唐菖蒲原生质体分离、纯化及培养的研究
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摘要
唐菖蒲(Gladiolus hybridus Hort)是鸢尾科(Iridaceae)唐菖蒲属(Gladiolus)多年生单子叶球根花卉，是世界著名的四大切花之一。唐菖蒲切花生产是我国花卉产业的重要组成部分。唐菖蒲是病虫害发生最为严重的花卉之一，利用原生质体导入外源基因为唐菖蒲抗病育种带来希望。目前我国唐菖蒲的栽培品种和生产用球绝大多数从国外引进，唐菖蒲的品种繁育工作不足，唐菖蒲原生质体分离、纯化及培养技术的研究将为唐菖蒲育种工作奠定基础。
本试验以唐菖蒲子球茎为材料，建立唐菖蒲原生质体分离、纯化及培养体系。主要结果如下：
1．唐菖蒲子球茎诱导愈伤的培养基为：“超级玫瑰”MS+2，4-D3.0mg／L+6-BA0.5mg／L：“超级粉”MS+2，4-D4.0mg／L+6-BA0.5mg／L；“蓝精灵”MS+2，4-D4.0mg／L+6-BA005mg／L
2．三种类型唐菖蒲愈伤组织：Ⅰ型为近胚性愈伤，Ⅱ型为非胚性愈伤，Ⅲ型为中间型愈伤，其中Ⅰ和Ⅲ型愈伤组织培养2～3月后可得到胚性愈伤。胚性愈伤在MS+2，4-D2.0mg／L+6-BA0.5mg／L培养基上培养有利于唐菖蒲胚性愈伤的保持。
3．唐菖蒲悬浮培养细胞系的培养方法为：悬浮细胞的初始接种量为2g／40ml，初始继代周期2～3d，一个月后继代周期为5～8d。在悬浮培养基中加入2mg／L的甘氨酸，100mg／L的谷氨酸、300mg／L的水解乳蛋白、100mg／L的酵母提取物、500mg／L的牛血清白蛋白有助于原生质体活力的提高。
4．露醇保持唐菖蒲原生质体渗透压稳定的浓度为0.6mol／L。
5．唐菖蒲叶片在0.6mol／L甘露醇中预处理1h分离得到的原生质体产量和活力较高；继代培养2个月左右的悬浮培养细胞系分离的原生质体产量和活力较高。
6．叶片和悬浮培养细胞分离原生质体所用酶液的配比浓度分别为Cellulase“Onzuka”RS1.5％+Pectinase Y-23 0.5％和Cellulase“Onzuka”RS 2.0％+Pectinase Y-23 0.5％，酶解时间为2h，pH5.6；酶解的条件为：黑暗下27℃，于摇床上振荡酶解(40r／min)。
7．原生质体培养的方法为先液体后包埋法，其培养基分别为：叶片“超级玫瑰”KM_8P+2，4-D1.0mg／L，+NAA0.2mg／L+KT0.2mg／L，“超级粉”KM_8P+2，4-D1.5mg／L+NAA0.2mg／L+ZT0.2mg／L，“蓝精灵”KM_8P+2，4-D2.5mg／L+KT0.2mg／L+6-BA0.1mg／L；悬浮培养细胞“超级玫瑰”KM_8P+2，4-D2.0mg／L+NAA0.2mg／L+KT0.2mg／L，“超级粉”KM_8P+2，4-D2.5mg／L+NAA0.2mg／L+ZT0.2mg／L，“蓝精灵”KM_8P+2，4-D4.0mg／L+KT0.2mg／L+6-BA0.1mg／L。
Gladiolus hybridus Hort, also called sword or gladiolus, is one of perennial monocotyledonous bulbous flowers of gladiolus Iridaceae, which is one of the four world-famous cut flowers. Cut flower production of gladiolus is the important parts of flower industry.
Gladiolus is one of the most serious diseases and insect pests of all flowers. It brings the new hope for the disease-resistance breeding of the gladiolus to transform foreign genes by protoplast method. Now most of planting varieties and corms used in production come from foreign and lack of varieties breeding of the gladiolus in our country. The study on separation, purification and culture technique of the gladiolus will lay the foundation for the breeding of the gladiolus.
