草坪草普通狗牙根成熟胚组织培养再生体系的创建及基因枪法遗传转化体系的建立
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摘要
草坪在现代生活中起着越来越重要的作用，以至已形成一项方兴未艾的产业――草坪业，对于草坪草的研究也逐渐形成了一门专门的学科――草坪学。草坪学的研究与发展离不开现代生物技术的发展和应用。由于传统育种手段的局限性，运用先进的分子生物学手段――植物遗传转化即转基因技术进行草坪草分子育种，培育理想的新品种，已成为草坪草育种研究的必然趋势。狗牙根是最重要、应用最广泛的暖季型草坪草之一，具备优良草坪草的诸多优点，但其抗寒性差、绿色期短、易感病等缺点极大地限制了它更广泛的应用，在高尔夫球场等运动休闲场地的应用上，矮化也是其亟待解决的问题之一。以目前技术而言，体外组织培养技术是草坪草转基因技术的基础。而狗牙根的组织培养再生非常困难，主要是因其胚性愈伤组织的诱导和分化及其胚性细胞再生能力的保持很困难，至今少有成功报道，这成为其遗传转化研究的瓶颈。
本文以普通狗牙根（Cynodon dactylon (L.) Pers. cv.‘suncity’）成熟颖果（成熟胚）为外植体，探讨了狗牙根胚性愈伤组织的诱导及分化条件，创建了一个操作简便、稳定有效的普通狗牙根组织培养再生体系。结果表明：用颖果（成熟胚）作外植体，以MS为基本培养基，phytagel 5 g/L，2,4-D浓度在2.0 mg/L-6.0 mg/L之间，都能诱导出高频率的胚性愈伤组织，而以2,4-D 4.0 mg/L为最佳，诱导率达88.5％。不同继代及分化途径对最终分化成苗的影响有显著差异。以在分化前由MS+2，4-D 4.0 mg/L转移到MS+2，4-D 2.0 mg/L及1/2MS+2,4-D 2.0 mg/L中继代，然后无激素的1/2MS中光照培养10天，最后MS+6-BA 3.0 mg/L分化为最佳继代、分化途径，分化率31.7％。环境扫描电镜、透射电镜观察到胚性愈伤组织与非胚性愈伤组织之间形态、结构上的差异，首次在细胞学水平上描述了狗牙根愈伤组织胚性细胞的超微结构特点，证实了再生小苗是从体细胞胚分化而来。
在建立起组织培养再生体系的基础上，利用Biolistic PDS-1000/He基因枪轰击胚性愈伤组织，导入BIN2和PSAG12-IPT两个外源基因，成功获得大量再生植株。PCR、Northern blot分子检测表明，外源基因已整合入普通狗牙根基因组内，并获得表达。初步表型分析亦显示外源基因在转基因植株中有所表达。从而证明基因枪法可用于狗牙根的遗传转化研究，成功建立起狗牙根遗传转化体系。基因枪轰击法是一种操作简便、无宿主限制的应用广泛的遗传转化方法，本文基因枪轰击参数为氦气压力1 100 psi，真空度27 Torr，射程9 cm，金粉直径1.0 μm，每平皿轰击两次。这是首次以成熟胚为外植体诱导胚性愈伤组织，以基因枪转化，成功获得转基因普通狗牙根植株。
本文所建立的以成熟颖果（成熟胚）为外植体，以基因枪轰击法直接导入外源基因的普通狗牙根遗传转化系统简便易行，容易获得大量再生植株，而且周期短，外植体来
源丰富，可随时进行研究工作，为大量、深入开展狗牙根以及类似的暖季型草坪草生物技术研究工作探索了一条便利、可行的道路。
Turf-grasses play more and more important roles in our modern lives and there are more and more people and companies run into the field of researching on them. As the limits of traditional method, using modern, advanced biotechnology to improve turf-grasses is becoming an unavoidable trend. Burmudagrass is one of the most important worm-season turf-grasses and widely used in the temperate and tropical regions of the world. As for most plant species, improvement of bermudagrass via biotechnology depends on improved tissue culture responses, especially in plant regeneration, and a successful scheme to introduce useful transgenes. Bermudagrass is a recalcitrant species in tissue culture and is highly recalcitrant with respect to the recovery of transgenic plants. This is the bottleneck of burmudagrass transformation research.
