In situ induction of chromosome doubling in vine cacti (Cactaceae)

Vine cacti have economic potential as exotic fruits in semi-arid and arid lands due to their high water use efficiency. The goal of this study was to attain autopolyploid plants by applying the antimitotic agents colchicine and oryzalin on axillary vegetative buds and germinating seeds. The diploid Hylocereus monacanthus and the tetraploid H. megalanthus; the interspecific triploid hybrid S-75, were studied. The effects of different concentrations and exposure times on bud survival and germination rate were recorded. A negative effect on vegetative bud survival was observed in the triploid hybrid. An inhibitory effect on germinating seeds was species specific, being greater on H. monacanthus with oryzalin and on H. megalanthus with colchicine. Putative polyploids were identified by comparing stomatal density between donor plants and plants arising from treated buds or seeds. Flow cytometric analysis and chromosome counts confirmed polyploidization in 14 autotetraploid H. monacanthus, 1 autohexaploid S-75 hybrid, and 14 autooctaploid H. megalanthus. This is the first report of artificial autopolyploidization in species of vine cactus, providing valuable novel plant material for further breeding programs.     

Tetraploid muskmelon alters morphological characteristics and improves fruit quality

Tetraploid muskmelon plants were induced successfully from diploid Cucumis melo inbred M01-3 (2n = 24) by colchicine. The morphological characteristics and fruit qualities of diploid and tetraploid muskmelons were investigated. The results showed that the leaves and flowers of the tetraploid plants were markedly larger, the plants were obviously higher, and the stems were thicker than those of the diploid plants. Transmission electron microscope observation revealed that the numbers of chloroplast, granule and grana, and the length of chloroplast and granule of the tetraploid plant leaves were significantly more or longer than those of the diploid plants. The soluble solid, soluble sugar and vitamin C contents in the tetraploid fruit were distinctly higher than those in the diploid fruit. The weight of the tetraploid fruit was 30% heavier than that of the diploid fruit. And the seeds from the tetraploid plants were broader and thicker than those from the diploid plants. Together, our results suggest that the tetraploid muskmelon exhibited better agronomical characteristics than the diploid muskmelon and the tetraploid muskmelon could be used as the improved variety and a potential germplasm for the development of triploid fruit.     

Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.)

The tips of cotyledon-stage seedlings of three crape myrtle cultivars (“Zi Wei”, “Hong Wei” and “Yin Wei”) were treated with colchicine. Various concentrations of colchicine and different treatment durations were tested. Seedlings of “Zi Wei” treated with 0.5% colchicine for 72 h and seedlings of “Yin Wei” treated with colchicine (0.2% for 96 h, 0.5% for 48 h and 0.8% for 72 h) demonstrated high rates of mutation; “Hong Wei” showed a slightly lower rate. The highest rate of morphological variation was 54.17% and this was achieved when tips were treated with 0.5% colchicine for 72 h. Putative tetraploid plants were identified with morphological and cytological variations, such as larger and thicker leaves, darker green coloration, larger stomata, lower density of stomata across the lower leaf epidermis and increased numbers of chloroplasts per stomata guard cell. Flow cytometric analysis demonstrated that the nuclear DNA content of 15 of these putative tetraploids (of which 7 were “Zi Wei”, 5 were “Yin Wei”, and 3 were “Hong Wei”) was indeed doubled relative to that of control diploid plants. The chromosome number of the tetraploid plants was 2n = 4x = 96, while that of the control diploid plants was 2n = 2x = 48. Ornamental characteristics were also enhanced in the tetraploid plants, with increased diameter of the individual flowers and greater basal length of the petals and claws. Pollen diameter and the size of capsules and seeds were also significantly greater than those of diploid plants.     