The study took the offspring corms of the gladiolus as materials and esTab.lished the system of the separation, purification and culture technique of the gladiolus. The results were as follows:
1. Culture medium of the induced callus system of the offspring corms of the gladiolus: 'Rose Supreme' MS+2.4-D 3.0 mg/L+6-BA 0.5 mg/L; 'Pink Supreme'MS+2,4-D4.0mg/L+ 6-BA0.5mg/L; and 'Blue Fairy' MS+2,4-D 4.0 mg/L+6-BA 0.5 mg/L.
2. The study gained the callus of three types: type I Embryonic callus; type II Non-embryonic callus; type III middle types of callus. Thereinto callus culture of type I and type III may get the embryonic callus after 2-3 months. Culture medium with MS, 2,4-D 2.0 mg/L and 6-BA 0.5 mg/L will be favour for the vigour of the embryonic callus.
3. Culture methods of suspending culture cells of the gladiolus were as following: First inoculation quantum was 2g/40ml, and first sub-culture cycle was 2-3d and 5~8d after a month. It was favour for the improving of protoplast vigour to the medium with 2mg/L Gly, 100mg/L Glu, 300mg/L LH, 100mg/L yeast extraction and 500mg/L BSA.
4. Confirmed 0.6mol/L mannitol to be concentration of the osmotic pressure.
5. Vigor and yields of lamina protoplast-pretreated 1h in 0.6mol/L mannitol were higher. Vigor and yields of protoplast, which was gained by the suspending culture system and sub-cultured for two month, were higher.
6. Determined that the enzyme concentrations of lamina and suspending cells of gladiolus to separate protoplast respectively were Cellulase "Onzuka" RS 1.5%+Pectinase Y-23 0.5% and Cellulase "Onzuka" RS 2.0% + Pectinase Y-23 0.5%, PH5.6, the digestion time was 2h, and the digestion conditions were incubated at 40rpm/min, temperature 27℃ in the dark.
7. Culture method of protoplast was first liquid and then embedded. Culture mediums were as follows: 'Rose Supreme' KM8P + 2,4-D1.0mg/L + NAA0.2mg/L + KT0.2mg/L; 'Pink Supreme' KM8P + 2,4-D 1.5mg/L + NAA0.2mg/L + ZT0.2mg/L; and 'Blue Fairy' KM8P +
本试验以唐菖蒲子球茎为材料，建立唐菖蒲原生质体分离、纯化及培养体系。主要结果如下：
1．唐菖蒲子球茎诱导愈伤的培养基为：“超级玫瑰”MS+2，4-D3.0mg／L+6-BA0.5mg／L：“超级粉”MS+2，4-D4.0mg／L+6-BA0.5mg／L；“蓝精灵”MS+2，4-D4.0mg／L+6-BA005mg／L
2．三种类型唐菖蒲愈伤组织：Ⅰ型为近胚性愈伤，Ⅱ型为非胚性愈伤，Ⅲ型为中间型愈伤，其中Ⅰ和Ⅲ型愈伤组织培养2～3月后可得到胚性愈伤。胚性愈伤在MS+2，4-D2.0mg／L+6-BA0.5mg／L培养基上培养有利于唐菖蒲胚性愈伤的保持。
3．唐菖蒲悬浮培养细胞系的培养方法为：悬浮细胞的初始接种量为2g／40ml，初始继代周期2～3d，一个月后继代周期为5～8d。在悬浮培养基中加入2mg／L的甘氨酸，100mg／L的谷氨酸、300mg／L的水解乳蛋白、100mg／L的酵母提取物、500mg／L的牛血清白蛋白有助于原生质体活力的提高。
4．露醇保持唐菖蒲原生质体渗透压稳定的浓度为0.6mol／L。
5．唐菖蒲叶片在0.6mol／L甘露醇中预处理1h分离得到的原生质体产量和活力较高；继代培养2个月左右的悬浮培养细胞系分离的原生质体产量和活力较高。
6．叶片和悬浮培养细胞分离原生质体所用酶液的配比浓度分别为Cellulase“Onzuka”RS1.5％+Pectinase Y-23 0.5％和Cellulase“Onzuka”RS 2.0％+Pectinase Y-23 0.5％，酶解时间为2h，pH5.6；酶解的条件为：黑暗下27℃，于摇床上振荡酶解(40r／min)。
7．原生质体培养的方法为先液体后包埋法，其培养基分别为：叶片“超级玫瑰”KM_8P+2，4-D1.0mg／L，+NAA0.2mg／L+KT0.2mg／L，“超级粉”KM_8P+2，4-D1.5mg／L+NAA0.2mg／L+ZT0.2mg／L，“蓝精灵”KM_8P+2，4-D2.5mg／L+KT0.2mg／L+6-BA0.1mg／L；悬浮培养细胞“超级玫瑰”KM_8P+2，4-D2.0mg／L+NAA0.2mg／L+KT0.2mg／L，“超级粉”KM_8P+2，4-D2.5mg／L+NAA0.2mg／L+ZT0.2mg／L，“蓝精灵”KM_8P+2，4-D4.0mg／L+KT0.2mg／L+6-BA0.1mg／L。
