紫花苜蓿和白花草木樨细胞融合技术研究
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摘要
利用原生质融合进行优良基因转移是一种短期而且非常有效的生物技术。目前国内外利用此项技术对番茄、烟草、水稻等植物进行了大量的研究。现已从许多种内、种间、属间甚至亚科间的体细胞杂交获得杂种细胞系或杂种植株。目前细胞融合技术已被广泛用于牧草研究的许多领域,并在紫花苜蓿和其他牧草抗性育种、减少膨胀病危害、提高干物质消化率、生物可降解材料开发、生物土壤改良、生物疫苗及活性制剂等方面取得了进展和突破。但是利用草木樨进行原生质体融合的研究国内外未见报道。本实验建立了完整的紫花苜蓿、草木樨组培体系,并对紫花苜蓿、白花草木樨原生质体融合的条件进行了初步分析,主要结果如下:
1.以MS为基本培养基,通过调整激素浓度等培养条件,摸索出紫花苜蓿和草木樨组织培养体系的最佳培养基。紫花紫花苜蓿的最佳培养基:愈伤组织诱导最佳培养为MS+2,4-D3.0mg-Lˉ1+6-BA0.3mg-Lˉ1;草木樨的最佳培养基:愈伤诱导最佳培养基为MS+2,4-D1.5mg- Lˉ1+6-BA0.3 mg-Lˉ1。
2.本研究对酶液最佳配方进行研究,得出分离紫花苜蓿原生质体酶液的组成以2%的纤维素酶、0.2%果胶酶、0.5%离析酶为好,产量可达1.9×107个/g;酶解时间为10h时原生质体的活力最大为60%;分离草木樨原生质体的酶液组成以2%的纤维素酶、0.3%果胶酶、1%离析酶为好,产量可达4.31×107个/g,酶解时间控制在15h时原生质体的火力最大为62.5%。
3.用不同浓度的IOA处理白花草木樨原生质体,得到以下结果:以3mmol/ml IOA处理10min,能有效地使白花草木樨原生质体失活。
4.用紫外线处理紫花苜蓿原生质原生质体,得到以下结果:以375 uw/cm2辐射量照射60s,能有效的使紫花苜蓿原生质原生质体失活。
5.通过PEG诱导原生质体融合,得到紫花苜蓿和草木樨的属间体细胞杂种,最佳融合条件为:PEG(6000)浓度为40%,融合率为12.5%。
It is a shot-term and effective biotechnology that make use of the protoplast fusion to transfer the good gene.now this technology was researched on tomatoes,tobacco,rice and other plants at home and abroad. Now many species between species and others or even a subfamily of somatic cell hybrid access to hybrid cell lines or hybrid plants.At present cell fusion technology has been widely used for forage in many areas of research and alfalfa and other forage resistance breeding,reduce swelling of hazards increase dry matter digestibility, biodegradable materials development,biological soil improvement, vaccines and biological agents such as the activity has made progress and breakthroughs But the protoplast fusion research has not been reported at home and abroad.In this study,the whole tissue cultivating systerm of Alfalfa and Melilotus suaveolen was established, and the condition of protoplasts fusion was primarily analyzed. The results of our experiment were shown as follows:
1. MS was used as the basic cultivating medium. The optimal cultivating conditions of Alfalfa and Melilotus suaveolen was found through adjusting the varieties and concentration of hormones according to the orthogonal design. The most appropriate cultivating medium of Alfalfa was MS + 2,4-D 3.0mg/L + 6-BA 0.3 mg/L for callus. The most appropriate cultivating medium of Melilotus suaveolen was MS + 2,4-D 1.5mg/L + 6-BA 0.3 mg/L.
2. The best combination of isolating protoplast of alfalfa was enzyme mixture containing 2% Cellulase R-10, 0.2% Pectinase and 0.5% Macerozyme R-10,and highest yield achieved to 1.9×107/g, hydrolysis time is 10h, the protoplast vitality achieved 60%; The best combination of isolating protoplast Melilotus suaveolen was enzyme mixture containing 2% Cellulase R-10, 0.3% Pectinase and 1% Macerozyme R-10 hydrolysis time is 15h, the protoplast vitality achieved 62.5%.
3. The protoplast of Melilotus suaveolen was treaed by IOA with different concentrations, the best of this experiment was shown as follows: 3mmol/ml IOA was used to kill the protoplast of Melilotus suaveolen by treating ten minutes.
