秋水仙素浓度、浸苗时间对烟草单倍体幼苗的加倍效应研究

为了提高烟草单倍体植株的染色体加倍率,采用二次饱和D-最优设计,以两片真叶期的烟草单倍体幼苗为材料,研究了秋水仙素浓度及其浸苗处理时间对染色体加倍的效应.结果表明,随着秋水仙素浓度的提高,加倍率呈开口向下的抛物线曲线变化趋势;加倍率与浸苗时间呈线性回归关系,加倍率随浸苗时间的延长而增加;0.346 9%的秋水仙素、浸苗72 h是烟草单倍体幼苗染色体加倍率最高(达54.18%)的最佳处理组合.     

半浸根法加倍小麦远缘杂交幼胚再生植株染色体

染色体加倍是小麦单倍体育种和克服远缘杂种不育的重要技术之一.多年来,应用秋水仙素对花培产生的单倍体植株的染色体加倍进行了大量研究[1～4],但对小麦远缘杂交幼胚再生植株的染色体加倍技术研究很少.传统的全浸根法染色体加倍技术成功率低,而且死苗严重.和现昌等[5]用半浸根法就地挖坑加倍小麦花粉植株,加倍率达80%以上,幼     

用气孔保卫细胞叶绿体计数法鉴定烟草染色体倍性方法初探

经不同浓度秋水仙碱液加倍处理的烟草花粉植株，用叶片气孔保卫细胞叶绿体计数法鉴定染色体倍性。研究表明：气孔保卫细胞叶绿体数平均值差异极显著，单倍体95％以上的叶绿体数在14个以下，双倍体95％以上的叶绿体数则在14个以上。经开花结实验证其准确率达91％，且展开到第5叶时就可鉴定。     

球茎大麦在大麦育种上的应用——Ⅰ.大麦单倍体的诱导与加倍单倍体的产生

以三种栽培大麦品种为母本,三种球茎大麦为父本进行杂交。诱导栽培大麦单倍体,结果杂交成胚率平均为47.9％,幼胚离体培养成苗率平均为54.0％。这些胚培苗的根尖细胞染色体数目分别含有7,14,21条,不同组合杂交后代含有的染色体数目不同,所有含有7条染色体的后代,其染色体C-带带型显示为父本栽培大麦的染色体,表明该后代为栽培大麦单倍体,用滴加0.04％秋水仙素的Knop溶液进行单倍体植株的染色体加倍,结果加倍率达90％以上,另外,我们还讨论了染色体消失法诱导单倍体的优越性及其机理。     

球茎大麦在大麦育种上的应用：Ⅰ.大麦单倍体的诱导与加倍单倍体的 …

以三种栽培大麦品种为母本,三种球茎大麦为父本进行杂交,诱导栽培大麦单倍体,结果杂交成胚率平均为４７．９％。幼胚离体培养成苗率平均为５４．０％。这些胚培苗的根尖细胞染色体数目分别含有７,１４,２１条,不同组合杂交含有的染色体数目不同,所有含有７条染色体的后代,其染色体Ｃ－带带型显示为父本栽培大麦的染色体,表明该后代为栽培大麦单倍体,用滴加０．０４％秋水仙素的Ｋｎｏｐ溶液进行单倍体植株的染色体加倍,结     

芦笋单倍体染色体加倍技术研究

研究了芦笋单倍体染色体加倍的方法。在离体培养条件下,以秋水仙素为诱变剂,分别用浸泡法和培养基添加法处理芦笋单倍体幼苗的茎尖,比较了秋水仙素不同浓度、不同处理时间的诱导效果。结果表明,培养基添加法的诱导效果好于浸泡法,当在培养基中添加0.3%秋水仙素并处理7天时诱导效果最佳,染色体加倍频率与成活率分别可达82.50%和80%。加倍后的二倍体植株与单倍体植株相比,茎干变粗,气孔与保卫细胞增大,保卫细胞内的叶绿体数增多。     

