秋水仙素诱导黄芩多倍体的初步研究

以浓度为0.01%,0.05%,0.1%,0.2%,0.5%的秋水仙素浸泡黄芩种子24、48、72 h和96 h,统计种子发芽率;此外,在黄芩愈伤组织诱导培养基中分别加入浓度为5、10 mg/L和15mg/L的秋水仙素,进行黄芩多倍体的诱导.种子发芽和诱导愈伤形成芽后,取二者幼芽分别固定和制片观察,统计秋水仙素诱导黄芩产生的多倍体细胞率.结果表明:0.01%浓度的秋水仙素处理(24、48、72 h)黄芩种子,随着时间的延长,种子发芽率提高;当浓度高于0.05%时,种子发芽率降低,但规律不明显.染色体压片观察表明,以浓度0.1%秋水仙素浸泡处理黄芩种子24h的多倍体细胞诱导率最高,可选49.2%;培养基中添加秋水仙素能够明显促进愈伤组织和芽的形成,但多倍体细胞诱导率仅为9.7%.以上结果表明,种子浸泡或培养基添加秋水仙素均能诱导黄芩根尖或愈伤产生多倍体细胞,但要获得纯合的四倍体或多倍体植株,还有待进一步深入研究.     

秋水仙素离体诱导大花蕙兰多倍体试验

在离体培养条件下,以大花蕙兰红宝石原球茎为材料,比较了秋水仙素浸泡法和混培法在不同浓度、不同处理时间诱导加倍的效果。结果表明:无论是用秋水仙素溶液浸泡还是将秋水仙素加入培养基中混培,均可诱导大花蕙兰多倍体的产生。但以混培法效果较好,在秋水仙素3%、处理15d的条件下,诱导率最高达30%。     

‘乐都紫皮大蒜’多倍体的诱导与鉴定

‘乐都紫皮大蒜’因其风味独特并具有高含量的碳水化合物,市场价值和开发利用前景广阔。利用多倍体诱导技术,可促进优良大蒜种质的创制。本研究通过秋水仙素浸泡‘乐都紫皮大蒜’的愈伤组织进行四倍体诱导,并利用流式细胞仪及农艺性状测定等方法进行鉴定。结果表明:在含有0.5 mg/L6-BA+1.5 mg/L2,4-D的B5培养基上培养大蒜愈伤组织的诱导率最高,愈伤组织长势最佳。以0.2%的秋水仙素浸渍4 h,愈伤组织的四倍体细胞诱导率最高,达39.42%。在此条件下,处理愈伤组织后诱导再生试管苗,愈伤组织存活率为54.44%,多倍体再生植株的诱导率为28.89%。与二倍体植株相比,四倍体试管苗的株高、叶长、叶宽、假茎长显著增加了183.44%、181.18%、133.33%和58.33%。本研究为培育大蒜新种质提供了技术方法与科学依据。     

落叶松多倍体体细胞胚的高效诱导途径

以日本落叶松×华北落叶松杂种无性系胚性细胞系Y68为材料,通过含有不同浓度的秋水仙素和氨磺灵水溶液、固体培养基和液体培养基进行处理,以体细胞胚分化能力和多倍体体细胞胚获得率为参数进行了对比研究.结果表明:秋水仙素水溶液浸泡法处理对落叶松多倍体体细胞胚诱导效果最显著;先对细胞系处理再进行体细胞胚分化较适合落叶松多倍体体细胞胚的发生.在500 mg/L的秋水仙素水溶液中浸泡36 h情况下多倍体诱导率最高,达79.8%,体细胞胚分化率为217.1个/g.     

多倍体大蒜胚状体及再生幼苗诱导研究

用秋水仙素诱导产生大蒜多倍体根尖,将其接种至诱导愈伤组织培养基中,诱导得到愈伤组织。将愈伤组织在继代培养基和MS培养基中继代培养,得到大小适宜且结构均为致密型的愈伤组织。将致密型愈伤组织移至细胞分化培养基和1/2 MS培养基中培养,得到有伸长的根状物并具有芽点的胚状体,1/2 MS培养基诱导胚状体的效果更好。最后将长出嫩叶的幼苗转入不同比例NAA和6-BA的1/2 MS培养基中,发现NAA浓度为0.5 mg/L,6-BA浓度为1、2、2.5mg/L时生长状况较好,作为对照组不加激素的1/2 MS培养基个别植株生长旺盛。     

