枇杷种质离体保存的初步研究
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摘要
本研究以枇杷(Eriobotrya japonica Lindl.)为材料，进行了种质资源保存的初步研究。包括枇杷快繁体系的建立；枇杷试管苗常规离体保存各影响因子的研究；枇杷种质资源低温保存和枇杷种质资源的超低温保存研究几个方面。主要结果如下：
1、枇杷快繁体系的研究
建立并比较了以茎尖、幼胚、成熟胚为材料的几种枇杷快繁途径。以茎尖为材料的快繁途径中，用饱和漂白粉上清液消毒20min，再用1g·L~(-1)的HgCl_2消毒8rain后接种，可大大降低污染率，外植体成活率达75％，并能有效地抑制枇杷茎尖培养中经常出现的褐化现象。培养基MS+BA1.0mg/L+NAA0.5mg/L可比较有效地促进芽苗形成。在幼胚和成熟胚培养中，培养基MS+BA2.0mg/L+NAA0.5mg/L有利于幼苗的形成并可以促进腋芽的增生。当小苗长至一定高度后，转移至激素含量降低的培养基MS+BA0.5mg/L+NAA0.1-0.2mg/L，则促进枇杷幼苗的增殖，根系正常生长。
2、枇杷种质常规离体保存
在试管包扎物选择实验中，发现白纸加聚丙烯塑料薄膜和锡箔纸作为封口材料较为合适。棉花、橡皮塞等作为封口材料明显不利于枇杷的种质保存。
低强度的光照处理有利于种质保存，但当光照强度＜700Lx时，对枇杷种质保存不利。
浓度为2-5mg/L的生长抑制剂多效唑(PP_(333))有利于枇杷种质保存，与对照组别(不加抑制剂)相比较，植株高度、新生叶片数目等方面明显受到抑制。高浓度的甘露醇(＞5g/L)不利于枇杷种质的离体保存，对枇杷试管苗会造成毒害，5g/L的甘露醇有较好的保存效果。矮壮素(CCC)在枇杷试管苗种质保存中的合适浓度为25mg/L左右。脱落酸(ABA)的适宜浓度为10mg/L左右，
三种生长抑制剂中，多效唑在枇杷试管苗种质保存中效果最为理想，5mg/L的浓度条件下，品种山里本保存8个月后存活率仍在90％以上。
3、枇杷种质低温保存
低温有利于枇杷种质的离体保存。在11±0.5℃的低温情况下，大部分品种都有很好的保存效果：生长表现因子植株高度、叶片数目等明显低于对照组别(25±1℃)。半年后存活率大部分品种都在70％以上。实验中也发现，个别品
种（香酣）在此低温条件下保存效果不理想，可能与个别品种的抗寒性有关。
4、超低温保存初步研究
采用干冻化法对批把花粉超低温保存进行了研究。结果表明：花粉的含水
量是决定批把花粉超低温保存成败的关键因素，30％左右的含水量能够保证超低
温保存后花粉的活力；解冻温度及方式对超低温保存后花粉的存活力没有明显
影响。干冻后染色及萌发检验表明，冻后花粉的生活力与未超低温保存花粉相
比没有明显降低；萌发率可达听％。
茎尖超低温保存研究发现，二步化冷冻法超低温保存的批把茎尖可获得愈
伤组织。
脱水至一定程度的幼胚超低温保存后培养获得了2株试管苗。
This paper explored the germplasm resources' preservation in vitro of loquat (Eriobotrya japonica Lindl.)- The study included the establishing of propagation system and the factors that affected the quality of the preservation in vitro of loquat's germplasm. The cryopreservation study of loquat was also been explored. The main results were as follows:
1. Propagation system of loquat
Three different propagation systems of loquat were compared, by shoot tip culture, immature embryo culture and mature embryo culture. In shoot tip culture, it was found that there were a low contamination ratio and a high survival ratio with about 75% in all the cultured materials disinfected with saturated bleaching powder for 20 minutes then 1 g-L-1 HgCl2_for 8 minutes. In this way the phenomenon of the tips' browning could be well avoided. The medium of MS+BA1.0 mg/L+NAA0.5 mg/L could well induce the germination of the plantlets. In the culture of the immature embryo and mature embryo, MS+BA2.0 mg/L+NAA0.5 mg/L was profitable for the germination of the seedlings and also arose the propagation ratio of the plantlets that germinated from axil buds. The plantlets were then transferred to the medium of MS+BA0.5 mg/L+NAA0.1-0.2 mg/L which with a reduced consistency of endocrine when they grew to some degree. In such medium the plantlets could propagate effectively and their roots could be well induced.
