利用小麦与玉米远缘杂交诱导小麦双单倍体的研究
摘要
小麦与玉米远缘杂交，利用玉米染色体的消失而获得小麦单倍体胚，经幼胚离体培养及染色体加倍诱导小麦双单倍体（Double Haploid DH）系，是小麦单倍体育种中一种高效率的途径。但目前普遍存在的问题是单倍体胚获得频率不高，研究者大都利用田间常规杂交法进行诱导，外界环境因子对其影响很大。本研究通过小麦与玉米在大田条件下的常规杂交及在人工控制环境条件的室内离体培养相结合诱导小麦单倍体，再经秋水仙素进行染色体加倍，以探索高效构建小麦双单倍体DH群体的途径，结果表明：
1． 通过夏季小麦人工春化后移栽配合分期播种，可与正常生育期的玉米花期相遇；冬季玉米温室栽培配合分期播种，也可与正常生育期的小麦花期相遇。
2． 不同小麦品种组合的平均成胚率差异显著，最高组合D021/Yang9为26.4%，最低组合仅2.6%；不同玉米品种的平均成胚率趋势表现为：爆烈玉米>糯玉米>普通玉米>甜玉米。
3． 2，4－D对单倍体的形成是必须的，且不同浓度之间存在显著差异，以100mg/L浓度溶液处理效果最为理想；同时，2，4－D重复处理的成胚率要高于一次处理。
4． T≥20℃条件下室内离体培养的平均成胚率（23.6％）要高于田间常规杂交法的平均成胚率（18.1％），且以20℃，光照3000Lx12h/d，相对湿度70％条件下离体培养14天的平均成胚率最高（28.6％）。说明人工控制环境条件的离体培养法是获得稳定成胚率的一种行之有效的方法。但就单个小麦组合与玉米杂交而言，田间常规杂交法的最高成胚率（53.9％）却比室内离体培养法的最高成胚率（34.1％）高出近20％。
5． 授粉后足够的T>10℃有效积温是提高成胚率的必要条件；获得最高成胚率的T>10℃有效积温应在187.9～254.9℃之间；授粉后适宜的环境温度是影响成胚率的重要因素，一般以20℃为最佳。
6． 用浓度为250mg/L、500 mg/L 、750 mg/L 、1000 mg/L的秋水仙素溶液对单倍体植株进行浸根加倍处理，平均加倍率（结实植株数／成活植株数×100％）分别为51.9％、83.2％、86.4％、82.9％；平均加倍处理效率（结实植株数／处理植株数×100％）分别为46.1％、68.8％、71.7％、70.9％，均以浓度为750 mg/L的秋水仙素溶液处理效果最好。用秋水仙素浸根处理时给予持续O2供应，获得79.9％的平均加倍率，要高于没有O2供应的处理（72.7％）；有O2供应的加倍处理效率（66.1％）也高于无O2供应的处理（62.2）％。
Haploid embryo is obtained due to the chromosome elimination of maize in wheat（Triticum aestivum L.） and maize(Zea mays L.) crosses. Producing a DH system by young embryo culture and chromosome doubling is a high efficiency way to haploid breeding in wheat.But the problem is that frenquent of producing embryo is still not perfect as we want at persent. Most researchers induced the embryo by general cross methods in field, but the effect was influnced seriously by environment factors. The purpose of this study was to investigate way of creating wheat DH plant groups by using isolated culture methods in green house and general cross methods in field for inducing wheat×maize haploid, and by chromosome doubling with colchicines solution. The results was showed as follows:
1. In order to meet the time of flower between wheat and maize, Using manual vernalization and planting wheat by stages in Summer; or growing maize in the green house and planting by stages in Winter.
2. The frenquencies of embryo formation in different cross combinations of wheat varieties was significantly different. The highest combination D021/Yang9 was 26.4%, and the lowest one D027/C98 was 2.6%. The frenquencies of embryo formation of different populations of maize was Puffed maize(9.9%)>Glutincus maize(8.9%)>Common maize(8.2%)>Sweet maize (4.6%).
3. The effect of 3 kinds of concentration of 2,4-D solution to the frenquencies of embryo formation was different too and it was highest when the concentration of 2,4-D solution was 100mg/L. In addition, 2,4-D repeating treatment in the frenquencies of embryo formation was better than no repeating.
4. Average frenquence of producing haploid was isolated culture methods(23.6%) at T≥20℃ was higher than the one of general cross methods in filed (18.1%), and the frenquence of producing haploid was the highest when isolated culture 14 days in the condition which temperture was 20℃，illumination was 3000Lx12h/d,and relatively humidity was 70%.
5. Enough effective accumulated temperature (T>10℃) was necessary to high frenquent of producing haploid after pollination, and effective accumulated temperature (T>10℃) to the highest frenquence of producing haploid should be in the range of 187.9~254.9℃; fitting environment temperature was important factor after pollination, and
the condition was the best when environment temperature was 20℃.
