萌发率及上胚轴长
Table 1. Epicotyl germination rate and length of Paeonia rockii embryo in different treatments
处理组 上胚轴萌发率/% 上胚轴长/cm 4 ℃低温处理的生根胚 74.44 ± 4.16 a 2.71 ± 0.44 a 未经低温处理的生根胚 0.00 ± 0.00 b 0.00 ± 0.00 b 4 ℃低温处理的生根去子叶胚 73.33 ± 4.71 a 2.81 ± 0.47 a 未经低温处理的生根去子叶胚 0.00 ± 0.00 b 0.00 ± 0.00 b 说明:同列小写字母不同表示差异显著(P < 0.05)
Primary studies with Paeonia rockii seed dormancy
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摘要: 为探索紫斑牡丹Paeonia rockii种子休眠原因,对其种皮透性、抑制物质强度(以豌豆Pisum sativum种子为材料)和种子不同部位外植体生长等进行了研究。结果表明:种皮透性对种子吸水率和呼吸速率有一定影响;用生根前的紫斑牡丹种子胚乳、胚、种皮浸提液处理的豌豆均有部分生根率,且生根率依次增大,但上胚轴萌发率均为0;用生根及发芽后的紫斑牡丹种子种皮、胚乳处理豌豆的生根率显著高于生根前的,且均有部分上胚轴萌发,但前两者生根率及上胚轴萌发率无显著差异;完整种子、种皮置于一旁的去皮种子、去皮种子、胚(裸胚或去子叶胚)的紫斑牡丹生根率依次提高,发芽率均为0;紫斑牡丹胚及去子叶胚均需要经过低温处理后上胚轴才可以萌发。由此说明:①造成紫斑牡丹种子下胚轴休眠的因素是种皮的透性、种皮及胚乳中的抑制物质。②紫斑牡丹种子上胚轴休眠的原因在胚内部,而与种子在生根后及发芽过程中种皮及胚乳中抑制物质强度的变化无关,去除种皮、胚乳及子叶无法解除其休眠,上胚轴休眠需要经过低温过程方可解除。Abstract: To determine causes of seed dormancy with Paeonia rockii seeds, this research studied seed coat permeability, inhibiting substance strengths using peas as the material, the growth status of different explant cultures and did difference analysis. Results showed that permeability of seed coat had certain effects on water absorption and respiration intensity of seed; some of the peas placed in endosperm, embryo, and seed coat extracts with P. rockii seeds before germination, took root with rooting rates of endosperm < embryo < seed coat; however, none of the epicotyls germinated. Rooting rates of peas in endosperm and seed coat extracts with germinated or sprouted P. rockii seeds were higher than those with seeds that had not germinated; they all had partial epicotyls sprouting, but no differences were found between those with germinated and sprouted P. rockii seeds. Rooting rates of P. rockii increased with complete seeds < peeled seeds with seed coats alongside < peeled seeds < embryos (embryos or embryos without cotyledons); however, none sprouted. Both embryos and embryos without cotyledons required low temperature treatment before they could germinate. Thus, (1) seed coat permeability, inhibitory substances in the seed coat and endosperm were reasons for hypocotyl dormancy with P. rockii seeds; (2) changes of inhibitory substances from rooted seeds to sprouted seeds had nothing to do with epicotyl dormancy meaning reasons for epicotyl dormancy were probably inside the embryo; and (3) low temperature could release epicotyl dormancy, but removing seed coat, endosperm, or cotyledons could not.
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表 1 不同处理紫斑牡丹种胚的上胚轴萌发率及上胚轴长
Table 1. Epicotyl germination rate and length of Paeonia rockii embryo in different treatments
处理组 上胚轴萌发率/% 上胚轴长/cm 4 ℃低温处理的生根胚 74.44 ± 4.16 a 2.71 ± 0.44 a 未经低温处理的生根胚 0.00 ± 0.00 b 0.00 ± 0.00 b 4 ℃低温处理的生根去子叶胚 73.33 ± 4.71 a 2.81 ± 0.47 a 未经低温处理的生根去子叶胚 0.00 ± 0.00 b 0.00 ± 0.00 b 说明:同列小写字母不同表示差异显著(P < 0.05) -
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https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2018.03.014