八仙花和艳果金丝桃再生与遗传转化体系研究
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摘要
八仙花[Hydrangea macrophylla (Thunb) Ser.]和艳果金丝桃(Hypericum androsaemum L.)分别为绣球科和金丝桃科的木本观赏植物。八仙花以花大色艳,花期长,成为庭园花境、林缘、基础种植、大型花卉装饰和盆花的极好材料；艳果金丝桃以果实美丽、果枝长、冬季枝条常绿、挂果期长成为冬季庭园花灌木的新型花灌木,也是最近几年来国内外新型的插花配材。它们在栽培管理中都容易出现病害问题,如八仙花的叶斑病和金丝桃的根结线虫病。基因工程是提高它们抗病性的有效途径之一。本文通过对外植体、培养条件、接种方式、激素种类、不同基因型等条件的比较试验,建立了八仙花和金丝桃稳定高效的再生体系,并对以根癌农杆菌介导的遗传转化体系进行了研究。另外,对八仙花叶斑病进行了分离和分子鉴定。其主要研究结果如下：
1.建立并完善了高效稳定的八仙花快繁与再生体系
获得了完全脱毒的八仙花Hydl、Hyd4和Hyd5三个基因型的无菌苗。以这三个基因型带芽茎段为外植体,对其组培快繁技术进行了研究,结果表明：三个基因型茎段均在B5+0.5 mg/L BA+0.1 mg/L NAA培养基上增殖效果最好,平均增殖芽数均达到了3个以上。其中以基因型Hydl为例,获得的最佳生根培养基为60 mL珍珠岩+30 mL液体培养基(1/2 B5+0.5 mg/L IBA),30d后生根率和移栽成活率达到100%。
以八仙花七个基因型Hydl、Hyd4、Hyd5、Hyd6、Hyd7、Hyd8和Hyd10的叶片和叶柄为外植体,通过培养基、激素和暗培养等因子比较试验得出,以叶片为外植体获得的最佳再生培养基为B5+2.25 mg/L BA+0.1 mg/L IBA+100 mg/L头孢霉素(Cef),暗培养20d后转入光照下培养,再生频率和平均再生芽数分别为100%和2.7个。不同基因型的再生能力差异较大,除基因型Hydl外,其他六个基因型叶片的再生频率均低于30%。
通过ISSR分子标记对直接再生苗进行的体细胞变异检测中,共得到1857个条带,相似度在0.980-0.983之间。表明直接再生苗不存在明显的体细胞变异。
2.初步建立了八仙花基因型Hydl的遗传转化体系
以再生率高的基因型Hydl叶片为试验材料,通过抗生素、预培养、侵染、共培养时间、农杆菌菌株等因素对比试验,探讨了相关因子对八仙花遗传转化的影响。研究表明：叶片不定芽再生的筛选浓度为20 mg/L卡那霉素(Km),再生苗生根的筛选浓度为200mg/LKm,200 mg/L Cef作为抑菌剂。叶片在预培养0d,侵染35 min和共培养3d条件下,GUS瞬间表达率最高可达100%。该转化条件结合延迟筛选7d可获得100%抗性愈伤率和12个抗性芽。抗性愈伤组织在后期分化抗性芽较为困难,需要进一步研究其分化条件。
3.建立了高效稳定的艳果金丝桃快繁与再生体系
以MS基本培养基和BA/NAA配比对比试验表明,以基因型Hyp3带腋芽茎段为外植体在MS+BA1.0mg/L+NAA0.1mg/L的培养基上获得了最优的腋芽诱导及增殖效果,丛生芽诱导率和平均增殖芽数分别为100%和3.7个。
以艳果金丝桃四个基因型Hyp1、Hyp3、Hyp4和Hyp7叶片为外植体对其再生体系进行的研究表明,MS+TDZ0.1mg/L+AgNO31.0 mg/L+蔗糖30mg/L为最优的再生培养基,在暗培养20d后转入光周期下培养,再生率达83.3%。不同基因型叶片再生效果差异较大,其中,以基因型Hyp3的再生能力最强。
4.初步建立了艳果金丝桃遗传转化体系
对影响艳果金丝桃基因型Hyp3遗传转化预培养时间、侵染时间等的几个因子进行了研究,结果表明：Hyp3叶片为外植体以EHA105为侵染菌株,预培养0d,侵染5min,在MS+TDZ 0.1 mg/L+NAA 0.01 mg/L+AS 40 mg/L+MES 2.0g/L培养基上共培养3d,然后转移到选择培养基(MS+TDZ 0.1 mg/L+NAA 0.01 mg/L+ AgN031.0 mg/L+cef200 mg/L)上培养。在该条件下能够得到79.2%的GUS瞬时表达率,并且能够得到少量抗性愈伤组织。
5.八仙花叶斑病病原菌的研究
通过对盆栽八仙花叶片感病症状的观察、并对导致叶斑病的病原菌进行分离、形态学鉴定和rDNA-ITS分析,确定该病原菌为八仙花尾孢菌(Cercospora hydrangeae)。所得ITS序列经测序后已上载到Genbank,序列号：JF495458.1。
Hydrangea macrophylla (Thunb.) and Hypericum androsaemum L. are woody ornamental plants belonging to the Hydrangeaceae and Hypericaceae families respectively. Hydrangea macrophylla (Thunb) is used for planting courtyard flower borders, forest fringes, and ornamental garden, and is used in large scale flower decorations and sold as potted flower due to its colorful and large flower and long-lasting florescence. Hypericum androsaemum L. is