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。     

不结球白菜小孢子胚植株再生及倍性研究

以不结球白菜子叶型小孢子胚为外植体,研究冷处理、活性炭及AgNO3对小孢子植株再生的影响,并对再生植株染色体倍性进行鉴定.结果表明:对小孢子胚进行5 d的4℃冷处理培养能提高其胚芽诱导率和胚芽数;培养基中添加1.0 g/L的活性炭对提高小孢子胚芽诱导率没有明显效果,但能有效减轻胚芽的玻璃化;添加5.0或7.0 mg/L的AgNO3对小孢子胚芽诱导有显著效果.染色体倍性鉴定结果表明:不结球白菜小孢子植株的染色体自然加倍率较高,在50%～100%之间;不同基因型不结球白菜小孢子植株的倍性变异具有多样性;在部分基因型中嵌合体占较高比例,最高达到42.86%.     

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.     

电子束辐照诱变烟草材料的小孢子培养研究

本研究以自有亲本材料B0与地方品种泉烟、柳烟杂交后与云85、红花大金元组配的三交材料为试材,用辐照剂量为100 Gy的电子束对其进行辐照处理,对辐照后代烟草材料小孢子培养技术进行研究。结果表明:不同基因型材料对游离小孢子培养的敏感性不同,所有的材料中产胚率最高的是(B0×柳烟)×云85组合,产胚率17.2;在材料4和材料6中随着秋水仙碱浓度的提高,两个材料的加倍率均增加,在浓度为100 mg/L时两个材料的加倍率最大。     

花药预处理对玉米游离小孢子存活率的影响

以大田种植的甜玉米“申甜1号”和糯玉米“沪玉糯1号”的花药为供试材料,比较了预处理波的渗透压(甘露醇浓度)、添加不同浓度秋水仙碱、脯氨酸和预处理温度对游离小孢子存活率的影响。结果表明:由低到高的渗透压变化处理(0.35mol/L 24h→0.40mol/L 24h→0.45mol/L 24h, 添加秋水仙碱(200mg/L)、脯氨酸(200mg/L)和15℃、48h的预处理分别能明显地提高游离小孢子的存活率。对上述处理的可能作用机理进行了推测。     

小麦花培苗染色体加倍技术研究

为了提高冬小麦花培苗染色体加倍效率,分别用不同浓度的秋水仙碱对参试的冬小麦材料的花药愈伤组织、再生植株根系和花培苗分蘖节进行了加倍处理。结果表明,用0.02‰和0.05‰秋水仙碱浓度处理的愈伤组织再生植株结实率达33.3%～61.5%。用0.2％的秋水仙碱浸根处理5 h,结实株率平均高达37.5％。用0.04％的秋水仙碱1%的二甲亚砜溶液浸泡分蘖节的时间应在5～10 h之间较为适宜,结实株率平均可达50％以上。     

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。     

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。     

花药预处理对玉米游离小孢子存活率的影响

以大田种植的甜玉米“申甜1号”和糯玉米“沪玉糯1号”的花药为供试材料,比较了预处理液的渗透压(甘露醇浓度)、添加不同浓度秋水仙碱、脯氨酸和预处理温度对游离小孢子存活率的影响。结果表明：由低到高的渗透压变化处理(0．35mol／L 24h→0.40mol／L 24h→0．45mol／L 24h,添加秋水仙碱(200mg／L)、脯氨酸(200mg／L)和15℃、48h的预处理分别能明显地提高游离小孢子的存活率。对上述处理的可能作用机理进行了推测。     

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。     

Efficient production of doubled haploid plants by immediate colchicine treatment of isolated microspores in winter Brassica napus

The effect of colchicine on embryogenesis induction and chromosomedoubling during microspore culture was evaluated in two F₁ hybridsofwinter oilseed rape (Brassica napus L.). Colchicinetreatment (50 and 500 mg/L) of isolated microspores during thefirst 15 h in culture stimulated embryogenesis and produced large amounts ofhealthy-looking embryos. These normal embryos germinated well at 24°C after being transferred to solid regeneration medium and aninitial period of low temperature (2 °C) for 10 days, andcoulddirectly and rapidly regenerate vigorous plants. A high doubling efficiency of84–88% was obtained from 500 mg/L colchicine treatment for15h with low frequency of polyploid and chimeric plants. Acolchicinetreatment duration of 6 h was less effective on embryogenesis anddoubling efficiency. The present experiment also showed that changing of induction medium 15h after microspore isolation produced higher spontaneous doublingefficiency, as compared with medium change 6 h after isolation.     

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.     

