甘蓝型油菜小孢子胚状体发生的细胞学观察

应用甘蓝型油菜ＤＨ系保６０４为材料研究小孢子胚发生过程,结果表明,在小孢子离体培养１～５ｄ内,随培养天数增加,小孢子的存活率迅速下降,部分小孢子培养后出现细胞膨大和分裂,并沿２－细胞。“ｆ”形３细胞,多细胞原体,胚柄球形胚,心形胚最终发育成鱼雷形胚,一般在心形胚阶段,胚柄脱离胚主体部分游离到培养基中,大多数膨大的细胞不能分裂或分裂后停止发育或发育异常。     

甘蓝型油菜小孢子胚状体发生的细胞学观察

应用甘蓝型油菜ＤＨ系保６０４为材料研究小孢子胚发生过程,结果表明,在小孢子离体培养１～５ｄ内,随培养天数增加,小孢子的存活率迅速下降,部分小孢子培养后出现细胞膨大和分裂,并沿２－细胞。“ｆ”形３细胞,多细胞原体,胚柄球形胚,心形胚最终发育成鱼雷形胚,一般在心形胚阶段,胚柄脱离胚主体部分游离到培养基中,大多数膨大的细胞不能分裂或分裂后停止发育或发育异常。     

影响羽衣甘蓝游离小孢子培养中胚状体发生的几个因素

以18个羽衣甘蓝(Brassica oleracea L.var.acephala DC.)F_1杂交种(表1)为试材,研究了几种影响小孢子胚发生的因素。试验于2005～2006年在本校蔬菜试验基地进行。分温室盆栽和露地栽培2部分。温室盆栽于2005年7月15日以穴盘播种育苗;8月25日移栽于花盆;11月5日移到     

几种影响羽衣甘蓝小孢子胚状体成苗的因素

羽衣甘蓝成熟小孢子胚转到固体培养基上可直接萌发成苗。成苗率与基因型、培养基成分和培养温度有关。MS+1.0%琼脂+3%蔗糖是适宜的成苗培养基;100MG·L-1活性炭对鱼雷形胚成苗起促进作用;10℃低温培养10D可提高成苗率。     

云南松小孢子发生的超微结构研究

云南松(Pinus yunnanensis Fr.)小孢子囊发育早期,原生质体发生收缩、同时形成胼胝质壁。小孢子母细胞减数分裂前互相分离,并被胼胝质壁包围。在壁上有约0.2微米的小孔。四分体小孢子形成后,核被膜及高尔基体显得非常地活跃,他们可能与外壁的沉积有关。这个现象在早期四分体阶段出现。内壁的形成是在自由小孢子时期,开始高尔基体和核被膜仍较活跃,但随之下降。内质网和线粒体增多,许多来自内质网的泡囊通过质膜被排放到内壁中去。乌氏体与花粉外壁之间观察到了孢粉素带。     

小麦小孢子发生过程的超微结构

采用透镜电镜技术对小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ）小孢子发生过程进行了超微结构观察。造孢细胞时期细胞质中含有丰富的核糖体,质体和线粒体。在小孢子母细胞减数分裂过程中,有核糖体数量存在逐渐减少的现象,质体和线袜体结构在双线期／终变期和中期１简化,二分体时期,质体和线粒体形态结构基本恢复正常。结果表明,啵麦小孢子母细胞减数分裂过程中存在质体和线粒体的脱人化与再分化过程,但该过程与核糖体数量增     

辣椒游离小孢子细胞团培养的胚状体形成

从预培养15天后的花药中机械游离小孢子及其细胞团,经28℃液体悬浮暗培养．30天后,获得了自球形期胚到子叶期胚发育程度不等的各类胚状体。从12个花药中可以形成高达22个胚状体,且子叶期胚的比例约为23％。显微镜检表明,这些胚状体来自游离的小孢子细胞．经核的对称分裂形成多核细胞或者早期形成多细胞团,最后经细胞的分裂分化形成。胚状体体表具毛,活力有差异。在适当培养基上,具活力的鱼雷期及子叶期胚状体均能发育成正常植株。7℃、32℃、35℃8天的胁迫处理均能诱导小孢子胚状体发生。但花药培养中7℃、35℃处理下的出胚率较32℃下高,而游离小孢子细胞团培养中以35℃、32℃下较好。7℃处理下获得的胚状体数很少．对产生这种现象的原因进行了探讨。出胚率在基因型间,不同胁迫处理温度间表现明显差异。而在温度处理的不同天数间差异不明显。流式细胞仪对再生株真叶的DNA含量分析表明．获得的再生株中具有单倍体、双单倍体以及单倍一双倍嵌合体植株。本结果为进一步开展辣椒雄性生殖途径的胚状体发育研究。提高辣椒成熟胚状体的频率提供了实验体系。     

