几种草坪草的组织培养与转基因研究
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摘要
草坪草在人类生活中的地位越来越重要，筛选与培育优良的草坪草新品种是草坪业的重要任务。本文对假俭草[Eremochloa ophiuroides(Munro.)Hack.]，高羊茅(Festuca arundinacea Schreb.)，狗牙根(Cynodon dactylon var.)，多年生黑麦草(Lolium perenne L.)四类草坪草进行了组织培养与植株再生的研究，建立了假俭草与高羊茅的遗传转化体系并对其进行了转基因研究，获得了抗除草剂与抗真菌病的假俭草与高羊茅转化植株。
草坪草组织培养体系的建立与完善将为传统与转基因育种奠定基础，本研究在这方面取得了以下结果：
1)在外植体的选取方面，发现高羊茅离体成熟种胚比完整种子作外植体愈伤组织的诱导率与质量都优，假俭草以含低浓度2，4—D的培养基上萌发的芽尖作外植体最佳。
2)高羊茅、假俭草、黑麦草诱导愈伤所需最佳2，4—D浓度不同，假俭草达到最高诱导率67.5％的2，4—D为3.5 mg/L，黑麦草达到最高诱导率85.6％的2，4—D为6.5mg/L。高羊茅的三个品种也具有不同的最佳2，4—D浓度，SAFari为2.5mg/L，Barleduc为4.0 mg/L，Firewar Ⅱ为4.5 mg/L。IAA对高羊茅三个品种的愈伤组织诱导率的影响趋势与2，4—D类似，但效应不及2，4—D显著。另外，0.5mg/L6—BA在一定程度降低了高羊茅愈伤组织的诱导率。
3)ABA对狗牙根和高羊茅在诱导愈伤时的种胚萌芽有一定的抑制作用，同时也降低了愈伤组织诱导率，但4.0mg/LABA与0.5mg/L ABA分别对高羊茅、狗牙根愈伤组织的外观质量有显著的提高。高羊茅、假俭草和狗牙根不同外观状态的愈伤组织内源的GA_3、ZT、IAA、ABA、IBA含量与比例不同，种间存在差异，种内也存在差异。
4)渗透调节剂甘露醇、蔗糖与PEG6000能提高培养基的渗透压，影响愈伤的出愈率与质量。1％甘露醇，1％PEG6000，3％蔗糖能改善愈伤的质量，提高胚胎发生的频率，大于5％的PEG6000则因其浓度过高，导致细胞缺水，降低了愈伤的诱导并致其褐化死亡。
5)高羊茅、假俭草在不同的基本培养基上愈伤组织诱导率有差异，但都以N_6最高，分别达90％与30.7％，高羊茅以MS最低，假俭草则以AA最低，分别为65％和20％。另外，固化剂种类与浓度对高羊茅、假俭草愈伤组织诱导率与质量具有不同的影响。
6)不同状态的愈伤组织体细胞形状不同，胚胎发生过程也有差异，高羊茅的胚性愈伤组织切片能观察到二细胞原胚、多细胞原胚、球形胚、梨形胚、胚芽等过程，细胞质浓核大，假俭草也类似，狗牙根的愈伤细胞则形状大，内有大液泡，观察不到胞核，为典型的非胚性细胞。
7)不同外观状态的假俭草与高羊茅愈伤分化长芽与长根的情况不同。黄色致密结节状的分化率高，浅白疏松水渍状的分化率低。一定量的CoCl_2和AgNO_3能提高假俭草愈伤分化长芽率。
草坪的建植与养护过程中存在许多问题，杂草防除与病害防治是最重要的两个方
面。通过转基因手段培育抗除草剂与抗病的新品种是一种较理想的方法。本实验在转
基因研究中，基因枪转pBar—Gus于高羊茅时，以轰击2枪，子弹距可裂圆片6cm，
子弹距轰击受体6cm为最佳轰击参数。农杆菌LBA4404／pCG—11与高羊茅愈伤组织共
培养转化，固体培养基表面垫上一层滤纸，愈伤组织周围滴加浸染菌液共培养一周，
后经甘露醇及抗生素液洗涤，提高了农杆菌的转化率。建立了基因枪与农杆菌转化PCG
—11与sBar—Gus的两类草坪草的优化转化系统，分别获得了转pBar—Gus的高羊茅。
假俭草及转扯G一11的高羊茅、假俭草。随机抽取的转叨G一11的高羊茅植株的mU的
PCR检测，阳性率为80兄随机抽取的转PCG—11的假俭草植株的Chi的PCR检测，阳
性率为100％。随机抽取的转pBar—Gus的高羊茅、假俭草植株的Bar的PCR检测，阳
性率都为100兄随机抽取的转PCG—11的高羊茅植株总DNA的GILI的点杂交分析，阳
性率为90儿随机抽取的转威G一11的假俭草植株总DNA的oU的点杂交分析，阳性率
为100仇 随机抽取的高羊茅、假俭草转化pBar—Gus获得的植株的总DNA杂交分析，
阳性率都为100趴转pBar—Gus植株的除草剂涂抹实验和转pCG—11的禾谷镰刀菌
（厂 8：rBmlnearun Schwabe）挑战接种实验，结果表明转化植株比对照都表现出了更强
的抗性。
Turfgrasses are playing a more and more important role in our daily life. Tissue
culture and plantlet regeneration of four turfgrasses, common
centipedegrass[Eremochloa ophiuroides (Munro.)Hack.], tall fescue ( Festuca
arundinaceaSchreb. ) , bermudagrass (Cynodondactylonvar. ) and perennial ryegrass
(Lolium perenne L. ) were studied in the second part of thesis. The genetic
transformation systems for common centipedegrass and tall fescue were established,
and Barus gene and Chilu gene were transferred into common centipedegrass
and tall fescue respectively by agrobacterium tumefaciesediated or biolistic
bombardment, and many transgenic plants resistant to herbicide and fungi disease
had been acquired.
