南瓜未受精子房和未受精胚珠离体培养研究
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摘要
为了明确南瓜( Cucurbita moschata D.)未授粉子房和未受精胚珠诱导的最佳培养条件,本文以“试验南瓜1号”为材料,以MS为基本培养基,主要研究了黑暗热激预处理时间、2,4-D浓度、激素组合、碳源种类和浓度等影响南瓜离体雌核发育的因素。同时应用石蜡切片技术观察了南瓜胚囊的发育过程,以期为南瓜单倍体培养奠定重要的实践和理论基础。本研究的结果表明:
1.未受精离体雌核的诱导需要黑暗热激的预处理,南瓜未受精子房培养的黑暗热激处理以在35℃条件下热激处理6d为最佳,出胚数和出胚率的均值分别达到了4.8个和22%以上,而且与热激5d、7d相比,在0.01水平上差异显著;不同胚囊时期间的诱导结果分析结果显示没有差异,说明不同胚囊时期对于热激诱导的敏感程度是相似的。未受精胚珠直接培养以黑暗热激处理5d的转绿率和胚状体诱导率最高,分别达到了80%和15%以上,说明黑暗热激5天处理能够有效的诱导未受精胚珠的雌核发育,提高胚状体诱导率。总结两个黑暗热激实验,南瓜未受精子房的培养热激6天能够有效地起动离体雌核的发育,南瓜未受精胚珠离体培养则热激5天的诱导效果最佳。
2.南瓜未受精子房离体培养的培养基筛选的第一步就是比较不同浓度的2,4-D对离体诱导的影响,结果显示:添加了3.5 mg.L~(-1)、4.0 mg.L~(-1)、4.5 mg.L~(-1)三个浓度的基本培养基能有效地刺激了离体雌核的发育。胚状体的诱导率在2,4-D浓度为3.5 mg.L~(-1)时达到最高,三个胚囊时期的诱导率都在4.5%以上,其次就是2,4-D浓度在4.5 mg.L~(-1)和4.0 mg.L~(-1)的诱导率;并在开花前0.5天(12小时)的未受精子房最终诱导出了子叶胚,三个浓度分别诱导出了4个、2个和1个。南瓜未受精胚珠离体培养在添加的2,4-D浓度为1.0 mg.L~(-1)和1.5 mg.L~(-1)的基本培养基上胚状体诱导率和转绿率较高,低浓度的和高浓度的诱导率都非常低。比较两个实验:较高的2,4-D浓度能够有效地启动南瓜未受精子房的雌核发育,抑制南瓜未受精胚珠胚状体的诱导;而低浓度的2,4-D能有效地诱导未受精胚珠的雌核发育。总之,基本培养基中添加的2,4-D应由培养方式的不同而进行选择不同的浓度水平。
3.筛选培养基中最优激素组合的试验结果显示:诱导南瓜未受精子房培养出胚状体效果最好激素组合是4.0 mg.L~(-1)2,4-D+0.5 mg.L~(-1)NAA+0.5 mg.L~(-1)6-BA;南瓜未受精胚珠离体培养试验中诱导效果最好的激素组合是: 1.0 mg.L~(-1)2,4-D+ 0.25 mg.L~(-1)NAA+1.0 mg.L~(-1)6-BA,诱导出子叶胚总数达到了6个。相比较而言,南瓜未受精子房离体培养需的激素组合为高浓度的2,4-D、NAA和低浓度的6-BA;南瓜未受精胚珠直接培养则需低浓度的2,4-D、NAA和高浓度的6-BA。
4.蔗糖和葡萄糖都可作为南瓜未受精子房离体培养的碳源,诱导雌核发育。不同蔗糖浓度间的诱导效果比较,30g/L蔗糖的胚状体诱导率最高;不同葡萄糖浓度间的诱导效果比较,40g/L葡萄糖的胚状体诱导率最高;不同碳源浓度间的胚状体诱导率比较,蔗糖的最高胚状体诱导率为8.52%,葡萄糖的最高诱导率为4.44%,蔗糖的诱导效果好于葡萄糖的诱导效果。南瓜未受精胚珠离体培养的不同蔗糖浓度胚状体诱导率比较实验,结果以25g/L的蔗糖作为碳源的诱导效果最好,胚状体诱导率为83.33%,子叶胚7个。南瓜未受精胚珠培养所需的蔗糖浓度与南瓜未受精子房培养诱导雌核发育所需的蔗糖浓度,处在低浓度水平。
5.应用石蜡切片技术观察到南瓜胚囊发育的整个发育过程。得出了明确的结论就是:开花前3天的胚囊处于单核期,开花前2天的胚囊处于双核胚囊期,开花前1天的,属于四核胚囊期,开花前0.5天,胚囊已经发育至八核期。
In order to obtain the best way to induce un-pollinated ovary and ovule in pumpkin, we used“NO.1”as test material which mainly studied the influencing factors of pumpkin in vitro gynogenesis ,such as heat pretreatment time, 2, 4-D concentration, hormone combination and while the process embryo sac development was observed by the technology of paraffin section. We expected to lay a theoretical and practical foundation on pumpkin in vitro gynogenesis. The result showed as:
1. The heat pretreatment in dark was essential for in vitro gynogenesis. The best pretreatment was under 35℃for 6 days on pumpkin in vitro culture of un-pollinated ovary, which had big differences compared with 5 days heat pretreatments and 7days pretreatment(p<0.01). There showed no difference between different development embryo sac, it proved that different development embryo sac has similar sensitivity under heat-inducible. The result of in vitro culture unpollinated ovule in pumpkin showed that: 5 days under this treatment turning green ratio reached 80% and embryoid induction rate reached 15%.was the best heat pretreatment period. The result showed that at 35℃heat treatment for 6 days can effectively improve the embryoid rate. In a word, the best heat pretreatment time was 6days on pumpkin in vitro culture un-pollinated ovary or 5 days on pumpkin in vitro culture un-pollinated ovule.
