The Use of Stomatal Chloroplast Number for Rapid Determination of Ploidy Level in Maize

The usefulness of using the chloroplast number in epidermal guard cells as an indirect ploidy indicator was evaluated on seed-grown and tissue culture-derived maize plants. For seed-grown plants, two maize genotypes (B89 and R75) which had both diploid and tetraploid seeds available were used as experimental materials. The ploidy levels of seed-grown plants were confirmed by flow cytometric analysis of nuclear suspensions from these plants. For regenerated plants, haploid and diploid levels were examined and the ploidy levels of these plants were determined by chromosome counts of cells from root tips. A positive relationship between the chloroplast number and ploidy level was observed for both seed-grown and regenerated plants. The stomatal guard cells of tetraploid plants had nearly double the number of chloroplasts as the diploid plants. Similar results were found from the regenerated plants. The differences in the mean chloroplast number between diploid and tetraploid seed-grown plants and between haploid and diploid regenerated plants were highly significant. The results of this study demonstrate that counting chloroplasts in guard cells can be an efficient means of screening a large number of plants for ploidy levels. In addition, this study suggests the possibilities of using this method for detecting contaminated seed lots by different ploidy seed and for distinguishing plants of different genotypes.     

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.     

Ploidy analysis in water yam, Dioscorea alata L. germplasm

Poor reproductive development in yams (Dioscorea spp.) has often been attributed to the polyploid nature of the crop. In this study, flow cytometry was used to determine the ploidy level of 53 accessions of Dioscorea alata, mostly from West African countries, Chad and Puerto Rico. Nuclei were isolated from young leaf material and stained with DAPI(4,6-diamidino-2-phenylindole). The nuclear genome size (2C) was measured as an indicator of the ploidy level. Dioscorea rotundata genotypes with known ploidy levels were used as standards. The results showed that the majority of plants were hexaploid (84.9%) with a smaller percentage of tetraploids (15.1%). A higher number of male plants were hexaploid than tetraploids. This is at variance with earlier findings, which reported that hexaploid male plants are rare. Higher ploidy levels were not directly related to sparse or erratic flowering as previously reported as profuse flowering occurred in some male hexaploid accessions. These findings have important implications for yam breeding in relation to yam genetic resources. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cytogenetic variation in somatic tissue cultures and regenerated plants of barley (Hordeum vulgare L.)

Summary Chromosome number of morphogenic and non-morphogenic calli and regenerated plants of barley were determined. Cultures were obtained from two kinds of explants, immature embryos and seedling leaves from three cultivars, Ingrid, Dissa and Golden Promise. Callus chromosome analyses were carried out during a 12 month period in a medium containing 2 mg/l of 2,4-D. Diploid cells were predominant in all cases; although in leaf-derived cultures, retraploid cells (2n=4x=28) showed a tendency to increase as time in culture increased and after more than six months in culture, diploid cells decreased to percentages of almost 70%. Aneuploid cells were generally infrequent in all cases. The source of explant has been more important than the genotype (cultivar) and the type of callus (morphogenic vs. non-morphogenic) in the chromosomal stability of cultures as time increases. From short term cultures, only 1.85% of the regenerated plants were tetraploid, the remaining were diploids. The ability of morphogenic calli to regenerate plants decreased before any significant reduction of diploid cells were observed.     

Somatic embryogenesis from floral tissue of cassava (Manihot esculenta Crantz)

The aim of this study was to examine the embryogenic potential of floral material of the cassava cultivar MCOL 1505. Macerated immature inflorescences were found to be highly embryogenic, with almost 78% of the explants producing somatic embryos. Somatic embryos were also produced from whole male florets and half florets although at much lower rates. No regeneration was obtained from anther, microspore or floret wall tissue. Somatic embryos derived from immature inflorescences were regenerated via organogenesis and the plants derived from this process were assessed in terms of phenotype and ploidy level. If haploid plants could be produced by this method, this would have significant implications in assisting traditional cassava breeding, as this would allow homozygosity to be reached more rapidly. In a crop such as cassava, which is highly heterozygous in nature, the use of haploids in a breeding programme could considerably shorten the time taken to produce new desirable cultivars. This is the first report on plant regeneration through somatic embryogenesis from floral tissue of cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosome doubling in Tripsacum: the production of artificial, sexual tetraploid plants

A collection of embryogenic diploid calli of Tripsacum was established and treated with colchicine to induce chromosome doubling. Sections containing duplicated cells in calli were identified using flow cytometry and ploidy level was determined in the regenerated plantlets. Tetraploid plants from several origins were obtained. In contrast to wild polyploid plants, which show apomictic development, the regenerated tetraploid plants reproduced sexually. By hybridizing these plants with wild tetraploid apomicts, various populations were established; these will allow a study of the inheritance of apomixis in Tripsacum.     

Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.     

甘蓝未受精子房离体培养及再生植株倍性的鉴定

甘蓝通过未受精子房离体培养诱导获得的再生植株,对再生植株的倍性进行有效的鉴定是将其进一步应用于优良品种选育的基础。本研究利用3种基因型的甘蓝材料(PMQM、QMF、RMQM)培育再生植株,优化甘蓝未受精子房离体培养体系,并通过形态学鉴定法、根尖染色体计数法、流式细胞仪鉴定法对组培植株进行倍性鉴定。结果表明:在0.4 mg/L ZT的分化培养基中,3种基因型材料的愈伤组织分化率明显高于1.0 mg/L 6-BA培养基中的组培苗,其中基因型RMQM的分化效果最好;最终确定诱导愈伤组织分化不定芽的最适培养基配方为MS+0.4 mg/L ZT+2.0 mg/L 2,4-D+0.1 mg/L NAA,且通过3种鉴定方法,得出再生植株倍性:单倍体3.4%,双倍体49.8%,四倍体15.9%,嵌合体35.3%。     

Identification of tetraploid regenerants from cotyledons of diploid watermelon cultured in vitro

Summary Adventitious shoots were obtained from the diploid watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] cultivars Dixielee, Jubilee II, Mickylee, Minilee, and Royal Sweet by culturing excised cotyledons on shoot regeneration medium for six weeks. Tetraploid and diploid regenerants were identified by counting the number of chloroplasts per guard cell pair from leaves of regenerated plants. Cross fertilization of putative tetraploids with diploid pollinators and the production of triploid seed confirmed the efficacy of this approach. The mean number of chloroplasts for tetraploid regenerants was 19.1 whereas diploids averaged 11.2. These values were similar to tetraploid and diploid plants from seed. Ovary diameter, petal, and anther diameter of male flowers, and leaf length by width ratio were also good indicators of plant ploidy. Progeny obtained from self-fertile tetraploids of Mickylee were crossed with various diploid pollinators to produce triploid hybrid seed. All triploid plants from tissue culture-derived tetraploids produced fruit comparable in quality to fruit produced by currently-available triploid hybrids, demonstrating that in vitro tetraploid induction can be used to produce high quality tetraploid plants for use in triploid hybrid seed production.     

不结球白菜小孢子胚成苗及倍性变异研究

以不结球白菜小孢子胚为外植体,对胚发育成苗过程中基本培养基、激素配比、基因型及生根条件进行了研究,并对再生植株的染色体倍性进行了鉴定。研究表明：适于小孢子胚芽分化的培养基为B5＋GA30.1 mg/L＋蔗糖3%＋琼脂1.2%,在此培养基上6号、14号、31号的子叶型胚状体的出芽率分别为53.33%,85.24%,75.55%,平均出芽数分别为5.66,3.83,3.28个;为了诱导获得健壮新根,提高植株再生率,最适宜的生根培养基为MS＋IBA 0.1 mg/L＋蔗糖3%＋琼脂0.6%,生根率达100%,平均根数9.70条。对133株再生植株进行了染色体倍性鉴定,结果发现不结球白菜自然加倍率很高,且倍性变异情况比较复杂,其中四倍体植株所占比例最高,达到56.39%,二倍体植株占39.10%,三倍体植株占3.01%,而单倍体植株仅占1.50%。     

中国水仙花药培养及植株再生体系建立

本研究以中国水仙花药为外植体,通过器官发生途径建立其植株再生体系,并通过染色体计数鉴定筛选变异个体。结果显示:在4℃下预处理3d有利于花药愈伤组织的形成;愈伤组织诱导培养基最适配比为:MS+2,4-D1.0mg/L+BA0.5mg/L+CH500mg/L+AC500mg/L;愈伤组织分化小鳞茎的最适培养基为:MS+BA0.5mg/L+NAA0.1mg/L+CH500mg/L+AC1000mg/L。通过染色体计数对38个再生植株进行倍性鉴定,结果显示其中30个为三倍体(2n=30),8个为非整倍体(2n=10,11,12,14,17,26)。以这些再生苗为外植体,经器官发生途径,建立了不同倍性的再生体系。     

Somatic embryogenesis and plant regeneration of Tripsacum dactyloides L.

