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。     

结球甘蓝游离小孢子胚胎发生

以结球甘蓝品种“强夏”为材料进行游离小孢子培养,对与胚胎发生关系密切的因子进行探讨。研究结果表明,在盛花前期取材最适宜;单核晚期至双核期的小孢子才能发育成胚状体;含17%蔗糖的培养液在培养初期有利于小孢子存活;培养3d后胚胎诱导则以14％蔗糖浓度为最好;高浓度（17％）蔗糖培养3d后添加低浓度（11％）蔗糖培养液能大大提高胚胎发生能力,比一直在14％蔗糖培养液培养的提高282．4％,比更新培养液培养的提高126．1％。     

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     

油菜小孢子胚发生的超微结构和胚状体形态

应用透射电镜和扫描电镜分别研究油菜游离小了包子培养后细胞的超微结构和胚状体的形态。单核晚期小孢子经培养后,具有胚状体发生能力的细胞中央液泡消失,积累淀粉,含丰富细胞器。     

初始培养条件对定兴芥小孢子胚胎发生的影响

本文探索了油菜花药培养中小孢子胚早期发育的适宜条件。花药分步悬浮法第一培养基的简化实验表明,小孢子胚在仅含有20%蔗糖的空白培养基上的诱导率(212%)与对照的(202%)无显著差异。这样,第一培养基便从原Keller培养基简化为不含大量、微量和有机元素而仅含有20%蔗糖的水溶液,并且,花药第一阶段的培养时间从原来的3—5天减少到1.5—2天。此外,研究了花药在蔗糖水溶液上的滞留时间对小孢子胚胎早期发育的影响。滞留36～48小时,小孢子胚的诱导率(135%)最佳。考察小孢子胚胎早期发育的状况与不同滞留时间的关系则发现,36—48小时内每100个活细胞里,膨大细胞约占70%。这表明,早期培养时,花粉只要膨大而不需分裂即可转入第二阶段培养,便可获得较高产率的胚状体。     

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。     

黄瓜胚性细胞悬浮培养及其原生质体的植株再生

黄瓜子叶原生质体来源的胚性愈伤组织,在液体培养中形成胚性悬浮细胞系,已继代两年,仍保持胚胎发生能力,不同的ABA和蔗糖浓度对胚状体的生长发育和同步化有明显影响。1mg/l的ABA和7－9％的蔗糖能显著地减少培养物的愈伤化,并同步地控制胚状体处于球形或球形后期,低浓度的蔗糖（1％）有利于胚状体的早期萌发,继代3－5天的胚性是浮细胞团酶解后,获得大量有活力的原生质体,原生质体在DPDK,液滴或浅层培养,或褐藻酸钠包埋培养中活跃分裂,形成细胞团和球形胚,转至固体培养基上或将包埋培养物直接转入液体振荡培养,胚状体可不断增殖,胚状体在大量元素减半,不加外源激素的MS培养基上发育成小植株。     

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。     

黄瓜胚性细胞悬浮培养及其原生质体的植株再生

黄瓜子叶原生质体来源的胚性愈伤组织,在液体培养中形成胚性悬浮细胞系,已继代两年,仍保持胚胎发生能力,不同的ABA和蔗糖浓度对胚状体的生长发育和同步化有明显影响。1mg/l的ABA和7－9％的蔗糖能显著地减少培养物的愈伤化,并同步地控制胚状体处于球形或球形后期,低浓度的蔗糖（1％）有利于胚状体的早期萌发,继代3－5天的胚性是浮细胞团酶解后,获得大量有活力的原生质体,原生质体在DPDK,液滴或浅层培养,或褐藻酸钠包埋培养中活跃分裂,形成细胞团和球形胚,转至固体培养基上或将包埋培养物直接转入液体振荡培养,胚状体可不断增殖,胚状体在大量元素减半,不加外源激素的MS培养基上发育成小植株。     

甘蓝花药培养胚状体诱导形成影响因子研究

用甘蓝F1、F2和自交系S33个世代6种基因型材料进行甘蓝花药培养诱导胚状体形成影响因子研究。结果显示:(1)高浓度蔗糖对甘蓝胚状体形成具有显著的诱导作用,6%蔗糖浓度是甘蓝花药培养的最适浓度,其胚状体的诱导率最高达12.2%;(2)材料基因型是影响花药培养的主要因素,F2和F1代材料胚状体诱导效果好,且胚状体诱导率F2代(F2P192和F2P194)18.9%比F1代(F1S17和F1S13)17.1%较高,但差异不显著,自交系S3代材料很难诱导出胚状体;(3)B5培养基比MS培养基更适合甘蓝花药胚状体的诱导培养。结果表明,甘蓝F2代是其花药诱导培养胚状体的最佳基因型材料,B5 2.0 mg/L 2,4-D 2.0 mg/L KT 6%Suc是甘蓝花药诱导培养胚状体的最适培养基。     

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.     