2. The first step of inducting medium for pumpkin in vitro gynogenesis is to compare the influence of 2, 4-D in different concentration to Inducement in Vitro, the result are as follows: 3.5 mg.L~(-1)、4.0 mg.L~(-1) and 4.5mg.L~(-1)of the 2, 4-D concentration can stimulate the growth of vitro gynogenesis effectively. Embryoid induction rate reached highest level when 2, 4-D concentration was 3.5mg.L~(-1), and the embryoid induction rates of the three stages embryo sac were above 4.5%. The next was successively 4.0mg.L~(-1) and 4.5mg.L~(-1). Cotyledon embryo was inducted on the medium with the three 2, 4-D concentration at pre-flowering 0.5d, and the number was four, two and one. On pumpkin in vitro culture un-pollinated ovule, medium supplemented with 1.0 mg.L~(-1) 2, 4-D and 1.5mg.L~(-1) 2, 4-D can effectively induct gynogenesis. In a word, the amount of adding 2, 4-D to vitro gynogenesis should be different according to different ways of growth of vitro gynogenesis.
3. We tried to screening the best hormone combination on MS basic medium. The result showed that: 4.0mg.L~(-1)2, 4-D + 0.5mg.L~(-1) NAA + 0.5 mg.L~(-1)6-BA was the optimal compounding for inducting unpollinated ovary in vitro gynogenesis. The best hormone was 1.0mg.L~(-1)2, 4-D+0.25mg.L~(-1)NAA+1.0mg.L~(-1)6-BA, which induced 6 cotyledon embryos from unpollinated ovule. Comparatively speaking, the medium supplemented with higher concentration 2, 4-Dand NAA and lower concentration 6-BA can effectively induce embryo from pumpkin unpollinated ovary. But inducing embryoid from pumpkin nupollinated ovule needed the medium supplemented with lower concentration 2, 4-D and NAA and higher concentration 6-BA.
4. Sucrose and glucose could be used as carbon source of pumpkin un-pollinated ovary in vitro culture, and used to induct gynogenesis. The medium containing 30mg.L~(-1) sucrose inducted embryoid of the highest rate, comparing induced embryoid between difference sucrose concentrations. Between the induced effects of different glucose concentration, the best induced effect was 40mg.L~(-1) glucose. Comparing the highest induction rate between different carbon sources, 8.52% was sucrose’s, 4.44% was glucose’s. The inducted effect of sucrose was better than glucose’s. We studied the induct effect of in vitro culture pumpkin unpollinated ovule with the medium containing sucrose. The best sucrose concentration was 25mg.L~(-1), which embryoid induction rate was 83.3% and 7 cotyledon embryo were inducted.