黄精多倍体诱导初报

通过人工诱变,使多花黄精染色体加倍,提高生物产量与品质,为大面积人工种植提供优良品种.种子经过人工打破休眠后,在MS+6-BA 1.0mg/L+2,4-D 0.5mg/L离体培养形成愈伤组织,再转入MS+TDZ 1.5mg/L+2,4-D1.0mg/L培养15～20d,用脱脂棉球浸泡于添加2%二甲基亚砜的0.05%～0.15%秋水仙素溶液中,在无菌条件下覆盖在愈伤组织上,进行染色体加倍诱导处理,再转移到MS+6-BA 1.0mg/L+NAA 2.0mg/L的分化培养基上,将分化出叶片的小苗转移到1/2MS+NAA1.0mg/L生根培养基上.结果表明:0.05%质量浓度的秋水仙素处理48h变异株诱导率最高达18.7%.0.1%和0.15%浓度处理24h的诱导率次之,为16.7%,0.1%浓度处理48h变异率虽然只有16.2%,但其变异株倍性稳定性较好,整倍率较高.     

影响组织培养诱导四倍体小果型西瓜三种因素的研究

以二倍体小果型西瓜自交系21，90，141，149,157的授粉后20～25d的未成熟种子中的子叶为材料，进行离体组织培养诱变四倍体，研究了不同的秋水仙素浓度、处理时间对组织再生，不同的秋水仙素浓度、处理时间与再生途径对加倍率的影响。结果表明，未成熟子叶组织再生有产生单芽、芽丛、愈伤组织3种方式；不经秋水仙素诱导处理，直接进行组织再生时，直接再生不定芽能力强；经秋水仙素诱导处理后，进行组织再生时不定芽产生受到抑制，愈伤组织发生数多于单芽或芽丛数；基因型不同，处理的秋水仙素浓度与时间不同，再生的组织类型与数量不同；还发现经一定时间的适宜浓度的秋水仙素处理后，有利于产生的愈伤组织分化成苗；对再生植株鉴定可知，0．03％秋水仙素处理9d或0．05％秋水仙素处理7d可诱导出较高比例的四倍体植株，并且通过愈伤组织途经的加倍率高于通过单芽和芽丛途经。     

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。     

豆科牧草沙打旺抗乙硫氨酸变异系筛选

以NaN3诱变处理的沙打旺胚性愈伤组织为材料,用甲硫氨酸类似物—乙硫 氨酸为选择剂,筛选到1株抗性稳定且能再生植株的抗0.6 mmol L－1乙硫氨酸 变异系。该变异系细胞对乙硫氨酸的抗性是野生型细胞的8倍。当把变异系愈伤组织传入 体细胞 胚胎诱导培养基上,平均体细胞胚胎发生频率为13.9%,平均每g鲜重愈伤组织可产生21 个体     

不同预处理对白菜型油菜花药愈伤组织诱导的影响

本试验以白菜型春油菜品种——‘天祝小油菜’为试验材料,研究不同花蕾部位、不同诱导培养基及不同低温等预处理时间对花药愈伤组织诱导率的影响。无预处理时,筛选不同花蕾部位接种到不同诱导培养基的最佳组合,结果表明：以主花序蕾盘中盘青绿色、长度2～3 mm的花药为接种材料,在添加1.5 mg/L2,4-D、1 mg/L KT的B5培养基上培养,其花药愈伤组织诱导率最高为27.26%,均高于内盘、外盘花蕾花药与其他诱导培养基组合诱导率。预处理结果表明：4℃预处理4 d对花药愈伤组织诱导最有效,诱导率为60.27%,显著高于同期对照1.21倍;0.2 mg/L甘露醇培养4 d对花药愈伤组织诱导率为48.07%,显著高于对照和其他甘露醇溶液处理。花蕾在0.1 mol/L蔗糖溶液浸泡处理30 min后,花药愈伤组织诱导率为54.8%,显著高于对照和其他蔗糖溶液处理。综上以白菜型油菜主花序蕾盘、中盘青绿色,长度2～3 mm的花药为接种材料,4℃预处理4 d后接种在添加1.5 mg/L 2,4-D、1 mg/L KT的B5培养基上愈伤组织诱导效果最好。     

人工诱变芦荟多倍体的初步研究

以秋水仙索为诱变剂，利用茎段浸泡和秋水仙素培养基2种方法对库拉索芦荟进行诱变，结果表明，茎段浸泡秋水仙素法诱导四倍体葡萄的效果优于秋水仙素培养基法，0．2％的秋水仙素处理4d变异效果最好，诱变率达到最高值41.5％。变异株叶片肥厚，叶色深绿，叶片表皮气孔保卫细胞中叶绿体数增多。     

