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.     

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。     

利用二硝基苯胺类除草剂离体诱导西瓜四倍体

 以二倍体西瓜单系Z-A和Z-R为试验材料,采用不同浓度的二硝基苯胺类除草剂——胺磺灵、二甲戊乐灵和氟乐灵处理其离体子叶,并以秋水仙素和不含任何诱导剂的液体培养基为对照,研究诱导西瓜四倍体的效果。结果表明,3种除草剂诱导西瓜四倍体的效果都比秋水仙素好,二甲戊乐灵、胺磺灵、氟乐灵和秋水仙素的四倍体诱导率分别为40.0%、33.3%、26.7%和13.3%。采用子叶直接处理比间接处理的四倍体诱导率高。通过 FDA荧光法对西瓜试管苗叶片表皮保卫细胞叶绿体计数,进行四倍体变异早期鉴定,准确率为90%以上。     

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.     

甜瓜组织培养再生植株中的四倍体变异

甜瓜未成熟子叶,幼苗子叶和真叶离体培养的再生植株中存在着明显的四倍体变异,比率分别为３０％,１６．２％和４２．９％,四倍体植株在花冠,气孔,花粉,种子和果实的形态和大小方面均与二倍体植株表现明显的差异。再生植株叶片气孔大小和气孔保卫细胞内叶绿体数目可以作为从再生植株中早期鉴别四倍体植株的可靠指标。组织培养可以成为获得四倍体甜瓜的有效途径。     

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。     

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.     

In vitro induction of the amphiploid in interspecific hybrid of blueberry (Vaccinium corymbosum × Vaccinium ashei) with colchicine treatment

Amphiploids were produced from pentaploid hybrids between Vaccinium corymbosum (4x) and Vaccinium ashei (6x) by colchicine treatment. Seeds of each parental species and those obtained by the interspecific crossing were treated with colchicine at 0, 500, 1000 and 2000 mg/l for 7 days, and the ploidy level of the seedlings was determined with flow cytometry. Either amphiploid (10x) or ploidy chimera (5x + 10x) was obtained from interspecific hybrid seeds treated with all colchicine concentrations, while no chromosome-doubled plants were obtained from both parents. Cross direction in interspecific hybridization affected the results of colchicine treatment and amphiploids were obtained only when V. corymbosum was used as the seed parent. In this cross, 17–25% of the seedlings turned to be amphidiploids or ploidy chimeras by treating with 500 mg/l colchicines for 7 days. These results indicate that susceptibility to colchicine may increase in the interspecific hybrid compared with the parental species but only when V. corymbosum was used as female parent.     

金冠苹果成熟胚离体诱导四倍体的研究

 以自然授粉的‘金冠’苹果种子为材料，研究了秋水仙素处理成熟胚离体诱导四倍体技术和嵌合体分离方法。结果表明：将完整胚在0.5%秋水仙素+1%二甲基亚砜的混合溶液中暗处理48 h，然后接种在MS+TDZ 1.0 mg·L^-1+IBA 0.5 mg·L^-1+蔗糖30 g·L^-1+琼脂6 g·L^-1的再生培养基中，变异率高达10.6%。与二倍体相比，四倍体试管苗生长缓慢，节间变短，叶片增大，叶形指数趋小化，气孔密度降低，保卫细胞增大。经压片法鉴定茎尖染色体数和流式细胞仪分析DNA含量，表明多数变异植株为同质四倍体。利用叶片离体再生器官技术，从诱导获得的嵌合体植株中分离出同质突变体。     

秋水仙素诱变薄皮甜瓜同源四倍体研究

为了获得四倍体薄皮甜瓜材料,研究薄皮甜瓜多倍体的特征特性,以二倍体薄皮甜瓜金美为材料,以0.2%、0.4%、0.6%的秋水仙素采用滴苗和浸种2种方法进行诱变四倍体试验,并通过染色体观察和细胞学鉴定对其进行倍性分析。结果表明,滴苗法比浸种法诱变率高,其中以0.4%秋水仙素滴苗诱变率最高,为6.67%;与二倍体相比,四倍体植株子叶和真叶长宽比变小,茎增粗,节间变短,花冠变大,果肉变厚,种子增宽,可育性降低,中心可溶性固形物含量增高;保卫细胞变大,气孔密度变小,保卫细胞内叶绿体数目增加;获得了同源四倍体材料。     