5. The embryo sac development process was observed by technology of paraffin section. The result showed that: at pre-flowering 3 d, embryo sac was at mononuclear stage; at pre-flowering 2 d, embryo sac was at binuclear stage; it became tetranucleate embryo sac, at pre-flowering 1 d; at pre-flowering 0.5 d, eight nuclears embryo sac cell could be observed in ovule.
1.未受精离体雌核的诱导需要黑暗热激的预处理,南瓜未受精子房培养的黑暗热激处理以在35℃条件下热激处理6d为最佳,出胚数和出胚率的均值分别达到了4.8个和22%以上,而且与热激5d、7d相比,在0.01水平上差异显著;不同胚囊时期间的诱导结果分析结果显示没有差异,说明不同胚囊时期对于热激诱导的敏感程度是相似的。未受精胚珠直接培养以黑暗热激处理5d的转绿率和胚状体诱导率最高,分别达到了80%和15%以上,说明黑暗热激5天处理能够有效的诱导未受精胚珠的雌核发育,提高胚状体诱导率。总结两个黑暗热激实验,南瓜未受精子房的培养热激6天能够有效地起动离体雌核的发育,南瓜未受精胚珠离体培养则热激5天的诱导效果最佳。
2.南瓜未受精子房离体培养的培养基筛选的第一步就是比较不同浓度的2,4-D对离体诱导的影响,结果显示:添加了3.5 mg.L~(-1)、4.0 mg.L~(-1)、4.5 mg.L~(-1)三个浓度的基本培养基能有效地刺激了离体雌核的发育。胚状体的诱导率在2,4-D浓度为3.5 mg.L~(-1)时达到最高,三个胚囊时期的诱导率都在4.5%以上,其次就是2,4-D浓度在4.5 mg.L~(-1)和4.0 mg.L~(-1)的诱导率;并在开花前0.5天(12小时)的未受精子房最终诱导出了子叶胚,三个浓度分别诱导出了4个、2个和1个。南瓜未受精胚珠离体培养在添加的2,4-D浓度为1.0 mg.L~(-1)和1.5 mg.L~(-1)的基本培养基上胚状体诱导率和转绿率较高,低浓度的和高浓度的诱导率都非常低。比较两个实验:较高的2,4-D浓度能够有效地启动南瓜未受精子房的雌核发育,抑制南瓜未受精胚珠胚状体的诱导;而低浓度的2,4-D能有效地诱导未受精胚珠的雌核发育。总之,基本培养基中添加的2,4-D应由培养方式的不同而进行选择不同的浓度水平。
3.筛选培养基中最优激素组合的试验结果显示:诱导南瓜未受精子房培养出胚状体效果最好激素组合是4.0 mg.L~(-1)2,4-D+0.5 mg.L~(-1)NAA+0.5 mg.L~(-1)6-BA;南瓜未受精胚珠离体培养试验中诱导效果最好的激素组合是: 1.0 mg.L~(-1)2,4-D+ 0.25 mg.L~(-1)NAA+1.0 mg.L~(-1)6-BA,诱导出子叶胚总数达到了6个。相比较而言,南瓜未受精子房离体培养需的激素组合为高浓度的2,4-D、NAA和低浓度的6-BA;南瓜未受精胚珠直接培养则需低浓度的2,4-D、NAA和高浓度的6-BA。
4.蔗糖和葡萄糖都可作为南瓜未受精子房离体培养的碳源,诱导雌核发育。不同蔗糖浓度间的诱导效果比较,30g/L蔗糖的胚状体诱导率最高;不同葡萄糖浓度间的诱导效果比较,40g/L葡萄糖的胚状体诱导率最高;不同碳源浓度间的胚状体诱导率比较,蔗糖的最高胚状体诱导率为8.52%,葡萄糖的最高诱导率为4.44%,蔗糖的诱导效果好于葡萄糖的诱导效果。南瓜未受精胚珠离体培养的不同蔗糖浓度胚状体诱导率比较实验,结果以25g/L的蔗糖作为碳源的诱导效果最好,胚状体诱导率为83.33%,子叶胚7个。南瓜未受精胚珠培养所需的蔗糖浓度与南瓜未受精子房培养诱导雌核发育所需的蔗糖浓度,处在低浓度水平。
5.应用石蜡切片技术观察到南瓜胚囊发育的整个发育过程。得出了明确的结论就是:开花前3天的胚囊处于单核期,开花前2天的胚囊处于双核胚囊期,开花前1天的,属于四核胚囊期,开花前0.5天,胚囊已经发育至八核期。
In order to obtain the best way to induce un-pollinated ovary and ovule in pumpkin, we used“NO.1”as test material which mainly studied the influencing factors of pumpkin in vitro gynogenesis ,such as heat pretreatment time, 2, 4-D concentration, hormone combination and while the process embryo sac development was observed by the technology of paraffin section. We expected to lay a theoretical and practical foundation on pumpkin in vitro gynogenesis. The result showed as:
1. The heat pretreatment in dark was essential for in vitro gynogenesis. The best pretreatment was under 35℃for 6 days on pumpkin in vitro culture of un-pollinated ovary, which had big differences compared with 5 days heat pretreatments and 7days pretreatment(p<0.01). There showed no difference between different development embryo sac, it proved that different development embryo sac has similar sensitivity under heat-inducible. The result of in vitro culture unpollinated ovule in pumpkin showed that: 5 days under this treatment turning green ratio reached 80% and embryoid induction rate reached 15%.was the best heat pretreatment period. The result showed that at 35℃heat treatment for 6 days can effectively improve the embryoid rate. In a word, the best heat pretreatment time was 6days on pumpkin in vitro culture un-pollinated ovary or 5 days on pumpkin in vitro culture un-pollinated ovule.
