枸杞悬浮培养条件下的胚状体发生

在MS培养基上诱导宁夏枸杞下胚轴切段形成愈伤组织,并进行细胞悬浮培养。观察了悬浮培养条件下胚状体的发生过程。<1>观察发现,细胞经悬浮培养几天以后,先形成胚性细胞团,再有胚性细胞团块形成一个或几个胚状体。较大的胚状体也可以形成新的次生胚状体。<1>影响胚状体形成的关键因素是激素的种类及其含量。实验采用的基本培养基为MS,比较适宜胚状体发生的激素是0.2mg/L 2,4-D。同样浓度的2,4-D,会抑制胚状体的进一步发育,用0.2mg/L6-BA代替2,4-D, 则胚状体可以进一步发育并形成小植株。     

金花茶子叶在离体培养中胚状体的发生和小植株的形成

研究了金花茶(Camellia chrysantha(Hu)Tuyama)子叶在离体培养中体细胞胚状体发生的条件。在MS基本培养基中附加苄基嘌呤(BA)或苄基嘌呤与萘乙酸(NAA)组合,诱导了胚状体发生。组织学观察表明,胚状体起源于子叶的表皮细胞。在增添细胞分裂素和生长素的MS或改良B_5液体培养基里振荡培养,明显地促进了胚状体根的生长和茎的发育。胚状体在继代培养中能保持旺盛的再生能力。已得到两个繁殖率较高的胚状体无性系。在合适的条件下,胚状体能长成正常的小植株。     

辣椒花药培养胚状体发生的组织学和细胞学研究

采用荧光显微镜、扫描电镜和透射电镜技术．系统研究了辣椒花药培养胚状体发生的组织学和细胞学变化特征。辣椒单个花药中花粉发育具有强烈的不同步性。随着培养时期的变化．不同时期花粉的百分率也发生变化。处于单核靠边期的小孢子培养以后按两种发育途径之一进行发育。在多数情况下,孢子体不对称分裂,产生典型双核花粉。胚性花粉粒是由营养核的重复分裂形成的。当小孢子从四分体中释放出来．特殊类型的外壁已经形成。在随后的花粉发育过程中．小孢子体积增大,外壁继续加厚。培养24h后,小孢子体积增大。胚性发生的小孢子表现出两种不同的形态变化。当胚状体发育到心形胚时．胚状体的表皮细胞排列规则。用光学和电子显微镜分析了小孢子胚状体形态形成过程．及胚状体诱导后细胞组织发生的一系列结构变化的时序性特征,这些变化主要影响质体、液泡室、细胞壁和细胞核,进一步分化的程序模拟合子胚的发育。     

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。     

伊贝母体细胞无性系的建立及其胚状体的发生

本文报道了伊贝母体细胞无性系的建立及其胚状体的发生。已继代培养三年零六个月共30多代的鳞芽愈伤组织,目前仍有分化能力。通过愈伤组织形态细胞学的观察,发现伊贝母体细胞无性系形成小鳞茎的途径有二:一是由特化了的愈伤组织表皮细胞。经多次分裂发育成不定芽而形成小鳞茎;二是由愈伤组织表层或内层特化了的胚性细胞,经多次分裂发育成胚状体而形成小鳞茎。不定芽和胚状体的形态发生是有区别的。     

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。     

乔纳金叶片培养胚状体发生体系的建立

利用乔纳金无菌苗叶片培养,成功地诱导出胚状体并获得再生植株。具体步骤如下:Ⅰ.在MS BA2.0mg.L-1 IAA6.0mg.L-1 2,4-D0.3mg.L-1培养基上预诱导6d;Ⅱ.在MS BA2.0mg.L-1培养基上胚性细胞发生胚状体;Ⅲ.在MS BA0.5mg.L-1 IAA0.1mg.L-1上壮苗培养20d;Ⅳ.在MS IBA0.8mg.L-1 IAA0.7mg.L-1培养基上小植株生根。     

三七胚培养中的胚胎发生

三七成熟胚培养于MS 1 mg/l IAA或NAA或2,4-D的培养基上。二月后,在MS 1mg/l IAA或NAA的培养基上由外植体可诱导产生胚状体,但在含2,4-D的培养基上只产生愈伤组织而无器官分化。胚状体转入MS GA_3 1mg/l IAA0.5 mg/l培养基上可发育成具胚根、根芽的小植株。     

三七胚培养中的胚胎发生

三七成熟胚培养子MS+1 mg／L 1AA或NAA或2,4 — D的培养基上。二月后,在MS+1 mg／L 1AA或NAA的培养基上由外植体可诱导产生胚状体,但在含2,4 — D的培养基上只产生愈伤组织而无器官分化。胚状体转入MS+CA3 1 mg／L+IAAo.5 mg／L培养基上可发育成具胚根、根芽的小植株。     

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。     

Manipulation of division symmetry and developmental fate in cultures of potato microspores

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell architecture during gametophytic and embryogenic microspore development in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

In this work, the cell architecture of the microspore following both gametophytic and embryogenic developmental pathways in vitro was compared with the gametophytic development in vivo in Brassica napus, at both light and electron microscopy level. The microspore reprogramming to embryogenesis involves defined changes affecting cell activities and structural organization which can be considered as markers of the microspore embryogenic pathway, but less is known about others developmental programmes followed by the microspore in vitro after both, inductive and non-inductive conditions. Low-temperature processing of the samples, cytochemical and immunocytochemical approaches to identify various cell components were performed. Differences in specific cellular features such as cellular size and shape, nuclear architecture, starch accumulation, presence of vacuoles and ribosomal population were studied to characterize sequential stages of microspore embryogenesis and other pathways occurring in vitro. The presence of abundant starch grains in a defined cytoplasmic region appeared as a specific feature of the in vitro gametophytic development, as well as of the non-induced microspores of in vitro cultures under embryogenic-inductive conditions.     