2. Preservation in vitro of Loquat's germplasm at regulation temperature
It was found that both the packing with white paper, then covered with polypropylene membrane and the packing with tinfoil paper were suitable for the germplasm preservation. As packing materials, cotton plug and rubber plug were obviously not suitable.
Although lower intensity light may be good for the preservation of germplasm in vitro, when the light intensity was below 700 Lx the preservation in vitro results of loquat's germplasm were not as well as those of a higher light intensity.
PP333, a kind of inhibitor, was in favour of the preservation in vitro of loquat's germplasm with the consistency scopes from 2 mg-L-1 to 5 mg-L-1. The existence of high consistency mannitol (>5 g-L-1) was harmful for the plantlets in vitro. CCC and ABA which also be used as inhibitors, for the preservation in vitro of loquat's germplasm, were found with suitable consistency of about 25 mg-L-1 and 10 mg-L-1 respectively.
Of all the three inhibitors, PP333 was most suitable for the preservation in vitro of loquat's germplasm. Loquat's survival ratio was above 90% after being preserved for 8 months with PPaaa at the consistency of 5 mg-L-1.
3. Preservation in vitro of Loquat's germplasm at low temperature
Low temperature was in favour of the preservation in vitro of loquat's germplasm. Almost all the species had pretty well effects of preservation at a low temperature of 11 ±0.5℃. The height of plantlets and the number of the leaves et al., which were as the statistic of the vital powers of preserved plantlets, were obviously below that of the check groups which preserved at the temperature of 25 + 1癈. And they were found having a high survival ratio of above 70% after being preserved in vitro for more than half a year. But it was also found that not every species preserved was adapt to this low temperature, Xiangtian, one species of loquat, being harmed at this temperature (11±1℃). This maybe mainly attributed to the different characteristics and cool-resistances of various species of loquat.
4. Preliminary study of cryopreservation of Loquat's germplasm
A procedure had been studied on cryopreservation of loquat pollen by the method of dry freezing. It indicated that the major factor that determined the cryopreserved loquat pollen longevity was its water content, the adequate water content for its survival was about 30%. The thaw temperature and methods after being cryopreserved were not significant to the percentage of stored pollen's germination in vitro. Staining and sprouting test indicated that the viability of cryopreserved pollen had littl
1、枇杷快繁体系的研究
建立并比较了以茎尖、幼胚、成熟胚为材料的几种枇杷快繁途径。以茎尖为材料的快繁途径中，用饱和漂白粉上清液消毒20min，再用1g·L~(-1)的HgCl_2消毒8rain后接种，可大大降低污染率，外植体成活率达75％，并能有效地抑制枇杷茎尖培养中经常出现的褐化现象。培养基MS+BA1.0mg/L+NAA0.5mg/L可比较有效地促进芽苗形成。在幼胚和成熟胚培养中，培养基MS+BA2.0mg/L+NAA0.5mg/L有利于幼苗的形成并可以促进腋芽的增生。当小苗长至一定高度后，转移至激素含量降低的培养基MS+BA0.5mg/L+NAA0.1-0.2mg/L，则促进枇杷幼苗的增殖，根系正常生长。
2、枇杷种质常规离体保存
在试管包扎物选择实验中，发现白纸加聚丙烯塑料薄膜和锡箔纸作为封口材料较为合适。棉花、橡皮塞等作为封口材料明显不利于枇杷的种质保存。
低强度的光照处理有利于种质保存，但当光照强度＜700Lx时，对枇杷种质保存不利。
浓度为2-5mg/L的生长抑制剂多效唑(PP_(333))有利于枇杷种质保存，与对照组别(不加抑制剂)相比较，植株高度、新生叶片数目等方面明显受到抑制。高浓度的甘露醇(＞5g/L)不利于枇杷种质的离体保存，对枇杷试管苗会造成毒害，5g/L的甘露醇有较好的保存效果。矮壮素(CCC)在枇杷试管苗种质保存中的合适浓度为25mg/L左右。脱落酸(ABA)的适宜浓度为10mg/L左右，
三种生长抑制剂中，多效唑在枇杷试管苗种质保存中效果最为理想，5mg/L的浓度条件下，品种山里本保存8个月后存活率仍在90％以上。
3、枇杷种质低温保存
低温有利于枇杷种质的离体保存。在11±0.5℃的低温情况下，大部分品种都有很好的保存效果：生长表现因子植株高度、叶片数目等明显低于对照组别(25±1℃)。半年后存活率大部分品种都在70％以上。实验中也发现，个别品
种（香酣）在此低温条件下保存效果不理想，可能与个别品种的抗寒性有关。
4、超低温保存初步研究
采用干冻化法对批把花粉超低温保存进行了研究。结果表明：花粉的含水
量是决定批把花粉超低温保存成败的关键因素，30％左右的含水量能够保证超低
温保存后花粉的活力；解冻温度及方式对超低温保存后花粉的存活力没有明显
影响。干冻后染色及萌发检验表明，冻后花粉的生活力与未超低温保存花粉相
比没有明显降低；萌发率可达听％。
茎尖超低温保存研究发现，二步化冷冻法超低温保存的批把茎尖可获得愈
伤组织。
脱水至一定程度的幼胚超低温保存后培养获得了2株试管苗。
This paper explored the germplasm resources' preservation in vitro of loquat (Eriobotrya japonica Lindl.)- The study included the establishing of propagation system and the factors that affected the quality of the preservation in vitro of loquat's germplasm. The cryopreservation study of loquat was also been explored. The main results were as follows:
