砀山酥梨脱病毒苗快繁体系的研究
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摘要
梨树在长期的无性繁殖过程中，感染并积累了多种病毒。分布较广泛的有梨环纹花叶病毒、梨脉黄病毒、苹果茎沟病毒，榲桲矮化病毒等。梨树一旦被病毒侵染，将终生带毒，持久受害，树势衰弱，严重影响梨果的产量和质量。茎尖离体培养可以获得无病毒苗。本试验以砀山酥梨带病毒植株为试材，采用了四种不同的茎尖脱病毒方法进行处理，经检测，获得了脱病毒试管苗。同时还系统研究了砀山酥梨脱病毒试管苗快速繁殖体系的相关技术环节，并对继代培养脱病毒苗的遗传稳定性作了深入探讨。
通过分析比较不同接种时期和不同接种外植体的污染率，褐化率及成活率，选定病毒含量少的春季旺盛生长的梢尖为茎尖脱病毒离体培养的外植体。并采取不同防褐变措施将春梢茎尖褐变率降低到5.7％。
采用正交优化设计和单因素试验相结合，筛选出茎尖分化最佳培养基配方：改良MS+2.0mg.L~(-1)BA+0.5mg.L~(-1)NAA+0.5％AC。初代培养，以无机盐浓度全量的MS为基本培养基，试管苗的生长状况最好。试管苗继代增殖阶段，BA浓度在0～2.0mg.L~(-1)范围内，随外源BA浓度增加，内源ZR含量上升，芽有效增殖系数增大。在评价芽增殖效果时，内源IAA／ZR值比内源ZR绝对量能更好说明问题。在适量浓度范围内，随培养基中添加外源激素NAA、GA_3浓度的增加能使相应的内源激素IAA、GA_(1+3)含量升高，从而促进试管苗的生长。通过4代继代苗的培养，总结出砀山酥梨脱病毒试管苗最佳继代间隔时间为45d。理论上，1个梨芽1年可繁殖4.06×10~6株试管苗。
试管苗生根阶段，通过正交优化设计，分析出各因素对瓶内直接生根率的影响程度大小依次为：间苯三酚＞IBA＞NAA＞基本培养基。采用瓶内双步生根法，试管苗的综合生根效应都优于直接培养生根，生根率高达87.5％。梨试管苗的瓶外生根率较低，但研究其生根规律对于试管苗的扩大繁殖，降低生产实际成本有极重要的应用价值。试管苗出瓶移栽，需先驯化、炼苗。
试验选用酶联免疫吸附法和草本指示植物鉴定法，快速检测田间对照株和脱病毒试管苗的带病毒情况。经分析比较，初步判断砀山酥梨不带烟草花叶病毒，带有苹果茎沟病毒和其它病毒。
试验采用了一次取尖，二次取尖以及试管苗恒温热处理、变温热处理后再取尖培养的四种脱病毒方法。经酶联免疫吸附法检测，四种脱病毒方法对于苹果茎沟病毒的脱除率分别为25.6％，44.4％，42.8％，62.5％，其中试管苗变温热处理后再取尖培养的方法脱病
毒效果最好。
通过分析杨山酥梨脱病毒试管苗的过氧化物同工酶、酯酶同工酶的酶谱，发现与田
间对照株相比，未发生酶带的缺失或增添。电泳结果表明，肠山酥梨茎尖脱病毒试管苗
在遗传上有相对的稳定性。
The yield and quality of pear are greatly taken from virus desease as viruses infecting and accumulating in the pear tree while continually asexual propagation. One of the best measures is selecting virus-free plants through meristem viruses elimination to treatment pear virus desease at present. The research reported four different virus elimination methods, virus detection techniques, rapid propagation system of virus-free plant and genetic stabilityof pear of continous generational culture. The results are as follows:
Growing buds in early May as explant would be easy to get the lowest contaminating rate, high surrival rate, better gemernation and high virus elimination rate. Growing buds in early May as explant with some measures of browning controlling can reduce browning rate to 5.7%.