秋水仙素和胺磺灵除草剂诱导小型西瓜四倍体研究

用0.2%秋水仙素水溶液和不同浓度胺磺灵除草剂滴苗处理诱导小型西瓜四倍体。结果表明,用秋水仙素处理,诱变频率基因型间差异较大,高的可以达到26.3%,低的仅有1.4%。胺磺灵诱导小型西瓜四倍体有一定的效果,但整体诱导频率不高。     

秋水仙素诱导三种紫薇多倍体的研究

 研究了秋水仙素处理浓度和时间对3种紫薇幼苗染色体加倍的诱导效应,通过染色体计数、流式细胞仪以及形态观察等方法进行了倍性鉴定。结果表明：秋水仙素诱导3种紫薇均得到了四倍体,0.5%～0.8%的秋水仙素处理紫薇和银薇48～96 h的变异率较高,其中以0.5%秋水仙素处理紫薇72 h的变异率最高,达54.17%。根尖染色体压片表明四倍体染色体数为：2n=4x=96,二倍体对照为：2n=2x=48。四倍体表现出叶片长、宽、厚均增大,气孔密度减小,气孔保卫细胞增大,保卫细胞内叶绿体数增加,花粉粒直径增大,花径、花瓣长、花瓣基部爪长均较二倍体明显增加,单朵花观赏价值大大提高。     

穿心莲四倍体的诱导及其愈伤组织对肉桂酸胁迫的响应

以不同倍性穿心莲诱导的愈伤组织为试材,采用秋水仙素对穿心莲种子进行四倍体诱导,通过在穿心莲四倍体及二倍体诱导愈伤组织培养基中添加化感自毒物质肉桂酸进行共培养的方法,研究了肉桂酸对不同倍性穿心莲愈伤组织的褐化率、细胞鲜质量、超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量的影响,以期为筛选穿心莲抗化感自毒物质的优良种质资源提供参考依据。结果表明:用0.05%的秋水仙素处理48h,穿心莲种子诱导成活率达89%,经过植株形态、气孔及染色体数目检测,确定了穿心莲四倍体株系PZ-1、PZ-2、PZ-3、PZ-4、PZ-5、PZ-6、PZ-7、PZ-8,其染色体数为4x=96,二倍体为2x=48。在1.00mmol·L^-1肉桂酸的愈伤组织诱导培养基中培养7d,穿心莲四倍体愈伤组织的褐化率和MDA含量较二倍体低,而愈伤组织鲜质量、SOD、POD、CAT活性较二倍体高,显示四倍体较二倍体具有对肉桂酸更强的抵御能力,其中四倍体PZ-7表现出较显著的优异性状。     

In vitro tetraploid induction and generation of tetraploids from mixoploids in Astragalus membranaceus

This paper describes an efficient colchicine-mediated technique for the in vitro induction of tetraploids in Astragalus membranaceus and its confirmation by flow cytometry. Buds immersed in 0.2% colchicine solution for 36 h prior to culture induced as high as 35.3% tetraploid plants. Colchicine-induced tetraploids remained stable after 6 months in soil. Leaf characteristics of diploids and tetraploids in A. membranaceus were compared. It was determined that leaf sizes of glasshouse-grown plants and stomatal sizes of both in vitro and glasshouse-grown plants were suitable parameters for identifying putative tetraploids in A. membranaceus. As well as generating tetraploids, this technique generated mixoploids in A. membranaceus. Calli derived from mixoploid leaves, were induced to form buds and shoots. Individual shoots were classed as diploid, mixoploid and tetraploid by flow cytometry. This callus-based technique can be employed when a genome-doubling agent generates mixoploids but no tetraploid.     

