栓皮栎体细胞胚胎诱导的研究
摘要
栓皮栎（Ｑｕｅｒｃｕｓ ｖａｒｉａｂｉｌｉｓ Ｂｌ）是我国最重要的软木资源树种。其种子
不耐贮藏，扦插繁殖困难，目前主要依靠天然更新。本研究以栓皮栎未成熟合子
胚、成熟合子胚和２～４周龄的实生幼苗茎段作为外植体，诱导体胚发生。系统地
研究了栓皮栎体细胞胚胎发生和植株再生所涉及的各种影响因素和体胚发生发
育过程中的组织学和细胞学特征。研究成果将为栓皮栎优良无性系繁殖、基因保
存、遗传改良，以及人工种子生产奠定基础。具体结果如下：
1. 未成熟合子在 ＭＳ 和 ＷＰＭ 基本培养基上均能诱导出体胚。两者相比，培养于
ＭＳ 上的合子胚不但生长健壮，诱导出的体胚数量较多，个体较大，而且体胚
诱导率也较高。１６ｈ．ｄ-1 光照培养和暗培养条件，体胚诱导率无显著差异，但
光培养下的体胚生长迅速而且健壮，暗培养下的部分体胚在发育的过程中会
愈伤组织化且诱导出的胚性愈伤组织在去掉激素后需要较长的时间才能分化
出体胚。
2. ＢＡＰ 与 ＴＤＺ 单独使用时，均以直接体胚发生为主，但前者诱导出的体胚数量
较多，质量高，发生早，发育快。二者分别与 ２，４－Ｄ 配合使用可以显著提高
诱导效率，以间接体胚发生为主。
3. 7 月中旬采的未成熟合子胚体胚诱导率较高，6 月中旬和 8 月中旬的未成熟合
子胚体胚诱导率较低，只有个别成熟合子胚诱导出体胚而幼苗茎段未能诱导
出体胚。不同发育时期的未成熟合子胚对激素配比要求也有一定差异。7 月
中旬的未成熟合子胚对激素浓度适应范围较广，但以同时附加 ０．１ ｍｇ．Ｌ-1
２，４－Ｄ 和 ０．５ ｍｇ．Ｌ-1 ＢＡＰ 诱导效果为佳，8 月中旬的未成熟合子胚要求较高浓
度的 2,4-D，而 6 月中旬未成熟合子胚则正好相反。
4. 同一时间采自同一母树上的不同大小的未成熟合子胚体胚诱导率无显著差
异，但采自不同母树上的未成熟合子胚体胚诱导率差异很大。未成熟合子胚
的手术不利于体胚发生，全胚接种不仅诱导率最高，而且长出的体胚数量也
多，品质也好。胚轴是体胚发生的重要部位，具胚轴的合子胚片段体胚诱导
率显著高于无胚轴片段。合子胚的手术对体胚诱导率的影响可以通过在培养
剂中添加 ２，４－Ｄ 而得以部分补偿。
5. 栓皮栎体胚增殖较为适合的培养基为附加 ０．５ ｍｇ．Ｌ-1 ＢＡＰ 的 ＭＳ 培养基；体
胚成熟较为适合的培养基为附加含有 ４～５％的蔗糖的 ＭＳ 培养基，而 ＡＢＡ 并不
能有效地促进体胚成熟；体胚萌发较为适宜的培养基为 １／２ＭＳ 培养基。光培
养和暗培养下萌发率无显著差异，但暗培养不利于体胚苗的展叶，所以
１６ｈ．ｄ-1的光照条件对栓皮栎体胚萌发较为适宜。萌发培养基中添加ＢＡＰ和ＩＢＡ
均不利于体胚的正常萌发。０．３ 和 ０．７ｍｏｌ．Ｌ-1 的山梨醇渗透预处理 １５ｄ 和冷
处理 １５ｄ、７０ｄ、９０ｄ 对体胚萌发率均无显著影响。
6. 组织学观察表明栓皮栎次生体胚和间接体胚为单细胞外起源。个别原胚具有
“胚柄” 状结构。个别成熟体胚的子叶中有成片的多核细胞存在，核数最多
达 34 个。
7. 观察 ８ 个多月的胚性愈伤组织染色体标本，其中四倍体细胞占 1. 87%，单倍
体细胞占 0.93%，二倍体细胞占 97.20%，未发现有异形核和明显的染色体形
态结构变异和非整倍性变异。
Quercus variabilis is one of the primary sources of industrial cork in China.