In vitro polyploidization of Mecardonia tenella, a native plant from South America

Mecardonia tenella is an herbaceous plant widely distributed in the temperate region of South America. Both plant architecture and flower size are characteristics that can be improved to become a viable new ornamental plant. Chromosome doubling by the use of agents such as colchicine is an available methodology to this end. Nodal segments from in vitro grown plants of M. tenella were submerged in the following doses of colchicine in 1% (v/v) dimethyl-sulfoxide (DMSO) solution (%, v/v): 0.0, 0.001 and 0.01 (24 and 48 h). The DNA content of the regenerated plants was measured by flow cytometer. A total of 68 tetraploid plants were detected out of 126 colchicine treated plants. The flowers and leaves of the tetraploid plants were bigger compared to those from the wild diploid type (control). Under field conditions, the selected tetraploid plants showed a more compact shape than the control plants.     

‘月月粉’连续开花习性遗传规律分析

连续开花(Perpetual blooming,PB)是观赏植物的重要性状之一,是中国月季对现代月季的重大贡献。然而月季连续开花性状的遗传基础至今尚未清晰。为研究中国古老月季品种‘月月粉'(Rosa chinensis‘Old Blush',用OB表示)连续开花习性的遗传规律,以其为母本,以一季开花(Single seasonal blooming,SB)园艺品种‘无刺光叶蔷薇'(Rosa wichuriana'Basye's Thornless',用W表示)为父本,构建了OB、W、F_1、F_2、BC_1OB和BC_1W等6个世代827个株系的二倍体遗传分离群体。遗传分离群体表型测定将开花表型分为连续开花和非连续开花(Non-perpetual blooming)2种类型。296株F_1和150株BC_1W群体均表现为非连续开花,表明OB连续开花性状受隐性基因位点调控。300株BC_1OB群体中83株为连续开花,223株为非连续开花,χ~2适合性测验符合1:3的遗传分离规律(P0.05),表现为双隐性基因遗传,表明‘月月粉'连续开花可能受双隐性基因位点共同调控。     

君子兰未成熟胚四倍体诱导及染色体数鉴定

 以君子兰（Clivia miniata Regel.）品种‘胜利’为试材,采用一种新型外植体未成熟胚,利用秋水仙素与组织培养相结合的方法,研究秋水仙素浓度、处理时间和不同胚龄对未成熟胚诱导加倍的影响。结果表明：在MS + NAA 2.0 mg · L-1 + BA 1.5 mg · L-1培养基上,0.01%秋水仙素处理180 d胚龄的未成熟胚20 d诱变效果最佳,四倍体诱变率最高,达30.8%,形态变化率达36.5%,死亡率为34.1%。经0.03%秋水仙素处理120 d胚龄的未成熟胚30 d诱变效果也较好,四倍体诱变率较高,达24.0%,形态变化率达21.1%,死亡率为46.7%。经染色体数观察,加倍处理后代中存在2x、4x和混倍体植株。随机调查了267株加倍处理植株,得到30株四倍体（2n = 4x = 44）、194株二倍体（2n = 2x = 22）和43株混倍体,平均四倍体诱变率为11.2%。秋水仙素处理出现的植株生长缓慢,生根明显较晚,叶片变宽变短等形态变化可以作为加倍的初期有效判断指标。     

Influence of the antimitotic agents colchicine and oryzalin on in vitro regeneration and chromosome doubling of diploid bananas (Musa spp.)

Summary

In vitro cultures of the four diploid banana cultivars, Sannachenkadali (AA), Anaikomban (AA), Kunnan (AB) and Thattillakunnan (AB) were treated with two antimitotic agents, colchicine (C₂₂H₂₅NO₆) and oryzalin (3,5-dinitro-N4,N4-dipropylsulfanilamide) to induce ploidy alterations, particularly the induction of tetraploids, which would serve as useful breeding material for creating newer, improved triploids. Both antimitotic agents, but particularly colchicine, had a negative effect on the in vitro regeneration of the four cultivars. This was reflected in terms of delay in regeneration, reduced multiple shoot regeneration rates, regeneration of smaller microshoots with lower fresh weights, and reduced response to rhizogenesis. Colchicine, as expected, had a negative effect on the number of multiple shoots regenerated. However, oryzalin at lower concentrations (10 mM and 20 mM) resulted in the regeneration of more microshoots per culture than the untreated control. The chromosome doubling capacity of colchicine was equal to that of oryzalin only at 125–200 times higher concentrations. The cultivars exhibited a genome-related response.     