Induction of <Emphasis Type="Italic">Anthurium andraeanum</Emphasis> “Arizona” tetraploid by colchicine in vitro

Tetraploids plants of Anthurium andraeanum “Arizona” were successfully induced after treating diploid tissue masses with colchicine. Masses originating from diploid aerial roots were treated with colchicine at three different concentrations (i.e., 0.1, 0.2, 0.3%) for about 3, 5 and 7 h, and then were transferred into Murashige and Skoog medium containing 3 mg/l BAP + 0.2 mg/l 2,4-D. After 60 days, the survival rate and numbers of regenerative shoots were scored. The high concentration and longer duration sharply reduced survival rate. In contrast, the regeneration of plantlets was not noticeably affected by colchicine. Tetraploid plants were obtained in all treatments, but the percentage of induced tetraploids ranged from 0.2 to 7.6%. The best induction was obtained with a 5-h, treatment with 0.3% colchicine. The stomatal size of tetraploid plants was larger than in diploid plants; however, the stomatal density was lower than in diploid plants. Tetraploid plants possessed stronger petioles, thicker and deeper green leaves, and thicker and longer lived spathes in comparison with diploid plants. Abnormal spathes, such as double spathes or those lacking pedicels, were observed in tetraploid plants. Tetraploid plantlets could be regenerated via aerial roots; this technique could be applied to tetraploid plant propagation.     

Chromosome doubling of Allium fistulosum × A. cepa interspecific F1 hybrids through colchicine treatment of regenerating callus

Regenerating calluses of Allium fistulosum × A. cepa interspecific F₁ hybrids were treated in vitro with colchicine. A factorial experiment was designed to test the effects of colchicine concentration and time on the recovery of tetraploid plants from in vitro-colchicine-treated calluses. Shoot production of regenerating calluses following in vitro colchicine treatment decreased with increasing colchicine concentration and treatment time. Cytological analyses of root tip cells from regenerated plantlets showed that chromosomes of control plantlets (not treated with colchicine) were not doubled. Chromosome number of some plantlets regenerated from in vitro-colchicine-treated calluses were doubled, resulting in tetraploids. Calluses treated with 0.1 or 0.2% colchicine in BDS liquid medium for 48 or 72 hours yielded the highest numbers of tetraploid plantlets. Chromosome bridges at anaphase or early telophase were observed in diploid and tetraploid plants; their potential use is discussed. These results demonstrate that in vitro-colchicine treatment of regenerating calluses of interspecific F₁ hybrids is effective in recovering tetraploid plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro production of autotetraploid Ponkan mandarin (<Emphasis Type="Italic">Citrus reticulata</Emphasis> Blanco) using cell suspension cultures

An efficient protocol for colchicine mediated production of in vitro autotetetraploids from Ponkan mandarin using cell suspension cultures is described. Cells were treated with 1 g l⁻¹ colchicine for 4 or 8 days before transfer into solid EME medium supplemented with 5% maltose. Colchicine treated cells were placed in medium with or without an overlay of 1:2 medium–mixture of liquid 0.6 M BH3 medium and 0.15 M EME + maltose liquid media. It was observed that modifying the immediate cell environment by addition of the liquid overlay played a positive role in cell differentiation and subsequent plant regeneration. Ploidy levels were determined with a flow cytometer and confirmed by chromosome staining using the enzymatic maceration method. A large number of non-chimeric autotetraploids were generated using this method. Such plants have great value in a breeding program for the development of seedless triploid citrus, as very few available tetraploid breeding parents are easy to peel.     

In vitro induction of tetraploids in Phlox subulata L.

Tetraploid plants of Phlox subulata L. were induced successfully by treating shoot tips in vitro with colchicine. Shoot tips excised from in vitro shoots were treated with four different concentrations of colchicine (0.005, 0.01, 0.02, 0.04%) in solid MS medium supplemented with 4.54 μM TDZ and 0.49 μM IBA for 10, 20 or 30 days, respectively. The survival rates of shoots tips were affected by the concentration of colchicine and the duration of treatment. High concentration and longer duration reduced survival of the shoot tips, but the effect of duration of colchicine was more than that of concentration. Tetraploid plants were obtained in all of the treatments, but the percentages of tetraploids varied among different treatments, from 25.0% to 75.0%. The most efficient condition for inducing tetraploids was to treat shoot tips with 0.005% colchicine for 20 days, with 30.0% survival rate of shoot tips and 6 tetraploid plants out of 10 plants examined. The rooted tetraploid plants were transplanted successfully in a solar greenhouse. Under the same growing condition, significant varieties in flower bud and flower sizes were detected between 2x and 4x plants. The flower diameters of tetraploid and diploid plants were 2.91 cm and 2.24 cm, respectively.     

