影响葡萄花药愈伤组织产生和分化频率的因素

葡萄花药培养能产生体细胞胚。体细胞胚的诱导及其频率高低与材料基因型、培养基和培养条件有关。试验结果表明,对花药进行低温和药物处理不能明显地促进体细胞胚胎发生,但通过改进培养基成份,添加AgNO_3和用液-固培养均能促进葡萄花药胚性愈伤组织的产生。供试的14个基因型材料中仅有3个能产生体细胞胚,而体细胞胚仅部分能萌发成正常苗。     

小麦组织培养体细胞胚胎的次生胚状体发生研究

本研究首次通过幼胚和成熟胚培养，对小麦体细胞胚胎的次生胚状体发生潜力进行了探讨。结果表明，幼胚培养效益显著优于成熟胚，主要体现为成熟胚外植体存在严重的难以克服的微生物污染难题，其平均污染率达50％以上，而幼胚仅为0．97％。同时．叶状体化愈伤组织发生率和植株再生率．幼胚极显著高于成熟胚（P〈0．001）。对愈伤组织再分化培养物的组织学切片观察发现，成熟胚愈伤组织的细胞大、多呈长形或不规则形、胞质稀薄或空囊状、胞间排列松散，没有明显的分生组织区；而幼胚愈伤组织的细胞小、多圆形、胞质浓密、染色深、胞间排列紧密、具有明显的分生组织区，能分化形成体细胞胚胎，并且还发现材料“Fan-33”具有体细胞胚胎的次生体细胞胚胎发生潜力。这种优异的体细胞无性克隆特性，对优化小麦遗传转化系统，提高转基因工程效率具有重要意义。     

栓皮栎体胚再生与增殖能力的保持研究

【目的】提高栓皮栎体胚的再生与增殖能力。【方法】将栓皮栎不同发育阶段合子胚诱导的胚性愈伤组织及不同发育时期的体胚,分别接种于含不同组合激素的MS培养基上,在不同光照条件下培养,探讨影响栓皮栎体胚再生与增殖能力保持的因素。【结果】随着继代次数的增加,胚性愈伤组织再生体胚能力呈下降趋势;6-BA与NAA配合使用可延缓继代过程中胚性愈伤组织再生体胚能力的下降,1.00mg/L 6-BA与0.25mg/L NAA是继代中保持胚性愈伤组织再生体胚能力的最佳培养基激素组合;胚性愈伤组织再生体胚需要自然散射光,强光及全程暗培养均不利于体胚再生;合子胚外植体发育程度影响胚性愈伤组织再生体胚能力,处于心形期及早子叶形期的幼嫩合子胚诱导的胚性愈伤组织,其再生体胚能力强于成熟子叶形合子胚诱导的胚性愈伤组织;体胚发育阶段越老,再生与增殖体胚能力越差,胚性愈伤组织及处于球形期的体胚,再生增殖体胚能力强于其他发育时期的体胚。【结论】选择处于心形期的幼嫩合子胚诱导胚性愈伤组织,然后在含1.00mg/L 6-BA与0.25mg/L NAA的MS培养基上于自然散射光下培养,可有效保持栓皮栎的体胚再生与增殖能力。     