1. Propagation system of loquat
Three different propagation systems of loquat were compared, by shoot tip culture, immature embryo culture and mature embryo culture. In shoot tip culture, it was found that there were a low contamination ratio and a high survival ratio with about 75% in all the cultured materials disinfected with saturated bleaching powder for 20 minutes then 1 g-L-1 HgCl2_for 8 minutes. In this way the phenomenon of the tips' browning could be well avoided. The medium of MS+BA1.0 mg/L+NAA0.5 mg/L could well induce the germination of the plantlets. In the culture of the immature embryo and mature embryo, MS+BA2.0 mg/L+NAA0.5 mg/L was profitable for the germination of the seedlings and also arose the propagation ratio of the plantlets that germinated from axil buds. The plantlets were then transferred to the medium of MS+BA0.5 mg/L+NAA0.1-0.2 mg/L which with a reduced consistency of endocrine when they grew to some degree. In such medium the plantlets could propagate effectively and their roots could be well induced.
2. Preservation in vitro of Loquat's germplasm at regulation temperature
It was found that both the packing with white paper, then covered with polypropylene membrane and the packing with tinfoil paper were suitable for the germplasm preservation. As packing materials, cotton plug and rubber plug were obviously not suitable.
Although lower intensity light may be good for the preservation of germplasm in vitro, when the light intensity was below 700 Lx the preservation in vitro results of loquat's germplasm were not as well as those of a higher light intensity.
PP333, a kind of inhibitor, was in favour of the preservation in vitro of loquat's germplasm with the consistency scopes from 2 mg-L-1 to 5 mg-L-1. The existence of high consistency mannitol (>5 g-L-1) was harmful for the plantlets in vitro. CCC and ABA which also be used as inhibitors, for the preservation in vitro of loquat's germplasm, were found with suitable consistency of about 25 mg-L-1 and 10 mg-L-1 respectively.
Of all the three inhibitors, PP333 was most suitable for the preservation in vitro of loquat's germplasm. Loquat's survival ratio was above 90% after being preserved for 8 months with PPaaa at the consistency of 5 mg-L-1.
3. Preservation in vitro of Loquat's germplasm at low temperature
Low temperature was in favour of the preservation in vitro of loquat's germplasm. Almost all the species had pretty well effects of preservation at a low temperature of 11 ±0.5℃. The height of plantlets and the number of the leaves et al., which were as the statistic of the vital powers of preserved plantlets, were obviously below that of the check groups which preserved at the temperature of 25 + 1癈. And they were found having a high survival ratio of above 70% after being preserved in vitro for more than half a year. But it was also found that not every species preserved was adapt to this low temperature, Xiangtian, one species of loquat, being harmed at this temperature (11±1℃). This maybe mainly attributed to the different characteristics and cool-resistances of various species of loquat.
4. Preliminary study of cryopreservation of Loquat's germplasm
A procedure had been studied on cryopreservation of loquat pollen by the method of dry freezing. It indicated that the major factor that determined the cryopreserved loquat pollen longevity was its water content, the adequate water content for its survival was about 30%. The thaw temperature and methods after being cryopreserved were not significant to the percentage of stored pollen's germination in vitro. Staining and sprouting test indicated that the viability of cryopreserved pollen had littl
引文
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