a relatively new winter courtyard shrub and is used as new cut flower material because of its attractive looking fruits, long evergreen shoots and long lasting fruiting stage. However, both crops are vulnerable to plant diseases during the growing season, namely Hydrangea leaf spot and Hypericum root-knot nematode. Genetic engineering is potentially an efficient way of improving their disease resistance. By investigating the factors such as choice of genotype and explants, incubation conditions, inoculation methods, and different plant growth regulators, we established a highly efficient regeneration system and studied. An agrobacterium tumefaciens-mediated genetic transformation of Hydrangea macrophylla was developed as well. In addition, the pathogen causing leaf spot was isolated and identified as Cercospora hydrangeae. The major results were as follows:
1. The establishment of a rapid propagation and regeneration system for Hydrangea
Sterile shoots of Hydrangea macrophylla were obtained for genotypes Hydl, Hyd4 and Hyd5. The stems with axillary buds of these three genotypes were used as explants for the establishment of a micropropagation system. The optimum medium for proliferation of the three genotypes was B5+0.5 mg/L BA+0.1 mg/L NAA with a resulting multiplication coefficient of 3.0. Hydl terminal buds were used as explants for rooting. The rooting medium with 60 mL perlite plus 30 mL liquid medium of 1/2 B5+ 0.5 mg/L IBA gave the best results for the rooting rate (100%). Rooted shoots transplanted to mixed substrates of peat soil:perlite (1:1) had a survival rate as high as 100% after 30 days.
Leaves and petioles of seven genotypes Hyd1, Hyd4, Hyd5, Hyd6, Hyd7, Hyd8 and Hyd10 were used as explants for experiments involving direct and indirect regeneration. The results showed that for leaf explants of Hydl on the B5 medium supplemented with 2.25mg/L BA,0.1 mg/L IBA and 100 mg/L cefotaxime, the regeneration rate was 100% and average number of shoots per explant was 2.7. There were large differences in regeneration capacity among different genotypes. On the same medium, the regeneration rate in leaf explants of Hyd4 and Hyd8 were lower than 30%.
Somaclonal variation of Hydrangea macrophylla regenerants were tested by ISSR markers. There were in total 1857 scorable bands. The observed similarity was between 0.980-0.983. No significant somaclonal variation was abserved.
