Chromosome doubling through adventitious shoot formation on in vitro cultivated leaf explants from diploid interspecific potato hybrids

Summary Nearly 450 plantlets were produced from 51 diploid Solanum etuberosum x S. pinnatisectum F₁ hybrids through adventitious shoot formation on in vitro cultivated rachis and petiole explants.On the basis of phenotypical assessment of the ploidy level of 425 plants, 84.7% of the plants were scored as doubled or doubled twice. A cytological analysis of ploidy in the three layers L₁.L₂ and L₃ of 112 plants revealed 83.9% complete doubling: periclinal ploidy chimeras were not found and only two sectorial ploidy chimeras were detected. Doubled plants were obtained from all 51 clones.Various flower colours and epinastic leaves (in 1 clone) may be indications of mutagenesis through the treatment.     

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.     

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.     

Analyses of phenotype and ARGOS and ASY1 expression in a ploidy Chinese cabbage series derived from one haploid

The aim of this research was to improve our understanding of how ploidy level influences phenotype and gene expression in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Haploid plants (2n = 10) was induced by 0.2% colchicine to produce diploid (2n = 20) and tetraploid plants (2n = 40). The aneuploid (2n = 24) was also obtained by hybridization between diploid plants as the female and tetraploid plants. The ploidy levels of all plants were identified through chromosome counts and flow cytometry. Leaves and petals became larger as the ploidy level increased from haploid to diploid, and from aneuploid to tetraploid. Similarly, expression of ARGOS was regulated by genome size, increasing in parallel with the level of ploidy. Among the four ploidy types, expression was stronger in the floral buds than in the leaves. Expression by ASY1 also differed according to ploidy level, being highest in diploid plants, followed in order by tetraploids. Expression was similar between haploids and aneuploids at two stages—prior to and after meiosis—but was higher in the haploids during meiosis. When buds were compared within the same ploidy type at different stages, ASY1 expression was obviously higher during meiosis than either before or after. Our study demonstrated the generation and phenotype of a ploidy Chinese cabbage series derived from one haploid. Expression of genes ARGOS and ASY1 were modulated by genome size in this ploidy series, and the regulated patterns of the two genes was different.     

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.     

Allelopathic Effect of Conyza albida on Avena sativa and Spirodela polyrhiza

The allelopathic influence of four plant tissues of Conyza albida (stems, leaves and inflorescences) on oat growth were investigated using in vivo tests. Oat growth (fresh and dry weights of above and underground parts) was inhibited from phytotoxic activity of upper leaves and inflorescence tissues of C. albida in pot studies. The inhibition was significantly higher than in the other tissues studied. The inhibiting action of crude extracts and volatile compounds from young plants (rosette) and mature plants were examined using two bioassay methods: (a) seed germination and radicle growth of oat and (b) fresh weight of duckweed plants. Both bioassayed species exhibited greater phytotoxic response from the young plants, collected in winter, than from the mature ones.     

转Bt基因棉杀虫蛋白含量时空分布及对棉铃虫产生抗性的影响

采用ＥＬＩＳＡ法（酶链免疫法）、室内初孵棉铃虫生测法和田间棉铃虫为害调查方法，研究和分析了中国构建的 Ｂｔ（Ｂａｃｉｌｌｕｓ ｔｈｕｒｉｎｇｉｅｎｓｉｓ）基因抗虫棉品系ＧＫ３和美国构建的Ｂｔ基因棉品系新棉３３Ｂ的不同生育期以及花铃期不同器官的杀虫蛋白含量、校正死亡率和田间受害率变化趋势以及它们之间的关系。结果表明，转Ｂｔ基因棉Ｂｔ杀虫蛋白含量在棉花生育过程中呈时空动态变化，在时间分布上，各生育期顶尖平展叶表现为：初花期＞蕾期、花铃期＞苗期＞吐絮期；在花铃期，各器官表现为：功能叶、茎尖＞小蕾、幼铃＞花蕊、花瓣、苞叶、老叶。室内生测幼虫校正死亡率与棉株Ｂｔ杀虫蛋白含量高度一致；田间表现与Ｂｔ杀虫蛋白含量有一定的差异，除主要受Ｂｔ杀虫蛋白影响外，棉铃虫取食选择性，以及残存高龄幼虫为害和棉株各部位营养结构等也影响其抗虫性，造成后期棉铃虫主要为害花、蕾和幼铃，两材料抗虫性表现较为一致。     

Unreduced 3x gamete formation of allotriploid hybrid derived from the cross of Primula denticulata (4x)?×?P. rosea (2x) as a causal factor for producing pentaploid hybrids in the backcross with pollen of tetraploid P. denticulata