Presently its stands were established mostly by natural regeneration as a result of
difficult to storage seeds and conventional vegetative propagation such as cutting.
Immature and mature zygotic embryos as well as young stem segments of seedlings
were used as a source of initial explants. We studied on the effects of factors and the
histological and cytological characteristics that related to the somatic embryogenesis
and plant regeneration of Q. variabilis. The results would lay a good foundation for
propagation excellent clones, gene preservation, genetic improvement and large-scale
artificial seed production for Q. variabilis. The results were as follows:
1. Somatic embryos can be induced on MS and WPM medium with immature
zygotic embryos. MS medium with 16 hours light condition was more favorable
to embryogenesis. Higher embryogenic responses were gained on MS medium
than on WPM medium. Although the embryogenic responses were not significant
with light conditions, better quality was reached at 16 hours light condition than at
dark condition.
2. Direct somatic embryos were gained on MS medium with BAP or TDZ
individually. And the quality of the somatic embryos induced on the former was
higher than on the latter. Indirect embryos were also gained on MS medium
containing 2,4-D, BAP or TDZ individually. Higher inducing rates were obtained
on MS medium with BAP and 2,4-D.
3. More efficient induction of embryogenic cultures was achieved from the immature
zygotic embryos sampled in middle of July. The embryogenic responses from
mature zygotic embryos were very low and there was no somatic embyogenesis
responses from young stem segments of seedlings. The requirment for
combination and concentration of phytohormones changed with the developing
time of immature zygotic embryos: immature zygotic embryos sampled in middle
of July adapted larger range combination and concentration of phytohormones,
but 0.1 mg.L-12,4-D combined with 0.5 mg.L-1 BAP got a better result; those
sampled in August needed higher 2,4-D concentration, while those sampled in
June needed lower 2,4-D concentration.
4. The immature zygotic embryos sampled from different individuals were
significant in the embryogenic responses. But there was not significant with
different scales sampled from the same individual. Surgery on the immature
zygotic embryos was not favorable to embryogenesis and inoculating entire
zygotic embryos got higher embryogenic responses. The hypocotyl was important
section to embryogenesis, and the embryo segments joined to the hypocotyls got
higher embryogenic responses than those without the hypocotyls. It could
compensate the decrease of embryogenic response by adding 2,4-D in cutting
treats culture.
5. MS with 0.5 mg. L-1 BAP could meet the need of proliferation of somatic embryos.
Somatic embryos cultured on MS media contained 4～5% sugar got higher
maturation rates, while ABA had not significant effect on maturation. The mature
somatic embryos were transferred into 1/2MS medium with 16 hours light
condition got a better germination rate. Although the rooting rates, shooting rates
and geminating rates were no significant at 16 hours light condition and at dark
condition, the dark culture was not favorable to leaf spread. 0.3 and 0.7mol.L-1
sorbitol desiccation lasted for 15 days and 4℃ chilling lasted for 15,70 and 90
days had not significant effect on germination rate.
6. Histological study showed that secondary embryos derived from epidermal cells
and from subjacent cells of somatic embryos by unicellular pathway and
multinuclear cells were very common in cotyledon of some somatic embryos and
34-nuclear-cell was found occasionally.