不同新型除草剂加倍糯玉米单倍体的效率研究

通过比较五氟黄草胺和二氯喹磷酸、双草醚3种除草剂在不同浓度下对糯玉米单倍体的加倍效率,选择最为合适的加倍方法.以京科糯2000、中糯2号、吉香糯87三种白糯基因型的单倍体籽粒为材料,用浓度为40,60,80 μmol/L的五氟黄草胺和二氯喹磷酸、双草醚作为加倍药剂,采用浸芽法对芽长1～2 cm的糯玉米单倍体种子进行处理,处理后移栽至大田,观察其散粉情况,统计单倍体植株的加倍率.结果表明,40～80μmol/L的3种加倍药剂对糯玉米单倍体加倍都有效果.五氟黄草胺、二氯喹磷酸和双草醚的加倍率依次为3.46％～10.17％,6.49％～14.87％,2.98％～10.05％;其中以80μ mol/L的二氯喹磷酸加倍效率最高,3种糯玉米单倍体种的平均加倍率分别为13.23％、14.63％、14.87％.方差分析表明,3种白糯基因型玉米单倍体种的加倍率呈显著差异;由此可见,五氟黄草胺、二氯喹磷酸和双草醚可以提高糯玉米单倍体的加倍效率,但不同基因型的糯玉米单倍体对加倍药剂的敏感性存在差异.     

高效糯玉米单倍体加倍方法的研究

以DR 5为父本诱导5份糯玉米杂交种获得的单倍体籽粒为试验材料,探究处理方法、浓度、处理时间和温度对糯玉米单倍体的加倍效果.结果表明,浸芽法的加倍效果最好,加倍率均值达11.02％.秋水仙素浓度为0.8 mg/mL时,加倍率最高,均值达10.47％.处理时间以8h最好,加倍率均值达7.58％.处理温度为21℃时达到最佳,加倍率均值达10.96％.     

巴西橡胶树自根动态无性系的试管微繁

带有腋芽的橡胶花药苗切段中在ＭＳ＋６－ＢＡ２ｍｇ／Ｌ的培养基上，３０～４０天新芽可长至３～５ｃｍ高，并可切割再次增殖，切取３～５ｃｍ高带叶片的增殖芽作为插条，基部经５０～１００ｍｇ／ＬＩＢＡ预处理再插到生根培养基上，生根率达９５％，诱发的根系与茎杆之间愈合良好，输导组织连接紧密，生极植株移栽成活率达８５％以上。     

由染色体重组制造小麦族的染色体组 Ⅰ.预备试验与可能性

单倍体小黑麦的不正常减数分裂将使 A、B、D、R4个染色体组的28条染色体进行随机分配,形成一系列具有不同大小、不同生活力、不同染色体数目和不同染色体组合的小孢子。对单倍体小黑麦花药培养的预备试验已经说明,如果选用具有高诱导率和绿苗高分化率的单倍体小黑麦品系或细胞系作为花药供体,由此获得大量愈伤组织和花粉植株     

Chromosome doubling of androgenic haploid plantlets of rice (Oryza sativa) using antimitotic compounds

The regeneration of haploid plantlets is considered as a bottleneck in rice anther culture. In this study, an antimitotic chromosome doubling method, simple and efficient, of androgenic haploid plantlets resulted in an efficient doubled haploid obtainment. Through chromosome doubling capacity comparison of the three antimitotic compounds (colchicine, trifluralin and oryzalin), colchicine at 500 and 625 mg/L without supplementing with DMSO was found to be the best antimitotic treatment, with a chromosome doubling capacity of 40%. Furthermore, the in vitro growth of plantlets was followed to analyse the effects of antimitotic compounds. Colchicine treatments were more toxic than dinitroanilines, and colchicine DMSO-supplemented treatments had significant lower values on shoot growth. On the other hand, dinitroaniline compounds impeded root growth, provoked helical growth of shoot and caused the apparition of white nodules in the base of the plantlet due to sprouting abortion. In this study, a protocol for doubled haploid plant recovery was established taking advantage from androgenic haploid plantlets in order to increase the number of doubled haploid plantlets produced after an anther culture protocol.     

