【作者】 徐清锋；

【导师】 赖钟雄；

【作者基本信息】 福建农林大学 ， 果树学， 2010， 硕士

【摘要】 龙眼(Dimocarpus longan Lour.)是我国南方大面积种植的重要热带亚热带果树。本研究主要以长期保存的龙眼胚性培养物为材料,对以下6个方面进行研究:①对三种类型龙眼EC进行限制生长保存条件的优化;②17个龙眼品种的体胚发生以及植株再生;③选择并优化龙眼愈伤组织转基因受体系统;④采用根癌农杆菌介导法进行ACS反义基因转化龙眼并优化转化体系;⑤对抗性龙眼EC进行筛选并进行其生长状况观察;⑥对转ACS反义基因的抗性EC进行GUS稳定表达检测和GUS基因的PCR检测。主要研究结果如下:1三种类型龙眼胚性愈伤组织限制生长保存条件优化试验比较了不同浓度山梨醇、甘露醇、PEG、肌醇、麦芽糖以及光质的差异对三种类型龙眼胚性EC生长的影响,并对上述三种类型龙眼EC最佳保存条件进行验证。试验结果表明: CⅢI和CⅡa两种类型的龙眼EC在相应的培养基中可以将其保存时间延长到60d,CⅡb也能够将其保存时间延长到50d,并且能够保持旺盛的生长及再生能力。2 17个龙眼品种的体胚发生和植株再生为检测长期限制性离体保存的龙眼EC的体胚发生能力,试验以尚未完成植株再生的17个龙眼胚性愈伤组织为材料,对其进行体胚诱导、成熟以及植株再生的相关研究,与此同时本试验的完成标志着所有长期限制性保存的龙眼EC均实现植株再生。试验结果表明,龙眼品种的差异对体胚发生和植株再生有一定的影响。对17个龙眼品种进行体胚诱导的结果表明:不同品种、不同麦芽糖浓度、甘露醇浓度、PEG浓度对体胚出现时间、胚状体数量以及质量都具有一定的影响,体胚出现时间一般在15-35d,形成胚状体的数量从大约在1080-14380个?g,分化频率为100%,颜色多为白色小子叶胚;对17个龙眼品种进行体胚成熟的研究表明:不同龙眼品种、不同比例的蔗糖和麦芽糖、不同高渗试剂、干燥处理对胚状体的大小、形态以及褐化情况都有一定的影响,其中胚状体大小在5-15mm不等,多白色增大;对17个龙眼品种进行植株再生的研究表明:不同龙眼品种、不同胚状体都对体胚萌发时间、体胚萌发率以及体胚再生苗的长势有一定的影响,萌发率也存在着一定的差异一般在从35.67%-90.00%不等,正常成熟体胚的萌发时间最早在13 d左右就萌发,最晚需要20 d才能萌发。3选择并优化龙眼愈伤组织转基因受体系统由于愈伤组织受体系统具有较强的分化和再生能力,遗传稳定性良好。试验以红核子的愈伤组织作为根癌农杆菌介导ACS反义基因转化龙眼的受体系统。通过对龙眼EC进行生长量的测定,发现在继代培养15 d左右的龙眼EC其生长速度最快,生活力最强,最适宜作为转基因的受体材料;试验过程中采用生长量测定和定性观察相结合的方法,试验发现100 mg·L-1卡那霉素浓度适合于龙眼愈伤组织进行转化操作的筛选抗生素浓度,而头孢霉素浓度范围在0-400 mg·L-1时,对愈伤组织的正常生长和再生均无较大影响。4农杆菌介导的ACS反义基因转化龙眼本试验在上述研究的基础上,对农杆菌介导的龙眼受体系统的遗传转化策略进行研究。试验结果表明,高效稳定的转化条件为将龙眼受体材料在转化前置于0.15mol·L-1的液态甘露醇中6 h,农杆菌在1/2 MS附加葡萄糖浓度80 g·L-1的重悬液中,将菌液的浓度稀释成0.9(OD600),受体材料侵染30 min,洗菌后在共培养基pH为5.6,温度为22℃时进行共培养5-6 d。在对最佳转化条件的验证试验中我们发现,以上述条件进行遗传转化可获得GUS瞬时表达率达90%以上的龙眼抗性EC,经过后期筛选本试验得到龙眼抗性EC 55份。5筛选和观察抗性EC以及GUS基因的分子检测对转化ACS反义基因的龙眼抗性EC进行筛选及观察,并对前期抗性EC进行GUS瞬时表达检测、后期抗性EC的GUS基因的PCR检测。试验采用逐步加压法作为抗性EC的筛选方法,其最终以附加100 mg﹒L-1卡那霉素、1 mg﹒L-1 2,4-D以及0.5 mg﹒L-1NAA的筛选培养基对共培养后的龙眼EC进行筛选,用DNA微量提取法对抗性EC进行PCR检测,结果表明GUS基因已整合进龙眼基因组。但是龙眼的抗性EC生长较为缓慢,体胚发生能力较差。更多还原