2. Study on development of a genetic transformation system of Hydrangea macrophylla
Agrobacterium tumefaciens-mediated genetic transformation was studied by using leaf explants of Hydl. The concentration of Kanamycin selection pressures was determined as 20 mg/L for shoot regeneration of leaves and 200 mg/L for rooting. A. tumefaciens could be inhibited with 200 mg/L cefotaxime. GUS transient expression analysis revealed that the leaf explants with 0 d pre-culture, infected for 35min and co-cultured for 3 day could reach the highest transient GUS expression rate of 100%. Delayed kanamycin selection for seven days benefited the formation of resistant callus and shoots. Resistant callus (100%) and 12 regenerated plants were obtained under the optimal transformation conditions。Resistant callus was difficult to induce for regeneration on kanamycin medium. Further research should focus on callus differentiation.
3. Establishment of a rapid micro-propagation and regeneration system of Hypericum androsaemum
Basal medium and BA/NAA were tested in order to establish a rapid micro-propagation sytem of genotype Hyp3. The highest shoot induction percentage (100%) and mean number of shoot per explant (3.7) were found on 1/2MS plus BA1.0mg/L.
A regeneration system for Hypericum androsaemum was studied using explants of four genotypes Hypl, Hyp3, Hyp4 and Hyp7. A stable and highly efficient regeneration system of Hyp3 leaf explants was established, viz., media:MS+TDZ0.1 mg/L+AgNO3 1.0 mg/L+sucrose 30 mg/L, initial 20d dark culture followed by photoperiod culture. The highest regeneration rate was 83.3%. Genotype was a significant factor in Hypericum androsaemum regeneration. Medium with AgNO3 supplied a highly efficient and stable regeneration system.
4. Study on genetic transformation of Hypericum androsaemum
The factors of genetic transformation were tested in Hypericum androsaemum genotype Hyp3. The optimal protocol was as follows. Genotype Hyp3 leaf explants without pre-culture was immerged into EHA105 for 5 min, co-cultured on MS+ TDZ 0.1 mg/L+NAA 0.01 mg/L+AS 40 mg/L+MES 2.0g/L medium for 3d, selected on MS+ TDZ 0.1 mg/L+NAA 0.01 mg/L+AgNO3 1.0 mg/L+cef200 mg/L medium. High transient GUS expression rated (79.2%) was gained.
5. Study on hydrangea leaf spot
Pathogen was isolated from diseased potted hydrangea leaves and was identified as Cercospora hydrangeae by morphological and rDNA-ITS way. The ITS sequence was deposited to Genbank, accessing No. JF495458.1.
1.建立并完善了高效稳定的八仙花快繁与再生体系
获得了完全脱毒的八仙花Hydl、Hyd4和Hyd5三个基因型的无菌苗。以这三个基因型带芽茎段为外植体,对其组培快繁技术进行了研究,结果表明：三个基因型茎段均在B5+0.5 mg/L BA+0.1 mg/L NAA培养基上增殖效果最好,平均增殖芽数均达到了3个以上。其中以基因型Hydl为例,获得的最佳生根培养基为60 mL珍珠岩+30 mL液体培养基(1/2 B5+0.5 mg/L IBA),30d后生根率和移栽成活率达到100%。
以八仙花七个基因型Hydl、Hyd4、Hyd5、Hyd6、Hyd7、Hyd8和Hyd10的叶片和叶柄为外植体,通过培养基、激素和暗培养等因子比较试验得出,以叶片为外植体获得的最佳再生培养基为B5+2.25 mg/L BA+0.1 mg/L IBA+100 mg/L头孢霉素(Cef),暗培养20d后转入光照下培养,再生频率和平均再生芽数分别为100%和2.7个。不同基因型的再生能力差异较大,除基因型Hydl外,其他六个基因型叶片的再生频率均低于30%。
通过ISSR分子标记对直接再生苗进行的体细胞变异检测中,共得到1857个条带,相似度在0.980-0.983之间。表明直接再生苗不存在明显的体细胞变异。