Cross- and self-compatibility in various species of the genus Rosa

Summary

Self- and cross-compatibility was evaluated under field conditions in 48 taxa belonging to all sections of subgenus Rosa and subgenus Platyrhodon in the genus Rosa. Four species exhibited fruit set through self-pollination out of the 28 diploid species tested. In contrast, nine out of 12 tetraploid species, all of pentaploid species, and four out of five hexaploid species exhibited fruit set through self-pollination. Thus, it may be generally said that the self-incompatibility in the genus Rosa breaks down with polyploidization. The relationship between self-incompatibility and ploidy level is discussed.     

秋水仙素诱导象鼻兰种子产生多倍体

以象鼻兰种子为试验材料,采用浸泡法,研究了秋水仙素处理对蝴蝶兰种子染色体加倍的影响,以期获得诱导多倍体的适宜条件。结果表明:秋水仙素浸泡法诱导种子染色体加倍时,种子在液体培养基中培养5d,再采用0.20%秋水仙素处理1d的处理效果最好,萌发率达到6.41%,高于1.96%的自然萌发率,四倍体、嵌合体诱导率分别达到了27.75%、34.84%;四倍体植株与二倍体植株相比,形态与气孔特征均发生了明显变化,形态特征表现为,植株粗壮、根系发达且粗壮、植株生长较快等;气孔特征表现为单位面积气孔数减少,气孔增大,长宽分别增长25.19%和26.19%。该研究得到了秋水仙素浸泡象鼻兰种子诱导染色体加倍的适宜条件且提高了象鼻兰种子的萌发率,获得了四倍体植株和嵌合体植株,为把象鼻兰优异基因高效导入栽培品种奠定了基础,为蝴蝶兰体细胞无性系变异新品种的培育提供了参考依据。     

抗菌肽MB39基因导入‘皇家嘎啦’苹果及其四倍体植株的培育

 采用农杆菌介导法, 将抗菌肽MB₃₉基因转入‘皇家嘎啦’苹果, 获得了7 个转基因株系。通过秋水仙素诱变染色体组工程育种技术, 由转化体叶片获得了20 个四倍体植株。经PCR 检测和Southern Blotting 杂交证明, 抗菌肽MB₃₉基因已经整合到皇家嘎啦苹果的染色体组中。转基因株系TR21、TR23 提高了对火疫病的抗性。采用流式细胞技术( Flow Cytometry) 确定植株为四倍体。     

Somaclonal variation in Tricyrtis hirta plants regenerated from 1-year-old embryogenic callus cultures

The Liliaceous perennial Tricyrtis hirta, sometimes called ‘Japanese toad lily’, has recently become popular as an ornamental for pot and garden uses. Highly embryogenic callus cultures of this plant predominately consisted of diploid cells but also contained tetraploid cells after 1 year of establishment. In the present study, plans regenerated from the 1-year-old embryogenic callus cultures were subjected to ploidy level analysis and morphological characterization following 3 years of cultivation. Among 37 plants examined, 28 kept the diploid level (2n = 2x = 26) but nine were tetraploid (2n = 4x = 52) as indicated by FCM analysis and chromosome observation. Although no morphological alterations were detected in 26 out of 28 diploid regenerants, the remaining two showed noticeable variations: both were severely dwarf and had crimped leaves and many malformed flowers. The tetraploid regenerants had several horticulturally attractive characteristics compared with the diploid controls, such as longer shoots, thicker stems, and larger flowers. Thus regeneration of tetraploid plants from 1-year-old embryogenic callus cultures offers a possibility to improve the horticultural value of T. hirta, although regeneration of trueness-to-type plants is essential for utilizing the cultures for micropropagation and genetic transformation.     

Germination in Rosa

It has been confirmed that immaturity in the embryo is not the cause of dormancy in Rosa; nor do the testa and pericarp in R. arvensis prevent access of water. In addition to the mechanical restriction which is imposed on the expansion of the embryo by the massive pericarp, the achene contains growth and germination inhibitors. The effect, on the germination of embryos, of these non-toxic substances extracted from achenes of R. rugosa and R. canina has been examined.