芸苔属蔬菜小孢子胚状体再生成苗及倍性鉴定

研究影响大白菜、甘蓝和红菜薹小孢子胚状体再生成苗的几个生理因素的结果表明,在1．0％～1．2％的琼脂中胚状体再生成苗率显著高于0．8％琼脂的。4℃处理10d可显著提高大白菜和甘蓝胚状体再生成苗率。大白菜和红菜薹胚状体再生成苗的最适胚龄为20—29d,甘蓝则为30—35d。培养基B,和MS对再生成苗率影响不大。检测3种芸苔属蔬菜小孢子再生植株的倍性结果表明,大白菜和红菜薹小孢子植株自然加倍率较高,均超过70％;甘蓝较低,仅为30％左右。同一物种的不同品种间胚状体再生成苗所需的条件和加倍效率基本一致。     

秋水仙碱对甘蓝型油菜离体小孢子胚胎发生的影响

研究了秋水仙碱不同浓度和处理时间对甘蓝型油菜23个基因型离体小孢子胚胎发生的影响.3个基因型的小孢子被10、50和100mg/L秋水仙碱处理24h或48h,胚产量是2.55～14.75胚/蕾,10～50mg/L处理72h则是0.94～2.43胚/蕾.这表明处理72h对小孢子胚发生有抑制作用.用200、400、500和800mg/L处理2个基因型小孢子16～48h,胚产量为0.6～1.33胚/蕾,未处理对照是6.25和9.36胚/蕾.可见200～800mg/L浓度对胚再生有不同程度的阻碍效应.结果还证明,小孢子对秋水仙碱的反应与其基因型有关.当用10、20、50和100mg/L处理48h时,22B5-6和903-3小孢子的胚产量为37.09～69.47胚/蕾,而F1-29、W592和SF10-12是0.28～1.45胚/蕾,相互之间差异很大.秋水仙碱处理小孢子的目的是使其再生植株的染色体高频率加倍,因此应根据胚产量和染色体加倍率来确定秋水仙碱浓度和处理时间.本试验中,采用10～50mg/L处理48h或者用100mg/L处理24h,约80%基因型的小孢子胚产量在5胚/蕾以上,约70%基因型的再生植株加倍率达60%以上,可有效地用于油菜遗传和育种研究等领域.     

羽衣甘蓝的小孢子胚诱导和植株再生

以羽衣甘蓝10个品种的游离小孢子培养,研究其胚状体及其再生植株诱导方法的结果表明,琼脂糖和活性炭对诱导胚状体发生及发育有促进作用;改良MS培养基中添加0.01%的活性炭可促进植株再生;确定1/2MS NAA 0.1 mg·L-1是优化生根的培养基;小孢子再生植株成活率可达74.6%.     

Expression of Polarity during early Development of Microspore-derived and Zygotic Embryos of Brassica napus L. cv. Topas

Microspore derived (MS-)embryogenesis and zygotic embryogenesis of Brassica napus L. cv. Topas were investigated by light and scanning electron microscopy to reveal the expression of polarity during the transition phase from globular to heart and torpedo shape. During the first 5 days of MS-embryo formation, the cell wall of the former microspores remained intact and a globular mass of cells developed within. Pollen walls ruptured after 5 days of culture; embryos proceeded through heart-shape and torpedo-shape stages within 15 days in a way comparable to, but faster than observed during zygotic embryogenesis. Expression of polarity in globular and elongating MS-embryos was analyzed by detection of the distribution of activated calmodulin as well as of free cytosolic calcium by using confocal scanning laser microscopy, and by the detection of starch. Calmodulin was evenly distributed in globular embryos and only exhibited clear polar distribution in elongated embryos. Free cytosolic Ca²⁺ accumulated in the protoderm of globular embryos and in the central cylinder of torpedo shaped embryos, but never showed polar distribution. Accumulation of starch granules at the root poles of both sexual as well as MS-embryos, however, indicated polar distribution before the transition from globular to heart shape stage. Since the local rupture of the pollen wall of 6-day-old MS-embryos was never preceded by the decrease of starch at that site, it is likely that the rupture of the pollen wall plays an important role in the local activation of cell metabolism and thus in the determination of the polarity axis in MS-embryos.     