2.初步建立了八仙花基因型Hydl的遗传转化体系
以再生率高的基因型Hydl叶片为试验材料,通过抗生素、预培养、侵染、共培养时间、农杆菌菌株等因素对比试验,探讨了相关因子对八仙花遗传转化的影响。研究表明：叶片不定芽再生的筛选浓度为20 mg/L卡那霉素(Km),再生苗生根的筛选浓度为200mg/LKm,200 mg/L Cef作为抑菌剂。叶片在预培养0d,侵染35 min和共培养3d条件下,GUS瞬间表达率最高可达100%。该转化条件结合延迟筛选7d可获得100%抗性愈伤率和12个抗性芽。抗性愈伤组织在后期分化抗性芽较为困难,需要进一步研究其分化条件。
3.建立了高效稳定的艳果金丝桃快繁与再生体系
以MS基本培养基和BA/NAA配比对比试验表明,以基因型Hyp3带腋芽茎段为外植体在MS+BA1.0mg/L+NAA0.1mg/L的培养基上获得了最优的腋芽诱导及增殖效果,丛生芽诱导率和平均增殖芽数分别为100%和3.7个。
以艳果金丝桃四个基因型Hyp1、Hyp3、Hyp4和Hyp7叶片为外植体对其再生体系进行的研究表明,MS+TDZ0.1mg/L+AgNO31.0 mg/L+蔗糖30mg/L为最优的再生培养基,在暗培养20d后转入光周期下培养,再生率达83.3%。不同基因型叶片再生效果差异较大,其中,以基因型Hyp3的再生能力最强。
4.初步建立了艳果金丝桃遗传转化体系
对影响艳果金丝桃基因型Hyp3遗传转化预培养时间、侵染时间等的几个因子进行了研究,结果表明：Hyp3叶片为外植体以EHA105为侵染菌株,预培养0d,侵染5min,在MS+TDZ 0.1 mg/L+NAA 0.01 mg/L+AS 40 mg/L+MES 2.0g/L培养基上共培养3d,然后转移到选择培养基(MS+TDZ 0.1 mg/L+NAA 0.01 mg/L+ AgN031.0 mg/L+cef200 mg/L)上培养。在该条件下能够得到79.2%的GUS瞬时表达率,并且能够得到少量抗性愈伤组织。
5.八仙花叶斑病病原菌的研究
通过对盆栽八仙花叶片感病症状的观察、并对导致叶斑病的病原菌进行分离、形态学鉴定和rDNA-ITS分析,确定该病原菌为八仙花尾孢菌(Cercospora hydrangeae)。所得ITS序列经测序后已上载到Genbank,序列号：JF495458.1。
Hydrangea macrophylla (Thunb.) and Hypericum androsaemum L. are woody ornamental plants belonging to the Hydrangeaceae and Hypericaceae families respectively. Hydrangea macrophylla (Thunb) is used for planting courtyard flower borders, forest fringes, and ornamental garden, and is used in large scale flower decorations and sold as potted flower due to its colorful and large flower and long-lasting florescence. Hypericum androsaemum L. is a relatively new winter courtyard shrub and is used as new cut flower material because of its attractive looking fruits, long evergreen shoots and long lasting fruiting stage. However, both crops are vulnerable to plant diseases during the growing season, namely Hydrangea leaf spot and Hypericum root-knot nematode. Genetic engineering is potentially an efficient way of improving their disease resistance. By investigating the factors such as choice of genotype and explants, incubation conditions, inoculation methods, and different plant growth regulators, we established a highly efficient regeneration system and studied. An agrobacterium tumefaciens-mediated genetic transformation of Hydrangea macrophylla was developed as well. In addition, the pathogen causing leaf spot was isolated and identified as Cercospora hydrangeae. The major results were as follows:
1. The establishment of a rapid propagation and regeneration system for Hydrangea
Sterile shoots of Hydrangea macrophylla were obtained for genotypes Hydl, Hyd4 and Hyd5. The stems with axillary buds of these three genotypes were used as explants for the establishment of a micropropagation system. The optimum medium for proliferation of the three genotypes was B5+0.5 mg/L BA+0.1 mg/L NAA with a resulting multiplication coefficient of 3.0. Hydl terminal buds were used as explants for rooting. The rooting medium with 60 mL perlite plus 30 mL liquid medium of 1/2 B5+ 0.5 mg/L IBA gave the best results for the rooting rate (100%). Rooted shoots transplanted to mixed substrates of peat soil:perlite (1:1) had a survival rate as high as 100% after 30 days.
