卧牛锦的组织培养及其种质创新初探

以具有嵌合性状的卧牛锦幼嫩花葶为外植体,进行了组培再生及其种质创新的初步研究。外植体在MS +4.0 mg·L-1 6\|BA +0.3 mg·L-1 IBA培养基上诱导形成的愈伤组织,在添加了不同浓度的6-BA和NAA的MS培养基上诱导分化,其中MS+0.01 mg·L-1 NAA培养基最适宜卧牛锦愈伤组织的分化及不定芽的形成;再生获得的不定芽有绿色、黄色和嵌合3种类型,其中嵌合类型的诱导率为14.77%,且植株能够正常生长。研究结果表明,通过愈伤途径对卧牛锦的不定芽诱导能够得到新类型的卧牛锦嵌合体,这对于嵌合体植物的种质创新具有重要的应用价值。     

果树嫁接杂交及其应用

介绍了目前果树嫁接杂交研究的概况,并对嫁接杂交在果树遗传研究和育种实践中的重要作用作了论述     

鸡恒定链功能片段跨物种增强小鼠对新城疫F2抗原免疫作用的研究

【目的】研究恒定链(Invariant chain,Ii)功能片段作为载体的跨物种免疫增强作用及其潜在机制。【方法】构建基于鸡Ii功能片段与新城疫病毒抗原表位F2的嵌合体(Ii-F2、Cyt/Tm/Ii-key/F2/AP、Tm/Ii-key/F2/AP、Ii-key/F2/AP、Ii-key/F2),将其定向克隆至原核表达载体pET-32a中,转化大肠杆菌Rosetta(DE3)并用IPTG诱导表达,嵌合体融合蛋白纯化后免疫小鼠,用ELISA法测定血清抗体效价。同时,分别构建含鸡Ii和小鼠MHCⅡ类分子基因的真核表达载体,并将其共转染COS7细胞,通过激光共聚焦显微镜观察其在细胞内的共定位。【结果】用F2与Ii-key等Ii功能片段连接的融合蛋白免疫的试验组小鼠的抗体效价,较单独用F2抗原肽免疫的对照组小鼠产生的抗体效价提高了1.5~3倍。显微观察发现,鸡Ii和小鼠MHCⅡ分子在细胞内可以共定位。【结论】Ii具有跨越物种限制增强免疫的作用。     

Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene

Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line.     

榨菜(Brassica juncea)和紫甘蓝(Brassica oleracea)种间周缘嵌合体光合特性研究

对榨菜和紫甘蓝种间嫁接嵌合体的光合作用、叶绿素荧光、叶绿素含量、Rubisco的活性以及Rubisco酶大亚基和小亚基基因的转录水平等进行了测定分析。研究发现,种间周缘嵌合体TCC(茎尖分生组织细胞层LI-LII-LIII=TCC,T代表榨菜,C代表紫甘蓝;)的净光合速率为18.09μmol CO2·m-2·s-1,与亲本榨菜相当,但比亲本紫甘蓝高出24.8%。嵌合体的气孔导度和胞间CO2浓度显著高于2个亲本。叶绿素荧光参数中光系统II的实际电子传递量子效率(ФPSⅡ)和光化学猝灭系数(qP)在榨菜中最高,而嵌合体和紫甘蓝的这2个参数基本一致。对叶绿素含量测定后发现,嵌合体的叶绿素a、b及总含量与榨菜比较接近,比紫甘蓝高97%。TCC嵌合体Rubisco酶的初始活性和总活性处于榨菜(最高)和紫甘蓝(最低)之间,为1.76和3.75μmol CO2·g-1·min-1。而TCC嵌合体的Rubisco酶大亚基和小亚基基因的mRNA转录丰度与榨菜和紫甘蓝相比明显增高。以上结果说明,与TCC嵌合体光合机构的层源亲本——紫甘蓝相比,其叶绿素含量升高、Rubisco酶的活性以及其大小亚基基因的转录水平的增强可能是导致其净光合速率提高的原因。可见,TCC嵌合体的异源表皮(来自榨菜)对改善其内部光合组织(来自紫甘蓝)的光合能力有很大的促进作用。     

Histogenic layer manipulation in chimeral ‘Thornless Evergreen’ trailing blackberry

Summary Thornless Evergreen blackberry (Rubus laciniatus Willd.) is a periclinal chimera in which the epidermis has mutated to a thornless phenotype while the internal portions of the plant possess the wild thorny genotype. Shoot tips were used to establish a source of experimental material. Nine hundred plants of Thornless Evergreen were proliferated and rooted in vitro in an effort to locate a chimeral rearrangement and/or a pure thornless plant. When these tissue culture propagated plants were grown in the greenhouse, two predominant plant types were observed; about 53% of the propagules showed a normal vining growth habit while the other 47% of the plants were dwarfed due to shortened internodes. Adventitious shoots from isolated root segments of the normal plants were thorny, while those from many of the dwarfed plants had developed from epidermal cells of the parent. Such plants of epidermal origin are no longer chimeral but are of pure thornless genotype.Graduate Assistant and Associate Professor, respectively. Funds for this research were provided by the Illinois Expreiment Station. We thank Mel Chu and Alan Otterbacher for technical assistance.     

