Nuclear DNA content and in vitro induced somatic polyploidization cassava (Manihot esculenta Crantz) breeding

Summary The diploid (2C) amount of DNA in cassava (Manihot esculenta Crantz) is 1.67 picograms (pg) per cell nucleus. This value corresponds to 772 mega-base pairs in the haploid genome. The size of the nuclear genome in cassava is very small in comparison with other Angiosperms. Flow cytometry techniques were used to screen ploidy levels in a large population of in vitro plantlets treated with colchicine and oryzalin (3,5-dinitro-N⁴,N-dipropylsulphate). Culture of axillary node cuttings for 48 hours in liquid medium supplemented with 2.5 to 5.0 mM colchicine in combination with 2% dimethyl sulfoxide (DMSO) resulted in a high frequency (23 to 42%) of non-chimeric tetraploids in the V₃ generation. Although mixoploidy may persist in as many as four cycles of vegetative propagation of node cuttings, solid (non-chimeric) tetraploids can be identified by flow cytometry among in vitro plantlets and then rapidly propagated for field testing. A somatic polyploidization system is proposed for implementation in cassava breeding programmes.Dedicated to the memory of the late Dr. Novak. Correspondence to M. van Duren     

Oryzalin as an Efficient Agent for Chromosome Doubling of Haploid Apple Shoots in vitro

In an outbreeding species such as apple, haploid plants may be especially useful in breeding programmes for the production of homozygous material. However, methods must be available to induce chromosome doubling in the haploid plants. Two antimitotic agents, colchicine and oryzalin, were compared as regards their efficiency in inducing chromosome doubling of in vitro haploid apple shoots. Three colchicine levels (0.025, 0.25 and 1.25 mM) and three oryzalin levels (5, 15 and 30 μM) were evaluated. Three techniques were also used and compared. Survival rate and chromosome counts were determined. Differences were observed between the two antimitotic agents and between the three techniques. This study demonstrates that oryzalin could be a better choice than colchicine for chromosome doubling on haploid apple shoots in vitro.     

Effects of Microtubule Polymerization Inhibitor on the Hypersensitive Response of Wheat Induced by the Non-Host Pathogen Sphaerotheca fuliginea

The plant cytoskeleton is a highly dynamic and versatile intracellular scaffold composed of microtubules and microfilaments, serving a multiplicity of functions in plant cells. To reveal the relationship between the cytoskeleton in wheat （Triticum aestivum L.） cv. Suwon 11 attacked by the non-host pathogen Sphaerotheca fuliginea and the initiation of the hypersensitive response, the microtubule inhibitor oryzalin was injected into the wheat leaves immediately prior to inoculation. The incidence of hypersensitive cell death was significantly lower than that in water-treated control. In addition, the occurrence of hypersensitive cell death was also delayed and S. fuliginea was able to penetrate and form haustoria in epidermal tissues of wheat. All the results above indicated that hypersensitive cell death was associated with depolymerisation of microtubules, suggesting that microtubules might play an important role in the expression of non-host resistance of wheat.     

Oryzalin离体诱导小果型西瓜四倍体及倍性鉴定研究

以3个二倍体小果型西瓜优良自交系048、032、047-1为试验材料,研究培养方式、Oryzalin浓度、处理时间对离体诱导西瓜四倍体的诱变效果,并对变异植株进行形态学比较、气孔测定及染色体鉴定。研究结果表明,采用液体培养的四倍体诱导率比固体培养高。在含50 mg/L Oryzalin的液体培养基中振荡处理2 d,四倍体诱导率最高达33.3%。变异植株通过染色体计数法鉴定得出染色体数为44,确定为四倍体植株。再生植株表现为叶片肥厚,叶色深绿,茎节缩短,株型紧凑矮小,茎蔓挺直,叶形指数变小;叶片气孔密度明显减小,保卫细胞体积增大,叶绿体数目增多;花瓣颜色深黄,且增宽、增厚。此结果说明Oryzalin对不同基因型的四倍体诱变效果存在一定差异。     

