崇明水仙试管成球及离体脱毒技术研究
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摘要
崇明水仙(Narcissus tazetta var. chinensis)是上海市的特有花卉品种之一,具有悠久的栽培历史。但目前生产上分球是唯一的繁殖方式,增殖系数低,且病毒积累,造成种性退化,产量降低,阻碍其产业发展。
本研究对影响崇明水仙试管球诱导和增殖的多种因素进行了探讨,建立了试管球高效诱导体系和高频增殖体系;采用DHT+高温处理、DHT+病毒唑处理等技术手段进行了离体脱毒研究。主要结果如下:
1.以带鳞茎盘的鳞片为外植体时,离体诱导前应对鳞茎进行4周的冷处理。
2.将鳞茎盘切成4块,在1000mg/L头孢拉定溶液中浸泡3h,污染率降至8.3%。
3.适合花序轴诱导试管球的最佳培养基为MS+BA10mg/L+NAA5mg/L+蔗糖30g/L,有效诱导率达到171.3%。适合带鳞茎盘的鳞片诱导试管球的最佳培养基为MS+BA1mg/L+NAA1mg/L+蔗糖30g/L,成球率为163.6%。
4.冷处理3周的内层鳞片是诱导愈伤的最佳材料,在MS+BA1mg/L+2,4-D0.1mg/L+蔗糖30g/L培养基上,诱导率和再分化率分别为48.4%和23.3%。子房愈伤诱导和再分化的最佳培养基为MS+BA10mg/L+NAA3mg/L+蔗糖30g/L,诱导率和再分化率分别为51.9%和22.2%。
5.试管球增殖的最佳培养基为MS+BA5mg/L+NAA1mg/L+蔗糖40g/L,增殖率为175.0%。适当提高蔗糖浓度以40g/L为宜,AC显著降低试管球的增殖率。
6.适合试管球生根的最佳培养基为1/2MS+NAA0.5mg/L+AC1g/L+蔗糖30g/L,生根率为90.4%。AC有利于提高试管球的生根率。
7.利用DHT+高温处理后病毒含量明显降低。DHT30mg/L+病毒唑30mg/L处理能使NMAV含量降低到极微弱的水平,且不影响试管球存活;DHT10mg/L+病毒唑10mg/L处理可以完全脱除NDV和NCLV病毒,脱毒效果通过RT-PCR技术检验证实。
Chongming Daffodil (Narcissus tazetta var. chinensis) is one of the most famous flowers in Shanghai which has a long cultivatural history. Now, there are many disadvantages, such as low rate of propagation, hurt of virus, reduce of products, etc., which have hindered the development of industrialization of Chongming Daffodil.
In this study, several factors affected the induction and multiplication of bulblets in vitro were discussed to obtain higher frequency, and a higher-efficient induction and multiplication system were established; the bublets in vitro treated with DHT+thermotherapy or DHT+virazole were used to eliminate virus diseases.
Main results were as follows:
1. Pretreating the bulbs at 4℃for 4 weeks before inoculation could improve the induction rate remarkably when leaf lamina with basal plate was used as explant.
2. The pollution rate decreased to 8.3% through marinating cutting bulbs in 1000mg/L Cefradine liquor for 3 hours.
3. Rachis was the most fitting explant to induce the bulblets in vitro directly. The MS medium with 10mg/LBA, 5mg/LNAA and 30g/L sucrose was founded to be optimum for induction bulblets from rachis, the effective induction rate reached 171.3%. The medium containing 1mg/LBA, 1mg/L NAA and30g/L sucrose was fit for bulblets developed from leaf lamina with based plate.
4. The inner leaf lamina pretreated under low temperature (4℃) for 3 weeks was the feasible explant to induce callus, the rate of induction was 48.4%, and the differentiation rate was 23.3% when inoculated on the MS medium with 1mg/L BA, 0.1mg/L 2,4-D and 30g/L sucrose. 51.9% callus can be induced from ovary on MS medium containing 10mg/L BA, 3mg/L NAA, and 22.2% was successfully differentiated to bulblets.
5. The medium with 5mg/LBA, 1mg/LNAA , 40g/L sucrose and without AC was fit for propagation. The higher sucrose concentration was good for bulblets proliferation and growth, but 40g/L was suitable. And AC reduced the propagation rate observably.
6. 1/2MS medium with 0.5mg/LNAA and 1g/LAC was the most efficient medium for rooting, 90.4% bulblets had grown roots after 20 days.
7. DHT with thermotherapy treatment could decrease the content of viruses. The content of NMAV in bulblets in vitro treated with 30mg/L DHT +30mg/L virazole was reduced to a very low level, and the treatment didn’t have impact on bulblet’s survival. NDV-free and NCLV-free plants were obtained by the treatment of combination of 10mg/L DHT and 10mg/L virazole, the viruses could not be detected by RT-PCR.