A triploid hybrid, which was obtained from interspecific crosses between tetraploid Primula denticulata (2n = 4x = 44) and P. rosea (2n = 2x = 22), successfully produced 11 plants by backcrossing with pollen of tetraploid P. denticulata. Analysis of ploidy level using flow cytometry and chromosome counting in the 11 BC₁ plants revealed that all progeny had much larger DNA contents and chromosome number than both parents. In this triploid-tetraploid (3x–4x) crossing, progeny was predominantly true or near pentaploid presumably produced by the fertilization between true or near triploid female gamete produced from triploid hybrid and diploid pollen of tetraploid P. denticulata. These results suggest that unreduced (3x) or near triploid female gametes were partially produced by single step meiosis, either first-division restitution or second-division restitution process. The zygotes formed by the fertilization between true or near triploid egg produced by single step meiosis in triploid hybrid and diploid pollen produced by normal meiosis of tetraploid P. denticulata might be the only survivors in embryogenesis.     

The effect of autopolyploidy on biomass production in homozygous lines of Brassica rapa and Brassica oleracea

In Brassica species contradictory results on the effect of polyploidization on vegetative biomass production have been reported. This might result from the fact that the level of heterozygosity in diploid and tetraploid lines as a major factor of plant performance in Brassicas is often disregarded. Therefore in this study autotetraploid lines were developed from seven completely homozygous lines of B. rapa and B. oleracea in order to analyse the unconfounded effect of polyploidy on vegetative biomass production of young plants. The tetraploid lines yielded 2–64% less than the corresponding diploid lines and there was no significant effect of the ploidy level on the dry matter content. These results indicate that the direct effect of polyploidization in B. rapa and B. oleracea is negative while in some other studies this negative effect could be compensated by a higher level of heterosis in tetraploid populations compared to their diploid reference populations.     

Characterization of natural orchardgrass (Dactylis glomerata L.) populations of the Thrace Region of Turkey based on ploidy and DNA polymorphisms

Determining the ploidy and geneticdiversity of a germplasm is necessarybefore initiating breeding or geneticstudies. This study was conducted tocharacterize the ploidy level of 57 naturalpopulations of orchardgrass (Dactylisglomerata L.) collected from the ranges ofThrace region of Turkey and the diversityamong populations based on RAPD (RandomAmplified Polymorphic DNA) markers. Flowcytometry was used to determine nuclear DNAcontent (pg 2C^-1 = DNA content of adiploid somatic nucleus) of 6 plants foreach population. Nuclear DNA contents werecorrelated to ploidy level with root tipchromosome counts on selected plants. Onthe basis of this study, mean nuclear DNAcontent of orchardgrass was determined as9.57 ± 0.33 (with 95% confidenceinterval) while all the plants used inchromosome counting were determined to betetraploid, with 2n = 28 mitoticchromosomes, suggesting that diploidorchardgrass plants are likely very rare orabsent in ranges of Thrace region ofTurkey. In the RAPD assay, over 40polymorphic fragments were generated whichallowed some populations to bedistinguished from the rest by uniquemarkers. A cluster analysis was performedusing Nei's (1972) genetic distance indexwith an unweighted pair group method witharitmetic mean (UPGMA). The clusteranalysis indicated that there is a highlevel of gene flow among naturalorchardgrass populations and thereforegenes distributed quite homogeneouslythrough out the region. The results of thisstudy can be useful in the development ofDactylis germ plasm collectionstrategies in Thrace region for breedingpurpose.     

Comparison of diploid and tetraploid potato families derived from Solanum phureja x dihaploid S. tuberosum hybrids and their vegetatively doubled counterparts