秋石斛同源四倍体诱导与鉴定

 为了创制秋石斛同源四倍体新种质, 以秋石斛同株异花授粉种子为材料, 用组织培养方法,通过种子非共生萌发与原球茎诱导, 在原球茎发育的原胚期, 以低浓度混合诱变剂进行多倍体诱导。结果用0.01%秋水仙素+ 5 mg·L ^{- 1}氨磺灵处理8～10 d, 四倍体(2n = 4X = 76) 诱变率达90%以上, 未发现嵌合体。与二倍体对照比较, 四倍体植株粗壮, 叶色深绿, 叶片宽厚。     

Chromosome doubling of Lychnis spp. by in vitro spindle toxin treatment of nodal segments

Lychnis (Caryophyllaceae) consists of about 30 species distributed throughout the temperate regions of the Northern Hemisphere, from East Asia to Europe. Many Lychnis spp. have high ornamental value and cultivated as pot or garden plants. In the present study, in vitro chromosome doubling of several Lychnis spp. was examined in order to widen their variability in horticultural traits. Initially effect of various spindle toxin treatments [100, 500 or 1000 mg l⁻¹ colchicine (COL), 10, 20 or 50 mg l⁻¹ oryzalin (ORY), or 1, 5, 10 mg l⁻¹ amiprophos-methyl (APM)] of nodal segments of a triploid genotype of L. senno (3x) was investigated on survival of nodal segments and chromosome doubling in nodal segment-derived plantlets. Significantly higher percentage (75.0%) of surviving segments after spindle toxin treatment was obtained in 10 mg l⁻¹ ORY treatment. Flow cytometry (FCM) analysis of leaf tissues showed that 9.4–13.8% of plantlets, which were derived from 10 to 20 mg l⁻¹ ORY, or 5 mg l⁻¹ APM treatments, were hexaploid (6x) or ploidy chimera (3x + 6x, 4x + 6x, 5x + 6x, 3x + 4x + 6x). The results obtained by FCM analysis were confirmed by chromosome observation in root tip cells. Thus 10 mg l⁻¹ ORY treatment of nodal segments is suitable for in vitro chromosome doubling of triploid L. senno. Efficient chromosome doubling was also achieved in diploid L. fulgens (2x) and L. sieboldii (2x) by treating nodal segments with 10 mg l⁻¹ ORY: 68.9–88.7% of nodal segments survived after ORY treatment, and 24.7–26.5% of plantlets derived from ORY-treated nodal segments were tetraploid (4x) or ploidy chimera (2x + 4x) in both species. These results indicate that the in vitro chromosome doubling method established for triploid L. senno may be applicable to a wide range of Lychnis spp. Tetraploid L. fulgens and L. sieboldii showed a compact plant form, and had thick stems and deep green leaves compared with the diploid mother plants. On the other hand, hexaploid L. senno showed very poor growth and died before flowering.     

离体诱导‘美人’梅多倍体初报

为了获得抗逆性强、观赏性好的梅花多倍体,该文以秋水仙素和氨磺乐灵为诱变剂,利用浸泡和培养基两种方法对二倍体‘美人’梅的丛生芽进行离体诱变,并采用流式细胞分析仪鉴定了形态变化植株细胞的染色体倍性。结果表明,秋水仙素和氨磺乐灵对丛生芽的生长有不同程度的抑制作用,但氨磺乐灵毒性比秋水仙素小。秋水仙素诱变丛生芽的半致死剂量为4000mg/L、72h或8000mg/L、60h,在浓度为8000mg/L溶液中浸泡48h后,继代3~5次,植株形态发生明显变化,为混倍体;氨磺乐灵诱变丛生芽的半致死剂量是28mg/L、36h或56mg/L、24h,在浓度为28mg/L溶液中浸泡96h后,继代3~5次,植株形态发生变化,为二倍体与四倍体的嵌合体。     

