Effects of ethylene on the viability of cryopreserved Dendrobium protocorm-like bodies
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摘要:目的 为探究乙烯对PLBs超低温保存存活率的影响，以期为提高其存活率开拓思路。方法 以春石斛‘红梦幻’类原球茎（PLBs）为材料，分别在其玻璃化超低温保存的预培养和装载阶段添加外源乙烯供体乙烯利和乙烯合成抑制剂硝酸钴，测定PLBs超低温保存过程中内源乙烯前体1-氨基环丙烷羧酸（ACC）含量、ACC氧化酶（ACO）活性及PLBs液氮保存后细胞存活率。结果 结果显示：无论在预培养还是装载阶段，外源乙烯供体乙烯利处理后，超低温保存过程中PLBs内源ACC含量和ACO活性均显著提高，同时液氮保存后存活率也显著提高；在这两个阶段添加外源乙烯合成抑制剂硝酸钴后，超低温保存过程中PLBs内源ACC含量、ACO活性显著降低，液氮保存后存活率也显著降低。相关分析显示，PLBs液氮冻后存活率与其内源ACC含量和ACO活性呈极显著正相关。结论 本研究表明，外源乙烯调节剂乙烯利和硝酸钴通过调节内源乙烯含量而对玻璃化超低温保存的春石斛类原球茎存活率产生显著影响。Abstract:Objective This paper aims to explore the effects of ethylene on the survival rate of PLBs under cryopreservation to develop ideas for improving the survival rate of PLBs.Method The protocorm-like bodies (PLBs) of Dendrobium nobile ‘Hamana Lake Dream’ were used as materials, exogenous ethylene donor ethephon and ethylene synthesis inhibitor cobalt nitrate were added during the preculture and loading stages of its vitrification cryopreservation, respectively to study the effects of ethylene on cell survival rate, endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) content and ACC oxidase (ACO) activity of the cryopreserved PLBs.Result The addition of exogenous ethylene ethephon can significantly improve the endogenous ACC content, ACO activity and cell survival rate of Dendrobium PLBs after cryopreservation by vitrification. While, the addition of exogenous ethylene inhibitor cobalt nitrate significantly reduced the endogenous ACC content, ACO activity and the cell survival rate of PLBs after cryopreservation by vitrification. In addition, the survival rate of PLBs after cryopreservation was significantly positively correlated with endogenous ACC content and was extremely significantly positively correlated with endogenous ACO activity.Conclusion This study shows that the exogenous ethylene regulator ethephon and cobalt nitrate has a significant effect on the viability of Dendrobium PLBs after vitrification-cryopreservation by regulating endogenous ethylene.
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Keywords:
- Dendrobium protocorm-like body /
- ethephon /
- cobalt nitrate /
- ACC /
- ACO /
- cryopreservation /
- survival rate
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鸢尾属育种历史久远，但多是近缘种间、品种间杂交后代，性状变异单一，育种价值较小。种间远缘杂交是观赏植物的重要育种手段[1]。1999年，清水弘用花菖蒲(Iris ensata var. hortensis)品种和黄菖蒲(I.pseudacorus)杂交，获得一系列具有眼影型眼斑的品种群，统一命名为Pseudata Irises, 即眼影鸢尾[2]。德国育种学家Tomas研究鸢尾种间杂交多年，利用金脉鸢尾亚类和山鸢尾(I.setosa)间杂交，杂交后代垂瓣喉部为黄色，后代‘Berlin tiger’网脉明显、遍布垂瓣，花量大、观赏价值极高；金脉鸢尾亚类原种和马蔺(I.lactea var. chinensis)杂交得到的后代稍有香味，一些后代品种垂瓣变窄，网脉似马蔺，但是花径比马蔺大[3]。Chimphamba[4]于1973年进行野鸢尾(Iris dichotoma)和射干(Belamcanda chinensis)的育种工作，后经一系列回交、杂交得到花色丰富、当年开花的可育后代群体，命名Candy lily，即糖果鸢尾。