Culture media are seleted by L9(34) orthogonal design and single factor experiment. The results showed the optimum medium for shoot tip differentiation inducting was the improved MS medium with BA 2.0mg/L, NAA 0.5 mg.L-1. However, MS medium with high mineral salt concentration is favorable to the growth of virus-free test-tube plantlet. Effects of exogenous hormones on the shoot propagation and the growth of the bud and for endogenous hormones in the course of tube plantlet subculture indicated that the content of endogenous ZR and the shoot effective proliferation were increased while the value of IAA/ZR and the height of the plantlet decreased markly after BA treated. Exogenous NAA showed significant effective on the amount of endogenous IAA and the height of plantlet. There are positively relative between the height of plantlet and the amount of IAA and IAA/ZR. Exogenous NAA and GA also revealed stimulating rule for content of endogenous IAA and GA1+3. The optimum time of subculture for virus-free plantle
t is 45 days. In theory, 4.06 × 106 times of tube plantlets per year can be obtained from one virus-free pear shoot through rapid propagation.
In rooting stage, we tested the effects of factors such as PG, IBA NAA and content of basal medium. The results showed that their effect is PG > IB A > NAA > content of basal medium. Two steps rooting method was more efficient and practical than direct rooting
method with rooting rate 87.5%. Higher concentration of IDA was needed for rooting. Aditionally rooting out of vessel technique was studied. Though the plantlets rooting rate out of vessel was very low, it could save production cost. The acclimatization of plantlets is important before transplanting.
Four methods (meristem culture, re-meristem-culture, meristem culture combining with heat treatment of constant temperature and variation temperature) for eliminating viruses have been compared. The examining result by ELISA showed that meristem culture combining with heat treatemnt of variation temperature is the most efficient for the elimination rates of apple stem grooving virus.
Herbaceous indicators and ELISA were applied in detecting virus elimination of tube plantlet. By analyzing, we drew a conclusion that Dangshan pear carried apple stem grooving virus and other viruses and unearned TMV.
The isozyme analyzing results showed that there are no change on band number of the peroxidase and estarase isozyme between virus-free plants and the contrast, which confirmed the genetic stabilities of Dangshan pear and virus-free plantlet.
通过分析比较不同接种时期和不同接种外植体的污染率，褐化率及成活率，选定病毒含量少的春季旺盛生长的梢尖为茎尖脱病毒离体培养的外植体。并采取不同防褐变措施将春梢茎尖褐变率降低到5.7％。
采用正交优化设计和单因素试验相结合，筛选出茎尖分化最佳培养基配方：改良MS+2.0mg.L~(-1)BA+0.5mg.L~(-1)NAA+0.5％AC。初代培养，以无机盐浓度全量的MS为基本培养基，试管苗的生长状况最好。试管苗继代增殖阶段，BA浓度在0～2.0mg.L~(-1)范围内，随外源BA浓度增加，内源ZR含量上升，芽有效增殖系数增大。在评价芽增殖效果时，内源IAA／ZR值比内源ZR绝对量能更好说明问题。在适量浓度范围内，随培养基中添加外源激素NAA、GA_3浓度的增加能使相应的内源激素IAA、GA_(1+3)含量升高，从而促进试管苗的生长。通过4代继代苗的培养，总结出砀山酥梨脱病毒试管苗最佳继代间隔时间为45d。理论上，1个梨芽1年可繁殖4.06×10~6株试管苗。
试管苗生根阶段，通过正交优化设计，分析出各因素对瓶内直接生根率的影响程度大小依次为：间苯三酚＞IBA＞NAA＞基本培养基。采用瓶内双步生根法，试管苗的综合生根效应都优于直接培养生根，生根率高达87.5％。梨试管苗的瓶外生根率较低，但研究其生根规律对于试管苗的扩大繁殖，降低生产实际成本有极重要的应用价值。试管苗出瓶移栽，需先驯化、炼苗。
试验选用酶联免疫吸附法和草本指示植物鉴定法，快速检测田间对照株和脱病毒试管苗的带病毒情况。经分析比较，初步判断砀山酥梨不带烟草花叶病毒，带有苹果茎沟病毒和其它病毒。
试验采用了一次取尖，二次取尖以及试管苗恒温热处理、变温热处理后再取尖培养的四种脱病毒方法。经酶联免疫吸附法检测，四种脱病毒方法对于苹果茎沟病毒的脱除率分别为25.6％，44.4％，42.8％，62.5％，其中试管苗变温热处理后再取尖培养的方法脱病
毒效果最好。
通过分析杨山酥梨脱病毒试管苗的过氧化物同工酶、酯酶同工酶的酶谱，发现与田
间对照株相比，未发生酶带的缺失或增添。电泳结果表明，肠山酥梨茎尖脱病毒试管苗
