梨品种及砧木离体叶片再生体系建立的研究
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摘要
本研究以优良梨栽培品种喜水和秦丰以及西洋梨矮化砧木BA29试管苗为试材,对影响叶片离体再生不定芽的因素如基因型、基本培养基成分、生长调节剂种类及浓度配比、接种材料类型、接种方式以及培养条件等进行了系统研究,成功建立了喜水梨、秦丰梨和西洋梨矮化砧木BA29的离体叶片再生体系。
主要研究结果如下:
1.影响梨品种及砧木离体叶片再生的内在因素:
(1)基因型是影响梨及砧木离体叶片再生的重要因素。
(2)离体再生能力叶片高于节间嫩梢和叶柄;再生效果整个叶片优于叶块;喜水梨和BA29以试管苗顶端幼嫩叶片为适宜外植体,秦丰梨适宜使用顶端幼嫩叶片或中部叶片诱导再生。
(3)继代次数对试管苗叶片再生能力有很大影响。喜水梨和秦丰梨15代左右的试管苗叶片再生能力强,矮化砧木BA29的8～15代试管苗叶片再生能力强。
2.影响梨品种及砧木离体叶片再生的外部因素:
(1)培养基:NN_(69)培养基为喜水梨再生培养基;秦丰梨对基本培养基的差异反应不敏感;BA29适宜应用MS或NN_(69)培养基诱导再生。
(2)生长调节剂:喜水梨宜采用TDZ1.5mg/L+IAA(0.1～0.5)mg/L或IBA(0.1～0.5)mg/L;秦丰梨宜采用6-BA5.0mg/L+IBA0.3mg/L;BA29宜采用TDZ1.5mg/L+NAA 0.3mg/L。
(3)接种前宜采用对叶片垂直主脉均匀切割三次的处理方式。秦丰梨和BA29宜采用远轴面接触培养基接种;喜水梨叶片再生诱导对接种方向不敏感。
(4)暗培养时间对叶片再生不定芽有影响:秦丰梨和BA29适宜经暗培养2～3周;喜水梨经1～3周暗培养与对照没有显著性差异,4～5周暗培养对再生不定芽不利。
(5)适当提高NN_(69)培养基中NO_3~--N的比例可显著提高BA29叶片再生的效率。最适NH_4~+-N与NO_3~--N比例为1:5,再生率可达100%。
(6)AgNO_3使不定芽发生高峰提前、不定芽再生效率提高,以1～2mg/L最适宜;胰蛋白胨可促进叶片再生,最佳使用浓度为800～1200mg/L。蛋白胨不能促进BA29叶片再生,且当浓度为800 mg/L时会抑制其再生。
3.再生植株的继代培养:将长有不定芽的部分叶片适时转移至继代培养基对不定芽的伸长有利。
喜水和砧木BA29再生植株的增殖培养基以MS+6-BA 0.5mg/L+IBA0.1mg/L适宜;秦丰梨再生植株增殖的培养基为AS+6-BA0.8mg/L+IBA 0.1mg/L。
用牛皮纸与封口膜作为封口材料最佳,经济方便,再生植株生长良好,且接种间隔时间适宜。
4.再生植株的生根培养:喜水梨再生植株的生根培养基为1/2MS+IBA1.0mg/L+NAA0.5mg/L+AC0.5mg/L,培养5d后转入不含生长素的培养基中;适宜秦丰梨再生植株生根的培养基为1/2MS+IBA0.5mg/L+NAA 1.0mg/L,培养5～10d后转入不含生长素的培养基中。
5.抗生素敏感性:BA29叶盘法基因转化中抑制农杆菌生长的抗生素选用头孢霉素(Cef)300mg/L。以卡那霉素(Kan)25mg/L作为转化时抗性芽的选择压。
This article was mainly involved in the cultivars of Pear named "Xishui" and "Qinfeng" and rootstock of Western Pear named "BA29".Regeneration system was studied using the leaves of plants in vitro.The adventitious bud regeneration system had been modified by studying the effect factors on regeneration such as the basic medium,the types and concentration of plant growth regulators,cultivate condition etc.
The main results were as followed:
1.Internal influence factors of regeneration from leaves of Pear Cultivars and Rootstock:
(1)Genotype plays an important role in the efficiency of regeneration.
(2)The regeneration ability in vitro of leaf was higher than stem segment's and leaf stalk's.Regeneration ability of whole leaf was greater than part of leaf.Young leaf at the tip of plantlet was suitable material part for regeneration of Xishui and BA29.Leaf from the tip or middle part of plantlet could be used to regeneration of Qinfeng.
(3)Subculture generation plays another important role in leaf regeneration.Leaf from plantlet in the subculture of 15th time was suitable for leaf regeneration of Xishui and Qinfeng,while 8th to 15th was suitable for BA29.
