甘蓝型油菜和白菜型油菜种间杂种的小孢子培养

利用分离小孢子培养从甘蓝型油菜（Ｂｒａｓｓｉｃａ ｎａｐｕｓ）和白菜型油菜（Ｂ． ｃａｍ ｐｅｓｔｒｉｓ）种间杂种中获得了胚和再生植株。所用的培养程序是,将甘蓝型油菜和白菜型油菜杂种小孢子在蔗糖浓度为１７％ 、ＢＡ 为０．１ ｍ ｇ／Ｌ的液体ＮＬＮ 培养基中３２ ℃下暗培养４８ ｈ,再转入蔗糖浓度为１０％ 的ＮＬＮ 培养液中２５ ℃下暗培养３ 周。不同杂种间小孢子胚胎发生能力存在差异,其中ＵＭ９２１（白菜型油菜）×９１１１８６（甘蓝型油菜）正反交杂种的胚产量显著高于供试的其它组合。供体植株种植在１０ ℃／５ ℃（昼／夜）条件下能显著改善杂种小孢子胚产量和质量。杂种小孢子胚产量和杂种植株每荚种子数存在极显著正相关,但杂种植株的花粉育性和胚产量间相关不显著。大多数甘蓝型油菜和白菜型油菜杂种小孢子胚衍生植株为非整倍体,２２．８％ 的植株起源于具亲本染色体数的小孢子,几乎全部为ｎ＝ １９ 的类型。讨论了影响种间杂种小孢子胚胎发生的因素以及种间杂种小孢子培养技术的可能用途     

甘蓝和青花菜杂种小孢子培养

对甘蓝(Brassicaoleraceavar.capitata)×青花菜(Brassicaoleraceavar.italica)的20个杂种及相应的父母本进行游离小孢子培养,并对影响甘青杂种小孢子胚胎发生的主要因子进行探讨,适于小孢子培养的培养基为1/2NLN,附加0.5mgL-1NAA、0.05mgL-1BA、5mgL-1AgNO3、0.2mgL-12,4-D和0.1mgL-1活性炭。结果有14个杂种能产生胚状体,诱导率70%;不同杂种间小孢子胚胎发生频率存在很大差异,最高的是绿洲808×夏宝,平均每蕾16.2个胚。诱导杂种胚状体发生的最佳时期是小孢子单核靠边期至双核期,34℃热激2d有利于小孢子细胞对称分裂。在含糖170gL-1的液体培养基中培养3d,添加低糖(含糖110gL-1)的培养液,可显著提高出胚率。     

甘蓝型油菜游离小孢子培养的胚胎发生

以生长在非控温控光下的４个冬性甘蓝型油菜（ＢｒａｓｓｉｃａｎａｐｕｓＬ．）品种为供体,进行游离小孢子培养。研究发现,多数品种在开花３－７天取材最为适宜。在甘蓝型油菜小孢子培养中只有单核晚期的小孢子才可能发育成胚状体,而花药培养时处于单核早期的小孢子易于发育成胚状体。在适当花期选取发育比较一致的单核晚期小孢子培养,经数小时后,部分小孢子便开始膨大,这是小孢子发育成胚的最早标志,膨大的小孢子中,有部分形成多细胞球并进一步发育成胚。用春性甘蓝型油菜为材料进行蔗糖浓度的实验结果表明：培养３天后,在１６％蔗糖培养基中存活的小孢子最多,达１６．１３％;培养３０天后,胚状体诱导频率则以１３％蔗糖浓度为最高,每花蕾可达１４４个胚状体。如果在１６％蔗糖培养基中培养３天后,添加等体积的１３％蔗糖培养基,能够大大提高胚状体的诱导频率,为仅用１３％蔗糖培养基培养的３．７倍。这一实验体系正在用于抗菌核病的诱变与筛选,并作为外源基因导入的实验体系。     