7. By analyzing chromosomes with 8 months of continuous subculture somatic
callus tissue cell demonstrated that there were 1.87%
不耐贮藏，扦插繁殖困难，目前主要依靠天然更新。本研究以栓皮栎未成熟合子
胚、成熟合子胚和２～４周龄的实生幼苗茎段作为外植体，诱导体胚发生。系统地
研究了栓皮栎体细胞胚胎发生和植株再生所涉及的各种影响因素和体胚发生发
育过程中的组织学和细胞学特征。研究成果将为栓皮栎优良无性系繁殖、基因保
存、遗传改良，以及人工种子生产奠定基础。具体结果如下：
1. 未成熟合子在 ＭＳ 和 ＷＰＭ 基本培养基上均能诱导出体胚。两者相比，培养于
ＭＳ 上的合子胚不但生长健壮，诱导出的体胚数量较多，个体较大，而且体胚
诱导率也较高。１６ｈ．ｄ-1 光照培养和暗培养条件，体胚诱导率无显著差异，但
光培养下的体胚生长迅速而且健壮，暗培养下的部分体胚在发育的过程中会
愈伤组织化且诱导出的胚性愈伤组织在去掉激素后需要较长的时间才能分化
出体胚。
2. ＢＡＰ 与 ＴＤＺ 单独使用时，均以直接体胚发生为主，但前者诱导出的体胚数量
较多，质量高，发生早，发育快。二者分别与 ２，４－Ｄ 配合使用可以显著提高
诱导效率，以间接体胚发生为主。
3. 7 月中旬采的未成熟合子胚体胚诱导率较高，6 月中旬和 8 月中旬的未成熟合
子胚体胚诱导率较低，只有个别成熟合子胚诱导出体胚而幼苗茎段未能诱导
出体胚。不同发育时期的未成熟合子胚对激素配比要求也有一定差异。7 月
中旬的未成熟合子胚对激素浓度适应范围较广，但以同时附加 ０．１ ｍｇ．Ｌ-1
２，４－Ｄ 和 ０．５ ｍｇ．Ｌ-1 ＢＡＰ 诱导效果为佳，8 月中旬的未成熟合子胚要求较高浓
度的 2,4-D，而 6 月中旬未成熟合子胚则正好相反。
4. 同一时间采自同一母树上的不同大小的未成熟合子胚体胚诱导率无显著差
异，但采自不同母树上的未成熟合子胚体胚诱导率差异很大。未成熟合子胚
的手术不利于体胚发生，全胚接种不仅诱导率最高，而且长出的体胚数量也
多，品质也好。胚轴是体胚发生的重要部位，具胚轴的合子胚片段体胚诱导
率显著高于无胚轴片段。合子胚的手术对体胚诱导率的影响可以通过在培养
剂中添加 ２，４－Ｄ 而得以部分补偿。
5. 栓皮栎体胚增殖较为适合的培养基为附加 ０．５ ｍｇ．Ｌ-1 ＢＡＰ 的 ＭＳ 培养基；体
胚成熟较为适合的培养基为附加含有 ４～５％的蔗糖的 ＭＳ 培养基，而 ＡＢＡ 并不
能有效地促进体胚成熟；体胚萌发较为适宜的培养基为 １／２ＭＳ 培养基。光培
养和暗培养下萌发率无显著差异，但暗培养不利于体胚苗的展叶，所以
１６ｈ．ｄ-1的光照条件对栓皮栎体胚萌发较为适宜。萌发培养基中添加ＢＡＰ和ＩＢＡ
均不利于体胚的正常萌发。０．３ 和 ０．７ｍｏｌ．Ｌ-1 的山梨醇渗透预处理 １５ｄ 和冷
处理 １５ｄ、７０ｄ、９０ｄ 对体胚萌发率均无显著影响。
6. 组织学观察表明栓皮栎次生体胚和间接体胚为单细胞外起源。个别原胚具有
“胚柄” 状结构。个别成熟体胚的子叶中有成片的多核细胞存在，核数最多
达 34 个。
7. 观察 ８ 个多月的胚性愈伤组织染色体标本，其中四倍体细胞占 1. 87%，单倍
体细胞占 0.93%，二倍体细胞占 97.20%，未发现有异形核和明显的染色体形
态结构变异和非整倍性变异。
Quercus variabilis is one of the primary sources of industrial cork in China.