除草剂Oryzalin诱变西瓜四倍体及倍性鉴定

 以两个二倍体西瓜自交系为实验材料,研究除草剂Oryzalin不同滴苗浓度（30,40,50,60mg/L）和不同处理时间（1,2,4,6d）对西瓜四倍体的诱变效果,同时进行变异植株的外部形态和气孔观察及染色体鉴定。结果表明：以50mg/L浓度,2d滴苗处理诱变效果最佳,两个材料四倍体诱变率分别达到1563%和1719%。其变异植株通过染色体计数法鉴定得出染色体数为44,确定为四倍体植株,植株表现为叶片钝圆而厚,叶色深绿,叶缘多锯齿,花瓣颜色深黄,花瓣增宽、增厚;叶片气孔密度明显减小,保卫细胞体积增大。Oryzalin对不同基因型的四倍体诱变效果具有一定差异。     

不同诱变剂对大蒜四倍体诱导的影响

为探讨不同诱变剂对大蒜茎尖试管苗成苗率和四倍体率的影响,采用二次饱和-D最优设计方法,以氨磺乐灵、二甲戊乐灵和秋水仙素作为诱变剂,以大蒜品种金乡紫皮茎尖为材料,采用流式细胞仪分析和根尖压片染色体计数相结合的方法进行染色体倍性鉴定。结果表明,氨磺乐灵、二甲戊乐灵对大蒜茎尖试管苗平均成苗率和四倍体率的影响均显著高于秋水仙素。分别为62.58%和20.92%、60.95%和23.20%、44.28%和12.25%,但3种诱变剂不同处理组合对大蒜茎尖成苗率影响存在显著差异,均以低浓度处理较短时间成苗率较高,而在较高浓度长时间处理下,成苗率降低。3种诱变剂诱导大蒜四倍体率的最佳处理浓度和时间也不同,且以氨磺乐灵、二甲戊乐灵诱变处理显著优于秋水仙素。经模拟运算,三者诱变大蒜茎尖的最佳处理浓度和时间分别为4.17~8.52 mg·L-1和71.14~118.03 h、4.68~8.90 mg·L-1和72.96~106.18 h、3.87~6.94 g·L~(-1)和67.92~114.43 h。本试验初步明确了3种诱变剂对大蒜茎尖试管苗四倍体的诱变效果,为进一步开展大蒜四倍体新品种选育奠定了基础。     

Microspore culture of white cabbage, Brassica oleracea var. capitata L.: Genetic improvement of non-responsive cultivars and effect of genome doubling agents

For the efficient application of haploid induction procedures in cabbage breeding, a sufficient number of regenerants should be achieved in a broad spectrum of genotypes. However, the majority of genotypes are somewhat recalcitrant. The efficiency of microspore culture was tested by crossing a responsive (28.7 embryos per Petri dish) and a non- responsive (0.1 embryo) cabbage cultivar. The embryo yield of one progeny was intermediate (18.9) while two were superior to the best parent cultivar (52.9 and 64.0 embryos). Thus, genes for haploid embryogenesis, present in responsive lines, can be effectively transmitted to responsive × non-responsive hybrids. Abscisic acid-induced desiccation of embryos was used for the efficient regeneration of plants. High germination percentages (54.7-70.6%) followed by normal plantlet development were achieved. Spontaneous genome doubling measured at the plantlet stage differed markedly in untreated genotypes. The percentage of diploids ranged from 21 to 67%. The effects of two antimitotic drugs applied to freshly isolated microspores were determined in two experiments. In the first experiment, trifluralin (0.5 and 1.0 mg:l) had no effect on embryo induction while oryzalin partly (0.125-0.25mg/l) or completely (0.5.mg/l) inhibited the formation of embryos. In the second experiment, higher concentrations of trifluralin increased the proportion of diploidized plants. Application of anti-mitotic drugs to microspores did generally not improve the overall production of haploid plants, which was higher in an untreated control.