外源性Wnt3a持续作用对小鼠胚胎干细胞Wnt／β-catenin信号通路的调控作用

目的观察Wnt/β-catenin信号通路是否在体外以外源性Wnt3a持续作用小鼠胚胎干细胞后被激活,并进一步调控该通路下游基因的表达。方法应用外源性Wnt3a持续作用ES-E14TG2a小鼠胚胎干细胞21d,通过细胞免疫荧光及Western Blotting检测细胞内β-catenin蛋白,以观察该蛋白的胞内积聚情况;同时QRT-PCR检测WNT下游靶标基因的表达量,采用完全随机F检验并用LSD法进行两两比较,来确定经典WNT／β-catenin信号通路是否被激活。结果ES-E14TG2a小鼠胚胎干细胞经Wnt3a连续培养21d后,β-catenin蛋白的细胞荧光明显较强,而对照组中的荧光强度较弱,说明细胞内β-catenin蛋白没有被降解而是在胞内大量积累;Western Blotting检测结果显示Wnt3a连续培养21d后ES-E14TG2a细胞内β-catenin蛋白条带明显比空白对照的蛋白条带粗;ES—E14TG2a细胞经wnt3a培养后Pitx2、Frizzled、Sox17的表达量均持续上升,Pitx2在培养7d、14d、21d分别为4．17±0．20、7．27±0．35、8．59±0．21（F=222．757,P=0．000）;Frizzled在培养7d、14d、21d分别为1．01±0．06、2．93±0．22、5．44±0．30（F=302．703,P=0．000）;Sox17在培养7d、14d、21d分别为8．45±0．41、18．35±0．17、34．93±0．16（F=7217．083,P=0．000）;Oct4培养到7d、14d的表达量持续增加分别为1．22±0．21、1．56±0．04,而连续培养21d后Oct4基因的表达量下降为1．15±0．07（F=8．827,P=0．016）。结论Wnt3a持续作用可激活Wnt／β-catenin信号通路,并调控下游基因的表达。     

木霉突变体T1010变异特性分析

木霉突变体T1010与出发菌株实验比较,其生长速度、产孢量、菌丝重量分别提高13．62％、48．52％、29．03％;T1010对培养液中氨态氮的吸收率比出发菌株仍2提高37．09％,T1010对无机磷的吸收率比出发菌株提高15．17％,T1010对K’吸收率比出发菌株高16．49％,在pH值为7．34的培养液中培养3d后,T1010的pH值为5．77,出发菌株的pH值为6．67;突变体T1010对培养液中葡萄糖的吸收能力比出发菌株提高19．64％,突变体T1010利用培养液中的氨基酸量较少,比出发菌株降低18．26％。拮抗作用结果显示,T1010对病菌瓜萎蔫镰刀菌抑制作用、覆盖作用分别比出发菌株提高87．45％、57．38％。     

外源生长素对烟草(Nicotiana tabacum L.)小孢子早期胚胎发生的影响

烟草（Nicotlana tabacum L．）小孢子胚胎发生系统,不仅可以提供大量可供处理的小孢子胚,还由于小孢子胚胎发生的不同步性,可同时提供从2-3细胞原胚到分化胚一系列胚胎以供研究。利用这一便利系统,探讨了外源生长素处理对小孢子胚胎发育的影响。使用3种浓度的IAA：1、3、10μmol／L,分别对不同发育时期烟草小孢子胚进行了处理,结果发现,对不同发育时期的小孢子胚,生长素处理的效果明显不同。外源生长素对胚胎发生有促进作用,表现为2-3细胞比例与非处理组相比升高,而当小孢子发育到小球形胚后,加入外源生长素对小孢子胚的进一步发育却表现出明显抑制作用。这说明在小孢子胚胎发育过程中早期和晚期发育对生长素的需求是不同的,且对生长素的敏感程度亦不同。反映了生长素调控机制在两个不同发育时期的差异。     

基因型与培养条件对羽衣甘蓝小孢子胚胎发生的影响

本文详细地研究了小孢子发育时期、基因型与培养条件对羽衣甘蓝小孢子胚胎发生的影响,建立了一个稳定、高频地获得小孢子胚胎的有效体系。结果表明,不同基因型材料相同大小的花蕾其小孢子发育时期存在很大差异,需针对不同基因型材料选取适合大小的花蕾。供试的37个基因型中,有20个获得了胚状体,占供试材料的54．1％,其中基因型‘桃舞’获得了最高的出胚率,为123．6个·皿-1。自交系的出胚率比商业品种和F,代杂种的出胚率要低得多,且自交代数越高,小孢子的胚胎发生能力就越弱。在热激培养48hN加液培养对小孢子的发育能起到积极作用,向培养基中添加激素和活性炭对小孢子的胚胎发生无促进作用。     