4. The protoplast of alfalfa was treaed by UV, the best of this experiment was shown as follows: RadiationOf UV is 375 uw/cm2 was used to kill the protoplast of alfalfa by treating one minutes.
5 Hybrid cells of alfalfa and Melilotus suaveolen were obtained using protoplast usion by PEG method, The best fusion condition 40% PEG(6000) can obtain the hybrid cell ,the Fusion rate is 12.5%.
1.以MS为基本培养基,通过调整激素浓度等培养条件,摸索出紫花苜蓿和草木樨组织培养体系的最佳培养基。紫花紫花苜蓿的最佳培养基:愈伤组织诱导最佳培养为MS+2,4-D3.0mg-Lˉ1+6-BA0.3mg-Lˉ1;草木樨的最佳培养基:愈伤诱导最佳培养基为MS+2,4-D1.5mg- Lˉ1+6-BA0.3 mg-Lˉ1。
2.本研究对酶液最佳配方进行研究,得出分离紫花苜蓿原生质体酶液的组成以2%的纤维素酶、0.2%果胶酶、0.5%离析酶为好,产量可达1.9×107个/g;酶解时间为10h时原生质体的活力最大为60%;分离草木樨原生质体的酶液组成以2%的纤维素酶、0.3%果胶酶、1%离析酶为好,产量可达4.31×107个/g,酶解时间控制在15h时原生质体的火力最大为62.5%。
3.用不同浓度的IOA处理白花草木樨原生质体,得到以下结果:以3mmol/ml IOA处理10min,能有效地使白花草木樨原生质体失活。
4.用紫外线处理紫花苜蓿原生质原生质体,得到以下结果:以375 uw/cm2辐射量照射60s,能有效的使紫花苜蓿原生质原生质体失活。
5.通过PEG诱导原生质体融合,得到紫花苜蓿和草木樨的属间体细胞杂种,最佳融合条件为:PEG(6000)浓度为40%,融合率为12.5%。
It is a shot-term and effective biotechnology that make use of the protoplast fusion to transfer the good gene.now this technology was researched on tomatoes,tobacco,rice and other plants at home and abroad. Now many species between species and others or even a subfamily of somatic cell hybrid access to hybrid cell lines or hybrid plants.At present cell fusion technology has been widely used for forage in many areas of research and alfalfa and other forage resistance breeding,reduce swelling of hazards increase dry matter digestibility, biodegradable materials development,biological soil improvement, vaccines and biological agents such as the activity has made progress and breakthroughs But the protoplast fusion research has not been reported at home and abroad.In this study,the whole tissue cultivating systerm of Alfalfa and Melilotus suaveolen was established, and the condition of protoplasts fusion was primarily analyzed. The results of our experiment were shown as follows:
1. MS was used as the basic cultivating medium. The optimal cultivating conditions of Alfalfa and Melilotus suaveolen was found through adjusting the varieties and concentration of hormones according to the orthogonal design. The most appropriate cultivating medium of Alfalfa was MS + 2,4-D 3.0mg/L + 6-BA 0.3 mg/L for callus. The most appropriate cultivating medium of Melilotus suaveolen was MS + 2,4-D 1.5mg/L + 6-BA 0.3 mg/L.
2. The best combination of isolating protoplast of alfalfa was enzyme mixture containing 2% Cellulase R-10, 0.2% Pectinase and 0.5% Macerozyme R-10,and highest yield achieved to 1.9×107/g, hydrolysis time is 10h, the protoplast vitality achieved 60%; The best combination of isolating protoplast Melilotus suaveolen was enzyme mixture containing 2% Cellulase R-10, 0.3% Pectinase and 1% Macerozyme R-10 hydrolysis time is 15h, the protoplast vitality achieved 62.5%.
3. The protoplast of Melilotus suaveolen was treaed by IOA with different concentrations, the best of this experiment was shown as follows: 3mmol/ml IOA was used to kill the protoplast of Melilotus suaveolen by treating ten minutes.
4. The protoplast of alfalfa was treaed by UV, the best of this experiment was shown as follows: RadiationOf UV is 375 uw/cm2 was used to kill the protoplast of alfalfa by treating one minutes.
5 Hybrid cells of alfalfa and Melilotus suaveolen were obtained using protoplast usion by PEG method, The best fusion condition 40% PEG(6000) can obtain the hybrid cell ,the Fusion rate is 12.5%.
引文
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