Establishment and optimization of tissue culture systems of turfgrasses would lay a solid foundation for traditional and transgenic breeding of turfgrasses. The major results studied in the part were abstracted as follows:
1) According to selection of explants, excised mature seederived embryos as explants had higher induction and differentiation frequency of calli compared to seeds as explants in tall fescue, and in common centipedegrass, epicotyl from seeds buds germinated on induction medium containing low 2,4 ?D concentration was the best explant for callus induction and differentiation compared with other explants such as mature seeds.
2) Tall fescue, common centipedegrass and ryegrass required different optimum 2,4 - D concentrations for callus induction, and the optimum 2,4 ?D concentration is 3. 5 mg/L and 6. 5 mg/L , respectively in common centipedegrass with the highest induction frequency 67.5% and in ryegrass with 85. 6%. Three cultivars of tall fescue also required different optimum 2,4 - D concentrations for callus induction, with 2.5 mg/L, 4.0 mg/L and 4.5 mg/L respectively in SAFari, Barleduc and Firewar II. There were similar effects on callus induction of SAFari, Barleduc and Firewar II between IAA and 2, 4, but more preminent with 2, 4. In addition, 0. 5 mg/L 6-BA decreased to an extent rate of callus induction in tall fescue,
3) ABA could suppress to an extent germination of seeds and decrease callus induction frequency in tall fescue and bermudagrass, however, 4. 0 mg/L ABA for tall fescue and 0. 5 mg/L ABA for bermudagrass increased somatic embryogenisis frequency from appearance. Callus with different apparent characteristics had different indogenous GA3, ZT, IAA, ABA and IBA content and proportions.
4) Mannitol, sucrose and PEG6000 could increase osmotic pressure of induction medium and affect induction frequency and quality of callus. 1% mannitol,3% sucrose and 1% PEG6000 could improve quality and increase somatic
embryogenisis frequency, and 5% or higher PEG6000 caused cell drought , and decreased callus induction frequency , finally caused it to be brown and dead.
5) There were different calli induction frequency on different basal mediums in tall fescue and common centipedegrass. There were the highest induction frequency (90%)on N6 medium and the lowest (65%) on MS in tall fescue, and highest(30.7%)and lowest(20%) in common centipedegrass. In addition, categories and concentrations of gelling agents had different effects on callus induction frequency and quality in tall fescue and common centipedegrass.
6) Callus with different characteristic had different cell shapes and somatic embryogenisis. Bicellular proembryos, multicellular proembryos, globular embryo id, pear haped embryoid , heart haped embryoid and plumule could be observed by microscopy in embryogeni-c callus of tall fescue and common centipedegrass. Callus cells in bermudagrass showed characteristics of non -embryogenic cells with big shape and big vacuoles,but no nucleolus .
