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核桃(Juglas regia L.)是核桃科核桃属植物,在植物分类中属于落叶阔叶型高大乔木。体细胞胚(下称:“体胚”)具有数量多、繁殖快、结构完整、植株再生率高以及不受季节影响等特点,是遗传转化的优良试材,也是人工种子制备的基础[1]。自Tuleche等首次报道核桃的体细胞胚发生以来,相继在核桃属的其他种和杂交品种中成功应用[2]。核桃体胚的诱导和发生阶段,外植体与培养基(包含植物生长调节剂)起决定作用。研究表明,DKW培养基是核桃体胚发生的首选培养基,幼胚及其组成成分作为外植体时胚性感受态水平较高[3]。不同果实发育时期的幼胚及其组成成分具有不同的胚性感受态水平,果实发育早期,体胚诱导效果不明显,易产生愈伤组织,而果实发育成熟期,子叶更易于产生不定根[4-6]。前人对采样时间及幼胚形态已有相关报道[7],然而,不同取材时间的形态特征与对应的外观表型的关系还需深入研究。此外,随着胚性愈伤组织培养时间的延长和继代次数的增多,体胚畸变率逐渐升高,再生能力逐渐下降[8]。在核桃的继代培养过程中,通常出现连体胚、子叶畸形胚及玻璃化胚等几种类型的畸形胚,在球形胚阶段人工分离子代体胚或筛选畸形子叶胚可在一定程度降低畸变率,但效率较低[1]。适量浓度的ABA处理不仅能抑制针叶树畸形体胚的产生,而且能提高植株再生率[9]。探索ABA在抑制核桃体胚畸变中的作用,将提高核桃体胚发生及再生效率。在核桃的体胚萌发阶段,存在萌发率较低的问题。低温或者不同干燥处理可提高萌发率。Tuleche等对核桃2℃低温暗培养8~10周后获得核桃的完整植株[2]。Tang等[10]用3~4℃处理了2~3个月的核桃体胚,仅4%~19%萌发为完整植株。张启香等[11]用饱和Ca(NO3)2·4H2O对山核桃体胚进行脱水处理,可将预处理时间缩短至3 d,最高萌发率达39.03%。
早实核桃是我国核桃主栽类型之一。《中国果树志·核桃卷》记载,我国有104个早实核桃类群,尤其以新疆地区较多[12]。早实核桃实生苗的幼龄期为1~3 a,具有结实早、丰产性强等特点[13]。然而,由于其开花年龄提前,树体较小,生命周期较短;同时,早实核桃种子形成过程中胚发育程度也存在差异,在生产中易出现播种出苗率低的现象;在试管继代培养中发现早实核桃容易瓶内开花,从而影响嫩茎的生长[14]。因此,研究早实核桃的体胚发生和再生技术对核桃快繁体系的建立具有重要实践意义,同时也为核桃的基因功能研究奠定了基础。
本研究以‘林早香’(J. Regia)和‘中林6号’(J. regia ‘Zhonglin 6’)花后42~77 d的幼果为试验材料,分别研究了果实发育时期对诱导体胚发生的影响,体胚继代培养过程中ABA对中畸形胚的调节作用以及适度脱水对体胚萌发的促进作用,以期建立高效、稳定的体胚发生和植株再生体系,为早实核桃的工厂化繁育提供实验平台,为早实核桃的遗传转化奠定基础。
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核桃体胚发生与幼果的发育时期有密切关系,不同发育时期的幼果诱导产生体细胞胚的能力存在显著差异。随着‘林早香’和‘中林6号’幼果的生长,体胚诱导率呈先升高后降低的趋势,分别于花后约49 d和56 d达到最高,最大诱导率分别为88.32%±2.28%和86.67%±1.57%(图 1)。
图 1 幼胚发育时期对体胚发生的影响
Figure 1. The effect of immature embryo development stages on the somatic embryogenesis
不同发育时期幼果的外观表型也不同。‘中林6号’幼果发育比‘林早香’晚7 d左右。图 2所示,‘中林6号’5个采样时期的幼果,花后42 d,果实膨大,果皮密被黄绿色柔毛,果顶渐尖,柱头和雌蕊未脱落,种室内胚乳胶状,部分果实出现球形胚或鱼雷胚;花后49 d,果皮密被柔毛变少,果顶变圆,柱头枯萎未脱落,幼胚为乳白色翅状;花后56 d,果实迅速膨大,果皮变光滑,果壳变硬,子叶伸展,柱头逐渐脱落;花后63 d,子叶迅速伸展,出现多个内褶;花后72 d,子叶充满种室,柱头完全脱落。
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随着继代培养次数的增多,子代体胚出现大量畸形胚,从而影响体胚的萌发。‘中林6号’继代20代时,体胚的畸变率约为8.67%,继代培养40代时,畸变率上升到约43.33%,而‘林早香’畸变率略低(图 3A)。适宜浓度的ABA可提高体胚诱导率并降低体胚畸变率。随着ABA浓度的增加,体胚畸变率呈下降趋势,ABA浓度>1.0 mg·L-1时,ABA的调节效应不明显;而体胚诱导率呈现先升高后降低的趋势,ABA浓度>1.0 mg·L-1时,体胚子叶干皱,呈现老化状态,体胚诱导率降低(图 3B)。因此,最佳ABA调节浓度为1.0 mg·L-1。
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随着成熟体胚脱水时间的延长,体胚萌发率呈现先升高后降低的趋势(表 1)。脱水处理0~24 h的体胚,胚根伸长,但难成苗;脱水处理48 h‘中林6号’和‘林早香’的体胚萌发率显著提高,分别为16.67%±0.80%和18.82%±0.22%;脱水72 h后,‘中林6号’和‘林早香’体胚萌发率最高,分别为53.33%±0.13%和56.67%±0.39%;若干燥时间继续延长,体胚的萌发率下降,部分体胚的子叶和生长点焦枯,甚至坏死,影响体胚的萌发。
表 1 脱水对两种早实核桃体细胞胚植株再生的影响
Table 1. Effect of deciccation on germination of two precocious walnuts
处理
Treament脱水时间/h
Desiccation length萌发率/%
Germination percentage‘林早香’ ‘中林6号’ 1(对照Control) 0 6.67±0.06 F 3.36±0.12 e 2 24 8.78±0.12 D 3.34±0.05 e 3 48 18.82±0.22 C 16.67±0.80 c 4 72 56.67±0.39 A 53.33±0.13 a 5 96 35.56±0.14 B 35.56±0.20 b 6 120 7.12±0.13 E 6.67±0.07 d 将脱水处理后的体胚置于2.0 mg·L-16-BA+0.01 mg·L-1 IBA的固体培养基进行光照培养,4~5 d之后胚根伸长,茎端生长点变为绿色(图 3A),1个月后可发育成完整植株(图 3B)。
早实核桃体细胞胚发生和植株再生的影响因素研究
Analysis on Key Factors Affecting Somatic Embryogenesis and Plant Regeneration in Precocious Walnuts
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摘要:
