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%.     

羽衣甘蓝小孢子胚胎发生观察及再生植株倍性鉴定

以5个不同基因型羽衣甘蓝品种为试材进行游离小孢子培养,研究小孢子发育时期与花蕾形态的关系、基因型对小孢子出胚率的影响、胚成苗及再生植株加倍和倍性鉴定。结果表明:(1)适宜于羽衣甘蓝游离小孢子培养的花蕾形态指标为:蕾长3.5～4.5mm、花瓣/花药为0.85～1.10。(2)在5个供试品种中,‘4105’出胚率最高,每蕾2.45个,其次是‘7341’,每蕾2.18个,‘7340’和‘7348’小孢子出胚情况较差,分别为每蕾1.36和0.51个,而‘7342’没有出胚。(3)采用150mg.L-1秋水仙碱浸泡幼苗根部24h,结合流式细胞仪快速检测植株倍性,发现二倍体总加倍率为78.4%,大大提高了小孢子再生植株的利用效率。     

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。     

大麦不同基因型游离小孢子的直接培养再生及培养体系的优化

以微搅拌法建立了小孢子直接游离的预处理和培养程序。在大田生长的４个对培养反应不同的大麦基因型上,以新鲜幼穗游离小孢子进行直接培养,均成功地诱导了胚状体并获得再生绿色植株。小孢子的发育进程说明,直接游离的小泡子在预处理过程中的发育要慢于在花药中预处理的小孢子,而且其培养效率也较低。直接游离小孢子的培养密度以０．８～１．０×１０５／ｍｌ较理想,至少应不低于６×１０４／ｍｌ．８％－１０％的糖浓度可明显提高小孢子分裂频率和胚状体诱导频率。实验结果也表明两种培养基ＦＨＧ和ＭＮ６无明显差异,均适宜于直接游离的小孢子培养,并对游离小孢子直接培养在理论和应用上的意义进行了讨论     

大白菜与结球甘蓝异源三倍体小孢子植株的获得与鉴定

以6个不同基因型的大白菜四倍体(AAAA,2n=4x=40)品系9401、9402、9403、9404、9405、9406为母本,结球甘蓝二倍体(CC,2n=2x=18)自交系9501为父本配制杂交组合得到的6个杂种一代为试材,进行了游离小孢子培养研究,成功诱导出胚状体,获得了再生植株,并对部分再生株进行了染色体数鉴定和性状调查。结果表明:不同组合小孢子胚胎发生能力不同,各组合产胚率均较低;小孢子再生植株中,染色体数为18的个体所占比例最大,达46.7%;小孢子植株减数分裂行为复杂,终变期除二价体和单价体外,还有三价体等联会形式;小孢子植株性状表现各异。     

用激光微束穿刺法转化甘蓝型油菜小孢子的研究

建立了油菜小孢子再生胚状体的实验体系,掌握了受体最佳发育时期,使再生频率高达２１．２个胚状体／花蕾。以油菜游离小孢子为受体,用微束激光穿刺法导入芜菁花叶病毒外壳蛋白基因（ＴｕＭＶ）,成功地获得了转基因植株。研究结果表明,用预培养３天的小孢子进行转化得到的１５个胚状体,再生出一株绿苗,２株白苗。而未经预培养的小孢子未能得到转基因植株。用植物总ＤＮＡ以ＰＣＲ法扩增ＴｕＭＶ基因产物片断,然后进行电泳检测,证明该绿苗带有芜菁花叶病毒外壳蛋白基因。     

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。     

禾谷类花器官机械游离小孢子培养研究

以大田及温室生长的植株为材料,成功地建立了直接从禾谷类花器官(大麦穗切段、水稻颖花、小麦小穗)机械游离小孢子的程序及培养系统。从供试的二个大麦材料上重复获得大量游离小孢子再生植株,从一个水稻广亲和品种上得到游离小孢子再生植株,以及从三个小麦品种(系)上获得小孢子形成的多细胞结构(MCS)和早期胚状体(ELS)。相对较长时间的低温预处理有利于提高ELS(大麦)及MCS(小麦)的得率,改善培养物的通气状况,以及提早再分化有利绿色植株再生。     

辣椒花药培养的胚状体分化及其与成苗率的关系

12个辣椒品种的花药培养的结果表明,所有品种均可诱导出胚状体,其中9个品种可获得健壮的再生植株。每一辣椒品种的适宜植物生长调节利配比不同。不同品种的胚状体诱导率和成苗率有差异。成熟的胚状体均能分化成苗,根先分化或停止发育的胚状体很少成苗。     

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。     

Microspore culture of radish (Raphanus sativus L.): influence of genotype and culture conditions on embryogenesis