【Abstract】 Longan(Dimocarpus longan Lour.)is an important southern subtropical fruit which has a large-scale cultivation in China. In this experiment, The long-term conserved embryogenic calli (EC) of longan were used as the materials to study the following aspects: the optimization of the restricting conservation conditions for the longan EC of three types; plant regeneration via somatic embryogenesis in 17 cultivars of longan; shoosing and optimizing the transgenic receptor system of longan; optimizing the transformation system of antisense ACS gene into longan;screening of the resistant longan EC and observation of the growth status; detection of the resistant longan EC. The main results were as follows:1. The optimization of the conditions for minimal growth conservation of longan EC of three typesThe effects of the concentrations of mannitol, sorbitol, PEG, inositol, maltose and the lights quality on the growth of embryogenic callus (EC) were compared in three types of longan EC, and the optimization of the minimal growth conservation conditions was validated in three types of longan EC. The obtained results showed that the suitable medium for CⅢEC was the MS medium supplemented with 1mg·L-1 2,4-D, 1.5% sucrose, 0.6% agar, 20g·L-1 mannitol, 0.1 g·L-1 inositol and 5g·L-1 maltose; the suitable medium for CⅡa was the MS medium supplemented with 1mg·L-1 2,4-D, 1.5% sucrose, 0.6% agar, 20g·L-1 mannitol, 0.2g·L-1 inositol and 5g·L-1 maltose; the suitable medium for CⅡb was the MS medium supplemented with 1mg·L-1 2,4-D, 1.5% sucrose, 0.6% agar, 20g·L-1 mannitol, 0.1g·L-1 inositol; the subculture cycle could be further prolonged from 20 d to more than 60 d for CⅢI and CⅡa on the suitable medium. For CⅡb, it could be prolonged to more than 50 d. At the same time, the EC could maintain strong growth and regeneration ability.2. Plant regeneration via somatic embryogenesis from longan EC of 17 longan cultivarsIn order to checkout the capacity of somatic embryogenesis of the minimal growth conservation of longan EC, in this experiment, longan EC of 17 cultivars were used as the materials for the studies on the induction, maturation and regeneration. And at the same time, when it was finished, it meaned the regeneration of all the conserved EC. The results showed that the somatic embryogenesis and the regeneration were affected by different varieties of longan. During the induction of somatic embryogenesis, different cultivars, concentrations of maltose, mannitol, PEG, affected the time, quantity and quality of somatic embryogenesis. And the somatic embryogenesis occurred from 15 d to 35 d, the frequency of somatic embryogenesis was 100%, the colors of the embryoids were white, the number of differentiated embryoids was 1080-14380 per gram callus. During the stage of embryoid maturation, different cultivars, the ratio of sucrose and maltose, concentrations of mannitol, PEG, and different time of desiccation effected the size, browning and the shape of matured embryoids.The embryoids were from 5 mm to 15 mm in size, white and larger. During the stage of plantlet regeneration, the results showed that the germination rate of somatic embryos and plantlet growth were affected by different cultivars, different types of somatic embryogenesis. The germination rate was 35.67% to 90.00% with a great difference. The time of normal matured somatic embryos germinated is 13-15 d.3. Choosing and optimization the transgenic receptor systems for Dimocarpus longan Lour.Because the receptor system of calli was provided with strong ability of differentiation, regeneration and favorable genetic stability. In the experiment, the EC of Honghezi was used as material for genetic transformation. Calli pre-cultured for about 15 d were proved to the appropriate genetic transformation materials for their rapid-growth and strong vitality stage. Through measuring the fresh weight increase of EC, it was suitable for transgenic. Through measuring the fresh weight increase combined with the qualitative obserbation, 100mg·L-1 kanamycin concentration was suitable for transgenic operations on EC, both the growth and regeneration were normal, while the cephamycin concentration range was 0-400 mg·L-1.4. The introduction of antisense ACS gene into longan mediated by Agrobacterium tumefaciens.Based on the above results, new transgenic strategies about transformation of longan were performed. The highly efficient and stable parameters of transgenic operations on longan showed that the receptor material of longan was pretreated with 0.15 mol·L-1 mannitol in liquid for 6 h , taking 1/2 MS liquid culture medium as re-suspension liquid, containing 80.0 g·L-1 glucose, and with 0.9 OD600 value of Agrobacterium suspension, the receptor material was infected for 30 min, then being co-cultured onto the MS medium (pH 5.6) supplemented with 1 mg·L-1 2,4-D for 6 days at 22℃in dark. The transient expression of GUS could reach 90% of the resistant EC, Through the latter part of screening in this study, 55 accessions of resistant EC were chalked up.5. The selection and screening of longan resistant EC, and the molecular detection of the GUS gene.In this experiment, the transient expression of GUS at the prior period and the detection by PCR assays of GUS for the resistant EC at the later period were examined. The gradual enhancement of concentration for kanamycin was used. At last, the resistant EC were selected on MS medium supplemented with 100 mg﹒L-1 kanamycin, 1 mg·L-1 2,4-D and 0.5 mg·L-1 NAA. 55 accessions of longan germplasm were maintained for further studies. The rapid method for trace genomic DNA extraction for identification of transgenic EC of longan with PCR, which proved that the GUS gene was integrated into the genome of these resistant EC. The resistant EC grew slowly, at the same time the capacity of somatic embryogenesis was weak.更多还原