菊花嵌合体‘Su-07’花色性状的组培分离及变异分析

[目的]分离和固定菊花嵌合花色性状,并探讨其花色变异机制。[方法]以紫红-黄色相嵌的菊花花色嵌合体‘su-07’为材料,通过花瓣组织培养再生植株的花色表型,分析花色嵌合体性状的稳定性和分离状况;同时,采用徒手切片的方法,检测不同花色花瓣的细胞内色素分布状况。[结果]在MS+6-BA 2.0 mg/L+NAA 0.2 mg/L培养基上,不同花色的花瓣均可诱导形成愈伤组织,进而分化形成不定芽,经继代培养、生根培养及大田栽培,成功获得了性状稳定遗传的黄色株系;在紫红色花瓣和嵌合花瓣的表皮细胞中都观察到了黄色花色素的存在。[结论]分离的黄色性状来源于嵌合体花瓣的表皮细胞,而未能获得紫红色性状的再生植株也与此有关。     

条斑紫菜种内杂交优良品系的筛选与特性分析

以条斑紫菜(Pyropia yezoensis)野生型品系(Py-WT,特性:颜色好,生长慢,藻体厚,主要光合色素含量低,耐高温性差)的叶状体作为父本,红色突变型品系(Py-HT,特性:颜色较红,生长快,藻体薄,主要光合色素含量高,具一定的耐高温性)的叶状体作为母本进行种内杂交,从杂交丝状体的子一代叶状体中分离出1个耐高温的重组优良品系(HW-4)。在24℃下培养13 d,父本Py-WT和母本Py-HT品系的壳孢子存活率分别为18.3%和65.2%,而HW-4品系的壳孢子存活率为77.5%。在25℃下培养13 d,3个品系(Py-WT、Py-HT和HW-4)的壳孢子存活率分别为16.9%、47.1%和67.3%。在高温(24℃和25℃)下培养的HW-4品系的壳孢子存活率分别是Py-WT品系的4.2倍和4.0倍,是Py-HT品系的1.2倍和1.4倍。常温(19℃)培养50 d的壳孢子萌发体,在24℃再继续培养25 d,Py-WT、Py-HT和HW-4品系的叶状体平均体长分别增加了0.6、0.6和2.3倍,HW-4品系的平均体长分别是Py-WT和Py-HT品系的5.9倍和2.6倍;在25℃再培养25 d,Py-WT、Py-HT和HW-4品系的平均体长分别增加了0.3、0.4和1.5倍,HW-4品系的平均体长分别是Py-WT和Py-HT品系的5.3倍和2.1倍。另外,两个亲本品系的叶状体在高温(24℃和25℃)下培养10~15 d会卷曲和腐烂,培养至25 d时藻体严重卷曲且大面积腐烂;而HW-4叶状体在高温下培养25 d时藻体仅轻度卷曲,35 d后仍未见腐烂现象。常温(19℃)培养的HW-4叶状体分别在常温(19℃)和高温(24℃和25℃)继续培养15 d,总藻胆蛋白含量分别为76.0、111.1和132.3 mg/g,分别是Py-WT品系的3.1、2.2和2.0倍,是Py-HT品系的1.2、1.1和1.1倍,均显著高于双亲品系(P0.05)。3个品系的壳孢子放散量之间无显著性差异(P0.05)。上述结果表明,与亲本品系相比,HW-4品系的壳孢子和叶状体的耐高温性均明显提高,叶状体的生长速度和色素含量也大幅度提高,是一个生产适用性好的优良品系。     

Production of germ-line chimeras by the transfer of cryopreserved gonadal germ cells collected from 7- and 9-day-old chick embryos

Gonadal germ cells (GGC) were collected from the gonads of 7‐ or 9‐day‐old White Leghorn chick embryos and suspended in freezing medium containing 10% dimethylsulfoxide (DMSO). The cell suspension was frozen at ?1°C/min. until the temperature reached ?80°C. Then, the cells were immersed in liquid nitrogen at ?196°C and stored for 3–4 months. Approximately 50 frozen/thawed GGC were injected into the dorsal aorta of each 2‐day‐old Rhode Island Red (RIR) embryo, from which blood was drawn before germ‐cell injection. The injected embryos were incubated until they hatched and the chicks were raised until sexually mature. On reaching sexual maturity, a progeny test was performed by mating recipient chicks with normal RIR of the opposite sex. Progenies were obtained from male germ cell recipients that were injected with germ cells collected from 7‐ and 9‐day‐old embryos. The results demonstrated that frozen/thawed GGC collected from 7‐ or 9‐day‐old fertilized eggs can be used to produce male germ‐line chimeras.