鸢尾属种内亲和性较高，多可得到杂种，但种间杂交障碍显著，杂交亲和性低，目前获得杂种较少，杂种后代部分可育[5]。植物杂交障碍可分为受精前障碍、受精后障碍、杂种不育3种情况[6]：杂种不育可通过回交、染色体加倍获得后代；鸢尾受精前障碍主要表现为柱头和花粉管胼胝质明显[7]、花粉不萌发[8]等，可通过涂抹法[8]、提前或延后授粉[9]等促进花粉管进入胚珠；鸢尾受精后障碍主要表现为胚囊解体[10]、胚乳退化[11]等，通过胚拯救得到杂种，目前已成功得到的种间杂种有燕子花(I. laevigata)×玉蝉花(I. ensata)[12]、暗黄鸢尾(I. fulva)(4x)×燕子花[13]、黄菖蒲×花菖蒲[14]。
黄菖蒲属于无髯鸢尾类的燕子花鸢尾类(Series Laevigatae)，2n=32[15]；蓝花喜盐鸢尾(I. halophila var. sogdiana)属于无髯鸢尾类的拟鸢尾类(Series spuriae)，2n=44[16]；西伯利亚鸢尾(I. sibirica)属于无髯鸢尾类的西伯利亚鸢尾类(Series sibiricae)，所选的5个品种中，分为两种类型：‘Dancing Nanou’、‘Lichter feidills’和‘Fourfold Lavendel’为二倍体，2n=28, ‘Big’s child’和‘Viel Creme’为四倍体，2n=4x=56[17]。为获得变异丰富的杂种后代，分析染色体条数不同的种间杂交障碍以及二倍体和四倍体在种间杂交中的区别，进行鸢尾种间常规杂交，观测子房发育动态和花粉原位萌发，分析鸢尾种间杂交障碍，为鸢尾种间杂交育种提供科学理论依据。
1. 材料与方法
实验材料为黄菖蒲、蓝花喜盐鸢尾、西伯利亚鸢尾的5个品种：‘Dancing Nanou’(2x)、‘Lichter feidills’(2x)、‘Big’s child’(4x)、‘Fourfold Lavendel’(2x)、‘Viel Creme’(4x)。材料均栽培在国家花卉工程中心小汤山苗圃(40°15″N，116°44″E)。
分别以黄菖蒲、喜盐鸢尾为亲本和5种西伯利亚鸢尾品种正反交。对于选定的杂交亲本，在开花前一天去除垂瓣。开花当天08:00—10:00授粉。授粉后2、4、6、12、24、48 h，每个杂交组合采集3朵授粉后花的花柱和子房，卡诺固定液(V(乙醇):V(冰醋酸)=3:1)固定3 h，后转移至70%酒精中，4 ℃冰箱储存备用。水溶性苯胺蓝(苯胺蓝以0.1%的比例溶于0.1 mol/dm3 K3PO4)染色后，LEICA荧光显微镜下观察。
授粉后每3 d，用游标卡尺记录子房的宽度。种子成熟后统计结实率，即采即播，并随时观察种子生长情况，统计种子萌发率。
2. 结果与分析
2.1 子房发育动态观测
黄菖蒲和西伯利亚鸢尾杂交，授粉16 d左右发现黄菖蒲为母本时子房明显膨大，若子房与果柄的连接处出现白色横线，子房很快脱落。以西伯利亚鸢尾为母本时，‘Dancing Nanou’、‘Fourfold Lavendel’、‘Viel Creme’的子房膨大明显，部分子房在5 d左右时萎蔫，膨大的子房生长分成两种情况：(1)不发生败育，子房发育比自交慢；(2)子房膨大到12 d左右时，子房变黄，横切子房，胚珠未膨大，仅子房璧膨大；‘Lichter feidills’的部分子房发生膨大，子房发育比自交慢，10 d左右变黄后脱落；‘Big’s child’子房膨大明显，膨大后不败育，杂交子房和亲本自交的子房前期长势基本一致(图 1)。
喜盐鸢尾和西伯利亚鸢尾杂交，喜盐鸢尾为母本，子房全部膨大，但生长势不均匀。5个杂交组合中子房发育情况有两种：(1)稍有膨大，7 d左右变黄，干枯不脱落；(2)膨大一定程度后，即停止，表皮保持绿色。‘Dancing Nanou’、‘Lichter feidills’为母本，部分子房膨大，9 d左右，子房下部变黄皱缩，出现败育迹象，横切子房，胚珠未膨大；‘Big’s child’、‘Fourfold Lavendel’、‘Viel Creme’为母本，子房全部膨大，15 d左右，子房棱部变黄，出现败育迹象，未完全败育的子房中获得有少量种子(图 1)。
2.2 花粉管原位萌发与杂交障碍分析
杨占辉[7]曾对该研究所用亲本花粉活力进行鉴定，均较高，可排除花粉活力的影响。
以黄菖蒲为母本，和西伯利亚鸢尾杂交2 h后，花粉萌发量均较少(图 2A)，虽然萌发量随时间增加，但是回折缠绕明显(图 2B)，进入花柱的少数花粉生长缓慢，仅‘Dancing Nanou’、‘Fourfold Lavendel’的花粉管进入胚珠(图 2C)。以西伯利亚鸢尾为母本，和黄菖蒲杂交时，‘Dancing Nanou’、‘Big’s child’、‘Fourfold Lavendel’、‘Viel Creme’柱头上的花粉萌发量较大，少量花粉管在48 h后到达胚珠(图 2D~G)；‘Lichter feidills’柱头上花粉极少数萌发，且相互缠绕，48 h后仅到达子房顶部，未到达胚珠(图 2H)。