一种快速生长的甘蓝型油菜DH系的获得和鉴定

从快速生长的甘蓝型油菜的小孢子培养中共获得23个再生植株。经倍性鉴定,其中自发加倍成二倍体的有10株,单倍体13株。单倍体再用秋水仙碱处理后获得DH系,所得材料对油菜功能基因组学的研究可能有一定的价值。     

甘蓝型油菜小孢子培养技术的几项改进

本研究在NLN-16和NLN-13的培养基中分别加入0.1mg/L 6-BA和0.05%的活性碳,结果表明6-BA对小孢子再生胚有明显地促进作用,再生胚的频率比对照增加26枚/皿,经分析达到显著水平;而0.05%的活性碳对小孢子再生胚促进作用不显著。对甘蓝型油菜小孢子培养再生植株的染色体加倍及移栽的研究结果表明,在小孢子培养初期加50mg/L秋水仙碱加倍效率最佳,加倍率达到67.6%。小孢子培养的再生苗移栽至大田后,采用遮阳网覆盖小苗,移栽成活率达到87.6%。Abstract:The application of microspore culture technique was restricted because of its low frequency of embryogenesis and chromosome doubling.Two methods of enhancing the frequency of embryogenesis were employed in the study,namely,activated charcoal treatment in NLN-13 media and 6-BA treatment in NLN-16 media.The treatment with 0.05% activated charcoal produced 24 embryos per plate,which increased 1.7 embryos per plate,as compared with the treatment without activated charcoal.However,the analysis of T-test showed that it was not significant.After adding 0.1mg/L 6-BA in NLN-16 media,the frequency of embryogeny was 38.3 embryos per plate,and it was 26 embryos more per plate than that of CK.Analysis of T-test is significant.This indicates that 6-BA promotes embryogeny in microspore culture.Adding 50mg/L colchicines in NLN-16 media,the doubling frequency was 67.6%.The plantlets transplanted into field with two methods of light-covered net and plastic films were investigated.A survival rate of 87.6% was obtained using light-covered method whereas 57.7% survived using plastic film method.     

Cytological Characterization of Multicellular Structures in Embryogenic Microspore Cultures of Brassica napus L.

We investigated an embryogenic microspore culture from Brassica napus L. cv. “Topas”, 3 days after induction of embryogenesis, using light and electron microscopical techniques. According to our observations, 6 groups of uni- or multicellular structures could be distinguished by differences in size, wall structure, structure and distribution of organelles and the degree of vacuolisation. Only one multicellular group represents real proembryos which are able to form embryos and to regenerate plants. These 6 groups could be detected in living cultures using an inverted microscope. The cell size and the degree of vacuolization are especially useful markers to distinguish the groups. Separate cultivation of the multicellular complexes of the 6 groups in culture plate inserts from the third day of culture proved that only one group contains real proembryos.     

Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

Individual buds of Brassica napus cv. Topas, near the first pollen mitosis, were used for microspore culture. Bud and petal lengths were recorded. Microspores isolated from the individual buds were plated and small samples were fixed for cytology. Following embryo induction and three weeks of culturing, numbers of embryos were scored. Bud and petal lengths did not accurately indicate which buds would supply microspores that would form embryos at high frequencies. Fluorescence microscopy was used to examine nuclei stained with Hoechst 33258 and vacuolar morphology of microspores was revealed by the weaker fluorescence due to glutaraldehyde fixation. Following isolation, nuclear and vacuolar characteristics were used to stage the microspores as miduninucleate, late uninucleate vacuolate, late uninucleate, mitotic, or binucleate. The relationship of developmental stage to the frequency of microspore-derived embryos was evaluated. A classification scheme was developed which uses the relative proportions of microspores at each of the stages to identify microspore isolations that would form embryos at high frequencies. It was found that when 1 to 87% of the isolated microspores were binucleate, 21.4 ± 3.0% of the viable microspores developed into embryos. This was a significant ( P < 0.001) increase over the other 3 classes. The ability to select highly embryogenic microspore isolations is of great advantage for developmental cell biology studies.