农杆菌介导的小麦成熟胚遗传转化影响因素分析

为解决农杆菌介导的小麦成熟胚遗传转化效率较低的难题,以春小麦Bobwhite成熟胚为外植体,分析植物激素、愈伤组织预培养时间、侵染时间和共培养阶段干燥处理对小麦成熟胚愈伤组织遗传转化的影响,并在遗传转化处理中辅以3个组织再生相关基因(TaSERK1、TaSERK2、TaNiR)的qRT-PCR分析。结果表明,愈伤组织预培养阶段添加适量的2,4-D和Picloram均可诱导成熟胚愈伤组织的形成,但是2mg·L~(-1)Picloram培养基中愈伤组织的胚萌发率为20.78%,远高于2,4-D诱导的胚萌发率(11.38%);共培养阶段对农杆菌侵染的愈伤组织进行干燥处理能适当提高愈伤组织的转化效率;qRT-PCR分析发现再生相关基因TaNiR相对表达量的增加能适当提高愈伤组织的转化率。     

Somatic embryogenesis and regeneration of five multipurpose cassava landraces extensively integrated in African cropping system

Successful development and adoption of transgenic cassava (Manihot esculenta Crantz) varieties in Africa depend on in vitro regeneration of landraces because of their agronomic value and amenability to genetic modification. The study investigated somatic embryogenesis from axillary bud and immature leaf-lobe explant and regeneration via shoot organogenesis in five cassava landraces. Landraces exhibited significant (P < 0.05) differences in frequencies of primary somatic embryo production, number of embryos per explant, and frequency of secondary embryos. The frequency of primary somatic embryogenesis ranged from 7.8 to 14.5%, whereas the number of embryos per explant varied from 5.3 to 10.6. However, only frequency of primary somatic embryos and number of embryos per explant showed significant (P < 0.05) differences when immature leaf lobe was used as explant. The frequency of primary somatic embryogenesis ranged from 42.7 to 49.2%, whereas number of embryos per immature leaf-lobe explant varied from 9.5 to 15.2 per explant. Secondary embryogenesis was cultivar-independent in the case of immature leaf-lobe explant. Cyclic and green (mature) embryogenesis showed no significant (P < 0.05) landrace differences in both axillary bud and immature leaf-lobe explants. Shoot regeneration from cotyledon of somatic embryos had significant (P < 0.05) landrace differences. The mean shoot-bud formation frequency of axillary bud and immature leaf-lobe explants was 51.4% and 37.2%, respectively. Root formation was efficient, with greater than 70% of shoots forming root in all landraces. Similarly, landrace differences were detected for the survival of acclimatized regenerated plants, with a mean of 94.7% for both explants. In conclusion, somatic embryos were produced from immature leaf and axillary bud explants of the landraces and the embryos were converted to plantlets via shoot organogenesis.     

CPT1A基因在广西麻鸡的表达研究

该研究旨在阐明CPT1A基因在广西麻鸡组织表达和时序表达谱,为研究鸡体内脂肪代谢提供一定的基础资料.选取胚胎期到性成熟时的广西麻鸡,在不同发育时间点采集胸大肌、腓肠肌、心脏和肝脏组织样品,利用实时荧光定量方法,分析CPT1A基因在不同组织不同发育阶段表达规律.组织表达结果显示,CPT1A在广西麻鸡不同发育阶段4种组织中...     

绵羊体外受精的TCM199渐变培养体系的研究

应用TCM199作卵母细胞体外成熟、体外受精及早期胚胎培养的基础培养液,再添加不同组分组成TCM199体外受精渐变培养体系,进行绵羊体外受精的研究.结果表明:TCM199体外受精渐变培养体系(试验组)与目前沿用的常规体外受精体系(对照组)相比较,虽然成熟率差异不显著(81.3%,80.4%),但能显著提高绵羊卵母细胞体外受精后的卵裂率(72.1%,50.8%)和桑囊率(94.8%,24.5%).这说明TCM199体外受精渐变培养体系能有效地克服早期胚胎发育阻断.     