Gladiolus hybridus Hort, also called sword or gladiolus, is one of perennial monocotyledonous bulbous flowers of gladiolus Iridaceae, which is one of the four world-famous cut flowers. Cut flower production of gladiolus is the important parts of flower industry.
Gladiolus is one of the most serious diseases and insect pests of all flowers. It brings the new hope for the disease-resistance breeding of the gladiolus to transform foreign genes by protoplast method. Now most of planting varieties and corms used in production come from foreign and lack of varieties breeding of the gladiolus in our country. The study on separation, purification and culture technique of the gladiolus will lay the foundation for the breeding of the gladiolus.
The study took the offspring corms of the gladiolus as materials and esTab.lished the system of the separation, purification and culture technique of the gladiolus. The results were as follows:
1. Culture medium of the induced callus system of the offspring corms of the gladiolus: 'Rose Supreme' MS+2.4-D 3.0 mg/L+6-BA 0.5 mg/L; 'Pink Supreme'MS+2,4-D4.0mg/L+ 6-BA0.5mg/L; and 'Blue Fairy' MS+2,4-D 4.0 mg/L+6-BA 0.5 mg/L.
2. The study gained the callus of three types: type I Embryonic callus; type II Non-embryonic callus; type III middle types of callus. Thereinto callus culture of type I and type III may get the embryonic callus after 2-3 months. Culture medium with MS, 2,4-D 2.0 mg/L and 6-BA 0.5 mg/L will be favour for the vigour of the embryonic callus.
3. Culture methods of suspending culture cells of the gladiolus were as following: First inoculation quantum was 2g/40ml, and first sub-culture cycle was 2-3d and 5~8d after a month. It was favour for the improving of protoplast vigour to the medium with 2mg/L Gly, 100mg/L Glu, 300mg/L LH, 100mg/L yeast extraction and 500mg/L BSA.
4. Confirmed 0.6mol/L mannitol to be concentration of the osmotic pressure.
5. Vigor and yields of lamina protoplast-pretreated 1h in 0.6mol/L mannitol were higher. Vigor and yields of protoplast, which was gained by the suspending culture system and sub-cultured for two month, were higher.
6. Determined that the enzyme concentrations of lamina and suspending cells of gladiolus to separate protoplast respectively were Cellulase "Onzuka" RS 1.5%+Pectinase Y-23 0.5% and Cellulase "Onzuka" RS 2.0% + Pectinase Y-23 0.5%, PH5.6, the digestion time was 2h, and the digestion conditions were incubated at 40rpm/min, temperature 27℃ in the dark.
7. Culture method of protoplast was first liquid and then embedded. Culture mediums were as follows: 'Rose Supreme' KM8P + 2,4-D1.0mg/L + NAA0.2mg/L + KT0.2mg/L; 'Pink Supreme' KM8P + 2,4-D 1.5mg/L + NAA0.2mg/L + ZT0.2mg/L; and 'Blue Fairy' KM8P +
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