7) There was different differentiation vigor in tall fescue and common centipedegrass with different characteristics, and high differentiation frequency with yellow hard granalous callus and low differentiation frequency with light white loose callus. The suitable concentrations of CoC
草坪草组织培养体系的建立与完善将为传统与转基因育种奠定基础，本研究在这方面取得了以下结果：
1)在外植体的选取方面，发现高羊茅离体成熟种胚比完整种子作外植体愈伤组织的诱导率与质量都优，假俭草以含低浓度2，4—D的培养基上萌发的芽尖作外植体最佳。
2)高羊茅、假俭草、黑麦草诱导愈伤所需最佳2，4—D浓度不同，假俭草达到最高诱导率67.5％的2，4—D为3.5 mg/L，黑麦草达到最高诱导率85.6％的2，4—D为6.5mg/L。高羊茅的三个品种也具有不同的最佳2，4—D浓度，SAFari为2.5mg/L，Barleduc为4.0 mg/L，Firewar Ⅱ为4.5 mg/L。IAA对高羊茅三个品种的愈伤组织诱导率的影响趋势与2，4—D类似，但效应不及2，4—D显著。另外，0.5mg/L6—BA在一定程度降低了高羊茅愈伤组织的诱导率。
3)ABA对狗牙根和高羊茅在诱导愈伤时的种胚萌芽有一定的抑制作用，同时也降低了愈伤组织诱导率，但4.0mg/LABA与0.5mg/L ABA分别对高羊茅、狗牙根愈伤组织的外观质量有显著的提高。高羊茅、假俭草和狗牙根不同外观状态的愈伤组织内源的GA_3、ZT、IAA、ABA、IBA含量与比例不同，种间存在差异，种内也存在差异。
4)渗透调节剂甘露醇、蔗糖与PEG6000能提高培养基的渗透压，影响愈伤的出愈率与质量。1％甘露醇，1％PEG6000，3％蔗糖能改善愈伤的质量，提高胚胎发生的频率，大于5％的PEG6000则因其浓度过高，导致细胞缺水，降低了愈伤的诱导并致其褐化死亡。
5)高羊茅、假俭草在不同的基本培养基上愈伤组织诱导率有差异，但都以N_6最高，分别达90％与30.7％，高羊茅以MS最低，假俭草则以AA最低，分别为65％和20％。另外，固化剂种类与浓度对高羊茅、假俭草愈伤组织诱导率与质量具有不同的影响。
6)不同状态的愈伤组织体细胞形状不同，胚胎发生过程也有差异，高羊茅的胚性愈伤组织切片能观察到二细胞原胚、多细胞原胚、球形胚、梨形胚、胚芽等过程，细胞质浓核大，假俭草也类似，狗牙根的愈伤细胞则形状大，内有大液泡，观察不到胞核，为典型的非胚性细胞。
7)不同外观状态的假俭草与高羊茅愈伤分化长芽与长根的情况不同。黄色致密结节状的分化率高，浅白疏松水渍状的分化率低。一定量的CoCl_2和AgNO_3能提高假俭草愈伤分化长芽率。
草坪的建植与养护过程中存在许多问题，杂草防除与病害防治是最重要的两个方
面。通过转基因手段培育抗除草剂与抗病的新品种是一种较理想的方法。本实验在转
基因研究中，基因枪转pBar—Gus于高羊茅时，以轰击2枪，子弹距可裂圆片6cm，
子弹距轰击受体6cm为最佳轰击参数。农杆菌LBA4404／pCG—11与高羊茅愈伤组织共
培养转化，固体培养基表面垫上一层滤纸，愈伤组织周围滴加浸染菌液共培养一周，
后经甘露醇及抗生素液洗涤，提高了农杆菌的转化率。建立了基因枪与农杆菌转化PCG
—11与sBar—Gus的两类草坪草的优化转化系统，分别获得了转pBar—Gus的高羊茅。
假俭草及转扯G一11的高羊茅、假俭草。随机抽取的转叨G一11的高羊茅植株的mU的
PCR检测，阳性率为80兄随机抽取的转PCG—11的假俭草植株的Chi的PCR检测，阳
性率为100％。随机抽取的转pBar—Gus的高羊茅、假俭草植株的Bar的PCR检测，阳
性率都为100兄随机抽取的转PCG—11的高羊茅植株总DNA的GILI的点杂交分析，阳
性率为90儿随机抽取的转威G一11的假俭草植株总DNA的oU的点杂交分析，阳性率
为100仇 随机抽取的高羊茅、假俭草转化pBar—Gus获得的植株的总DNA杂交分析，
阳性率都为100趴转pBar—Gus植株的除草剂涂抹实验和转pCG—11的禾谷镰刀菌
（厂 8：rBmlnearun Schwabe）挑战接种实验，结果表明转化植株比对照都表现出了更强
的抗性。
Turfgrasses are playing a more and more important role in our daily life. Tissue
culture and plantlet regeneration of four turfgrasses, common
centipedegrass[Eremochloa ophiuroides (Munro.)Hack.], tall fescue ( Festuca
arundinaceaSchreb. ) , bermudagrass (Cynodondactylonvar. ) and perennial ryegrass
(Lolium perenne L. ) were studied in the second part of thesis. The genetic
transformation systems for common centipedegrass and tall fescue were established,
and Barus gene and Chilu gene were transferred into common centipedegrass
and tall fescue respectively by agrobacterium tumefaciesediated or biolistic
bombardment, and many transgenic plants resistant to herbicide and fungi disease
had been acquired.