PCIB an Antiauxin Enhances Microspore Embryogenesis in Microspore Culture of Brassica juncea

An efficient protocol to improve microspore embryogenesis is established in an important oleiferous crop, Brassica juncea (Indian mustard). Colchicine was used for enhancing microspore embryogenesis and also to obtain doubled haploid embryos. Colchicine at high concentrations (>10 mg l⁻¹), for 24 h, proved convenient for direct recovery of diploid embryos. Higher temperature treatment and an antiauxin PCIB (p-chlorophenoxyisobutyric acid) enhanced microspore embryogenesis significantly as compared to colchicine. An increase in temperature from 32°C to 35°C proved very efficient in increasing embryogenesis by 10-fold. The highest embryogenesis rate was obtained when PCIB was added at 35°C in the culture after 1 day of culture initiation. 20 μM PCIB could enhance microspore embryogenesis by 5-fold. Different abnormal shapes of embryos like lemon, banana, flask and fused cotyledons were observed. Both normal and fused cotyledonous embryos showed normal germination when transferred on the B₅ basal medium.     

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.     

Evaluation of Brassica rapa L. genotypes for microspore culture response and identification of a highly embryogenic line

Isolated microspore culture techniques are being widely used in Brassica breeding programs to generate haploid and doubled haploid plants. A number of factors influence regeneration response in vitro including genotype. In order to assess the effect of genotype on microspore embryogenesis in B. rapa L. var. oleifera, 17 cultivars and breeding lines were evaluated. Embryos developed from all but one genotype when using NLN medium with 17% sucrose, followed by a reduction in sucrose concentration to 10%, 48 h later. The number of embryos /100 buds differed between genotypes, ranging from 0 to 70. Further studies indicated that sucrose concentration and incubation time influenced embryogenesis. Selection studies carried out with an Agriculture and Agri-Food Canada breeding line have resulted in the identification of a highly embryogenic B. rapa line. This line produced thousands of microspore-derived embryos /100 buds and will be useful in mutant selection and gene transfer as well as biochemical and developmental studies.     

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.     

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.     

Isolated microspore culture of oat (Avena sativa L.) for the production of doubled haploids: effect of pre-culture and post-culture conditions

The production of doubled haploid (DH) plants from microspores is an important technique used in plant breeding programs and basic research. Although doubled haploidy efficiencies in wheat and barley are sufficient for breeding purposes, oat (Avena sativa L.) is considered recalcitrant. The objective of this project was to develop a protocol for the production of microspore-derived embryos of oat and further develop these embryos into fertile DH plants. A number of experiments were conducted evaluating the factors influencing microspore embryogenesis, i.e. donor plant conditions, pretreatments, media composition, and culture conditions. The initial studies yielded little response, and it was not until high microspore densities (10⁶ microspores/mL and greater) were used that embryogenesis was achieved. Depending on the treatment, yields of over 5,000 embryos/10⁶ microspores were obtained for breeding line 2000QiON43. The doubled haploidy protocol includes: a 0.3 M mannitol pretreatment of the tillers for 7 days, culture in W14 basal medium with a pH of 6.5–7.5, a microspore density of 10⁶ microspores/mL, and continuous incubation at 28 °C incubation. The resulting embryos observed after 28 days were plated onto solidified W14 medium with 0.8 or 1.0 g/L activated charcoal. A colchicine treatment of 0.2 % colchicine for 4 h resulted in conversion of 80 % of the plants from haploid to DH. This protocol was successful for the production of oat microspore-derived embryos and DH green plants with minimal albinism. DH seed was produced and planted for evaluation in a field nursery.     

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.     

Effects of colchicine on anther and microspore culture of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The aim of this work was to study the effects of colchicine application on chromosome doubling and androgenic response in anther and microspore culture of different bread wheat genotypes. Colchicine was applied during a mannitol stress pretreatment or during the first 48 h of culture at concentrations of 0, 150 and 300 mg l⁻¹. When colchicine was applied during stress pretreatment, the percentage of doubling depended on genotype and concentration. A significant increase in doubling was observed with 300 mg l⁻¹ in the low androgenic responding cv. Caramba. Colchicine incorporation during the first hours of culture improved percentage of doubling in all genotypes, in both anther and microspore culture. Application of 300 mg l⁻¹ colchicine improved the percentage of doubling in the two low responding genotypes, to 118% of control in DH24033, and 75% in Caramba in microspore and anther culture, respectively. Concerning the androgenic response, the effect of colchicine on embryo formation and percentage of green plants depended on the genotype and on the culture method. In cv. Pavon, a 2- and a 3-fold increase in percentage of embryogenesis and green plants, respectively, were obtained with 300 mg l⁻¹ colchicine in microspore culture. However, no significant differences in these two variables were observed in anther culture. The number of green doubled haploid (DH) plants reflects the index of success of the procedure. Regardless of the culture method, when colchicine was incorporated during the first hours of culture, the number of green DH plants increased significantly in three of four genotypes. These results confirm the usefulness of colchicine application during the first hours of culture in wheat breeding programs.