Presently its stands were established mostly by natural regeneration as a result of
difficult to storage seeds and conventional vegetative propagation such as cutting.
Immature and mature zygotic embryos as well as young stem segments of seedlings
were used as a source of initial explants. We studied on the effects of factors and the
histological and cytological characteristics that related to the somatic embryogenesis
and plant regeneration of Q. variabilis. The results would lay a good foundation for
propagation excellent clones, gene preservation, genetic improvement and large-scale
artificial seed production for Q. variabilis. The results were as follows:
1. Somatic embryos can be induced on MS and WPM medium with immature
zygotic embryos. MS medium with 16 hours light condition was more favorable
to embryogenesis. Higher embryogenic responses were gained on MS medium
than on WPM medium. Although the embryogenic responses were not significant
with light conditions, better quality was reached at 16 hours light condition than at
dark condition.
2. Direct somatic embryos were gained on MS medium with BAP or TDZ
individually. And the quality of the somatic embryos induced on the former was
higher than on the latter. Indirect embryos were also gained on MS medium
containing 2,4-D, BAP or TDZ individually. Higher inducing rates were obtained
on MS medium with BAP and 2,4-D.
3. More efficient induction of embryogenic cultures was achieved from the immature
zygotic embryos sampled in middle of July. The embryogenic responses from
mature zygotic embryos were very low and there was no somatic embyogenesis
responses from young stem segments of seedlings. The requirment for
combination and concentration of phytohormones changed with the developing
time of immature zygotic embryos: immature zygotic embryos sampled in middle
of July adapted larger range combination and concentration of phytohormones,
but 0.1 mg.L-12,4-D combined with 0.5 mg.L-1 BAP got a better result; those
sampled in August needed higher 2,4-D concentration, while those sampled in
June needed lower 2,4-D concentration.
4. The immature zygotic embryos sampled from different individuals were
significant in the embryogenic responses. But there was not significant with
different scales sampled from the same individual. Surgery on the immature
zygotic embryos was not favorable to embryogenesis and inoculating entire
zygotic embryos got higher embryogenic responses. The hypocotyl was important
section to embryogenesis, and the embryo segments joined to the hypocotyls got
higher embryogenic responses than those without the hypocotyls. It could
compensate the decrease of embryogenic response by adding 2,4-D in cutting
treats culture.
5. MS with 0.5 mg. L-1 BAP could meet the need of proliferation of somatic embryos.
Somatic embryos cultured on MS media contained 4～5% sugar got higher
maturation rates, while ABA had not significant effect on maturation. The mature
somatic embryos were transferred into 1/2MS medium with 16 hours light
condition got a better germination rate. Although the rooting rates, shooting rates
and geminating rates were no significant at 16 hours light condition and at dark
condition, the dark culture was not favorable to leaf spread. 0.3 and 0.7mol.L-1
sorbitol desiccation lasted for 15 days and 4℃ chilling lasted for 15,70 and 90
days had not significant effect on germination rate.
6. Histological study showed that secondary embryos derived from epidermal cells
and from subjacent cells of somatic embryos by unicellular pathway and
multinuclear cells were very common in cotyledon of some somatic embryos and
34-nuclear-cell was found occasionally.
