非洲菊（Gerbera hybrida）耐热变异品系的离体筛选与初步鉴定
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摘要
非洲菊(Gerbera hybrida)为世界十大切花之一，属半耐寒性花卉，忌高温，生产上急需培育耐高温的非洲菊新品种。本实验以Lilabella(F3)、Diablo(F25)、Vino(F30)、Sangria(F32)、Pinkas(F39)及深圳五号(S5)等六个非洲菊品种为材料，建立了非洲菊耐热变异的离体筛选体系；探讨了~(60)Co γ射线与EMS、PYM化学诱变处理对离体培养的影响和筛选耐热变异的可能性；确定了再生植株诱导与增殖过程中羟脯氨酸适宜的筛选剂量，并在此基础上开展了非洲菊抗羟脯氨酸变异系的筛选；初步分析了耐热和耐羟脯氨酸筛选获得的三个初选系在高温下耐热表现。主要研究结果如下：
1．45℃热激处理2h能较为稳定提高组培苗的增殖率。细胞膜相对电解质渗透率与存活率在大多数情况下具有良好的相关性，在很大程度上反应了植物的耐热性，可用于确定筛选的压力和鉴定非洲菊不同品种的耐热性。同一非洲菊品种在35℃与45℃胁迫下的热害表现不同，耐热性也不一致。耐热性不同的非洲菊需选取不同的胁迫压力，35℃下10到25d，45℃下20h左右为各品种适宜的筛选压范围。
2．5000rad适宜作为~(60)Co γ辐射诱变花托的处理剂量，对于组培苗来说，除了品种F3外，仍可适当提高诱变的剂量。EMS诱变处理中，非洲菊花托对诱变处理时间的变化比对EMS浓度的变化更为敏感，4h适宜作为0.4％EMS的处理时间。PYM作为一种新型诱变剂，40μg／ml浸泡处理1.5 h是预培养花托的适宜处理方法。诱变苗在进行高温筛选时，存活率出现变化、叶色叶型发生变异，表明诱变与筛选是有效的。经6至9次高温定向筛选后，获得了多个S5、F25、F32的耐热初选变异系。
3．不同浓度HYP对花托和组培苗增殖培养影响的研究表明，0.4mg／ml的HYP是适宜的筛选剂量，对于预培养25d的花托来说，HYP浓度增加到0.6mg／ml比较合适。对花托和组培苗多次HYP间隔胁迫筛选，获得了经5次和6次筛选的初选变异系各1个。
华南师范大学硕士学位论文
非洲菊耐热变异的离体筛选与初步鉴定
4.SR7、SHPS、SE6初选系组培苗在模拟高温下与对照的耐热性进行了比
较，发现它们在45℃和35℃两种高温处理下的存活率都有所提高。其中，SR，
与对照在35℃处理15d时的存活率具差异显著，是一个耐热变异新材料。SR7
的Pro含量、POD和SOD活性等指标在35℃处理后与对照55具有显著的差异，
可能与非洲菊耐热性的提高有关。
我们的研究结果表明，通过体细胞无性系变异离体筛选技术获得目的变异
是切实可行的，但需建立在大规模筛选的基础之上。
Gerbera hybrida, one of the ten greatest cut flowers in the world, is a kind of cold-half-resistant flower. Because almost all of the varieties in Gerbera are maladjusted to high temperature, it is necessary to breed thermotolerant varieties in practice. In this research, we established an in vitro screening procedure for thermotolerant variants with six varieties of Gerbera including Lilabella(F3), Diablo(F25), Vino(F30), Sangria(F32), Pinkas(F39) and No.5 Shengzheng( S5). 60Co γ irradiation and EMS/PYM treatment were used for mutation of the plants and the proper HYP dosages for screening in the in vitro culture were investigated. .The physiological and biochemical characteristics of the three thermotolerant variant lines from in vitro screening with high temperature or HYP were analyzed
comparing with the control. The main results are summarized as follows:
1. The proliferation rates of in vitro shoots increased steadily after 2 h heat shock of 45 ℃. The relative electrolyte leakage of cell membrane showed well relat-ionship to the survival rate of shoots in vitro. The appearance of heat damage and the thermotolerance at 35℃ or 45℃ were different among the different varieties of Gerbera and it is necessary to screen thermotolerant lines under different pressures for them. The results showed that 10 to 25 d at 35℃and 20 h at 45℃ were the proper intimidate pressure ranges for each variety.
2. The dosage of 60Co γ irradiation in 5000 rad was proper for thalami mutation and it could be moderately higher for shoots in vitro of all varieties except F3. The thalami were more sensitive to the treatment time than the concentration when EMS was used as a mutagen and 4 h was the proper treatment time under 0.4% EMS. PYM is another mutagen we used and 1.5 h soak with 40 g/ ml PYM was proper for pre-cultured of thalami. The survival rates and the variation of the color and shape in leaves after screening with high temperature showed that our experimental procedure
was effective and several primary thermotolerant lines in S5, F25, F32 were selected after 6 to 9 heat screening.
3. The effects of HYP with different concentrations on the culture of thalami and shoots in vitro were investigated. The results showed that 0.4mg/ml HYP was the proper dosage for thalami and shoots in vitro and 0.6mg/ml was appropriate for 25 d precultured thalami. The primary variation lines were isolated from S5 after 5 and 6 HYP screening.
