摘要
本研究在前人工作的基础上,以贡蕉(Musa AA Pisang Mas cv.Mas)品种为试材,对其再生体系、超低温保存和遗传转化进行了比较深入的研究。其主要试验结果如下:
1、以贡蕉(Musa AA Pisang Mas cv.Mas)的未成熟雄花序为外植体,分别利用不同浓度的2,4-D和Picloram诱导胚性愈伤组织,发现4 mg/L的Picloram诱导效果最好,比同浓度的2,4-D诱导的胚性愈伤组织率高出一倍以上。但是Picloram诱导的胚性愈伤组织由于含水量较高难于建立悬浮系,而用2,4-D诱导的胚性愈伤组织比较容易建立悬浮系。比较了不同浓度的ABA对体细胞胚诱导、成熟和萌发的影响,其中0.5 mg/L的ABA能够促进体细胞胚的成熟、萌发和植株再生,其成熟体胚数量为37600个/mL PCV,其处理的体胚萌发率和植株转换率分别为30.73%、23.42%。
2、建立了贡蕉悬浮细胞的玻璃化法保存技术体系。具体方法为:取处于悬浮培养对数期的胚性细胞,在含有0.5 mol/L蔗糖的培养液中预培养2 d,在室温下用装载液(ML+2 mol/L甘油+0.4 mol/L蔗糖)处理40 min,在0℃用玻璃化液PVS_2处理30 min,迅速投入液氮,保存24 h后,37℃水浴解冻,用含有1.2 mol/L蔗糖的洗涤液洗涤3次,每次10 min,TTC法检测细胞生活力最高可达80.46%。将细胞转移到含有0.5 mol/L的过渡培养基上,24 h后转移到恢复培养基中,对悬浮细胞进行再培养,通过体细胞胚胎发生途径获得了再生植株。
3、应用透射电镜对香蕉胚性悬浮细胞玻璃化法超低温保存过程中细胞超微结构变化进行了观察。发现细胞在经过预培养、装载、脱水的过程中,细胞质壁分离程度逐渐加重,液泡体积变小,部分细胞器逐渐膨大。在液氮中保存后,部分细胞的细胞壁、细胞核以及细胞器受到了严重的损伤,是导致细胞致死的可能原因之一。部分细胞结构完整,虽然细胞结构发生了变化,但是程度不深,在恢复培养时会自动修复,可以再生出植株。
4、用含质粒载体pCAMBIA1301的根癌农杆菌(Agrobacterium tumefaciens)EHA105对贡蕉悬浮细胞进行了转化,通过测定GUS基因瞬时表达率,对转化初期的主要影响因素进行了研究。结果表明,取处于对数期的悬浮细胞和浓度为OD_(600)0.2菌液混合,在室温、8 000r/min离心5 min,然后在110 rpm的旋转摇床上摇5 min,在体胚诱导和成熟培养基上共培养8 d,瞬时表达率达到最高值25.3%。通过对体细胞胚的进一步选择和再培养,得到了转基因抗性芽。
Based on pre-researches,further studies were carried out on the establishment of regeneration system,cryopreservation and genetic transformation of Musa AA Pisang Mas cv.Mas.The main results are as follows:
1.Using immature male flower of Musa AA Pisang Mas cv.Mas as explant,the embryogenic calli were induced with different concentration of 2,4-D and picloram. The results showed that the induction percentage of embryogenic calli was doubled when 4 mg/L 2,4-D in the callus induction medium was substituted by 4 mg/L picloram.It was difficult to establish embryogenic cell suspension by picloram, because the moisture content of embryogenic calli is too high.But embryogenic cell suspension was easy to establish if induced by 2,4-D.The effect of different concentration of ABA was compared on induction,muturation and germination of somatic embryo.The results were that 0.5 mg/L ABA could promote the induction, maturation and germination of somatic embryo.The number of mature embryo somatic was 37600/mL PCV.The percentage of germination and the plant conversion were 30.73%and 23.42%,respectively.
2.Embryogenic cell suspension of Musa AA Pisang Mas cv.Mas was successfully cryopreserved with the vitrification technique.A suitable procedure was established as follows:the embryonic cell suspensions in the logarithmic phase were precultured for 2 days on liquid medium with 0.5 mol/L sucrose.Then cells were loaded in liquid medium with a mixture of 2 mol/L glycerol plus 0.4 mol/L sucrose for 40 min at room temperature.These exercised cells were sufficiently dehydrated in a highly concentrated vitrification solution for 30 min at 0℃,then plunged directly into liquid nitrogen and conserved for at least 24h.Then rapid thawing in water at 37℃for 90 s, the cells were rinsed in a 1.2 mol/L sucrose cleaning solution for three times.The highest rate of cell viability detected by TTC method is 80.46%,then plated on a transition culture medium supplemented with 0.5 mol/L sucrose.After 24 h,the cells were transferred to recovery culture medium.The plantlets can be obtained after further culturing.
3.Ultrastructure of embryogenic cell suspension of banana during vitrification cryopreservaton was observed by using the transmission electron microscopy(TEM). The results showed that the extent of cell plasmolysis became more and more severe during the pretreatment and dehydration after cells were precultured.Meanwhile,cells with numerous smaller vacuoles and a few organelles intumescentiae.Cell walls,cell membranes and organelles were lethally injured after liquid nitrogen preservation, which may account for the death of some cells.And although there were some extent changes in many other cells structure,these were kinds of reversible damage,which may be rehabilitated automatically during recovery culture and regenerable plantlets.
4.Embryogenic cell suspension(Musa AA Pisang Mas cv.Mas) was used as the receptors for transformation by Agrobacterium tumefaciens containing pCAMBIA1301. By detecting the transient expression of GUS gene,the factors affecting the early phase of Agrobacterium-mediated transformation of banana were investigated.The results showed that the the highest rate of transient expression is 25.30%,when mixing the bacterium OD_(600) 0.2 concentration with the embryonic cell suspension in the logarithmic phase,then centrifugating at room temperature,8000 r/min for 5 minutes, and then shaking at 110 rpm in orbital shaker for 5 minutes,finally co-culturing for 8 days in somatic embryo induction and maturation medium.The transgentic resistance buds can be obtained after the somatic embryo was reelected and cultured.
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