Summary This article reports the culture and plant regeneration of Tripsacum dactyloides. Mature embryos of Tripsacum dactyloides dactyloides were used to obtain embryogenic callus cultures. Currently, 180 normal plants have been regenerated from these cultures. Callus was initiated on MS medium supplemented with dicamba (10 mol or 20 mol) and sucrose (3% or 6%), and plants were regenerated on hormone free MS medium containing 2% sucrose. No significant differences were found in callus initiation frequency or in embryogenic response of cultures on the four combinations of sucrose and dicamba tested. The embryogenic cultures have been maintained for 9 months (12 subcultures) and have retained regeneration capacity. Plants regenerated from tissue culture of maize-by-Tripsacum hybrids could be useful in maize improvement.     

Ploidy Manipulation and Introgression of Resistance to Alternaria Helianthi from Wild Hexaploid Helianthus Species to Cultivated Sunflower (H. annuus L.) Aided by Anther Culture

The present investigation discusses the scope for transferring of resistance to leaf spot disease incited by Alternaria helianthi from two hexaploid wild species (H. tuberosus and H. resinosus) to diploid cultivated sunflower. Interspecific hybrids produced between sunflower and these two hexaploid species were partially fertile with tetraploid chromosome status. Backcrosses of these interspecific hybrids with cultivated sunflower resulted in the formation of sterile triploid plants. To overcome the problem of sterility and facilitate backcrosses with cultivated sunflower, anther culture of the tetraploid interspecific hybrids was carried out to bring down their chromosome number to diploid status. Anthers from both interspecific hybrids were cultured on basal Murashige and Skoog media supplemented with varying concentrations of organics and the growth regulators benzyladenine and naphthaleneacetic acid. Anthers of interspecific hybrids involving H. resinosus responded well and regenerated through an embryogenic route at a frequency of 98.7%. But in interspecific hybrids with H. tuberosus, anthers formed callus and subsequently regenerated shoots through an organogenic pathway. DNA ploidy analysis of anther culture plants of interspecific hybrids derived from H. tuberosus crosses was carried out to identify plants with desired diploid status. In vitro screening of parents, interspecific hybrids and anther culture plantlets against A. helianthi showed resistance in 68.5% of the anther culture plants of interspecific hybrids from H. tuberosus and in 24.3% of the plants derived from interspecific hybrids involving H. resinosus.     

辣椒花药培养再生株群体染色体倍性构成的多样性

采用流式细胞分析术和染色体计数法对辣椒花药培养再生株群体的染色体倍性构成情况进行了鉴定。显示了花药培养再生株中染色体倍性构成的多样性。观察到染色体倍性在不同检测组织器官中的差异现象,说明对同一材料不同器官进行倍性检测以确定植株倍性的必要性,以及植株上部器官的染色体倍性对于结籽能力的决定性。观察到再生株中个别细胞染色体的丢失现象。对流式细胞检测技术和染色体计数法的相关性进行了研究,得出2种检测技术下二者的吻合度为0.95,并对流式检测技术中的偏峰现象进行了初步的分析。     

The induction of tetraploidy in chicory (Cichorium intybus L. var. Magdebourg) by protoplast fusion

Summary Fusions of chicory leaf protoplasts were realized in order to obtain a higher number of tetraploid plants compared to the traditional doubling techniques with colchicine. Thanks to the fusion technique, using a mixture of PEG, DMSO and a solution of CaCl₂ (pH 10.5), 25% tetraploid (2n=36) plants were obtained. Among the 167 regenerated plants, only one aneuploid of a near tetraploid level (2n=33) was identified.     

