苹果组培苗多代保存过程中生长特性及生长节奏调控技术研究
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摘要
本研究以苹果(Malus domestica Borkh.)品种金矮生、乔纳金、富士和嘎拉试管苗为试材,对不同继代次数苹果组培苗的芽增殖能力、生根能力和叶片再生能力进行研究,以确定长期继代保存的苹果组培苗的生长繁殖能力是否发生变化;通过改变培养温度、增加培养基的渗透压或在培养基中加入生长抑制剂等处理,延缓苹果组培苗生长,以延长继代间隔时间、减少继代次数;同时对延缓生长和恢复生长后的苹果组培苗SOD、POD酶活性及MDA含量进行了测定,以检测苹果组培苗逆境下延缓生长的生理代谢变化,为确定苹果组培苗适宜的种质保存时间,筛选适合的延缓生长方法提供依据。试验结果表明:
1.苹果组培苗在继代5代以内时,继代增殖倍数、生根和叶片不定芽再生能力均较低,不适合进行快速繁殖和离体再生。
2.苹果种质保存组培苗继代13次以后,其芽增殖能力,生根能力和叶片再生能力均稳定在较高水平,经20年左右100多次(富士200多次)继代培养保存后,4个品种器官生长、繁殖能力无明显变化。
3.低温条件抑制试管苗生长效果明显,苹果试管苗适宜的低温处理为接种后在25℃条件下缓苗5～10d再移至3～8℃培养,这样可保存试管苗1～1.5年继代一次,茎尖成活率达100%。
4.提高培养基琼脂、蔗糖浓度或添加甘露醇都能提高培养基渗透压,抑制试管苗生长,且随着浓度增加,抑制作用愈发明显,但琼脂、甘露醇处理中有部分茎尖死亡;综合比较,60/L蔗糖处理延缓试管苗生长效果较好。同一处理下品种间有差异,富士延缓生长的效果好于乔纳金。
5.在室温下添加B_9、CCC、PP_(333)、ABA等生长抑制剂的处理中,以80m/LB_9效果较好,不但存活率高,而且试管苗的生长状况较好。
6.在培养基中提高蔗糖、琼脂浓度,添加甘露醇增加渗透压,或者在培养基中添加B_9、CCC、PP_(333)、ABA等生长抑制剂,均能使乔纳金和富士的试管苗SOD、POD活性提高,MDA含量降低。不同处理的不同浓度对保护酶活性和MDA含量影响不同。对恢复生长后的试管苗生理活性测定表明,当代SOD、POD活性和MDA含量就恢复到处理前水平。
The organogenesis characteristics of apple (Malns domestica Borkh.) shoot explants repeatedly subcultured in vitro were examined by analysis of the shoot multiplication, rooting and adventitious bud regeneration rate from leaves, to make sure whether the growth and reproduction ability of apple in vitro has changed after subcultured a long time; Changing temperature, improving osmotic pressures , supplying growth inhibitor to delay the growth of apple in vitro and reduce subculture times, the changes of the number of buds and the growth amount, SOD、POD activity and MDA amount during conservation period in subculture media and recovery process were applied to explants during conservation in vitro in order to achieve the optimal status for retardant growth. The result showed:
1. The results showed low propagation ability for the shoot explants subcultured in vitro for five times or less at the beginning of inoculation in terms of its shoot multiplication, rooting and adventitious bud regeneration rate from leaves.
2. Apple shoot plantlets being subcultured for 13 times, shoot multiplication, rooting and adventitious bud regeneration rate from leaves of the organs enhanced and remained at a high level, the growth and regeneration ability of the organs don not have changed after 20 years more than 100 times subculture(Fuji moer than 200 times).
3. A low incubation temperature can significantly retard the growth of explants. The optimal incubation temperature was 8℃after an incubation at 25℃for 5～10 d succeeding on inoculation of new plantlets, and these plantlets could be persevered for one and a half years with a survival rate of 100%.
4. The increase of osmotic pressure in subculture media by enhancing the concentration of Agar, Sugar, and Mannitol could retard the growth of the explants. An increase of pressure retarded the amount of explant growth. But some explants died in media with a high concentration of Agar and Mannitol. The combination of osmotic pressure and temperature suggests that explants under incubation at a temperature at 8℃and supplying 60 g/L Sugar in a medium would maintain their healthy status at the lowest growth rate. The effect had variation between cultivars with favorable results in Fuji when compared to Jonagold.
5. The effect of supplying 80mg/L B_9 is better than supplying other kinds of plant-growth regulators such as B_9、CCC、PP_(333)、ABA at room temperature. The conditions and survival rates are better than supplying CCC、ABA and PP333.