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图 2 花粉管萌发A.I.pseudacorus בLichter feidills’授粉后2 h；B. I. pseudacorus בLichter feidills’授粉后6 h；C.I. pseudacorus בFourfold Lavendel’授粉后48 h；D. ‘Dancing Nanou’ × I. pseudacorus授粉后48 h；E. ‘Big’s child’ × I. pseudacorus授粉后48 h；F. ‘Fourfold Lavendel’ × I. pseudacorus授粉后48 h；G.‘Viel Creme’ ×I. pseudacorus授粉后48 h；H. ‘Lichter feidills’ × I.pseudacorus授粉后48 h；I. I. halophila var. sogdiana × ‘Lichter feidills’授粉后2 h；J. I. halophila var. sogdiana × ‘Viel Creme’授粉后2 h；K. I. halophila var. sogdiana × ‘Dancing Nanou’授粉后48 h；L. I. halophila var. sogdiana × ‘Lichter feidills’授粉后48 h；M. I. halophila var. sogdiana בFourfold Lavendel’授粉后48 h；N. ‘Dancing Nanou’ × I. halophila var. sogdiana 授粉后2 h；O. ‘Big’s child’ × I. halophila var.sogdiana授粉后48 h；P. ‘Lichter feidills’ × I. halophila var. sogdiana授粉后24 h。Figure 2. Germination of pollenA, 2 hours after pollination for I. pseudacorus × 'Lichter feidills'; B, 6 hours after pollination for I. pseudacorus × 'Lichter feidills'; C, 48 hours after pollination for I. pseudacorus × 'Fourfold Lavendel'; D, 48 hours after pollination for 'Dancing Nanou' × I. pseudacorus; E, 48 hours after pollination for 'Big's child' × I. pseudacorus; F, 48 hours after pollination for 'Fourfold Lavendel' × I. pseudacorus; G, 48 hours after pollination for 'Viel Creme' × I. pseudacorus; H, 48 hours after pollination for 'Lichter feidills' × I. pseudacorus; I, 2 hours after pollination for I. halophila var. Sogdiana ×'Lichter feidills'; J, 2 hours after pollination for I. halophila var. sogdiana × 'Viel Creme'; K, 48 hours after pollination for I. halophila var. sogdiana × 'Dancing Nanou'; L, 48 hours after pollination for I. halophila var. sogdiana × 'Lichter feidills'; M, 48 hours after pollination for I. halophila var. sogdiana ×'Fourfold Lavendel'; N, 2 hours after pollination for 'Dancing Nanou' × I. halophila var. sogdiana; O, 48 hours after pollination for 'Big's child' × I. halophila var. sogdiana; P, 24 hours after pollination for 'Lichter feidills' × I. halophila var. sogdiana.以喜盐鸢尾为母本，‘Big’s child’为父本时，附着花粉较少，未萌发(图 2I)；‘Viel Creme’为父本时，有大量花粉附着，但几乎不萌发(图 2G)；其他3种西伯利亚鸢尾授粉6 h后附着的花粉均萌发，在48 h后均到达胚珠(图 2K、L、M)。以西伯利亚鸢尾为母本，和喜盐鸢尾杂交2 h后，花粉在柱头上大量萌发(图 2N)，虽有胼胝质的产生，但是并不影响花粉管的生长，仍到达胚珠(图 2O)，其中‘Lichter feidills’为母本时，花粉管生长较快，在授粉后24 h，大量花粉管进入胚珠(图 2P)。5组杂交组合中，导致杂交障碍的原因有4种：(1)花粉附着困难：I. halophila var. sogdiana × ‘Big’s child’；(2)花粉萌发率低：I. halophila var. sogdiana × ‘Viel Creme’、‘Lichter feidills’ × I. pseudacorus；(3)花粉管伸长障碍：I. pseudacorus × ‘Lichter feidills’、I. pseudacorus × ‘Big's child’、I. pseudacorus × ‘Viel Creme’；(4)胚囊败育的受精后障碍：I. pseudacorus × ‘Dancing Nanou’、I. pseudacorus × ‘Fourfold Lavendel’、‘Dancing Nanou’×I. pseudacorus、‘Big's child’×I. pseudacorus、‘Fourfold Lavendel’×I. pseudacorus、‘Viel