Leaves and petioles of seven genotypes Hyd1, Hyd4, Hyd5, Hyd6, Hyd7, Hyd8 and Hyd10 were used as explants for experiments involving direct and indirect regeneration. The results showed that for leaf explants of Hydl on the B5 medium supplemented with 2.25mg/L BA,0.1 mg/L IBA and 100 mg/L cefotaxime, the regeneration rate was 100% and average number of shoots per explant was 2.7. There were large differences in regeneration capacity among different genotypes. On the same medium, the regeneration rate in leaf explants of Hyd4 and Hyd8 were lower than 30%.
Somaclonal variation of Hydrangea macrophylla regenerants were tested by ISSR markers. There were in total 1857 scorable bands. The observed similarity was between 0.980-0.983. No significant somaclonal variation was abserved.
2. Study on development of a genetic transformation system of Hydrangea macrophylla
Agrobacterium tumefaciens-mediated genetic transformation was studied by using leaf explants of Hydl. The concentration of Kanamycin selection pressures was determined as 20 mg/L for shoot regeneration of leaves and 200 mg/L for rooting. A. tumefaciens could be inhibited with 200 mg/L cefotaxime. GUS transient expression analysis revealed that the leaf explants with 0 d pre-culture, infected for 35min and co-cultured for 3 day could reach the highest transient GUS expression rate of 100%. Delayed kanamycin selection for seven days benefited the formation of resistant callus and shoots. Resistant callus (100%) and 12 regenerated plants were obtained under the optimal transformation conditions。Resistant callus was difficult to induce for regeneration on kanamycin medium. Further research should focus on callus differentiation.
3. Establishment of a rapid micro-propagation and regeneration system of Hypericum androsaemum
Basal medium and BA/NAA were tested in order to establish a rapid micro-propagation sytem of genotype Hyp3. The highest shoot induction percentage (100%) and mean number of shoot per explant (3.7) were found on 1/2MS plus BA1.0mg/L.
A regeneration system for Hypericum androsaemum was studied using explants of four genotypes Hypl, Hyp3, Hyp4 and Hyp7. A stable and highly efficient regeneration system of Hyp3 leaf explants was established, viz., media:MS+TDZ0.1 mg/L+AgNO3 1.0 mg/L+sucrose 30 mg/L, initial 20d dark culture followed by photoperiod culture. The highest regeneration rate was 83.3%. Genotype was a significant factor in Hypericum androsaemum regeneration. Medium with AgNO3 supplied a highly efficient and stable regeneration system.
4. Study on genetic transformation of Hypericum androsaemum
The factors of genetic transformation were tested in Hypericum androsaemum genotype Hyp3. The optimal protocol was as follows. Genotype Hyp3 leaf explants without pre-culture was immerged into EHA105 for 5 min, co-cultured on MS+ TDZ 0.1 mg/L+NAA 0.01 mg/L+AS 40 mg/L+MES 2.0g/L medium for 3d, selected on MS+ TDZ 0.1 mg/L+NAA 0.01 mg/L+AgNO3 1.0 mg/L+cef200 mg/L medium. High transient GUS expression rated (79.2%) was gained.
5. Study on hydrangea leaf spot
Pathogen was isolated from diseased potted hydrangea leaves and was identified as Cercospora hydrangeae by morphological and rDNA-ITS way. The ITS sequence was deposited to Genbank, accessing No. JF495458.1.
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