7. By analyzing chromosomes with 8 months of continuous subculture somatic
callus tissue cell demonstrated that there were 1.87%
引文
［１］任宪威．树木志（北方本） ［Ｍ］．北京：中国林业出版社，１９９６：１８０－１８３．
［２］中国森林编辑委员会编辑．《中国森林》（第三卷）［Ｍ］．北京：中国林业出版社，１９９９．
［３］李俊英，冯天杰．对麻栎和栓皮栎果实成熟期的商榷［Ｊ］．河北林果研究，１９９８，１３（１）：５８－５９．
［４］闻天声．栓皮栎的培养与用途［Ｊ］．中国林副特产，１９９１，１７（２）：２９．
［５］ 李 文 英 ， 王 冰 ． 栎 类 树 种 的 生 态 效 益 和 经 济 价 值 及 资 源 保 护 对 策 ［Ｊ］． 林 业 科 技 通
讯，２００１（８）：１３－１５．
［６］张存旭，姚增玉．栎属植物体细胞胚胎发生研究现状［Ｊ］．西北植物学报，２００４，２４（２）：３５６－３６２．
［７］陈夜江 ，赖钟雄．果树和林木体细胞胚胎发生的研究与利用［Ｊ］．福建农业大学学报，２００１，
３０（３）：４２０－４２６．
［８］上海植物生理研究所细胞生理研究室．植物细胞培养与细胞杂交研究的进展［Ｊ］．植物学
报，１９７３，１５（２）：２８５－３０７．
［９］汤浩茹，王永清，任正隆．果树的体细胞胚胎发生［Ｊ］．四川农业大学学报，１９９９，１７（１）：６９－７９．
［１０］ 唐 巍 ，杨 映 根， 桂耀林等 ． 松柏类植物体细胞胚胎发生的研究与应用 ［Ｊ］． 植物学通
报，１９９６，１３（１）：２５－３１．
［１１］ 黄 健 秋， 卫志明 ， 许智宏 ． 云南松成熟胚的体细胞胚胎的发生研究［Ｊ］． 实验 生 物学
报，１９９５，２８（４）：３７１－３７５．
［１２］ 邱 德 有 ， 李 如 玉 ， 李 铃 ． 红 豆 杉 及 南 方 红 豆 杉 体 细 胞 胚 胎 发 生 的 研 究 ［Ｊ］．林 业 科
学，１９９８，３４（６）：５０－５４．
［１３］王影，黄敏仁，陈道明等．杨树细胞悬浮培养及体细胞胚胎发生的研究［Ｊ］．南京林业大学学
报，１９９１，１５（３）：３１－３６．
［１４］吴蝴蝶，王泽云．影响橡胶体细胞胚萌发成植株的几个因素［Ｊ］．热带作物研究，１９９７，（２）：５－８．
［１５］ 施季森．迎接 ２１ 世纪现代林木生物技术育种的挑战［Ｊ］．南京林业大学学报 ２０００，２４（１）：１－６．
［１６］ 郑万均．中国树木志（第二卷）［Ｍ］。北京：林业出版社，１９８５：２３２４－２３５７．
［１７］王傲雪，李景富．植物体细胞胚状体的诱导研究及应用［Ｊ］．黑龙江农业科学，１９９９，（２）：３９－４１．
［１８］韩碧文，刘淑兰．植物离体体细胞胚胎发生［Ｊ］．植物生理学通讯，１９８８，（１）：９－１５．
［１９］ 杨 映 根 ， 桂 耀 林 ， 郭 仲 琛 ． 裸 子 植 物 体 细 胞 胚 胎 发 生 和 人 工 种 子 的 研 究 ［Ｊ］． 种
子，１９９５，（３）：２５－３０．
［２０］夏海武，宋雨青．植物人工种子及其应用［Ｊ］． 昌雄师专学报，２００１，２０（２）：２８－２９．
［２１］达克东，李雅志，束怀瑞．苹果离体叶片特定部位体细胞胚胎发生和人工种子研究［Ｊ］．核农学
报，１９９８，１２（１）：１６－２０．
44 栓皮栎体细胞胚胎诱导的研究
［２２］白淑霞，刘立秋，陈文龙．植物人工种子高质量体细胞胚胎发生的研究［Ｊ］．河北农业大学学
报，１９９８，２１（１）：９７－１０１．
［２３］叶克难，黄俊潮，李宝健．杂种一代番木瓜人工种子制作的研究［Ｊ］．植物学报，１９９３，３５（增
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［２４］ 陈 德 富 ， 陈 喜 文 ， 程 炳 嵩 ． 人 工 种 子 几 个 问 题 的 讨 论 ［Ｊ］． 山 东 农 业 大 学 学
报，１９９５，２６（２）：２４９－２５６．
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［２６］郑国锠．生物显微技术［Ｍ］．北京：人民教育出版社，１９７８．
［２７］季道藩，遗传学实验［Ｍ］．北京：农业出版社，１９９２：３－７．
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［３１］ 赖钟雄，陈春玲．龙眼体细胞胚胎高质量浓度蔗糖成熟培养后的超微结构变化［Ｊ］．福建农林大