Influence of Genotype, Plant Growth Temperature and Anther Incubation Temperature on Microspore Embryo Production in Brassica napus ssp. oleifera

Eleven F₁ hybrid genotypes of winter rape (Brassica napus ssp.oleifera) were used in a study of induction and growth of microspore-derivedembryos. Plants of each genotype were grown in controlled environmentsat either a constant 15°C or a constant 20°C, both witha 16 h photoperiod. Equal numbers of buds, approximately 2.5mm in length, containing uninucleate microspores were harvestedfrom each genotype and either pretreated (14 d at 4°C) ordissected immediately after harvest. Anthers were cultured onliquid medium based upon that of Murashige and Skoog (1962)and containing 8% sucrose, 0.5 mg dm^–3 naphthylaceticacid and 0.05 mg dm^–3 benzylaminopurine. Anthers fromequal samples of buds were incubated at 35°C for 0, 1, 2or 3 d before transfer to 30°C (21 d) and then 25°C.After a total of 42 d incubation, cultures were scored for thepresence of macroscopic embryos (1–2 mm in length) andfor the presence of anthers containing aborted embryoids whichhad not developed further. The results showed first that bud pretreatment completely inhibitedinduction and secondly that anthers of all genotypes had anabsolute requirement for a 35°C treatment (optimal duration2 d) in order to induce embryoid formation. In the great majorityof genotypes plants grown at 15°C provided more productiveanthers than plants grown at 20°C. However, within eachtreatment there were great differences both in the frequencyof anthers showing induced embryoids and of the final yieldof embryos. There was evidence that hybrids with a common parentresponded similarly under certain treatments. This confirmedthe importance of genotypic control for some components of embryoyield. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

Induction and Growth of 'Microspore-Derived' Embryos of Brassica napus ssp. oleifera

Three cultivars of spring rape (Brassica napus ssp. oleifera),Tower, Willi and Duplo, were used for a study of induction andgrowth of ‘microspore-derived’ embryos, Buds, 2.0mm in length, containing uninucleate microspores were harvestedand stored for 14 d at 4 ?C in darkness. Anthers were then removedand cultured on a liquid medium based upon that of Murashigeand Skoog and containing 8% sucrose, 0.5 mg l^–1 naphthylaceticacid and 0.05 mg l^–1 benzylaminopurine. Cultures werepre-incubated at 35 ?C for 0–3 d and then incubated at30 ?C. After a total of 42 d incubation, cultures were scoredfor the presence of macroscopic embryos (1–2 mm in length)and for the presence of anthers containing abortive embryoidswhich had not developed further. The cultivars differed greatly in terms both of the frequencyof anthers showing induced embryoids and of the final yieldof embryos. Tower showed the highest frequency of induction(maximum 38% of cultured anthers with induced embryoids) whereasthe highest yield (equivalent to 1.1 embryo per cultured anther)was obtained from anthers of the cv. Duplo after a 3 d treatmentat 35 ?C. Yields from the other cultivars were much lower andwere relatively unaffected by the 35 ?C treatment. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

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.     

甘蓝型油菜双单倍体高效诱导技术体系的优化研究

本研究通过对甘蓝型油菜小孢子培养过程影响成胚成苗效果效率最关键的因素,包括取材时期、选蕾大小、培养密度、启始诱导培养基和加倍处理等技术环节及其流程的优化研究,建立了不受大田、温室和人工气候室生长条件影响和基因型的限制,规模化大批量获得甘蓝型油菜双单胚体的诱导技术体系,为甘蓝型油菜基础研究和生物技术育种奠定基础。     

The effect of different carbohydrate sources upon the initiation of embryogenesis from barley microspores

Isolated microspores of Hordeum vulgare L. cv. Igri were incubated in the presence of different sugars. In the presence of maltose, the optimum concentration for the development of embryoids or calluses from the microspores was 175 mM. At this concentration 0.2% of the cells developed into embryoids or calluses. Microspores cultured without a carbohydrate source died after three days' incubation. In contrast microspores incubated in the presence of sucrose, glucose or fructose died within three days. Moreover, microspores also died when incubated in the presence of a combination of 175 mM maltose with varying concentrations of either sucrose, glucose or fructose. It is concluded that incubation of microspores in the presence of sucrose, glucose or fructose results in the death of the cells via some unknown mechanism. In contrast to this, maltose can sustain development of embryoids and calluses from cultured microspores.