Summary To determine whether in potatoes the tetraploid level is preferable to the diploid level, especially regarding tuber yield, four diploid (2n=2×=24) Solanum phureja x dihaploid S. tuberosum hybrid parents and their vegetatively doubled, tetraploid (2n=4×=48) counterparts were intermated, which resulted in two F₁ hybrid families at both levels of ploidy. The parents and clones of the F₁ families and their offspring were used in crosses in such a way that in addition Sib₁, Sib₂, F₁×Sib₁, BC₁ and Sib₁×Sib₁ families were produced. Of the first clonal generation of the 12 2 x families and their 12 counterpart 4 x families two tubers per clone were planted in three replications in a field experiment at Sturgeon Bay in 1969; of the parents six tubers were planted in each replication. Data were recorded on 16 characters, including plant height at four different times.The ANOVA's showed significant clone effects within each family for all characters. Computed from all family means as well as from the family means per ploidy level, differences due to family were also significant for all characters except one.As at the 2 x level and at the 4 x level of ploidy the mean phenotypic correlations between characters were of similar magnitude, it is concluded that they are independent of ploidy level.With the exception of eye depth, the mean coefficient of variation was greater at the 2 x than at the 4 x level of ploidy, indicating the greatest response to selection for those characters at the 2 x level. From the differences in family means between the 4 x and the 2 x level of ploidy it was apparent that the 4 x families generally had significantly taller plants, later maturity, fewer tubers, higher mean tuber weight, more tuber yield and more dry matter yield than their counterpart 2 x families. In contrast, the 4 x parents had on the average shorter plants, lower mean tuber weight, much lower tuber yield and lower dry matter yield than their 2 x counterparts.The phenotypic correlation and Spearman's rank correlation between the family means of the 2 x and the 4 x level of ploidy were positive for almost all characters and significant for nearly half of them.From the results it is concluded that 1. in potatoes the 4 x level of ploidy is preferable to the 2 x level, and 2. the performance of 4 x families is predictable from the performance of their counterpart 2 x families.Based on results mentioned in the literature and on the present results, a continued use of S. tuberosum dihaploids in potato breeding needs to be dissuaded.     

荫蔽锻炼对大豆苗期光合特性的影响

荫蔽是影响间套作大豆产量进一步提高的限制因素。为探究荫蔽锻炼对大豆叶片光合、荧光特性的影响,本研究采用盆栽试验,分别用远红光LED灯(λ=730nm)、30%透光率遮阳网模拟荫蔽信号和荫蔽胁迫,分锻炼(S1)–恢复(S2)–胁迫(S3)3个阶段,以全过程自然光照为对照(LLLL),设荫蔽信号锻炼(LFLS)、荫蔽胁迫锻炼(LSLS)、不锻炼(LLLS) 3个处理,分析其S3阶段遭受荫蔽胁迫时叶片光合色素含量、光合参数以及叶绿素荧光参数的响应特征。结果表明, LFLS和LSLS较LLLS老叶和成熟叶叶绿素a、叶绿素b、类胡萝卜素含量以及叶绿素总含量显著增加,老叶和新叶叶绿素a/b显著下降。除LSLS成熟叶外,各叶位净光合速率、气孔导度均较未锻炼处理显著增加,老叶和成熟叶锻炼与未锻炼处理胞间二氧化碳浓度差异不显著。与对照相比,无论锻炼与否,后期荫蔽使Fo、qp、NPQ、ΦPSII、ETR降低,而F_v/F_m和F_v'/F_m'则升高,其中,S1阶段的锻炼处理较未锻炼处理Fo下降的幅度更小;老叶qp较对照降低幅度依次为LFLSLLLSLSLS,成熟叶为LSLSLFLSLLLS,新叶为LSLSLFLSLLLS;ΦPSII、ETR均较对照降低但处理间差异不显著; LLLS、LFLS、LSLS新叶中F_v'/F_m'则分别比对照增加6.51%、8.79%和12.05%(P0.05),锻炼后增加幅度更大。由此可见,通过荫蔽锻炼,大豆能通过光合特征的可塑性来适应光环境并表现出更强的荫蔽耐受能力。     

一种利用流式细胞术鉴定梨倍性的方法

流式细胞术鉴定植物染色体倍性的方法广泛应用于果树多倍体育种中,然而相关流式细胞术鉴定梨染色体倍性的方法还没有系统的研究。以梨树叶片为试材系统优化了各个流式细胞术关键实验节点,结果表明：实验材料选取当年春季新梢第3~5 片梨树嫩叶,尤其是大棚新梢嫩叶最好;叶片前处理时用蒸馏水、去离子水依次洗净叶片表面后,再用去离子水浸泡10 min;使用WPB解离液对梨叶片细胞核进行提取后,用500 目滤膜过滤2 次,离心1 次后用流式细胞仪进行检测可得到理想的实验结果。该方法步骤简单、结果可靠,是一种快速、高效的梨染色体倍性鉴定的方法,可广泛用于梨树多倍体鉴定。     

Morphological and RAPD analysis of poplar trees of anther culture origin

Our objectives were to improve the rate of haploid plant regeneration through increasing the rate of callus initiation on the anthers and sustaining shoot regeneration frequency, and to analyze the field population of anther culture origin by morphological and molecular methods. Regarding the callus initiation, the most responsive clones were ‘N-90’(59%) in P. nigra and ‘D-29’ (75%) in P. deltoides. The rate of shoot regeneration and number of shoots/calli ranged from 4%–79% and 1–9, respectively. From the 208 rooted plants 8 haploid, 179 diploid, 4 tetraploid and 17 aneuploid plants were found. In the field population, the haploid plants could be easily identified by their retarded development and morphological characteristics (size and shape of the leaves, strong branching, etc). Several diploid plants showed depressed developmental and morphological traits similar to the haploid ones. Three traits (growth rate, leaf blade length and shape of leaf base) of the 6 different morphological characteristics measured were in correlation with the ploidy level within the poplar field population. Six primers of the 48 primers tested were able to detect polymorphism among the field plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anther-culture response in different genotypes and F1 hybrids of pepper (Capsicum annuum L.)