A number of factors influencing embryogenesis from isolated microspores of radish (Raphanus sativus) were examined. Of 11 genotypes evaluated, six produced embryos ranging from 8.3 embryos per 10⁵ microspores for Chugoku-ao to 0.2 for Tenshun, but five genotypes were not responsive. An initial culture period at elevated temperature before incubation at 25°C was essential for induction of microspore embryogenesis. However, the optimum period of the treatment varied among genotypes and/or experiments. Bud size also influenced microspore embryogenesis. Though optimum bud size was different between genotypes, the microspore populations represented in these buds contained uninucleate and binucleate microspores. Selection of embryogenic microspores using percoll density gradient resulted in up to 1.3-fold increase of embryo yield. Though almost all embryos failed to develop directly into plantlets, plants were obtained by multiple subcultures. The regenerated plants had hyperploid chromosome numbers.     

Effects of pH, MES, arabinogalactan-proteins on microspore cultures in white cabbage

The objective of this study was to improve induction of embryogenesis in white cabbage (Brassica oleracea var. capitata) microspore cultures. The effect of NLN-13 liquid medium pH on isolated microspore embryogenesis was investigated in five white cabbage genotypes. Relatively high pH (6.2 or 6.4) was more effective on microspore embryogenesis in most of the white cabbage genotypes than the pH of 5.8, especially for inducing microspore-derived embryos in recalcitrant genotype ??Zhonggan No. 8??. Based on this, 2??(N-Morpholino) ethanesulfonic acid (MES) and the arabinogalactan-protein from gum arabic were tested on four out of five genotypes to see if they could increase embryo yield in microspore cultures. Adding MES or gum arabic alone was effective for these four genotypes, but the frequency of embryos derived from microspores was still low. However, the combination of 10?mg?l^?1 gum arabic and 3?mM MES in NLN-13 at pH 6.4 significantly enhanced microspore embryogenesis efficiency (with embryo production of 4.57?C222.97 embryos per bud), especially with recalcitrant genotype ??Zhonggan No. 8?? for which it was increased by about 35-fold.     

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。     

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.     

Non-ionic surfactants improved microspore embryogenesis and plant regeneration of recalcitrant purple flowering stalk (Brassica campestris ssp. chinensis var. purpurea Bailey)

As Brassicaceae species are mostly cross-pollinated, breeding homozygous parental lines by traditional approaches is time-consuming and costly. Alternatively, microspore culture has been widely applied to produce double haploid lines in a short time. This study aimed to establish a highly efficient microspore culture protocol for purple flowering stalk. Among the five genotypes studied, the highest and lowest embryo induction rates were observed in J18 and J17 (13.5 and 7.67 embryos per bud, respectively). Microspores of genotypes J17 and J18 were successfully induced to produce embryos in NLN-13 medium, but the frequency of microspore embryogenesis was low. Three non-ionic surfactants (Pluronic F-68, Triton X-100, Tween-20) were evaluated independently for their effect on microspore embryogenesis of purple flowering stalk. Microspores of the two genotypes were cultivated in NLN-13 medium supplemented with different concentrations (0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, and 1% (w/v)) of the three non-ionic surfactants to enhance microspore embryogenesis and plant regeneration. In both genotypes, supplementation with any of the three non-ionic surfactants at 0.0001% significantly increased the frequency of microspore embryogenesis; furthermore, at that concentration, Tween-20 significantly increased the number of plants regenerated from induced embryoids by 29.9% and 30% in J17 and J18, respectively. Moreover, the rate of double haploid formation among regenerated plants of the five genotypes was above 60%, which allowed the creation of 93 double haploid lines.

     

Influence of Preculture Variables on Microspore Embryo Production in Brassica napus ssp. oleifera cv. Duplo

The effect of four preculture variables on microspore embryoinduction and growth were examined: (1) the source of the budselected for culture (apical or axillary inflorescence; (2)the method of harvest (single harvest of whole inflorescenceor sequential harvest of individual buds; (3) the length ofthe bud (2, 3 or 4 mm); and (4) the application of a 4 ^°Cpretreatment to the bud after harvest. Microscopic and macroscopicanalysis of every anther used for culture permitted an assessmentof the following parameters: (1) the percentage of induced buds;(2) the number of induced anthers per induced bud; (3) the numberof productive buds (with macroscopic embryos) as a percentageof the induced buds; (4) the degree of induction per inducedanther (an estimate of the number of microspores in which initialembryogenic divisions had commenced); and (5) embryoid survival(the number of embryos as a proportion of the degree of induction). The product of parameters 1 and 2 gave the number of inducedanthers and all five parameters were components of the finalyield - the number of embryos produced per bud cultured. It was found that the maximum number of induced buds (67·0per cent) occurred with 2 mm sequentially harvested non-pretreatedbuds. Overall, the values decreased with increasing bud lengthand were lower for pretreated and axillary buds. In contrast,the two other estimates of induction - number of induced anthersper induced bud and degree of induction per induced anther -both had maximum values from 3 mm sequentially harvested, pretreatedbuds from apical inflorescences. The highest final yield ofembryos per cultured bud (44·9) was found with 2 mm non-pretreatedbuds taken from a single harvest of the apical inflorescence.The study therefore confirmed that the different componentsof the final embryo yield are differentially affected by thefour preculture variables tested. These variables must be controlledif reproducible results are to be achieved. Brassica napus, tape, anther culture, pollen, microspore, haploid     