In this thesis, a tissue culture system for embryogenic callus (EC) induction and plant regeneration of common bermudagrass using mature caryopses (embryos) as explants was developed. The results showed that embryogenic callus could be induced from caryopses with high frequency, in MS medium with 5 g/L phytagel, and with 2.0－6.0 mg/L 2,4-D, and the best concentration of 2,4-D was 4.0 mg/L with 88.5% induction percentage. The best method for maintaining EC and tissue differentiation was to subculture EC in MS+2,4-D 4.0 mg/L for 1－2 times, follwed by subculturing in 1/2 MS+2,4-D 2.0 mg/L for 1－2 times, then transfer EC to 1/2 MS without hormone for a 10-day-preregeneration in light, followed by transferring to MS+6-BA 3.0 mg/L for regeneration, with regeneration frequency 31.7%. The regenerated plantlets were transplanted to pots or the field and grew healthily. The morphological and micro-structural differences between EC and non-embryogenic callus (NEC) were observed by environmental scanning electron microscopy (ESEM) and transmission electron microscopy (TEM). And ultrastructrual characteristics of the EC cells are described.
On the basis of above tissue culture system, a useful bermudagrass transformation system was developed. Two foreign genes—BIN2 and PSAG12-IPT(a chimeric gene) have been transfered directly into EC cells derived from common bermudagrass mature caryopses(embryos) by particle bombardment(Biolistic PDS-1000/He) following such parameters: 1 100 psi helium pressure, 27 torr vacuum, 9 cm distance between the stopping screen and target tissue, two shots per target plate, and 1.0 μm gold particles. Numerous plantlets regenerated. PCR and Northern blot results showed that the foreign genes have integrated into common bermudagrass genome and have been expressed. Some transgenic regenerated plantlets’ phenotype also implicated that the foreign genes have likely played roles. This is the first time of recovery of transgenic common bermudagrass using mature caryopses as explant.
The common bermudagrass transformation system developed in this thesis is easy for manipulation and the explant—mature caryopse can be obtained quite conveniently and so research can be conducted all around the year, unlike immature inflorescences which could only be collected during the blooming season (May to September).
本文以普通狗牙根（Cynodon dactylon (L.) Pers. cv.‘suncity’）成熟颖果（成熟胚）为外植体，探讨了狗牙根胚性愈伤组织的诱导及分化条件，创建了一个操作简便、稳定有效的普通狗牙根组织培养再生体系。结果表明：用颖果（成熟胚）作外植体，以MS为基本培养基，phytagel 5 g/L，2,4-D浓度在2.0 mg/L-6.0 mg/L之间，都能诱导出高频率的胚性愈伤组织，而以2,4-D 4.0 mg/L为最佳，诱导率达88.5％。不同继代及分化途径对最终分化成苗的影响有显著差异。以在分化前由MS+2，4-D 4.0 mg/L转移到MS+2，4-D 2.0 mg/L及1/2MS+2,4-D 2.0 mg/L中继代，然后无激素的1/2MS中光照培养10天，最后MS+6-BA 3.0 mg/L分化为最佳继代、分化途径，分化率31.7％。环境扫描电镜、透射电镜观察到胚性愈伤组织与非胚性愈伤组织之间形态、结构上的差异，首次在细胞学水平上描述了狗牙根愈伤组织胚性细胞的超微结构特点，证实了再生小苗是从体细胞胚分化而来。
在建立起组织培养再生体系的基础上，利用Biolistic PDS-1000/He基因枪轰击胚性愈伤组织，导入BIN2和PSAG12-IPT两个外源基因，成功获得大量再生植株。PCR、Northern blot分子检测表明，外源基因已整合入普通狗牙根基因组内，并获得表达。初步表型分析亦显示外源基因在转基因植株中有所表达。从而证明基因枪法可用于狗牙根的遗传转化研究，成功建立起狗牙根遗传转化体系。基因枪轰击法是一种操作简便、无宿主限制的应用广泛的遗传转化方法，本文基因枪轰击参数为氦气压力1 100 psi，真空度27 Torr，射程9 cm，金粉直径1.0 μm，每平皿轰击两次。这是首次以成熟胚为外植体诱导胚性愈伤组织，以基因枪转化，成功获得转基因普通狗牙根植株。
本文所建立的以成熟颖果（成熟胚）为外植体，以基因枪轰击法直接导入外源基因的普通狗牙根遗传转化系统简便易行，容易获得大量再生植株，而且周期短，外植体来
源丰富，可随时进行研究工作，为大量、深入开展狗牙根以及类似的暖季型草坪草生物技术研究工作探索了一条便利、可行的道路。