学学报（自然科学版），２００２，３１（２）：１８８－１９１
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［３４］周俊彦．植物体细胞在组织培养中产生的胚状体Ⅱ影响植物胚状体发生和发育的因素［Ｊ］．植物
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［３５］ 张远记，钱迎倩，蔡起贵．毛花称猴桃原生质体再生植株根尖染色体数目变异与细胞多核现象
［Ｊ］． 植物学报，１９９７，３９（２）：１０２－１０５．
［３６］余龙江，张长河，梅兴国．红豆杉离体培养染色体数变异与细胞多核现象．华中理工大学学
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［３］李俊英，冯天杰．对麻栎和栓皮栎果实成熟期的商榷［Ｊ］．河北林果研究，１９９８，１３（１）：５８－５９．
［４］闻天声．栓皮栎的培养与用途［Ｊ］．中国林副特产，１９９１，１７（２）：２９．
［５］ 李 文 英 ， 王 冰 ． 栎 类 树 种 的 生 态 效 益 和 经 济 价 值 及 资 源 保 护 对 策 ［Ｊ］． 林 业 科 技 通
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［７］陈夜江 ，赖钟雄．果树和林木体细胞胚胎发生的研究与利用［Ｊ］．福建农业大学学报，２００１，
３０（３）：４２０－４２６．
［８］上海植物生理研究所细胞生理研究室．植物细胞培养与细胞杂交研究的进展［Ｊ］．植物学
报，１９７３，１５（２）：２８５－３０７．
［９］汤浩茹，王永清，任正隆．果树的体细胞胚胎发生［Ｊ］．四川农业大学学报，１９９９，１７（１）：６９－７９．
［１０］ 唐 巍 ，杨 映 根， 桂耀林等 ． 松柏类植物体细胞胚胎发生的研究与应用 ［Ｊ］． 植物学通
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［１１］ 黄 健 秋， 卫志明 ， 许智宏 ． 云南松成熟胚的体细胞胚胎的发生研究［Ｊ］． 实验 生 物学
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［１２］ 邱 德 有 ， 李 如 玉 ， 李 铃 ． 红 豆 杉 及 南 方 红 豆 杉 体 细 胞 胚 胎 发 生 的 研 究 ［Ｊ］．林 业 科
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［１５］ 施季森．迎接 ２１ 世纪现代林木生物技术育种的挑战［Ｊ］．南京林业大学学报 ２０００，２４（１）：１－６．
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［１９］ 杨 映 根 ， 桂 耀 林 ， 郭 仲 琛 ． 裸 子 植 物 体 细 胞 胚 胎 发 生 和 人 工 种 子 的 研 究 ［Ｊ］． 种
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［２０］夏海武，宋雨青．植物人工种子及其应用［Ｊ］． 昌雄师专学报，２００１，２０（２）：２８－２９．
［２１］达克东，李雅志，束怀瑞．苹果离体叶片特定部位体细胞胚胎发生和人工种子研究［Ｊ］．核农学
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44 栓皮栎体细胞胚胎诱导的研究
［２２］白淑霞，刘立秋，陈文龙．植物人工种子高质量体细胞胚胎发生的研究［Ｊ］．河北农业大学学
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［３１］ 赖钟雄，陈春玲．龙眼体细胞胚胎高质量浓度蔗糖成熟培养后的超微结构变化［Ｊ］．福建农林大
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［３２］刘庆昌，吴国良．植物细胞组织培养［Ｍ］． 北京：中国农业大学出版社，２００３．
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［３５］ 张远记，钱迎倩，蔡起贵．毛花称猴桃原生质体再生植株根尖染色体数目变异与细胞多核现象
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