甜(辣)椒单倍体培养研究进展

介绍了甜(辣)椒花药培养和游离小孢子培养的研究概况,花药培养应用相对成熟,游离小孢子培养尚未获得突破性进展。对影响花药培养的各关键因素(包括材料基因型、供体植株生长状态、小孢子发育时期、培养基、培养方法、变温处理、培养条件等)进行了综述,并讨论了甜(辣)椒单倍体培养存在的问题和进一步研究方向。     

Microspore culture of small grain cereals

Androgenesis of wheat, rice and triticale was studied in isolated microspore culture. It is the first publication which studies microspore culture reaction of Hungarian rice varieties. The effect of different basic media, lack and absence of growth regulators in culture media were tested on important parameters of microspore culture. Direct embryogenesis was observed in microspore culture of wheat and triticale genotypes. In the case of rice, calli were induced in isolated rice microspore culture and haploid rice plantlets were regenerated via organogenesis.In wheat, the effect of basic media (W₁₄, A2, CHB₃, P4-m) was compared and among them the W₁₄, and A2 had a superior effect on embryo production and albino and green plantlet regeneration. In rice the C, CHB₃ and MS_m media were tested in microspore culture and the significantly highest numbers of calli were achieved by using C and CHB₃ media depending on the genotypes. The lack of exogenous growth regulators was observed in isolated microspore culture of triticale and rice. Growth regulator-free medium had a positive effect on embryo production and plant regeneration of triticale genotypes, whereas in rice microspore culture multicellular structures did not continue their division without growth regulators from the third week of microspore culture. Developing of microspore-origin calli was maintained by supplement of 2,4-D and Kinetin combination in the microspore culture medium.     

Comparison of Anther and Microspore Culture in the Embryogenesis and Regeneration of Rye (Secale cereale)

Anther culture in solid and liquid medium and isolated microspore culture were compared in rye genotypes with potential agronomic characteristics. Some important factors influencing androgenic capacity were optimised. Three weeks cold pre-treatment of spikes and two days mannitol pre-treatment of anthers maximized callus and green plant yield in both culture methods. Intensity order of the culture methods in callus and green plant production was: isolated microspore culture, anther culture in liquid medium and anther culture in solid medium. Genotype ability of embryogenesis followed the same pattern in both cultivation methods. Kinetin (BA) with genotype dependent concentrations created the most effective regeneration conditions.     

Doubled haploid sunflower (Helianthus annuus) plant production by androgenesis: fact or artifact? Part 2. In vitro isolated microspore culture

Previously reported attempts to routinely produce certified doubled haploid sunflower plants were unsuccessful. Isolated microspore culture was assayed in order to overcome the high reactivity of somatic tissues such as anther wall, multicellular hair-type structures, anther connective and parenchymatous vascular bundle. Using filtration or density gradients in liquid medium (N6 supplemented with NAA 1 mg l^-1, BA 0.2 mg l^-1 and maltose 0.44 M), asymmetrical and symmetrical microspore divisions were obtained. However, contaminating cells or groups of cells coming from hairy type structures were very reactive, and able to develop into calluses. A second purification step was therefore necessary after two days in culture, to isolate a population consisting of highly viable swollen microspores. Using this procedure, besides avoiding somatic contaminants, the microspore response in terms of viability and initial division rate was increased, and sustained division and microcallus formation were achieved after addition of aminocyclopropane carboxylic acid, an ethylene precursor.Abbreviations ACC aminocyclopropane carboxylic acid - AOA aminooxyacetic acid - DAPI 4,6 diamidino-2-phenyl indole     

麦类作物小孢子培养与遗传转化研究进展

小孢子作为单个单倍体细胞,通过诱导培养能再生成纯合的二倍体植株,不仅为分子生物学、遗传学、形态发生学研究提供稳定的材料,也对小麦等作物的转基因研究提供新方向和新思路。本文主要介绍麦类作物小孢子培养的基本步骤及关键因素,简要介绍了以小孢子为材料进行小麦基因遗传转化的方法。     

Regeneration of androgenic embryos in apple (<Emphasis Type="Italic">Malus x domestica</Emphasis> Brokh.) <Emphasis Type="Italic">via</Emphasis> anther and microspore culture

Based on optimized protocols for anther and microspore culture in apple (Malus x domestica Borkh.), the regeneration phase and the efficiency of the processes in general were compared by using the same androgenic material of two experimental years. Microspore culture resulted in an increase in embryo induction depending on the genotype (Höfer 2004), however anther culture was superior to microspore culture in the total number of regenerated plants. The regeneration process in anther and microspore culture is similar. Two developmental pathways were observed: 1) secondary embryogenesis followed by adventitious shoot formation and 2) direct adventitious shoot formation from primary embryos. Induction and regeneration processes are delayed in microspore culture as compared with anther culture. The reasons for the reduced regeneration efficiency in microspore culture are discussed.