杭州部分地区头癣73例类型及病原菌分析

目的 了解近2年来杭州部分地区头癣类型及其病原菌分布情况。方法 采用回顾性方法对2003年5月～2005年4月在杭州市第三人民医院皮肤科门诊诊治的头癣患者进行分析。结果 73例头癣患者中白癣41例（56．16％）、黑癣23例（31．5％）、脓癣9例（12．33％）,未发现黄癣。51例行真菌培养。主要病原菌为犬小孢子菌22例（43．14％）、紫色毛癣菌17例（33．33％）、须癣毛癣菌8例（15．69％）、断发毛癣菌4例（7．84％）。结论 杭州地区头癣患者中白癣发病率占首位,犬小孢子菌为主要致病菌。     

大麦雄核发育的基因型效应及相关的RAPD标记

以具有不同花粉胚胎发生能力的两个大麦（Ｈｏｒｄｅｕｍ ｖｕｌｇａｒｅ Ｌ．）基因型为材料,对离体培养花药中不同类型小孢子频率的边续统计表明,两基因型的早期雄核发育过程无明显区别,最终出愈率（平均每个花药产生的愈伤组织块数）的显著差异是在小孢子形成多核花粉以后产生的。推测基因型之间胚胎发生能力的差异不受小孢子脱分化启动能力控制,而是由其多核花粉发育成愈伤组织的能力决定的,可能与细胞壁的形成有关。利用     

高温对大白菜小孢子培养的影响

应用 FDA和 DAPI荧光显微术观察了高温处理对大白菜 Brassica campestris spp.pekinensis L our. Olsson 小孢子培养的影响 .在 2 5℃恒温培养条件下 ,小孢子的成活率迅速下降 ,至第 7天基本无幸存小孢子 ;小孢子膨胀后呈椭圆形 ,核第一次分裂多为不对称型 ;小孢子丧失胚胎发生能力 .相反 ,在培养开始时 ,先用 33℃高温处理 2 4 h,然后转入 2 5℃培养 ,小孢子成活率虽在第 1天急剧下降 ,但随后仍保持一定水平 ;膨胀的小孢子多呈圆球形 ,小孢子核第一次分裂出现对称型 ,频率可达 4 0 %左右 ;小孢子可进行几种方式的细胞分裂 ,以类似于胚胎细胞的对称分裂方式为主 占总分裂数的 5 5 % ;小孢子经多次分裂可形成紧密的多细胞团 ,最终形成胚状体 .若以高温诱导的小孢子分裂频率为评价指标 ,小孢子接受高温处理的敏感期位于开始培养的 1 2 h内 ,但以胚胎发生频率为指标 ,敏感期位于开始培养的2 4 h内     

DNA Synthesis in Microspores in Anther Culture of Wheat (Triticum aestivum L.)

Anthers with mid-unlnucleate microspores were cultured on W5 medium supplemented with 0.5 mg/l kinetin, 2 mg/l 2,4-D and 9% or 3% sucrose. At a series of interval (0, 1, 1.5, 2, 14 days) after cultured, the anthers were labelled with 3H-thymidine (4 MCi/mi) for 24 h, fixed, and then performed autoradiography according to conventional method. Results show that after cultured for 24 h, 3H-thymidine was incorporated into some late-uninucleate microspores (see Plate I, 3), and after for 2.5 days, vegetative nuclei in pollen grains were la- belled (see Plate I, 4). Usually, vegetative nuclei were labelled frequently and generative ones were labelled rarely. Sometimes generative cell which could synthesis DNA might develop suspensor-like structure individually (see Plate I, 13). During early stage of development of a multicellular pollen grain, the DNA synthesis in the cells were synchronized. With pollen development, the synchronism of DNA synthesis was destroyed. When anthers cultured on medium with 3% sucrose, DNA in microspores could be synthesized normally, and the number of labelled microspores was more than that of anthers cultured on medium with 9% sucrose. However, on medium with 3% sucrose, the nuclei in microspores stopped dividing after one or two divisions and the cell wall of them could not be formed and multicellular pollen was not observed. It seems that the absence of multicellular pollen on medium with 3% sucrose was primarily due to the block of cell division and cell wall formation, not due to the interruption of DNA synthesis.     