不同高粱自交系幼胚组织培养及植株再生

以7份高粱自交系幼胚为外植体,研究了不同基因型对组织培养的反应和植株再生的差异。结果表明:不同高粱基因型间愈伤组织诱导率没有明显差异,但胚性愈伤组织百分率、不定芽分化率以及再生频率有明显差异。其中YN336A、YN267R、YN510R和YN338A的再生效果较好,其不定芽发生率、不定根发生率及移栽成活率分别为64.4%-72.8%,76.2%-100.0%和36.3%-80.0%。对只生根不生芽的基因型,加入1-2 mg/L KT有明显抑制生根、促进生芽的效应。     

Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.     

桂花幼胚培养及愈伤组织增殖诱导

采用不同培养基对桂花胚的培养进行了试验研究,结果表明:MS 1-1.5 mg.L-16-BA有利于桂花幼胚的萌发;MS 2 mg.L-16-BA 0.3 mg.L-12,4-D有利于幼胚苗的幼叶愈伤组织的诱导,诱导率高,且质地致密;B5 1.5 mg.L-16-BA 0.05 mg.L-1NAA有利于幼胚苗基部产生的愈伤组织的增值诱导,所形成的新愈伤组织呈淡绿色,且质地致密,生长很好,并能分化出根。     

不同培养液对昆明小鼠体外受精率及早期胚胎体外发育的影响

在TYH受精液中添加促卵泡素（FSH）和苯甲酸雌二醇（E2）,检测不同浓度的FSH和E2对昆明小鼠的体外受精率的影响;检测在M16、CZB两种培养液中添加胎牛血清（FCS）与否对昆明小鼠早期胚胎体外发育的影响．结果表明：在．rYH中添加1．0IU／mL FSH,受精率和2-细胞胚率显著高于对照组;在TYH中添加0．1μg／mL E2,受精率和2-细胞胚率也显著高于对照组．在CZB中添加体积分数为10％FCS,可显著减少2一细胞期体外发育阻断,并显著提高小鼠胚胎体外发育的囊胚率,可见,以TYH添加1．0IU／mL FSH和0．1μg／mL E2为受精液,以含体积分数为10％FCS的CZB为胚胎培养液,小鼠胚胎可获得较高的囊胚率．葡萄糖并不是小鼠胚胎形成囊胚所必需的．     

玉米花粉诱导不同基因型小麦单倍体得胚率的比较及幼胚离体培养方法对成苗率的影响

选用六倍体普通小麦JYP97系列、常规材料小披白粒,9312(Z)、贵农、恒丰1号和四倍体硬粒小麦、雄性不育系HB系列(12号、11号、13号、26号)等不同倍性或不同基因型的小麦与玉米加-1、超-1、加-6、综-1、701,703等杂交,采用1/2MS培养基,剥取胚龄为14 d、大小为0.4~1.0 mm的小麦杂种幼胚,接种于培养基上,放置24~25℃黑暗条件下2~3周,置于温度为(26±1)℃、光照为1800~2 000 lx的条件下进行幼胚离体培养。结果表明:玉米花粉诱导六倍体普通小麦得胚率为5.4%,明显高于四倍体,玉米花粉诱导雄性不育系的得胚率为3.2%,高于常规材料。混合玉米花粉诱导小麦单倍体得胚率4.6%,高于单一玉米花粉。采用未加任何激素的1/2MS培养基,选取14 d胚龄0.4~1.0 mm的幼胚离体培养,成苗率为81%,显著高于前人所采用的其它培养基进行幼胚离体培养的成苗率。     

百合远缘杂交的胚囊和胚胎发育及胚拯救

采用石蜡切片和子房切片培养方法,对百合杂交组合‘多安娜’ב西伯利亚’和‘普瑞头’ב雪皇后’授粉前后胚囊(胚胎)发育以及胚拯救效果进行了研究。结果表明:‘多安娜’子房内成熟胚囊高峰期滞后于‘普瑞头’3 d左右,而开花后8、10 d时其保持成熟胚囊的比例高于后者。两杂交组合的双受精作用开始于授粉后10 d左右,较亚洲百合自交亲和组合晚,且均存在不同程度的受精后障碍。子房切片培养和胚珠培养仅使‘多安娜’ב西伯利亚’组合获得幼苗。授粉70 d后,两杂交组合都收获少量成熟种子,并经MS培养基培养30 d后萌发成苗。