本研究对影响崇明水仙试管球诱导和增殖的多种因素进行了探讨,建立了试管球高效诱导体系和高频增殖体系;采用DHT+高温处理、DHT+病毒唑处理等技术手段进行了离体脱毒研究。主要结果如下:
1.以带鳞茎盘的鳞片为外植体时,离体诱导前应对鳞茎进行4周的冷处理。
2.将鳞茎盘切成4块,在1000mg/L头孢拉定溶液中浸泡3h,污染率降至8.3%。
3.适合花序轴诱导试管球的最佳培养基为MS+BA10mg/L+NAA5mg/L+蔗糖30g/L,有效诱导率达到171.3%。适合带鳞茎盘的鳞片诱导试管球的最佳培养基为MS+BA1mg/L+NAA1mg/L+蔗糖30g/L,成球率为163.6%。
4.冷处理3周的内层鳞片是诱导愈伤的最佳材料,在MS+BA1mg/L+2,4-D0.1mg/L+蔗糖30g/L培养基上,诱导率和再分化率分别为48.4%和23.3%。子房愈伤诱导和再分化的最佳培养基为MS+BA10mg/L+NAA3mg/L+蔗糖30g/L,诱导率和再分化率分别为51.9%和22.2%。
5.试管球增殖的最佳培养基为MS+BA5mg/L+NAA1mg/L+蔗糖40g/L,增殖率为175.0%。适当提高蔗糖浓度以40g/L为宜,AC显著降低试管球的增殖率。
6.适合试管球生根的最佳培养基为1/2MS+NAA0.5mg/L+AC1g/L+蔗糖30g/L,生根率为90.4%。AC有利于提高试管球的生根率。
7.利用DHT+高温处理后病毒含量明显降低。DHT30mg/L+病毒唑30mg/L处理能使NMAV含量降低到极微弱的水平,且不影响试管球存活;DHT10mg/L+病毒唑10mg/L处理可以完全脱除NDV和NCLV病毒,脱毒效果通过RT-PCR技术检验证实。
Chongming Daffodil (Narcissus tazetta var. chinensis) is one of the most famous flowers in Shanghai which has a long cultivatural history. Now, there are many disadvantages, such as low rate of propagation, hurt of virus, reduce of products, etc., which have hindered the development of industrialization of Chongming Daffodil.
In this study, several factors affected the induction and multiplication of bulblets in vitro were discussed to obtain higher frequency, and a higher-efficient induction and multiplication system were established; the bublets in vitro treated with DHT+thermotherapy or DHT+virazole were used to eliminate virus diseases.
Main results were as follows:
1. Pretreating the bulbs at 4℃for 4 weeks before inoculation could improve the induction rate remarkably when leaf lamina with basal plate was used as explant.
2. The pollution rate decreased to 8.3% through marinating cutting bulbs in 1000mg/L Cefradine liquor for 3 hours.
3. Rachis was the most fitting explant to induce the bulblets in vitro directly. The MS medium with 10mg/LBA, 5mg/LNAA and 30g/L sucrose was founded to be optimum for induction bulblets from rachis, the effective induction rate reached 171.3%. The medium containing 1mg/LBA, 1mg/L NAA and30g/L sucrose was fit for bulblets developed from leaf lamina with based plate.
4. The inner leaf lamina pretreated under low temperature (4℃) for 3 weeks was the feasible explant to induce callus, the rate of induction was 48.4%, and the differentiation rate was 23.3% when inoculated on the MS medium with 1mg/L BA, 0.1mg/L 2,4-D and 30g/L sucrose. 51.9% callus can be induced from ovary on MS medium containing 10mg/L BA, 3mg/L NAA, and 22.2% was successfully differentiated to bulblets.
5. The medium with 5mg/LBA, 1mg/LNAA , 40g/L sucrose and without AC was fit for propagation. The higher sucrose concentration was good for bulblets proliferation and growth, but 40g/L was suitable. And AC reduced the propagation rate observably.
6. 1/2MS medium with 0.5mg/LNAA and 1g/LAC was the most efficient medium for rooting, 90.4% bulblets had grown roots after 20 days.
7. DHT with thermotherapy treatment could decrease the content of viruses. The content of NMAV in bulblets in vitro treated with 30mg/L DHT +30mg/L virazole was reduced to a very low level, and the treatment didn’t have impact on bulblet’s survival. NDV-free and NCLV-free plants were obtained by the treatment of combination of 10mg/L DHT and 10mg/L virazole, the viruses could not be detected by RT-PCR.
引文
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