6. The increase of osmotic pressure in subculture media by enhancing the concentration of Agar, Sugar, Mannitol or supplying B9, CCC, PP333 all can increase SOD and POD activity and decrease MDA amount of Jonagold and Fuji. The effects of SOD and POD activity and MDA amount are different between different treatments. This difference of SOD and POD activity and MDA amount disappeared when the explants were transferred to the conventional medium for recovery growth culture.
1.苹果组培苗在继代5代以内时,继代增殖倍数、生根和叶片不定芽再生能力均较低,不适合进行快速繁殖和离体再生。
2.苹果种质保存组培苗继代13次以后,其芽增殖能力,生根能力和叶片再生能力均稳定在较高水平,经20年左右100多次(富士200多次)继代培养保存后,4个品种器官生长、繁殖能力无明显变化。
3.低温条件抑制试管苗生长效果明显,苹果试管苗适宜的低温处理为接种后在25℃条件下缓苗5～10d再移至3～8℃培养,这样可保存试管苗1～1.5年继代一次,茎尖成活率达100%。
4.提高培养基琼脂、蔗糖浓度或添加甘露醇都能提高培养基渗透压,抑制试管苗生长,且随着浓度增加,抑制作用愈发明显,但琼脂、甘露醇处理中有部分茎尖死亡;综合比较,60/L蔗糖处理延缓试管苗生长效果较好。同一处理下品种间有差异,富士延缓生长的效果好于乔纳金。
5.在室温下添加B_9、CCC、PP_(333)、ABA等生长抑制剂的处理中,以80m/LB_9效果较好,不但存活率高,而且试管苗的生长状况较好。
6.在培养基中提高蔗糖、琼脂浓度,添加甘露醇增加渗透压,或者在培养基中添加B_9、CCC、PP_(333)、ABA等生长抑制剂,均能使乔纳金和富士的试管苗SOD、POD活性提高,MDA含量降低。不同处理的不同浓度对保护酶活性和MDA含量影响不同。对恢复生长后的试管苗生理活性测定表明,当代SOD、POD活性和MDA含量就恢复到处理前水平。
The organogenesis characteristics of apple (Malns domestica Borkh.) shoot explants repeatedly subcultured in vitro were examined by analysis of the shoot multiplication, rooting and adventitious bud regeneration rate from leaves, to make sure whether the growth and reproduction ability of apple in vitro has changed after subcultured a long time; Changing temperature, improving osmotic pressures , supplying growth inhibitor to delay the growth of apple in vitro and reduce subculture times, the changes of the number of buds and the growth amount, SOD、POD activity and MDA amount during conservation period in subculture media and recovery process were applied to explants during conservation in vitro in order to achieve the optimal status for retardant growth. The result showed:
1. The results showed low propagation ability for the shoot explants subcultured in vitro for five times or less at the beginning of inoculation in terms of its shoot multiplication, rooting and adventitious bud regeneration rate from leaves.
2. Apple shoot plantlets being subcultured for 13 times, shoot multiplication, rooting and adventitious bud regeneration rate from leaves of the organs enhanced and remained at a high level, the growth and regeneration ability of the organs don not have changed after 20 years more than 100 times subculture(Fuji moer than 200 times).
3. A low incubation temperature can significantly retard the growth of explants. The optimal incubation temperature was 8℃after an incubation at 25℃for 5～10 d succeeding on inoculation of new plantlets, and these plantlets could be persevered for one and a half years with a survival rate of 100%.
4. The increase of osmotic pressure in subculture media by enhancing the concentration of Agar, Sugar, and Mannitol could retard the growth of the explants. An increase of pressure retarded the amount of explant growth. But some explants died in media with a high concentration of Agar and Mannitol. The combination of osmotic pressure and temperature suggests that explants under incubation at a temperature at 8℃and supplying 60 g/L Sugar in a medium would maintain their healthy status at the lowest growth rate. The effect had variation between cultivars with favorable results in Fuji when compared to Jonagold.
5. The effect of supplying 80mg/L B_9 is better than supplying other kinds of plant-growth regulators such as B_9、CCC、PP_(333)、ABA at room temperature. The conditions and survival rates are better than supplying CCC、ABA and PP333.
6. The increase of osmotic pressure in subculture media by enhancing the concentration of Agar, Sugar, Mannitol or supplying B9, CCC, PP333 all can increase SOD and POD activity and decrease MDA amount of Jonagold and Fuji. The effects of SOD and POD activity and MDA amount are different between different treatments. This difference of SOD and POD activity and MDA amount disappeared when the explants were transferred to the conventional medium for recovery growth culture.
引文
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