在遗传上有相对的稳定性。
The yield and quality of pear are greatly taken from virus desease as viruses infecting and accumulating in the pear tree while continually asexual propagation. One of the best measures is selecting virus-free plants through meristem viruses elimination to treatment pear virus desease at present. The research reported four different virus elimination methods, virus detection techniques, rapid propagation system of virus-free plant and genetic stabilityof pear of continous generational culture. The results are as follows:
Growing buds in early May as explant would be easy to get the lowest contaminating rate, high surrival rate, better gemernation and high virus elimination rate. Growing buds in early May as explant with some measures of browning controlling can reduce browning rate to 5.7%.
Culture media are seleted by L9(34) orthogonal design and single factor experiment. The results showed the optimum medium for shoot tip differentiation inducting was the improved MS medium with BA 2.0mg/L, NAA 0.5 mg.L-1. However, MS medium with high mineral salt concentration is favorable to the growth of virus-free test-tube plantlet. Effects of exogenous hormones on the shoot propagation and the growth of the bud and for endogenous hormones in the course of tube plantlet subculture indicated that the content of endogenous ZR and the shoot effective proliferation were increased while the value of IAA/ZR and the height of the plantlet decreased markly after BA treated. Exogenous NAA showed significant effective on the amount of endogenous IAA and the height of plantlet. There are positively relative between the height of plantlet and the amount of IAA and IAA/ZR. Exogenous NAA and GA also revealed stimulating rule for content of endogenous IAA and GA1+3. The optimum time of subculture for virus-free plantle
t is 45 days. In theory, 4.06 × 106 times of tube plantlets per year can be obtained from one virus-free pear shoot through rapid propagation.
In rooting stage, we tested the effects of factors such as PG, IBA NAA and content of basal medium. The results showed that their effect is PG > IB A > NAA > content of basal medium. Two steps rooting method was more efficient and practical than direct rooting
method with rooting rate 87.5%. Higher concentration of IDA was needed for rooting. Aditionally rooting out of vessel technique was studied. Though the plantlets rooting rate out of vessel was very low, it could save production cost. The acclimatization of plantlets is important before transplanting.
Four methods (meristem culture, re-meristem-culture, meristem culture combining with heat treatment of constant temperature and variation temperature) for eliminating viruses have been compared. The examining result by ELISA showed that meristem culture combining with heat treatemnt of variation temperature is the most efficient for the elimination rates of apple stem grooving virus.
Herbaceous indicators and ELISA were applied in detecting virus elimination of tube plantlet. By analyzing, we drew a conclusion that Dangshan pear carried apple stem grooving virus and other viruses and unearned TMV.
The isozyme analyzing results showed that there are no change on band number of the peroxidase and estarase isozyme between virus-free plants and the contrast, which confirmed the genetic stabilities of Dangshan pear and virus-free plantlet.
引文
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