6. Soaking roots of seeding for 5h using colchicines solution with concentration of 250mg/L、500 mg/L 、750 mg/L 、1000 mg/L，the frequencyes of doubling were 51.9％、83.2％、86.4％、82.9％ respectively； Efficiency of doubling were 46.1％、68.8％、71.7％、70.9％. Both of the effects were the best of the colchicines solution with concentration of 750mg/L. Afforting continuance O2 when Soaking roots of seedling for 5h using colchicines solution, the average frequencyes of doubling was 79.9%, which was higher than the average frequences of doubling without afforting continuance O2 when Soaking roots of seedling. Efficiency of doubling(66.1%)when affording O2 was higher than the one(62.2%) without afforting continuance O2 too.
1． 通过夏季小麦人工春化后移栽配合分期播种，可与正常生育期的玉米花期相遇；冬季玉米温室栽培配合分期播种，也可与正常生育期的小麦花期相遇。
2． 不同小麦品种组合的平均成胚率差异显著，最高组合D021/Yang9为26.4%，最低组合仅2.6%；不同玉米品种的平均成胚率趋势表现为：爆烈玉米>糯玉米>普通玉米>甜玉米。
3． 2，4－D对单倍体的形成是必须的，且不同浓度之间存在显著差异，以100mg/L浓度溶液处理效果最为理想；同时，2，4－D重复处理的成胚率要高于一次处理。
4． T≥20℃条件下室内离体培养的平均成胚率（23.6％）要高于田间常规杂交法的平均成胚率（18.1％），且以20℃，光照3000Lx12h/d，相对湿度70％条件下离体培养14天的平均成胚率最高（28.6％）。说明人工控制环境条件的离体培养法是获得稳定成胚率的一种行之有效的方法。但就单个小麦组合与玉米杂交而言，田间常规杂交法的最高成胚率（53.9％）却比室内离体培养法的最高成胚率（34.1％）高出近20％。
5． 授粉后足够的T>10℃有效积温是提高成胚率的必要条件；获得最高成胚率的T>10℃有效积温应在187.9～254.9℃之间；授粉后适宜的环境温度是影响成胚率的重要因素，一般以20℃为最佳。
6． 用浓度为250mg/L、500 mg/L 、750 mg/L 、1000 mg/L的秋水仙素溶液对单倍体植株进行浸根加倍处理，平均加倍率（结实植株数／成活植株数×100％）分别为51.9％、83.2％、86.4％、82.9％；平均加倍处理效率（结实植株数／处理植株数×100％）分别为46.1％、68.8％、71.7％、70.9％，均以浓度为750 mg/L的秋水仙素溶液处理效果最好。用秋水仙素浸根处理时给予持续O2供应，获得79.9％的平均加倍率，要高于没有O2供应的处理（72.7％）；有O2供应的加倍处理效率（66.1％）也高于无O2供应的处理（62.2）％。
Haploid embryo is obtained due to the chromosome elimination of maize in wheat（Triticum aestivum L.） and maize(Zea mays L.) crosses. Producing a DH system by young embryo culture and chromosome doubling is a high efficiency way to haploid breeding in wheat.But the problem is that frenquent of producing embryo is still not perfect as we want at persent. Most researchers induced the embryo by general cross methods in field, but the effect was influnced seriously by environment factors. The purpose of this study was to investigate way of creating wheat DH plant groups by using isolated culture methods in green house and general cross methods in field for inducing wheat×maize haploid, and by chromosome doubling with colchicines solution. The results was showed as follows:
1. In order to meet the time of flower between wheat and maize, Using manual vernalization and planting wheat by stages in Summer; or growing maize in the green house and planting by stages in Winter.
2. The frenquencies of embryo formation in different cross combinations of wheat varieties was significantly different. The highest combination D021/Yang9 was 26.4%, and the lowest one D027/C98 was 2.6%. The frenquencies of embryo formation of different populations of maize was Puffed maize(9.9%)>Glutincus maize(8.9%)>Common maize(8.2%)>Sweet maize (4.6%).
3. The effect of 3 kinds of concentration of 2,4-D solution to the frenquencies of embryo formation was different too and it was highest when the concentration of 2,4-D solution was 100mg/L. In addition, 2,4-D repeating treatment in the frenquencies of embryo formation was better than no repeating.
4. Average frenquence of producing haploid was isolated culture methods(23.6%) at T≥20℃ was higher than the one of general cross methods in filed (18.1%), and the frenquence of producing haploid was the highest when isolated culture 14 days in the condition which temperture was 20℃，illumination was 3000Lx12h/d,and relatively humidity was 70%.
5. Enough effective accumulated temperature (T>10℃) was necessary to high frenquent of producing haploid after pollination, and effective accumulated temperature (T>10℃) to the highest frenquence of producing haploid should be in the range of 187.9~254.9℃; fitting environment temperature was important factor after pollination, and
the condition was the best when environment temperature was 20℃.
6. Soaking roots of seeding for 5h using colchicines solution with concentration of 250mg/L、500 mg/L 、750 mg/L 、1000 mg/L，the frequencyes of doubling were 51.9％、83.2％、86.4％、82.9％ respectively； Efficiency of doubling were 46.1％、68.8％、71.7％、70.9％. Both of the effects were the best of the colchicines solution with concentration of 750mg/L. Afforting continuance O2 when Soaking roots of seedling for 5h using colchicines solution, the average frequencyes of doubling was 79.9%, which was higher than the average frequences of doubling without afforting continuance O2 when Soaking roots of seedling. Efficiency of doubling(66.1%)when affording O2 was higher than the one(62.2%) without afforting continuance O2 too.
引文
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