Dormancy in the seeds of R. arvensis maybe largely broken by 6-benzyl-aminopurine or gibberellic acid, and one possible function of the inhibitors may be so to affect the balance between endogenous growth promoters and inhibitors that germination is prevented until the protective pericarp has been weakened by decay.     

In vitro induced tetraploid of Dendranthema nankingense (Nakai) Tzvel. shows an improved level of abiotic stress tolerance

The tetraploid of Dendranthema nankingense (Nakai) Tzvel. was induced by the colchicine treatment using nodal segments. Ploidy level was determined by an analysis of flow cytometry and chromosome counting. The morphological characteristics such as the stomata, leaves, flowers and pollen grains of the tetraploid were significantly larger than those of the diploid. The tolerance responses of the diploid and tetraploid were compared under the imposition of heat, cold, drought and salinity stress. Semi-lethal temperatures suggest that cold tolerance is improved by polyploidization, but the heat tolerance is reduced. Under drought and salt stress, the activity of peroxidase (POD) and relative water content (RWC) in the tetraploid were higher than those in the diploid. Accordingly its malondialdehyde (MDA) content maintained a lower level. The content of chlorophyll (a + b) in the tetraploid was higher, and decrease of its content was postponed in tetraploid compared with the diploid under salt stress. It suggested that polyploidization could alleviate oxidative stress, maintain good water balance and higher chlorophyll (a + b) content, thereby enhanced the drought and salt tolerance in colchicine induced tetraploid.     

Induction of tetraploidy in meristematically active seeds of Japanese barberry (Berberis thunbergii var. atropurpurea) through exposure to colchicine and oryzalin

Japanese barberry (Berberis thunbergii DC) is an invasive shrub, widely naturalized across the United States, whose numerous cultivars remain an important horticultural commodity. Maintaining this crop for the future necessitates the development of sterile clones. Exposure to the mitotic inhibitors colchicine and oryzalin is a traditional method for inducing tetraploidy in breeding lines as a precursor to creating sterile genotypes. Treatments utilized pre-germinated B. t. var. atropurpurea seeds with emerged radicles. Seeds were immersed in aqueous solutions of colchicine (.02%, .05%, .1% and .2%) and oryzalin (.002%, .005%, .01% and .02%) dissolved in 1% DMSO for 6, 12 and 24 h durations. Seedling ploidy level was determined via flow cytometry following 6 and 52 weeks of growth in the greenhouse. Both anti-mitotic chemicals proved effective at inducing tetraploidy and produced comparable efficiency rates. The survival rate of treated seeds decreased in response to both increased mitotic inhibitor concentration and longer exposure duration. While exposure to oryzalin produced greater seed mortality than colchicine, most seedlings that survived had altered ploidy levels. The most efficient oryzalin concentration was 0.002% with a rating of 28%, while the most efficient colchicine concentrations were in the range from 0.05% to 0.2%. Duration of exposure to mitotic inhibitor was not a significant factor over the range from 6 to 24 h. Reversion of tetraploid plants to the diploid state occurred at a low frequency following a dormancy period. Some tetraploid seedlings derived from exposure to both chemicals displayed foliar abnormalities including irregular leaf margins and mottled lamina. The primary advantage of colchicine was low seedling toxicity, while oryzalin was notable for its ability to induce tetraploidy at low concentrations.     

秋水仙素诱导西瓜多倍体的研究

研究了不同浓度秋水仙素处理对3个西瓜品系幼苗染色体加倍的诱导效应,通过形态观察、染色体计数以及流式细胞仪等方法进行了倍性鉴定。结果表明,秋水仙素诱导3个西瓜品系均得到了四倍体,0.3%的秋水仙素处理幼苗的变异率较高,其中以0.3%秋水仙素处理黄小玉母本的变异率最高,达15.1%。四倍体表现出叶片长、宽、厚均增大,花瓣大小和果皮厚度均较二倍体明显增加。根尖染色体压片检查表明,四倍体染色体数为2n=4x=44,二倍体对照为2n=2x=22。流式细胞仪检测结果表明,诱导不仅产生四倍体,还有嵌合体。     

DNA ploidy level of colchicine-treated hops (Humulus lupulus L.)