Creme’ × I. pseudacorus、I. halophila var. sogdiana × ‘Dancing Nanou’、I. halophila var. sogdianaבFourfold Lavendel’、I. halophila var. sogdiana × ‘Lichter feidills’、‘Big’s child’× I. halophila var. sogdiana、‘Fourfold Lavendel’× I. halophila var. sogdiana、‘Lichter feidills’ × I. halophila var. sogdiana、‘Dancing Nanou× I. halophila var. sogdiana’、‘Viel Creme’ × I. halophila var. sogdiana。
2.3 杂交结实及萌发情况
在以黄菖蒲为母本时，结实率极低，仅‘Dancing Nanou’、‘Lichter feidills’、‘Fourfold Lavendel’、‘Viel Creme’得到了种子，种子萌发率低，而反交组合除‘Lichter feidills’未结实外，结实率明显增高，但种子多干瘪无胚，萌发率低。蓝花喜盐鸢尾为母本，除‘Viel Creme’外，其他杂交组合结实率相对较高，播种后，除I. halophila var. sogdiana בFourfold Lavendel’不萌发外，其他萌发率较高。反交组合中，‘Fourfold Lavendel’、‘Viel Creme’得到了种子，‘Fourfold Lavendel’为母本获得的种子萌发。
表 1 杂交结实萌发情况Table 1. Germination condition of hybrid seed setting杂交组合
Cross combination杂交数量
Pollination quantity结实率
Fruit setting rate/%种子数量
Seed quantity萌发率
Germination rate/%I.pseudacorus × ‘Dancing Nanou’ 50 8.00 83 43.37 I.pseudacorus × ‘Lichter feidills’ 53 3.77 5 0.00 I.pseudacorus × ‘Big’s child’ 33 0.00 0 0.00 I.pseudacorus × ‘Fourfold Lavendel’ 51 3.92 12 25.00 I.pseudacorus × ‘Viel Creme’ 43 2.32 2 0.00 ‘Dancing Nanou’ × I. pseudacorus 65 26.15 487 0.41 ‘Lichter feidills’ × I. pseudacorus 137 0.00 0 0.00 ‘Big’s child’ × I. pseudacorus 13 92.31 250 0.00 ‘Fourfold Lavendel’ × I. pseudacorus 90 10.00 169 11.83 ‘Viel Creme’ × I. pseudacorus 47 31.91 357 0.00 I.halophila var . sogdiana× ‘Dancing Nanou’ 104 95.96 518 55.21 I.halophila var . sogdiana× ‘Lichter feidills’ 34 26.47 34 14.71 I.halophila var . sogdiana× ‘Big’s child’ 16 87.50 14 92.86 I.halophila var . sogdiana× ‘Fourfold Lavendel’ 56 3.57 8 0.00 I.halophila var . sogdiana× ‘Viel Creme’ 50 0.00 0 0.00 ‘Dancing Nanou’ × I. halophila var. sogdiana 66 0.00 0 0.00 ‘Lichter feidills’ × I. halophila var. sogdiana 116 0.00 0 0.00 ‘Big’s child’ × I. halophila var. sogdiana 22 0.00 0 0.00 ‘Fourfold Lavendel’ × I. halophila var. sogdiana 117 3.42 80 27.50 ‘Viel Creme’ × I. halophila var. sogdiana 97 12.37 168 0.00 3. 结论与讨论
无髯鸢尾远缘杂交败育时间均集中在授粉后9~16 d。子房胚败育可以通过子房生长状况动态观察，确定败育时间范围，及时进行胚拯救，获得远缘杂交后代。同样，Yabuya[13]在燕子花×玉蝉花远缘杂交过程中，及时观测子房发育，正反交组合分别在授粉后6 d和15 d发现子房败育迹象，进行胚拯救获得杂种。
花粉原位萌发取材方便，观察过程短，可以作为远缘杂交前检测指标。无髯鸢尾种间杂交障碍，主要原因有花粉附着或萌发困难、花粉管伸长困难、胚囊败育等，其中花粉附着困难、花粉管发育不良等受精前障碍使结实率极低，可在检测后采取药物涂抹柱头、提前或延期授粉等方法克服受精前障碍，提高杂交工作效率。马蔺花粉在溪荪(I.sanguinea)柱头上不萌发，通过涂抹激素和提前授粉获得种间杂种[8]；马蔺和黄菖蒲杂交受精障碍显著，不结实，提前授粉后获得种子[9]；涂抹激素、切割柱头等授粉方式增加了百合(Lilium brownii var. viridulum)的结实率[18]。花粉顺利进入胚珠后，结实率和胚、胚乳发育相关性高；若胚珠发育不良，结实率极低，可动态观测子房发育，出现败育迹象后及时进行胚拯救，增大杂种获得率。百合在子房膨大一定程度后进行胚拯救得到杂种后代[18]。