Plant regeneration from isolated microspore cultures of Chinese cabbage (Brassica campestris spp. pekinensis)

Isolated microspores of Chinese cabbage (Brassica campestris ssp. pekinensis) were incubated in modified NN medium containing 10% sucrose in darkness at 33°C for one day followed by culture at 25°C. After 14 days of culture, microspores developed into embryos ranging from globular to cotyledonary stage. Plants were regenerated after transfer of embryos to medium containing 3% sucrose and no plant growth regulators.Abbreviations NN Nitsch and Nitsch - MS Murashige and Skoog - NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

Microspore culture of swede (Brassica napus ssp. rapifera) and the effects of fresh and conditioned media

Microspore culture was successfully carried out with four cultivars of swede (Brassica napus ssp. rapifera). Embryo yields increased markedly if the culture medium was replaced by fresh medium after a culture period of three days, with more than 200% increase with the highly responding cultivar Gry and more than 800% with the poorly responding cultivar Stenhaug. If the medium was changed at a later time, embryo production declined sharply. Transfer of microspores after 3 days of culture to a medium in which microspores of a different cultivar had been cultured for 3 days had no effect on embryogenesis. Bud size had a large effect on embryo production, particularly with the least responsive cultivar, Stenhaug.     

Microspore embryogenesis of high sn-2 erucic acid Brassica oleracea germplasm

Brassica oleracea accessions possess traits that would be useful in commercial Brassica crops. These traits can be studied more effectively through the production of doubled haploid plants. Nineteen B. oleracea accessions from several subspecies possessing significant sn-2 erucic acid were screened for suitability for microspore culture using techniques well established for Brassica. Fifteen of the 19 accessions produced embryos. Genotypic differences were observed with embryogenesis ranging from 0 to 3000 embryos/100 buds. Embryogenesis was improved for two of four accessions by initiating cultures in NLN medium with 17% sucrose, then reducing sucrose to 10% after 48 h. An increase in embryogenesis for the same two accessions was observed when microspores were cultured at a density of 100 000/ml rather than 50 000 microspores/ml. A culture temperature of 32 °C for 48 h was beneficial for three of the four accessions when compared to a longer incubation period (72 h) or a higher temperature (35 °C). One accession line, Bo-1, was found to produce microspore-derived embryos which contained triacylglycerols with significant proportions of erucic acid at the sn-2 position. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of various exogenous and endogenous factors on microspore embryogenesis in Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern and Coss)

Summary The influence of donor plant growth environment, microspore development stage, culture media and incubation conditions on microspore embryogenesis was studied in three Indian B. juncea varieties. The donor plants were grown under varying environments: field conditions, controlled conditions, or a combination of the two. The correlation analysis between the bud size and microspore development stage revealed that the bud size is an accurate marker for donor plants grown under controlled conditions, however, the same does not hold true for the field-grown plants. The buds containing late uninucleate microspores collected from plants grown under normal field conditions up to bolting stage and then transferred to controlled environment were observed to be most responsive with genotypic variability ranging from 10 to 35 embryos per Petri dish, irrespective of the other factors. NLN medium containing 13% sucrose was found to be most suitable for induction of embryogenesis The fortification of this medium with activated charcoal, polyvinylpyrrolidone, colchicine, or growth regulators (6-benzylaminopurine and 1-naphthaleneacetic acid) was observed to be antagonistic for microspore embryogenesis, while silver nitrate (10 μM) had a significant synergistic effect. A post-culture high-temperature incubation of microspores at 32.5±1°C for 10–15 d was found most suitable for high-frequency production of microspore embryos. The highest frequency of microspore embryogenesis (78 embryos per Petri dish) was observed from the late uninucleate microspores (contained in bud sizes 3.1–3.5 nm irrespective of genotype) cultured on NLN medium containing 13% sucrose and silver nitrate (10 μM), and incubated at 32.5°C for 10–15 d.     