Establishment and optimization of tissue culture systems of turfgrasses would lay a solid foundation for traditional and transgenic breeding of turfgrasses. The major results studied in the part were abstracted as follows:
1) According to selection of explants, excised mature seederived embryos as explants had higher induction and differentiation frequency of calli compared to seeds as explants in tall fescue, and in common centipedegrass, epicotyl from seeds buds germinated on induction medium containing low 2,4 ?D concentration was the best explant for callus induction and differentiation compared with other explants such as mature seeds.
2) Tall fescue, common centipedegrass and ryegrass required different optimum 2,4 - D concentrations for callus induction, and the optimum 2,4 ?D concentration is 3. 5 mg/L and 6. 5 mg/L , respectively in common centipedegrass with the highest induction frequency 67.5% and in ryegrass with 85. 6%. Three cultivars of tall fescue also required different optimum 2,4 - D concentrations for callus induction, with 2.5 mg/L, 4.0 mg/L and 4.5 mg/L respectively in SAFari, Barleduc and Firewar II. There were similar effects on callus induction of SAFari, Barleduc and Firewar II between IAA and 2, 4, but more preminent with 2, 4. In addition, 0. 5 mg/L 6-BA decreased to an extent rate of callus induction in tall fescue,
3) ABA could suppress to an extent germination of seeds and decrease callus induction frequency in tall fescue and bermudagrass, however, 4. 0 mg/L ABA for tall fescue and 0. 5 mg/L ABA for bermudagrass increased somatic embryogenisis frequency from appearance. Callus with different apparent characteristics had different indogenous GA3, ZT, IAA, ABA and IBA content and proportions.
4) Mannitol, sucrose and PEG6000 could increase osmotic pressure of induction medium and affect induction frequency and quality of callus. 1% mannitol,3% sucrose and 1% PEG6000 could improve quality and increase somatic
embryogenisis frequency, and 5% or higher PEG6000 caused cell drought , and decreased callus induction frequency , finally caused it to be brown and dead.
5) There were different calli induction frequency on different basal mediums in tall fescue and common centipedegrass. There were the highest induction frequency (90%)on N6 medium and the lowest (65%) on MS in tall fescue, and highest(30.7%)and lowest(20%) in common centipedegrass. In addition, categories and concentrations of gelling agents had different effects on callus induction frequency and quality in tall fescue and common centipedegrass.
6) Callus with different characteristic had different cell shapes and somatic embryogenisis. Bicellular proembryos, multicellular proembryos, globular embryo id, pear haped embryoid , heart haped embryoid and plumule could be observed by microscopy in embryogeni-c callus of tall fescue and common centipedegrass. Callus cells in bermudagrass showed characteristics of non -embryogenic cells with big shape and big vacuoles,but no nucleolus .
7) There was different differentiation vigor in tall fescue and common centipedegrass with different characteristics, and high differentiation frequency with yellow hard granalous callus and low differentiation frequency with light white loose callus. The suitable concentrations of CoC
引文
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38 洪亚辉主编．植物外源DNA直接导入技术．长沙：湖南科学出版社，2000．1～3
39 徐淑平，等．基因枪的使用方法介绍．植物生理学通讯，1998，34(1)：41～43
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50 Altpeter F, et al. Improved plant regeneration from cell suspensions of commercial cultivars, breeding and inbred lines of perennial ryegrass (Lolium perenne L.). J Plant Physiol, 2000, 156: 790～796
51 朱智甲，等．高效液相色谱紫外检测法测定内源激素．光谱实验室，1999，3(16)：
52 张政，等．荞麦幼苗内小麦幼苗中的源激素的高效液相色谱法．色谱，1994，12(2)：140
53 刘怀攀，等．渗透胁迫和外源ABA对芦苇愈伤组织中的3种保护酶活性的影响．植物生理学通迅，2002，38(1)：
54 易自力博士论文．2000，11
55 李洪清，等．几种影响木薯芽器官发生及植株再生的因素．植物生理学通迅，2000，36(4)：297～299
56 Simon EW, et al. Raja Harun RM. J Exp Bot 1972, 23:1076
57 Powell AE, et al, Matthews S. J Exp Bot 1978, 29: 1215
58 Woodstock LW, et al, Tao KL. Physiol Plant 1981, 51:133
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