黄菖蒲和西伯利亚鸢尾杂交时，西伯利亚鸢尾适合做母本；喜盐鸢尾和西伯利亚杂交时，喜盐鸢尾适合做母本，喜盐鸢尾和西伯利亚鸢尾的亲和性高于黄菖蒲。同一物种的不同品种和其他种的杂交亲和性不同，而且在染色体条数不同的种间杂交中，二倍体和四倍体的亲和性无明显区别。不同物种间杂交，正反交的杂交障碍不同，杂交结实情况差别较大，可以尝试不同品种类型并同时进行正反交，增加无髯鸢尾种间杂交亲和性。
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图 1 预培养阶段添加外源乙烯调节剂乙烯利和硝酸钴对超低温保存后春石斛‘红梦幻’PLBs存活率的影响
图中数据用3个重复的平均值 ± 标准误表示，同阶段下不同小写字母表示各处理间差异显著（P < 0.05）。下同。Each bar represents mean ± SE over four repetitions. Columns with different letters are significantly different (P < 0.05). The same below.
Figure 1. Effects of adding exogenous ethylene regulator ETH and Co at the preculture stage on the survival rate of Dendrobiumnobile ‘Hamana Lake Dream’ PLBs after cryopreservation
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图 2 装载阶段添加外源乙烯调节剂乙烯利和硝酸钴对超低温保存后春石斛‘红梦幻’PLBs存活率的影响
Figure 2. Effects of adding exogenous ethylene regulator ETH and Co at the loading stage on the survival rate of D. nobile ‘Hamana Lake Dream’ PLBs after cryopreservation
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图 3 外源乙烯调节剂乙烯利和硝酸钴的添加对玻璃化超低温保存中PLBs中内源ACC含量的影响
A、B分别表示在预培养阶段和装载阶段添加外源乙烯调节剂（乙烯利和硝酸钴）对玻璃化超低温保存下PLBs内源ACC含量的影响。Pr. 预培养阶段；L. 装载阶段；UNL. 去装载阶段；CK. 常规玻璃化程序；Pr+Eth和Pr+Co分别指预培养阶段添加乙烯利和硝酸钴的玻璃化程序；L+Eth和L+Co分别指装载阶段添加乙烯利和硝酸钴的玻璃化程序。下同。A, B are the effects of exogenous ethylene added in preculture medium or loading stage on endogenous ACC in PLBs under cryopreservation. Pr, preculture stage; L, loading stage; UNL, unloading stage; CK, common vitrification procedure; Pr+ETH and Pr+Co refer to vitrification procedure with ETH or Co addition at the preculture stage, respectively; L+ETH and L+Co refer to vitrification procedure with ETH or Co addition at the loading stage, respectively. The same below.
Figure 3. Effects of the addition of exogenous ethylene regulators ETH and Co on the content of endogenous ACC in PLBs during vitrification cryopreservation
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图 4 外源乙烯调节剂乙烯利和硝酸钴的添加对玻璃化超低温保存中PLBs中内源ACO活性的影响
Figure 4. Effects of the addition of exogenous ethylene regulators ETH and Co on the activity of endogenous ACO in PLBs during vitrification cryopreservation
表 1 存活率和内源ACC、ACO的简单相关系数矩阵
Table 1 Correlation coefficient matrix analysis of survival rate, endogenous ACC content and ACO activity
指标 Index 存活率 Survival rate ACC ACO 存活率 Survival rate 1.000 0.741* 0.956** ACC 1.000 0.732* ACO 1.000 注：**表示差异极显著(P < 0.01)，*表示差异显著(P < 0.05)。
Notes: ** indicates that the difference is extremely significant (P < 0.01), and * indicates that the difference is significant (P < 0.05).
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