2. The first step of inducting medium for pumpkin in vitro gynogenesis is to compare the influence of 2, 4-D in different concentration to Inducement in Vitro, the result are as follows: 3.5 mg.L~(-1)、4.0 mg.L~(-1) and 4.5mg.L~(-1)of the 2, 4-D concentration can stimulate the growth of vitro gynogenesis effectively. Embryoid induction rate reached highest level when 2, 4-D concentration was 3.5mg.L~(-1), and the embryoid induction rates of the three stages embryo sac were above 4.5%. The next was successively 4.0mg.L~(-1) and 4.5mg.L~(-1). Cotyledon embryo was inducted on the medium with the three 2, 4-D concentration at pre-flowering 0.5d, and the number was four, two and one. On pumpkin in vitro culture un-pollinated ovule, medium supplemented with 1.0 mg.L~(-1) 2, 4-D and 1.5mg.L~(-1) 2, 4-D can effectively induct gynogenesis. In a word, the amount of adding 2, 4-D to vitro gynogenesis should be different according to different ways of growth of vitro gynogenesis.
3. We tried to screening the best hormone combination on MS basic medium. The result showed that: 4.0mg.L~(-1)2, 4-D + 0.5mg.L~(-1) NAA + 0.5 mg.L~(-1)6-BA was the optimal compounding for inducting unpollinated ovary in vitro gynogenesis. The best hormone was 1.0mg.L~(-1)2, 4-D+0.25mg.L~(-1)NAA+1.0mg.L~(-1)6-BA, which induced 6 cotyledon embryos from unpollinated ovule. Comparatively speaking, the medium supplemented with higher concentration 2, 4-Dand NAA and lower concentration 6-BA can effectively induce embryo from pumpkin unpollinated ovary. But inducing embryoid from pumpkin nupollinated ovule needed the medium supplemented with lower concentration 2, 4-D and NAA and higher concentration 6-BA.
4. Sucrose and glucose could be used as carbon source of pumpkin un-pollinated ovary in vitro culture, and used to induct gynogenesis. The medium containing 30mg.L~(-1) sucrose inducted embryoid of the highest rate, comparing induced embryoid between difference sucrose concentrations. Between the induced effects of different glucose concentration, the best induced effect was 40mg.L~(-1) glucose. Comparing the highest induction rate between different carbon sources, 8.52% was sucrose’s, 4.44% was glucose’s. The inducted effect of sucrose was better than glucose’s. We studied the induct effect of in vitro culture pumpkin unpollinated ovule with the medium containing sucrose. The best sucrose concentration was 25mg.L~(-1), which embryoid induction rate was 83.3% and 7 cotyledon embryo were inducted.
5. The embryo sac development process was observed by technology of paraffin section. The result showed that: at pre-flowering 3 d, embryo sac was at mononuclear stage; at pre-flowering 2 d, embryo sac was at binuclear stage; it became tetranucleate embryo sac, at pre-flowering 1 d; at pre-flowering 0.5 d, eight nuclears embryo sac cell could be observed in ovule.
引文
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