Embryogenic Suspension Culture and Plant Regeneration from Suspension-Derived Proto-plasts of Cucumber (Cucumis sativus L.)

Fast growing embryogenic cell suspension culture was established when embryogenic callus derived from cotyledon protoplasts of cucumber was transferred into a liquid culture. So far the cell line has been subcultured for two years and retained the ability of embryogenesis and plant regeneration. Experimental data showed that the concentration of ABA or sucrose had a dramatic effect on embryogenesis and synchronization of embryoid development. Low level of sucrose concentration (1%) facilitated the precocious germination of the embryoids while 1 mg/l of ABA or 7–9% of sucrose was found to be effective for reducing callusing of the cultures and synchronisticly controlling the embryoids at globular or late globular stage. Embryogenic cells taken from 3–5 days after subculture were enzymatically digested. A large amount of viable protoplasts was isolated. Protoplasts were cultured in a DPDK1 medium either by means of drop or thin layer liquid culture or by means of sodium alginate encapsulation culture. Actively dividing cells formed cell colonies and globular embryoids which were transferred onto a solidified agar medium or directly into a liquid medium to form a shaken culture. The embryoids would proliferated continuously. Embryoids eventually developed into plantlets when they were transferred onto a 1/2 MSO medium devoid of phytohormones.     

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。     

A co-culture system leads to the formation of microcalli derived from microspore protoplasts ofBrassica napus L. cv. Topas

Summary This paper describes a procedure in which protoplasts are obtained from microspores and pollen of rapeseed to induce callus formation aided by a feeder cell system with embryogenic microspores. Microspores at late unicellular stage and pollen at early bicellular stage were isolated and precultured for 24 h at 32 °C before enzymatic treatment. Eleven enzymes were tested in various combinations and concentrations. The optimal enzyme combination was 1.0% cellulase, 0.8% pectinase, 0.3% macerozyme, and 0.02% pectolyase, in which 26.3% of the microspores released protoplasts. A successful co-culture system was set up by employing embryogenic microspores as feeder cells. To this end, microspores were cultured in a medium with high osmotic pressure at 32 °C. Up to 37% of the microspores exhibited cell division and embryos developed to the heart-shape stage without changing medium. Microspore protoplasts were cultured in Millicells surrounded by the embryogenic microspores as feeder. In growth-regulator-free medium 14.5% of the protoplasts divided but only formed budding-like multicellular structures. Only after pretreatment with 4 mg of 2,4-dichlorophenoxyacetic acid and 1 mg of naphthaleneacetic acid per liter protoplasts divided and formed microcalli. Pollen tubes or tubelike structures were not observed. The experiments reveal that selection of the specific developmental stage of microspores, which is a prerequisite for microspore embryogenesis, is also important in microspore protoplast culture. Compared to other methods used before, microculture fed with embryogenic microspores has obvious superiority.Abbreviations CPW basic protoplast washing medium according to Power and Chapman - CPW972 CPW basic medium supplemented with 9% mannitol and 7.2% sorbitol - DAPI 4,6-diamidino-2-phenylindole - NLN nutrient medium according to Lichter modified by Pechan and Keller - NLN13 NLN medium supplemented with 13% sucrose - NLNP NLN13 supplemented with 7.2% sorbitol     

黑穗醋栗(Ribes nigrum L.)未受精胚珠培养诱导体细胞胚状体和植株再生的研究

本文报告了黑穗醋栗(Ribes nigrum L.)三个栽培品种中的薄皮黑豆未受精胚珠(花粉发育到单核晚期)在MS基本培养基附加植物激素BA,2.4-D和GA_3中形成了体细胞胚状体。长度约为0.5—1.0cm大小的胚状体在生根培养基中可以形成完整的再生植株。经过筛选得到了体细胞胚性愈伤组织无性系,继代培养二年多仍能保持胚状体形成能力。试验结果表明:诱导体细胞胚胎发生受品种和接种时期的影响,适宜的接种时期为花粉发育到单核晚期。培养基中的BA为诱导胚胎发生和保持胚性愈伤组织无性系所必需。