Turf-grasses play more and more important roles in our modern lives and there are more and more people and companies run into the field of researching on them. As the limits of traditional method, using modern, advanced biotechnology to improve turf-grasses is becoming an unavoidable trend. Burmudagrass is one of the most important worm-season turf-grasses and widely used in the temperate and tropical regions of the world. As for most plant species, improvement of bermudagrass via biotechnology depends on improved tissue culture responses, especially in plant regeneration, and a successful scheme to introduce useful transgenes. Bermudagrass is a recalcitrant species in tissue culture and is highly recalcitrant with respect to the recovery of transgenic plants. This is the bottleneck of burmudagrass transformation research.
In this thesis, a tissue culture system for embryogenic callus (EC) induction and plant regeneration of common bermudagrass using mature caryopses (embryos) as explants was developed. The results showed that embryogenic callus could be induced from caryopses with high frequency, in MS medium with 5 g/L phytagel, and with 2.0－6.0 mg/L 2,4-D, and the best concentration of 2,4-D was 4.0 mg/L with 88.5% induction percentage. The best method for maintaining EC and tissue differentiation was to subculture EC in MS+2,4-D 4.0 mg/L for 1－2 times, follwed by subculturing in 1/2 MS+2,4-D 2.0 mg/L for 1－2 times, then transfer EC to 1/2 MS without hormone for a 10-day-preregeneration in light, followed by transferring to MS+6-BA 3.0 mg/L for regeneration, with regeneration frequency 31.7%. The regenerated plantlets were transplanted to pots or the field and grew healthily. The morphological and micro-structural differences between EC and non-embryogenic callus (NEC) were observed by environmental scanning electron microscopy (ESEM) and transmission electron microscopy (TEM). And ultrastructrual characteristics of the EC cells are described.
On the basis of above tissue culture system, a useful bermudagrass transformation system was developed. Two foreign genes—BIN2 and PSAG12-IPT(a chimeric gene) have been transfered directly into EC cells derived from common bermudagrass mature caryopses(embryos) by particle bombardment(Biolistic PDS-1000/He) following such parameters: 1 100 psi helium pressure, 27 torr vacuum, 9 cm distance between the stopping screen and target tissue, two shots per target plate, and 1.0 μm gold particles. Numerous plantlets regenerated. PCR and Northern blot results showed that the foreign genes have integrated into common bermudagrass genome and have been expressed. Some transgenic regenerated plantlets’ phenotype also implicated that the foreign genes have likely played roles. This is the first time of recovery of transgenic common bermudagrass using mature caryopses as explant.
The common bermudagrass transformation system developed in this thesis is easy for manipulation and the explant—mature caryopse can be obtained quite conveniently and so research can be conducted all around the year, unlike immature inflorescences which could only be collected during the blooming season (May to September).
引文
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