大白菜、青花菜和叶用芥菜体细胞杂交种形态学与细胞学特性鉴定

 为拓宽十字花科蔬菜育种种质资源,以大白菜（Brassica campestris L. ssp. pekinensis）、青花菜（B. oleracea L. var. varitalica）和叶用芥菜（B. juncea L. Czern）的子叶、下胚轴为材料,分离、纯化原生质体,采用40% 聚乙二醇（Polyethylene glycol,PEG）融合法进行原生质体融合。融合细胞在附加0.2 mg · L^-1 2,4-D + 0.5 mg · L^-1 6-BA + 0.1 mg · L^-1 NAA + 0.1 mg · L^-1 激动素（kinetin）的改良K8p培养基中液体培养,当细胞分裂至8 ~ 10细胞期时,将分裂的细胞包埋于0.15%琼脂糖,然后在添加0.3 mol · L^-1蔗糖和2 mg · L^-1 6-BA + 2 mg · L^-1 玉米素（zeatin）+ 1 mg · L^-1 NAA + 0.5 mg · L^-1 kinetin的Kao培养基中诱导愈伤组织。将培养获得的936个愈伤组织转到3种不定芽诱导培养基MS + 5 mg ? L-1 zeatin + 2 mg · L^-1 IAA;MS + 2 mg · L^-1 zeatin + 2 mg · L^-1 IAA;MS + 0.5 mg · L^-1 6-BA + 0.5 mg · L^-1 NAA上诱导芽分化,当芽长3 cm左右时,转到1/2 MS + 0.2 mg · L^-1 NAA的生根培养基上诱导生根,共获得了96株再生植株,对其进行形态学、细胞学和分子生物学鉴定。形态学观察显示再生植株表型变化大,包括3个亲本的中间型或两个亲本的中间型;PCR技术鉴定结果显示96株杂种植株中93株为细胞质雄性不育特性;染色体计数显示,再生的植株染色体数变化范围广（2n = 38 ~ 64）,但未发现染色体数总和与3种融合亲本染色体数总数（2n = 74）相一致的个体。基因组原位杂交（Genomic in situ hybridization,GISH）和扩增片段长度多态性（Amplified fragment length polymorphism,AFLP）分析结果表明,再生植株基因组中分别检测出大白菜、青花菜、叶用芥菜的DNA片段,证实了大白菜、青花菜、叶用芥菜种间体细胞杂交种的真实性。     

花椰菜—黑芥体细胞杂交获得抗黑腐病异附加系新材料

 以花椰菜—黑芥体细胞杂种的自交及回交后代为材料,建立了结合抗病性人工接种鉴定,流式细胞仪DNA含量分析,B基因组特异分子标记和染色体原位杂交的大规模群体分析技术,在50份高代自交及回交材料中筛选获得了20份形态学特征偏向花椰菜,30份呈花椰菜—黑芥中间类型的材料。细胞DNA含量检测（FLC）及染色体计数结果表明,偏花椰菜的类型,DNA含量基本在2.24 pg · cell^-1以下,染色体数在28条以下。经连续3年黑腐病病菌人工接种鉴定,获得高抗株系12个,抗性株系17个,占全部受试材料的14%。对其中3个（PFCN29 BC3-3-5、PFCN16-1 S2BC3-107和PFCN14-1 S1BC4-123）形态偏花椰菜且高抗黑腐病的植株进行基因组原位杂交,结果显示其分别为花椰菜染色体组附加了2、7和8条黑芥染色体的异附加系材料。这些植株可以用于后续的研究和作为育种桥梁材料,进一步获得黑腐病抗性的单体附加系或渐渗系。本研究表明通过体细胞杂交转育野生优异抗性资源,创新甘蓝类蔬菜育种种质的可行性。     

不同转染方法转染牛体细胞效率的比较

为了从新转染方法与传统转染方法的比较中找出更为高效的转染方法,本研究以绿色荧光蛋白(GFP)质粒DNA为外源基因,分别采用核转染法、电穿孔法和脂质体法转染牛的3种常用核移植供体细胞(胎儿成纤维细胞、胎儿输卵管上皮细胞、卵丘颗粒细胞).针对不同类型的细胞,实验首先对核转染法和电穿孔法的转染参数进行了系统的摸索和优化,然后将优化后的核转染法、电穿孔法与脂质体法在可控实验条件完全一致的情况下进行了转染对比实验,分析比较了转染48 h后的绿色荧光比率和细胞存活率.结果显示,核转染法转染胎儿成纤维细胞、胎儿输卵管上皮细胞和卵丘颗粒细胞的优化程序分别是V013、T023和U023;在电穿孔法中,当电场强度为90 V/mm,脉冲时间为10 ms时,胎儿成纤维细胞和卵丘颗粒细胞的转染效率最高,胎儿输卵管上皮细胞则在电场强度为80 V/mm,脉冲时间为5 ms时获得了最佳转染效率;最后通过转染对比实验得出,核转染法在3种细胞中都获得了最高的转染效率,分别为(98.78±0.30)％、(88.43±2.10)％和(71.31±0.77)％,均显著高于电穿孔法和脂质体法;转染后的存活率则为脂质体法最高,电穿孔法最低.研究结果说明,核转染法可以成为一种结合牛体细胞克隆技术生产转基因牛更有效的转染方法.     