Linamarin content and genetic stability of cassava plants derived by somatic embryogenesis

A protocol was established for high frequency cyclic somatic embryogenesis for different varieties of cassava. An efficient plant regeneration system was developed for the high cyanogenic variety PRC60a. Linamarin content and linamarase activity were determined in various tissues of secondary somatic embryos and regenerated plants of PRC 60a. Both linamarin and linamarase activity were not detected in embryogenic callus, roots induced from callus and somatic embryo tissues. The stems and leaves of regenerated plants (in vitro) and storage roots and leaves of mature plants (in vivo), however, contained variable amounts of linamarin and linamarase activity whereas in the non storage root tissues (in vitro) only linamarin was detected. The present study suggested that the linamarin biosynthetic pathway may be absent or not switched on in the embryogenic callus and somatic embryos. The ploidy level and somatic chromosome number of the regenerated plants were found to be same as the source plants. The availability of this regeneration system would be useful not only for investigating cyanogenesis but also for genetic manipulation in cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

Diploidization in Haploid Tissue Cultures of Sorghum

Conditions for the effective experimental regulation of ploidy level in regenerants from callus cultures derived from young, undifferentiated leaves and panicles of haploid sorghum were established. Diploidization depended on the ontogenetic age of the explant and the 2,4-D concentration in the medium. With a low 2,4-D concentration (0.5 mg/1) and segments of young panicles (< 35 mm long) the cultures produced only haploid regenerants. Diploid plants were formed from cultures derived from more mature panicles ( 35 mm long) and young leaves (15–65 mm long). Under a high 2,4-D concentration (2.5 mg/1) diploid plants were regenerated from cultures derived from young panicles (less than 35 mm) except the most young ones (5–15 mm). The majority of the diploid regenerants contained mutations, mainly affecting male fertility and plant height.     

Multivariate analysis of variation among Solanum commersonii (+) S. tuberosum somatic hybrids with different ploidy levels

In order to integrate the outcomes of interspecific somatic fusion experiments in breeding schemes, it is important to understand factors involved in the variability observed among regenerated plants. With such a purpose in mind, a population of Solanum commersonii (+) S. tuberosum somatic hybrids was examined by means of discriminant and cluster analyses. Data were collected on 56 hybrids with different ploidies and on the two diploid parents grown in a greenhouse. The ploidy group was used as discrimination criterion. Three significant canonical variables were extracted by discriminant analysis; they were mainly correlated with the number of leaflets and stems, degree of flowering, plant height, and leaf length. After cluster analysis carried out with the significant canonical variables, parental and hybrid clones were grouped in 7 clusters. In the canonical space of reduced dimensions, patterns of morphological variation depended mainly on ploidy level and non-additive gene interactions. Hybrids were in general more similar to the cultivated than to the wild species, suggesting a good chance of fast introgression of useful traits from S. commersonii into the S. tuberosum genetic background.     

‘细叶’芒(Miscanthus sinensis’Variegatus’)四倍体诱导与表型分析

本研究以观赏植物二倍体‘细叶’芒的幼穗为外植体诱导出愈伤组织,采用秋水仙素诱导处理获得其同源四倍体,分别通过流式细胞仪检测和根尖细胞染色体计数鉴定再生植株的倍性。进一步对四倍体‘细叶’芒的叶片气孔特征和植株表型特征分析,结果表明,与其二倍体亲本相比,四倍体叶片下表皮的气孔密度显著降低而气孔大小明显增大,四倍体植株的株高、分蘖和冠幅显著降低,但是叶片宽度比二倍体植株明显增加,而四倍体茎粗和叶长跟二倍体植株无明显差异。该研究获得的‘细叶’芒同源四倍体可为观赏植物新品种的培育提供新种质资源。     

A new method for selecting cytochimeras by the maximum number of nucleoli per cell in Allium wakegi Araki and A. fistulosum L.

Summary A new method for determining ploidy levels in cytochimeral plants was developed by examining the maximum number of nucleoli per cell. Colchicine-treated plants of Allium wakegi Araki and A. fistulosum L., which have different ploidy levels in the first (LI), the second (LII), and the third (LIII) germ layer, were used together with untreated 2-2-2 plants of the same species. Nucleoli in guard cells for LI and in mesophyll cells for LII were stained in a 50% AgNO₃ aqueous solution at 55° C for three hours under dark, humid conditions. In both species the ploidy levels of LI determined by the maximum number of nucleoli per guard cell accorded well with those determined by guard cell length. In A. fistulosum the ploidy levels of LII determined by the maximum number of nucleoli per mesophyll cell clearly agreed with those determined by pollen size. This method provided more precise and efficient identification for LI and LII ploidy than the conventional methods of using guard cell length for LI and pollen size for LII.