The genome doubling agent colchicine has been used effectively to obtain tetraploid plants when starting with diploid material. Since the plant meristem consists of many cells, as well as obtaining tetraploids, it is possible to obtain mixoploids (chimeras consisting of diploid and tetraploid tissue) after colchicine treatment. Flow cytometry was used to determine the DNA ploidy level of different tissues of individual hop plants following colchicine treatment of diploid hops. Plants that are shown to be mixoploid after analysis of leaf tissue can display higher levels of tetraploid nuclei in root tissue, such that they could be mistakenly classified as tetraploid. The hop variety Galena-4n, which was previously considered to be a tetraploid, is shown to be a mixoploid based on leaf material. However, Galena-4n consistently produces triploid progeny when crossed with a diploid hop variety, indicating that Galena-4n produces tetraploid reproductive tissues and is effectively a tetraploid from a breeding perspective. The results from this study indicate that care should be taken when determining the DNA ploidy level of an individual plant after exposure to genome-doubling agents based on single tissues and that leaf tissue is a choice material for flow cytometric analysis.     

Acquisition of triploid germplasms by controlled hybridisation between diploid and tetraploid in Chinese jujube

‘Chenguang’ is the first released tetraploid cultivar (2n = 4x = 48) of Chinese jujube (Ziziphus jujuba Mill.) induced by colchicine. The purpose of this study was to create triploid germplasms by using ‘Chenguang’ as a parent. The controlled crosses between autotetraploid ‘Chenguang’ and diploid ‘Dongzao’ were carried out from 2013 to 2015 by maintaining the 2 parents in a nylon net and employing bees as pollinator. A total of 606 fruits were produced, yet only 17 seeds were obtained from ‘Dongzao’ and none from ‘Chenguang’. Direct sowing of the seeds in greenhouse showed a higher seedling rate than embryo culture. Four progeny plants were eventually obtained in the greenhouse and all of them were confirmed as authentic hybrids by SSR marker technique. At last, 3 triploids and 1 diploid were identified by flow cytometric analysis. Consequently, tetraploid ‘Chenguang’ with high pollen vitality and pollen number per anther could be used as a reliable 2n pollen provider, and the 2x × 4x cross combination type is more suitable for triploid creation in Chinese jujube. This is the first successful production of triploid hybrids by controlled cross between diploids and tetraploids in jujube.     

四倍体黑芥的获得与鉴定

以黑芥为材料进行了同源多倍体的诱导和鉴定研究。结果表明,用0.1%秋水仙素药液处理黑芥籽苗生长点24 ~ 72 h均可诱导获得多倍体植株,其中处理48 h的诱导效果最好,四倍体株率达73.33%。四倍体黑芥长势良好,叶片和花变大,叶柄变短,叶色和花色变深,开花期较二倍体晚2 ~ 3 d。四倍体黑芥能正常开花散粉,但结籽率较低,仅为13.83%。     

In vitro colchicine-induced polyploid plantlet production and regeneration from leaf explants of the diploid pear (Pyrus communis L.) cultivar, ‘Fertility’

Summary

Polyploid plantlets, including triploid, tetraploid, and mixoploid, were induced from the European pear (Pyrus communis L.) cultivar ‘Fertility’ by in vitro colchicine treatment of leaf explants. The leaf explants were incubated in 0.4% (w/v) colchicine for 24, 48, or 72 h, then transferred to adventitious shoot-induction medium. Regenerated shoots were pre-selected according to their morphological characteristics when compared to control shoots from untreated shoot proliferation cultures. Shoots with putative polyploid morphological characteristics were maintained and proliferated. The ploidy levels of all putative polyploid individuals were analysed by flow cytometry and identified by chromosome counts of shoot tip tissue squashes. Polyploid shoots were rooted, and the resulting plantlets were transferred to the field. Polyploid plantlets had a higher specific leaf mass and larger stomata than those of diploid plantlets.