In vitro germination and seedling development of cryopreserved Dendrobium hybrid mature seeds

In vitro germination and seedling development from Dendrobium Swartz. hybrid ‘Sena Red’, ‘Mini WRL’, ‘Jaquelyn Thomas’, and ‘BFC Pink’ seeds cryopreserved through vitrification (PVS2) were evaluated. Germination percentages after cryopreservation (LN) were variable among different controls and treatments, despite the initial high seed viability for all hybrids. Seeds exposed to PVS2 at ice temperature from 1 to 3 h prior to LN exhibited significantly higher germination than seeds exposed to PVS2 at room temperature for the same time periods. No significant differences in germination percentages were observed for time exposure to PVS2 at 1, 2 or 3 h for ‘Sena Red’, ‘Mini WRL’, and ‘BFC Pink’. Seeds of ‘Jaquelyn Thomas’ exposed to 1 h prior to LN showed higher germination percentage than for exposure to 2 or 3 h. The combination of a pre-cooling treatment (ice) with a dehydration treatment (PVS2) for a period of time of 1–3 h was essential to allow proper germination of cryopreserved seeds. Although variability in seed germination among different hybrids and treatments was observed, germination was above 50% of the controls and all germinated seeds developed into normal seedlings with healthy shoot and root formation. No abnormalities, nutritional deficiencies, or diseases were observed in developed seedlings and no significant differences were observed for seedling growth and development from germinated seeds among the different hybrids. Seedlings transplanted to pots acclimatized well and developed into normal plants within 6–8 months in greenhouse. Transplanted seedlings exhibited 100% survival for all hybrids.     

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.     

苹果三倍体后代培养及倍性鉴定

以自然授粉的三倍体苹果品种乔纳金的种子为材料,研究了培养方式对苹果三倍体实生后代获得的影响及实生后代的倍性水平。结果表明,离体培养与常规播种在乔纳金种子成苗率上存在明显差异,其中离体培养的成苗率是48.1%,常规播种的成苗率是20.9%,离体培养获得的植株是常规播种获得的植株的2倍以上。本研究获得的690个乔纳金实生后代中以非整倍体植株占多数,共获得非整倍体452株,占植株总数的65.5%。在后代中有13个为多倍体,其中离体培养获得7株三倍体和4株四倍体,而常规播种只获得2株三倍体,表明离体培养可以获得较多的多倍体资源。乔纳金实生后代中不同倍性植株在形态上差异显著,根据植株形态和叶形指数,可以容易地将多倍体(三倍体、四倍体)植株与二倍体植株和非整倍体植株区分开来。     

Colchicine- and trifluralin-mediated polyploidization of Rosa multiflora Thunb. var. inermis and Rosa roxburghii f. normalis

Rosa multiflora Thunb. var. inermis and Rosa roxburghii f. normalis are two important diploid species for rose breeding. Interspecific crosses between diploid species and tetraploid rose cultivars result in triploids with generally decreased fertility. A most promising gene transfer strategy is the production of fertile tetraploid plants before mating with tetraploids. In our study, germinating seeds of R. multiflora and R. roxburghii were treated with colchicine, and cotyledon-stage seedlings of R. multiflora were treated with colchicine or trifluralin in order to obtain tetraploid plants. Also, various concentrations of antimitotic agents and different treatment durations were tested. The results showed the antimitotic agents affected the plant morphological characteristics (i.e. plant height, stem diameter) of R. multiflora and R. roxburghii. Twenty-three tetraploid plants were obtained from the germinating seed treatments (21 from R. multiflora and two from R. roxburghii), but no tetraploid plants were obtained from the seedling treatments. The highest rate of chromosome doubling (25.0%) was achieved when germinating seeds of R. multiflora were treated with 0.2% colchicine for 12 h. At the same time, marked morphological variation was observed and analyzed among tetraploid plants and their corresponding diploids of R. multiflora.     

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.