The effect of colchicine on triticale anther-derived plants: Microspore pre-treatment and haploid-plant treatment using a hydroponic recovery system

In cereals, chromosome doubling of microspore-derived haploid plants is a critical step in producing doubled haploid plants. This investigation was undertaken to study the effect of incorporation of colchicine in the induction medium for anther culture, and the effect of colchicine on anther culture-derived plants of triticale grown under controlled greenhouse conditions. In the latter case, chromosome doubling of adult sterile plants derived from anther culture of fourteen triticale populations was attempted, where androgenetic plants with non-dehiscent anthers were cloned and subjected to the colchicine treatment, and then grown with the aid of hydroponics. The hydroponic system provided optimal conditions for recovery of the affected haploids from the toxic effects of colchicine treatment and all colchicine-treated plants survived. A topcross-F₁ (TC₁F₁) population with timopheevii cytoplasm produced the highest percentage of plants with seed-set either due to chromosome doubling by colchicine (98%) or spontaneous doubling of chromosome number (15%). Colchicine-treated anthers performed inferior than control in both induction and regeneration phases. One of the key observation of this study was the reversal from reproductive stage back to the vegetative stage which in turn enabled further cloning of haploid plants under hydroponic conditions once they were identified as sterile. The one hundred percent survival rate of in vitro-derived plants, 100% survival rate of colchicine treated haploid plants and the high chromosome doubling success rate (X = 82.3) observed in this study imply that a temperature-controlled greenhouse with an hydroponic system provides an efficient environment for inducing chromosome doubling of haploid plants in cereals. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro chromosome doubling with colchicine during microspore culture in wheat (Triticum aestivum L.)

Isolated microspores of two DH lines of wheat were treated with 8 different colchicine concentrations up to 3 mM for either 24 h or 48 h during microspore culture. Untreated control cultures produced on average 220 embryos per spike (100,000 microspores), 68% of the regenerated plantlets were green, and 15% of the flowering plants were fertile. The colchicine treatments had a significant effect on chromosome doubling as measured by the percentage of fertile regenerants. Using colchicine concentrations around 1 mM the percentage of fertile plants among the regenerants was increased up to 53%. The highest number of embryos and regeneration rates were observed after 24 h colchicine treatment, while the highest frequencies of green plants and fertile plants were obtained with 48 h colchicine treatments. The highest number of DH plants per spike was found after treatment with colchicine concentrations of 300 to 1000 μM. Such treatments resulted in an estimated average between the two genotypes of 23 doubled haploid plants per spike. This revised version was published online in July 2006 with corrections to the Cover Date.     

Improving androgenesis‐mediated doubled haploid production efficiency of FCV tobacco (Nicotiana tabacum L.) through in vitro colchicine application

Production of doubled haploid plants through androgenesis in flue‐cured Virginia (FCV) tobacco is a promising and convenient alternative to conventional selfing techniques for the generation of absolute homozygous lines. Here, we show a robust in vitro haploid and doubled haploid development protocol in FCV tobacco with major emphasis on improving the efficiency of chromosome doubling using in vitro colchicine treatment. We used five FCV tobacco hybrids for comparison of colchicine treatments. The anther culture response varied with developmental stages of the buds, and the highest response was observed in stage 2 buds. The effect of cold pretreatment was significant, and 4 days of pretreatment was optimum for gametic embryogenesis. Among the methods used for determining the ploidy status of plants, flow cytometry was found to be easy, fast and reliable for high‐throughput screening of haploids. Doubled haploids regeneration percentage varied from 6.77 to 11.95 in in vivo treatment, while the range of variation was 22.11% to 28.40% in in vitro colchicine treatment. We observed a pronounced increase in plant survival and the proportion of doubled haploid plants in in vitro treatment compared with the standard in vivo approach.     

马铃薯高效染色体加倍方法建立与抗寒资源创制

In this work, different treatment combinations including colchicine concentrations and treatment methods as well as different time points was used to investigate the survival rate and chromosome doubling efficiency of potato tissue culture plantlets. The potato chromosome doubling by using colchicine had been successfully optimized. The potato plantlet stems treated with 0.1% colchicine for three days shaking at 120 r min^-1 showed the highest doubling efficiency due to its better contact to the colchicine solution. Compared with other potato chromosome doubling techniques, this method has much higher chromosome doubling rate, shorter time treatment and easier to operate, so that it could provide a higher efficient method for potato ploidy operation. In the meantime, compared with the diploid, the tetraploid interspecific hybrids showed differences in the morphological characteristic, which had higher plants, thicker stem, bigger petals and pollen grain. In addition, no significant difference was found between diploid and tetraploid interspecific hybrids in terms of cold resistance, but both significantly enhanced cold resistance compared with the common potato cultivar. Taken together, the doubled interspecific hybrids could sever for improving cold resistance of potato cultivars.     