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.     

A high throughput <Emphasis Type="Italic">Brassica napus</Emphasis> microspore culture system: influence of percoll gradient separation and bud selection on embryogenesis

Microspore culture for the purpose of developing doubled haploid plants is routine for numerous plant species; however, the embryo yield is still very low compared with the total available microspore population. The ability to select and isolate highly embryogenic microspores would be desirable for high embryo yield in microspore culture. To maximize the efficiency of canola microspore culture, a combination of bud size selection and microspore fractionation using a Percoll gradient was followed. This approach has consistently given high embryo yields and uniform embryo development. Microspores isolated from buds 1.5 to 4.4 mm in length of Brassica napus genotypes Topas 4079, DH12075, Westar and 0025 formed embryos at different frequencies. The most embryogenic bud size range varied with each cultivar: Topas 4079 3.5–3.9 mm, DH12075 2.0–2.4 mm, and Westar and 0025 2.5–2.9 mm. When the microspores from 2.0 to 2.4 mm buds of DH12075 were carefully layered on top of a discontinuous Percoll gradient of 10, 20 and 40%, and subsequently spun through the Percoll layers by centrifugation, bands were formed containing populations of microspores of uniform developmental stage. The middle layer of the gradient contained the late uninucleate and early binucleate microspores that were the most embryogenic. In addition, the relationship between the bud size, developmental stage of isolated microspores, Percoll gradient concentration and the embryogenic frequency of each cultivar were studied. Optimization of these factors is required for each genotype evaluated.     

High-frequency embryogenesis inBrassica campestris microspore culture

Microspore culture is a very important and useful tool in plant breeding for haploid production and has been developed for many years.Brassica campestris (Brassica rapa L. ssp.oleifera) is an important oilseed crop, but it is relatively recalcitrant in tissue culture including microspore culture. The microspore culture in our laboratory is based on the Canadian protocol. Thirty genotypes ofB. campestris were included in this study; twenty produced embryos. The highest yield was 5930 embryos per 100 buds from Canadian genotype Cv-2, this result was one of the best that had been reported in microspore culture inB. campestris. The buds measuring 2.0 mm to 3.9 mm in length responded best to produce embryos, the optimum timing for microspore culture was confirmed to be during the mid-late to very-late uninucleate stage. The buds could be removed from either the main raceme or lateral racemes. Activated charcoal (150 mg l^-1) was added to the liquid NLN medium, it promoted embryogenesis significantly; embryo development was faster and the embryo yield was significantly higher than those cultures without activated charcoal. The donor plant condition was considered an important factor influencing embryogenesis; older donor plants (older than five weeks) and a cold treatment are recommended.     

Activated charcoal induced high frequency microspore embryogenesis and efficient doubled haploid production in Brassica juncea

Three Indian Brassica juncea cultivars were studied for embryogenic response of microspores, microspore embryo regeneration, ploidy assessment of microspore-derived plants and their diploidization. Genotype dependence for microspore totipotency was observed and a significant effect of genotype by bud size selection was established. The addition of activated charcoal in NLN medium containing 13% (w/v) sucrose and 10 μM silver nitrate resulted in a fourfold increase in microspore embryogenesis, ranging from 100 to 405 embryos per Petri dish corresponding to 2,700–10,935 embryos per 100 buds. Conversion/germination of embryos produced in presence or absence of activated charcoal was similar but air-drying of microspore embryos was essential. Incubation of microspore embryos at 4 ± 1°C for 10 days in dark resulted in 82.3% conversion. The majority of plants produced from these embryos was haploid. Treating microspore-derived plants at the 3–4 leaf growth stage with 0.34% colchicine for 2–3 h resulted in greatest survival (70%) and chromosome doubling (75%) frequencies. Doubled haploid plants were self-pollinated and grown to maturity under field conditions.