2.External influence factors of regeneration from leaves of Pear Cultivars and Rootstock:
(1)NN_(69)Was confirmed as the best suitable medium of the leaf regeneration in vitro of Xishui.Qinfeng was less sensitive to the different medium.BA29 needed MS or NN_(69) medium to induce leaf regeneration in vitro.
(2)The best combination of plant growth regulators was confirmed on NN_(69)medium. TDZ1.5mg/L+IAA(0.1～0.5)mg/L or IBA(0.1～0.5)mg/L was suitable for Xishui,6-BA 5.0mg/L+ IBA 0.3mg/L was best for Qinfeng,and TDZ1.5mg/L+NAA0.3mg/L was optimum to BA29.
(3)The leaf should be cut by 3 times vertical to main vein before inoculated.The leaf of Qinfeng and BA29 should be inoculated with the method of abaxial side in contact with medium,while Xishui was less sensitive to the different inoculated method.
(4)For Qinfeng and BA29,2～3 weeks of dark culture was suitable.As to Xishui,it made no difference between 1～3 weeks of dark culture and light culture.What's more, 4～5 weeks of dark reduced the regeneration efficiency.
(5)It is favorable for leaf regeneration of BA29 to properly increase the ratio of NO_3~--N. The most ratio of NH_4~+-N/NO_3~--N was 1:5,and the leaf regeneration frequency could be increased to 100%.
(6)AgNO_3 and Tryptone could improve leaf regeneration of BA29.AgNO_3 could also advance the peak period of regeneration,the best concentration was 1～2mg/L.The best concentration of Tryptone was 800～1200mg/L.However,Peptone could not improve leaf regeneration,and 800mg/L of Peptone might reduce the regeneration efficiency.
3.Transferring the part leaf with adventitious buds into new subculture medium was suitable for elongation of adventitious shoots.MS+6-BA 0.5mg/L+IBA0.1mg/L was the best proliferation medium for Xixhui and BA29,and AS+6-BA0.8mg/L+IBA0.1mg/L is suitable for proliferation of Qinfeng.Kraft paper+seal membrane was the best seal material for multiplication of plantlets from regeneration,which was economy and convenient.It could make the plantlets grow up well,and could avoid mould contamination which from moisture of cotton plug.
4.The plantlets from regeneration of Xixhui should be cultured on MS+IBA 1.0mg/L+NAA0.5mg/L+AC0.5mg/L for 5days,and then be transferred to medium without Auxin.The plantlets from regeneration of Qinfeng should be cultured on MS+IBA0.5mg/L+NAA1.0mg/L for 5～10 days,and then be transferred to medium without Auxin.
5.The optimum concentration of Cefotaxime was 300 mg/L during the gene transformation to inhibit bacterial.25mg/L kanamyein could be used as a selecting pressure for the regeneration of adventitious buds with antibiotic-resistant.
主要研究结果如下:
1.影响梨品种及砧木离体叶片再生的内在因素:
(1)基因型是影响梨及砧木离体叶片再生的重要因素。
(2)离体再生能力叶片高于节间嫩梢和叶柄;再生效果整个叶片优于叶块;喜水梨和BA29以试管苗顶端幼嫩叶片为适宜外植体,秦丰梨适宜使用顶端幼嫩叶片或中部叶片诱导再生。
(3)继代次数对试管苗叶片再生能力有很大影响。喜水梨和秦丰梨15代左右的试管苗叶片再生能力强,矮化砧木BA29的8～15代试管苗叶片再生能力强。
2.影响梨品种及砧木离体叶片再生的外部因素:
(1)培养基:NN_(69)培养基为喜水梨再生培养基;秦丰梨对基本培养基的差异反应不敏感;BA29适宜应用MS或NN_(69)培养基诱导再生。
(2)生长调节剂:喜水梨宜采用TDZ1.5mg/L+IAA(0.1～0.5)mg/L或IBA(0.1～0.5)mg/L;秦丰梨宜采用6-BA5.0mg/L+IBA0.3mg/L;BA29宜采用TDZ1.5mg/L+NAA 0.3mg/L。
(3)接种前宜采用对叶片垂直主脉均匀切割三次的处理方式。秦丰梨和BA29宜采用远轴面接触培养基接种;喜水梨叶片再生诱导对接种方向不敏感。
(4)暗培养时间对叶片再生不定芽有影响:秦丰梨和BA29适宜经暗培养2～3周;喜水梨经1～3周暗培养与对照没有显著性差异,4～5周暗培养对再生不定芽不利。
(5)适当提高NN_(69)培养基中NO_3~--N的比例可显著提高BA29叶片再生的效率。最适NH_4~+-N与NO_3~--N比例为1:5,再生率可达100%。
(6)AgNO_3使不定芽发生高峰提前、不定芽再生效率提高,以1～2mg/L最适宜;胰蛋白胨可促进叶片再生,最佳使用浓度为800～1200mg/L。蛋白胨不能促进BA29叶片再生,且当浓度为800 mg/L时会抑制其再生。
3.再生植株的继代培养:将长有不定芽的部分叶片适时转移至继代培养基对不定芽的伸长有利。
喜水和砧木BA29再生植株的增殖培养基以MS+6-BA 0.5mg/L+IBA0.1mg/L适宜;秦丰梨再生植株增殖的培养基为AS+6-BA0.8mg/L+IBA 0.1mg/L。
用牛皮纸与封口膜作为封口材料最佳,经济方便,再生植株生长良好,且接种间隔时间适宜。
4.再生植株的生根培养:喜水梨再生植株的生根培养基为1/2MS+IBA1.0mg/L+NAA0.5mg/L+AC0.5mg/L,培养5d后转入不含生长素的培养基中;适宜秦丰梨再生植株生根的培养基为1/2MS+IBA0.5mg/L+NAA 1.0mg/L,培养5～10d后转入不含生长素的培养基中。
5.抗生素敏感性:BA29叶盘法基因转化中抑制农杆菌生长的抗生素选用头孢霉素(Cef)300mg/L。以卡那霉素(Kan)25mg/L作为转化时抗性芽的选择压。
This article was mainly involved in the cultivars of Pear named "Xishui" and "Qinfeng" and rootstock of Western Pear named "BA29".Regeneration system was studied using the leaves of plants in vitro.The adventitious bud regeneration system had been modified by studying the effect factors on regeneration such as the basic medium,the types and concentration of plant growth regulators,cultivate condition etc.