目的 建立2种早实核桃高效和稳定的体胚发生体系,为离体快繁技术及其基础研究提供技术和理论支撑,也为核桃遗传转化体系的构建奠定基础。 方法 分别以实生核桃‘林早香’和嫁接核桃‘中林6号’花后42~77 d的幼胚为试材进行离体培养,通过统计体胚诱导率,筛选体胚的最佳诱导时期;统计多次继代的体胚畸变率,并筛选最适ABA调节浓度;选取发育成熟的体胚进行饱和NH4NO3溶液脱水处理,分别处理0、24、48、72、96和120 h后统计失水量和转株率,探讨脱水程度对体胚植株再生的影响。 结果 外植体的发育时期对体胚发生至关重要,以‘林早香’和‘中林6号’幼胚为外植体进行体胚诱导,最佳诱导时期分别为花后约49 d和56 d,体胚诱导率分别为88.32%和86.67%。体胚经多次继代培养后产生较高畸变率,适量ABA处理不仅降低体胚畸变率,且提高体胚发生率,最佳ABA调节浓度为1.0 mg·L-1。随着体胚脱水时间的延长,体胚失水量逐渐增加,植株再生率先升高后降低,脱水72 h时,‘林早香’和‘中林6号’的体胚萌发率达到最高,分别为56.67%和53.33%,此时失水量分别为38.73%和40.56%。 结论 早实核桃幼果发育过程中,合子胚的体胚发生率随着发育时期变化,适宜发育时期是提高体胚诱导发生的关键;适量ABA在体胚多次继代培养过程中对畸形胚起重要调节作用;适度的脱水处理对核桃体胚再生具有良好的促进作用。 Abstract:Objective To establish an efficient and stable somatic embryogenesis system, to provide technical and theoretical support for in vitro rapid propagation and its further research, and to lay a foundation for the construction of gene transformation of walnut. Method Two early-bearing walnut young fruits were sampled during 42-77 days after blooming. Somatic embryogenesis percentages were counted to screen the optimum development stage of the fruit. After subcultured for several times, the somatic embryos distortion percentage was calculated and then the optimum ABA concentration was tested. The somatic embryos were desiccated respectively for 0, 24, 48, 72, 96 and 120 h with saturated NH4NO3, and the germination rate of somatic embryo can be obtained. Result The highest somatic embryogenesis percentage of 'Zao Lin Xiang' and 'Zhong Lin 6' reached 88.32% and 86.7%, respectively, when the young fruits were sampled at 49 d and 56 d after flowering, respectively. Moderate concentration of ABA supplement not only reduced the rate of somatic embryos aberration, resulted from multiple subcultivation, but also increased the rate of somatic embryogenesis, and the optimum ABA concentration was 1 mg·L-1. As the dehydration time was prolongated, the water loss increased gradually, while the germination rate increased at first and decreased later on. After dehydrated for 72 h, the germination rate of 'Zao Lin Xiang' and 'Zhong Lin 6' were up to the highest (56.67% and 53.33%), while the water loss was 38.73% and 40.56%, respectively. Conclusion In the process of young fruit development of early-bearing walnut, the inducing rate of somatic embryogenesis is changed with the development period. The suitable development period is the key to improve somatic embryo induction. Proper amount of ABA also plays an important role in regulating deformed embryo in the process of somatic embryo multiple subculture. What is more, moderate dehydration treatment has a well promoting effect on the regeneration of somatic embryo of walnut. -
Key words:
- precocious walnut
- / somatic embryogenesis
- / plant regeneration
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表 1 脱水对两种早实核桃体细胞胚植株再生的影响
Table 1. Effect of deciccation on germination of two precocious walnuts
处理
Treament脱水时间/h
Desiccation length萌发率/%
Germination percentage‘林早香’ ‘中林6号’ 1(对照Control) 0 6.67±0.06 F 3.36±0.12 e 2 24 8.78±0.12 D 3.34±0.05 e 3 48 18.82±0.22 C 16.67±0.80 c 4 72 56.67±0.39 A 53.33±0.13 a 5 96 35.56±0.14 B 35.56±0.20 b 6 120 7.12±0.13 E 6.67±0.07 d -
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