氮水平和形态配比对巴西橡胶树花药苗生长及氮代谢、光合作用的影响

采用砂培方法研究了不同供氮水平及NH4+/NO3-配比对巴西橡胶树花药苗生长、氮代谢及光合作用相关指标的影响。结果表明,随供氮水平的提高,橡胶花药苗茎秆、叶、地上部及单株总干重、植株各部位氮含量和氮积累量、叶绿素含量均相应升高,其中粗根、茎秆、叶片含氮量、单株吸氮量及叶绿素含量各处理间差异均达显著水平;株高、茎粗、细根干重、叶片光合速率先增后减,且以8 mmol/L氮水平时最大;低氮水平下细根硝酸还原酶（NR）活性较高。不同NH4+/NO3-配比试验中,纯NH4+营养下小苗各部位氮含量、氮积累量最高（除细根和叶柄氮积累量）;NH4+/NO3-为75/25时,小苗株高、茎粗、叶柄、叶片、植株地上部及株干重最大,NH4+/NO3-为50/50时,小苗细根干重、茎秆干重、叶绿素含量、光合速率最大,且显著大于纯NO3-营养处理;而NH4+/NO3- 50/50时,处理间差异不显著（除细根和茎秆N含量）;纯NO3-营养下小苗细根NR活性最高,但生长最弱。结果说明橡胶花药苗不适宜于纯NO3-环境,而适宜于纯NH4+及NH4+/NO3-混合营养,其适宜水平为8 mmol/L和NH4+/NO3-50/50。     

Cloned calves derived from somatic cell nuclear transfer embryos cultured in chemically defined medium or modified synthetic oviduct fluid

Somatic cell nuclear transfer (SCNT) is considered to be a critical tool for propagating valuable animals. To determine the productivity calves resulting from embryos derived with different culture media, enucleated oocytes matured in vitro were reconstructed with fetal fibroblasts, fused, and activated. The cloned embryos were cultured in modified synthetic oviduct fluid (mSOF) or a chemically defined medium (CDM) and developmental competence was monitored. After 7 days of culturing, the blastocysts were transferred into the uterine horn of estrus-synchronized recipients. SCNT embryos that were cultured in mSOF or CDM developed to the blastocysts stages at similar rates (26.6% vs. 22.5%, respectively). A total of 67 preimplantational stage embryos were transferred into 34 recipients and six cloned calves were born by caesarean section, or assisted or natural delivery. Survival of transferred blastocysts to live cloned calves in the mSOF and the CDM was 18.5% (to recipients), 9.6% (to blastocysts) and 42.9% (to recipients), 20.0% (to blastocysts), respectively. DNA analysis showed that all cloned calves were genetically identical to the donor cells. These results demonstrate that SCNT embryos cultured in CDM showed higher viability as judged by survival of the calves that came to term compared to blastocysts derived from mSOF cultures.     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.     

奶牛TMR饲料的研究进展

介绍了全混合日粮的概念,指出了使用全混合日粮的必要性;并对全混合日粮在奶牛生产中的应用进行了论述,提出全混合日粮是奶牛集约化养殖的必然趋势。