The main results were as followed:
1.Internal influence factors of regeneration from leaves of Pear Cultivars and Rootstock:
(1)Genotype plays an important role in the efficiency of regeneration.
(2)The regeneration ability in vitro of leaf was higher than stem segment's and leaf stalk's.Regeneration ability of whole leaf was greater than part of leaf.Young leaf at the tip of plantlet was suitable material part for regeneration of Xishui and BA29.Leaf from the tip or middle part of plantlet could be used to regeneration of Qinfeng.
(3)Subculture generation plays another important role in leaf regeneration.Leaf from plantlet in the subculture of 15th time was suitable for leaf regeneration of Xishui and Qinfeng,while 8th to 15th was suitable for BA29.
2.External influence factors of regeneration from leaves of Pear Cultivars and Rootstock:
(1)NN_(69)Was confirmed as the best suitable medium of the leaf regeneration in vitro of Xishui.Qinfeng was less sensitive to the different medium.BA29 needed MS or NN_(69) medium to induce leaf regeneration in vitro.
(2)The best combination of plant growth regulators was confirmed on NN_(69)medium. TDZ1.5mg/L+IAA(0.1～0.5)mg/L or IBA(0.1～0.5)mg/L was suitable for Xishui,6-BA 5.0mg/L+ IBA 0.3mg/L was best for Qinfeng,and TDZ1.5mg/L+NAA0.3mg/L was optimum to BA29.
(3)The leaf should be cut by 3 times vertical to main vein before inoculated.The leaf of Qinfeng and BA29 should be inoculated with the method of abaxial side in contact with medium,while Xishui was less sensitive to the different inoculated method.
(4)For Qinfeng and BA29,2～3 weeks of dark culture was suitable.As to Xishui,it made no difference between 1～3 weeks of dark culture and light culture.What's more, 4～5 weeks of dark reduced the regeneration efficiency.
(5)It is favorable for leaf regeneration of BA29 to properly increase the ratio of NO_3~--N. The most ratio of NH_4~+-N/NO_3~--N was 1:5,and the leaf regeneration frequency could be increased to 100%.
(6)AgNO_3 and Tryptone could improve leaf regeneration of BA29.AgNO_3 could also advance the peak period of regeneration,the best concentration was 1～2mg/L.The best concentration of Tryptone was 800～1200mg/L.However,Peptone could not improve leaf regeneration,and 800mg/L of Peptone might reduce the regeneration efficiency.
3.Transferring the part leaf with adventitious buds into new subculture medium was suitable for elongation of adventitious shoots.MS+6-BA 0.5mg/L+IBA0.1mg/L was the best proliferation medium for Xixhui and BA29,and AS+6-BA0.8mg/L+IBA0.1mg/L is suitable for proliferation of Qinfeng.Kraft paper+seal membrane was the best seal material for multiplication of plantlets from regeneration,which was economy and convenient.It could make the plantlets grow up well,and could avoid mould contamination which from moisture of cotton plug.
4.The plantlets from regeneration of Xixhui should be cultured on MS+IBA 1.0mg/L+NAA0.5mg/L+AC0.5mg/L for 5days,and then be transferred to medium without Auxin.The plantlets from regeneration of Qinfeng should be cultured on MS+IBA0.5mg/L+NAA1.0mg/L for 5～10 days,and then be transferred to medium without Auxin.
5.The optimum concentration of Cefotaxime was 300 mg/L during the gene transformation to inhibit bacterial.25mg/L kanamyein could be used as a selecting pressure for the regeneration of adventitious buds with antibiotic-resistant.
引文
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