催眠睡茄的离体快繁及四倍体的诱导

以催眠睡茄腋芽为外植体建立快繁体系,利用秋水仙素进行催眠睡茄四倍体诱导,并将其与二倍体进行了初步的形态学和细胞学方面的比较.结果发现:催眠睡茄二倍体(2n=2x=48)最适增殖培养基为MS 6-BA 0.5 mg/L NAA 0.05 mg/L,生根培养基为1/2 MS IBA 0.2 mg/L;四倍体(2n=4x=96)诱导率因秋水仙素浓度和处理时间的不同而异,其中用0.5%的秋水仙素对丛生芽浸泡处理48 h诱导率最高,诱导率可达13.79%.四倍体与二倍体相比:叶片明显变厚,颜色加深,叶片气孔及其保卫细胞大小增大,气孔密度减小,且保卫细胞内叶绿体数目增加.     

Spontaneous induction of tetraploidy in hop using adventitious shoot regeneration method

In the process of breeding triploid hop cultivars, diploid varieties need to be raised to the tetraploid level. Existing methods are predominantly based on treatment of the apices with antimitotic substances, in vivo or in vitro , resulting in a reported maximum of 26% tetraploids and a large proportion (over 60%) of mixoploids. This study was set-up to test whether an adventitious shoot regeneration protocol can be used as an alternative. In the first part of the experiments, regeneration media and a choice of explants (internodes, petioles and leaf discs) were optimized using three hop cultivars with putatively low, medium or high adventitious shoot regeneration capacity. On an optimized induction medium supplemented with 6 mg/l N ⁶-(2-isopentenyl) adenine (2iP), a regeneration rate up to 17.7% was obtained from cultured internodes, even of the most recalcitrant 'Aurora', while the regeneration rate of the most responsive 'Tettnanger' was 56.9%. Flow cytometric analysis of ploidy level revealed a high frequency of tetraploid induction, the highest (58.6%) being in 'Savinjski golding'. In 616 regenerants of all three cultivars, 30.4% were found to be tetraploid, the others were diploid. Cytokinin content was found to be of minor importance in tetraploid induction. The high frequency of spontaneous tetraploid induction and, in particular, the complete absence of mixoploids makes this protocol an alternative to the currently established methods of chromosome doubling based on the use of antimitotic substances. The occurrence of a high proportion of tetraploid adventitious regenerants in hop is discussed in view of certain other biotechnological applications.     

Improved techniques for the induction and isolation of polyploids in the genus Fragaria

Summary The sensitivity of Fragaria seedlings to colchicine is dependent on the plant organ that is treated. Complete immersion of seedlings in a 1.5% colchicine solution results in total lethality, whereas the survival rates were more than 75% even at concentrations of 3.0% when only shoot apices were treated. High proportions of polyploids were isolated by treating shoot apices of seedlings with a 2.0% colchicine solution for 24–28 h. The dropper method is preferred to the tube method as it involves a minimum of manipulation and requires simple equipment. A differential response to colchicine was observed within and between different diploid species, diploid and tetraploid hybrids.     

墨兰多倍体的离体诱导和鉴定

以墨兰‘企剑白墨’的根状茎为材料,研究了秋水仙素处理对墨兰根状茎生长和多倍体诱导的影响。结果表明,秋水仙素对根状茎有严重的毒害致死作用,在试验浓度和时间范围内,随着秋水仙素浓度的升高或处理时间的延长,根状茎存活率明显降低。秋水仙素能诱导‘企剑白墨’根状茎的染色体加倍,但不同浓度和时间处理的染色体加倍效率不一样。只在0.01%×3 d处理下诱导出四倍体,诱导率为11.11%,其余处理诱导出二、四混倍体,诱导率在0～19.44%之间。四倍体根状茎比二倍体粗壮,顶端更圆,再生出的四倍体植株株型紧凑,叶片质地变硬,根明显增粗。