4. The shoots in vitro of three thermotolerant lines of SR7, SHP5 and SE6 showed higher survival rates at 35 ℃and 45 ℃ comparing with the control. Among three lines SR7 had a remarkable difference with the control under the treatment of 15 days with
35 ℃ and it is a new material of thermotolerant variation we selected. The content of Pro and the activities of POD and SOD in SR7 also increased remarkably indicating
these biochemical changes may be related to the thermotolerance of Gerbera.
Our results showed above demonstrated that it was feasible to isolate thermotolerant variant lines by in vitro screening technique and a large scale of screening was required.
1．45℃热激处理2h能较为稳定提高组培苗的增殖率。细胞膜相对电解质渗透率与存活率在大多数情况下具有良好的相关性，在很大程度上反应了植物的耐热性，可用于确定筛选的压力和鉴定非洲菊不同品种的耐热性。同一非洲菊品种在35℃与45℃胁迫下的热害表现不同，耐热性也不一致。耐热性不同的非洲菊需选取不同的胁迫压力，35℃下10到25d，45℃下20h左右为各品种适宜的筛选压范围。
2．5000rad适宜作为~(60)Co γ辐射诱变花托的处理剂量，对于组培苗来说，除了品种F3外，仍可适当提高诱变的剂量。EMS诱变处理中，非洲菊花托对诱变处理时间的变化比对EMS浓度的变化更为敏感，4h适宜作为0.4％EMS的处理时间。PYM作为一种新型诱变剂，40μg／ml浸泡处理1.5 h是预培养花托的适宜处理方法。诱变苗在进行高温筛选时，存活率出现变化、叶色叶型发生变异，表明诱变与筛选是有效的。经6至9次高温定向筛选后，获得了多个S5、F25、F32的耐热初选变异系。
3．不同浓度HYP对花托和组培苗增殖培养影响的研究表明，0.4mg／ml的HYP是适宜的筛选剂量，对于预培养25d的花托来说，HYP浓度增加到0.6mg／ml比较合适。对花托和组培苗多次HYP间隔胁迫筛选，获得了经5次和6次筛选的初选变异系各1个。
华南师范大学硕士学位论文
非洲菊耐热变异的离体筛选与初步鉴定
4.SR7、SHPS、SE6初选系组培苗在模拟高温下与对照的耐热性进行了比
较，发现它们在45℃和35℃两种高温处理下的存活率都有所提高。其中，SR，
与对照在35℃处理15d时的存活率具差异显著，是一个耐热变异新材料。SR7
的Pro含量、POD和SOD活性等指标在35℃处理后与对照55具有显著的差异，
可能与非洲菊耐热性的提高有关。
我们的研究结果表明，通过体细胞无性系变异离体筛选技术获得目的变异
是切实可行的，但需建立在大规模筛选的基础之上。
Gerbera hybrida, one of the ten greatest cut flowers in the world, is a kind of cold-half-resistant flower. Because almost all of the varieties in Gerbera are maladjusted to high temperature, it is necessary to breed thermotolerant varieties in practice. In this research, we established an in vitro screening procedure for thermotolerant variants with six varieties of Gerbera including Lilabella(F3), Diablo(F25), Vino(F30), Sangria(F32), Pinkas(F39) and No.5 Shengzheng( S5). 60Co γ irradiation and EMS/PYM treatment were used for mutation of the plants and the proper HYP dosages for screening in the in vitro culture were investigated. .The physiological and biochemical characteristics of the three thermotolerant variant lines from in vitro screening with high temperature or HYP were analyzed
comparing with the control. The main results are summarized as follows:
1. The proliferation rates of in vitro shoots increased steadily after 2 h heat shock of 45 ℃. The relative electrolyte leakage of cell membrane showed well relat-ionship to the survival rate of shoots in vitro. The appearance of heat damage and the thermotolerance at 35℃ or 45℃ were different among the different varieties of Gerbera and it is necessary to screen thermotolerant lines under different pressures for them. The results showed that 10 to 25 d at 35℃and 20 h at 45℃ were the proper intimidate pressure ranges for each variety.
2. The dosage of 60Co γ irradiation in 5000 rad was proper for thalami mutation and it could be moderately higher for shoots in vitro of all varieties except F3. The thalami were more sensitive to the treatment time than the concentration when EMS was used as a mutagen and 4 h was the proper treatment time under 0.4% EMS. PYM is another mutagen we used and 1.5 h soak with 40 g/ ml PYM was proper for pre-cultured of thalami. The survival rates and the variation of the color and shape in leaves after screening with high temperature showed that our experimental procedure
was effective and several primary thermotolerant lines in S5, F25, F32 were selected after 6 to 9 heat screening.
3. The effects of HYP with different concentrations on the culture of thalami and shoots in vitro were investigated. The results showed that 0.4mg/ml HYP was the proper dosage for thalami and shoots in vitro and 0.6mg/ml was appropriate for 25 d precultured thalami. The primary variation lines were isolated from S5 after 5 and 6 HYP screening.
4. The shoots in vitro of three thermotolerant lines of SR7, SHP5 and SE6 showed higher survival rates at 35 ℃and 45 ℃ comparing with the control. Among three lines SR7 had a remarkable difference with the control under the treatment of 15 days with
35 ℃ and it is a new material of thermotolerant variation we selected. The content of Pro and the activities of POD and SOD in SR7 also increased remarkably indicating
these biochemical changes may be related to the thermotolerance of Gerbera.
Our results showed above demonstrated that it was feasible to isolate thermotolerant variant lines by in vitro screening technique and a large scale of screening was required.
引文
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[89] Park S, Shivaji R, Krans J V et al. Heat-shock response in heat-tolerant and nontolerant variants of Agrostis palustris Huds, Plant Physiology. 1996, 111: 515-524.
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