Relative Efficiency of Anther Culture and Chromosome Elimination Techniques for Haploid Induction in Triticale × Wheat and Triticale × Triticale Hybrids

Summary The study was undertaken to evaluate the relative efficiency of anther culture and chromosome elimination (by crosses with maize) techniques of haploid induction in intergenotypic triticale and triticale × wheat hybrids. For this, 15 triticale × wheat and 8 triticale × triticale F₁ hybrids were subjected to anther culture and were also simultaneously crossed with the `Madgran Local' genotype of maize (Zea mays L.) to induce haploids through the chromosome elimination technique. The haploid embryo formation frequency through the chromosome elimination technique was significantly higher in both, triticale × wheat (20.4%) and triticale × triticale (17.0%) F₁ genotypes, as compared to the calli induction frequencies through anther culture (1.6 and 1.4%, respectively). Further, four triticale × wheat and three triticale × triticale F₁ genotypes failed to respond to anther culture, whereas, all the F₁ genotypes formed sufficient number of haploid embryos through the chromosome elimination technique with no recovery of albino plantlets. The haploid plantlet regeneration frequencies were also significantly higher through the latter technique in both triticale × wheat (42.7%) and triticale × triticale (49.4%) F₁s as compared to anther culture (8.2 and 4.0%, respectively), where the efficiency was drastically reduced by several constraints like, high genotypic specificity, low regeneration frequency and albinism. The overall success rates of obtaining doubled haploids per 100 pollinated florets/anthers cultured were also significantly higher through the chromosome elimination technique (1.1% in triticale × wheat and 1.5% in triticale × triticale hybrids), proving it to be a highly efficient and economically more viable technique of haploid induction as compared to anther culture, where the success rates were only 0.2% and 0.1%, respectively.     

Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus

Three methods of chromosome doubling to produce doubled haploid plants from microspore cultures of Brassica napus were compared: colchicine treatment of microspore-derived plants, microspore-derived embryos, and isolated microspores. In the whole plant treatment, 53% of the treated plants set seed, but the treatment delayed plant growth and reduced seed set. When microspore-derived embryos were treated with colchicine, the doubling frequency was 32% (compared to 15% for spontaneous doubling). Direct colchicine treatment of isolated microspores resulted in a doubling efficiency of 70 % of the whole plants. This treatment also stimulated embryogenesis in microspore culture, leading to increased plant regeneration. Thus, direct chromosome doubling of isolated microspores is efficient and more than 10 000 doubled haploid plants have been produced in this manner in the past three years in order to accelerate the plant-breeding process.     

The production of homozygous diploid plants of Solanum verrucosum by tissue culture techniques

Summary Monohaploid plants of S. verrucosum (2n=x=12) were induced in anther culture. Axillary buds from these plants were treated with colchicine in shoot tip culture for 48 hours and then transferred to a colchicine free medium. The resulting plantlets were scored for diploidy by stomatal chloroplast counts and root tip cytology and it was found that doubling of the chromosome number had occurred.     

Development of novel chromosome doubling strategies for wheat × maize system of wheat haploid production

Two chromosome doubling strategies were evaluated for producing wheat doubled haploids from wheat x maize crosses: (i) in vitro colchicine application to haploid embryos and (ii) colchicine treatment through postpollination tiller injections. In the in vitro approach the haploid embryos were rescued on medium containing colchicine (at concentrations of 0.2, 0.3, 0.4 and 0.5%) and moved to a colchicine‐free regeneration medium 48 h later. Embryos exposed to 0.5% colchicine had 91.67% of their regenerated plants showing chromosome doubling. In the tiller injection approach, different concentrations (0.5, 0.75 and 1.0%) of colchicine solution, which also contained 2,4‐D (100 ppm), were injected into the uppermost inter‐node of crossed tillers 48 and 72 h after pollination. The chromosome doubling efficiency varied from 33 to 100%, with 1% treatment being the most effective. No chimeras of doubled/haploid sectors were observed in the case of the tiller injection treatment and all the florets showed seed set in the doubled plants. Stomatal guard cell length provided rapid, early‐stage and unambiguous analysis of ploidy level on the basis of 10 guard cell observations per plant.