An in vitro anther-culture method has been improved by using young mother plants and by using frequent subcultures, thus increasing the androgenic yield in different Capsicum annuum L. genotypes. An assortment of peppers was used, composed of 15 genotypes (four breeding lines, seven cultivars and four F₁ hybrids). A new system for qualifying the androgenic response was established. For use in practical breeding, a minimum of 5 % of plant regeneration was proposed as the criterion for a fair response. Accordingly, one excellent, one good and eight fair responses were identified among the genotypes investigated. As compared to the standard cultivar. 2 genotypes gave a significantly better response, i.e. ‘Fehérözön’ (75.8%) and ‘Szechuan 90716’ (21.0%). In comparative investigations, F₁ hybrids, produced from crosses between poor/non-responsive and responsive genotypes, showed a fair level of response, even the case of a poor response in donor parent. The ploidy level of the resulting plants was determined by flow-cytometric analysis.     

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.     

Colchicine-induced chromosome doubling in Platanus acerifolia and its effect on plant morphology

We report the production of tetraploid plants of Platanus acerifolia, with the ultimate aim of improving the ornamental qualities of this important urban landscaping tree. Chromosome doubling was achieved by the application of colchicine to either pre-soaked seed or to the apical meristems of young seedlings. Treatment of the ungerminated seed was the more efficient method in terms of numbers of tetraploid seedlings (up to 40%, as determined by chromosome counting of the root-tip nuclei) but this method produced no mature tetraploid plants due to the deleterious effect of colchicine on subsequent root growth. When colchicine was applied directly to the apical growing tip of cotyledon-stage seedlings, leaf and stem growth was temporarily affected but the plants eventually recovered. We conducted a preliminary screen for putative tetraploids based on the observation in other plant species of a correlation of stomatal size and distribution with ploidy. Plants containing significantly larger stomata and at a lower density across the lower leaf epidermis, were selected for further analysis by flow cytometry and chromosome counting. These techniques confirmed that, of the 12 putative polyploids, four were tetraploid, five were mixoploid and three were, in fact, diploid. Morphological differences of the tetraploids included a more compact growth habit and broader, thicker leaves. These plants are being grown to full maturity in order to test their potential for use in a breeding programme aimed at producing sterile triploid lines.     

Cytological and genetic analysis of a virescent mutant in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

It is well known that genetic mutation could be generated by physical treatment (for example, γ-irradiation) and chemical treatment (for example methylnitrosourea and ethyl methanesulfonate). Here we reported identification of a virescent mutation (vsp) after exposing the upland cotton (Gossypium hirsutum L.) CCRI58 seeds in space environments. vsp mutant was characterized at the morphological, agronomic, cellular and genetic levels. vsp mutant showed an earlier virescence and specific only to true leaves. Agronomic traits of vsp mutant, such as plant height, number of bolls, boll weight, yield and fiber quality were significantly lower than those of CCRI 58. Chlorophyll level, carotenoid level and photochemical efficiency of vsp mutant true leaves were significantly lower compared to CCRI 58 at young leave stage. Anatomical studies of chloroplasts showed that vsp mutant lacked grana in the thylakoids of the mesophyll cells at young leave stage, while CCRI 58 showed normal grana in the thylakoids of the mesophyll cells at young leave stage. This indicated that chlorophyll and carotenoid levels were related with chloroplast structure. Genetic analysis indicated that vsp was controlled by one recessive gene in nucleus. Allelic tests showed that vsp was nonallelic to 12 virescent genes currently available at Anyang, China. In summary, we identified a vsp mutant after exposing the upland cotton (Gossypium hirsutum L.) seeds in space environments. vsp could be a newly identified virescent gene. vsp may also be used as a marker in cotton breeding programs. Exposing seeds in space environments could cause new spectrum of genetic mutations and could be used for breeding programs.     

Chromosomal instability in cell- and tissue cultures of tomato haploids and diploids

Summary The effect of the tissue culture system, the genotype and the ploidy level of the plant material used as explant source on the stability of the ploidy level of plants regenerated fromcell and tissue cultures of tomato was investigated. In addition the use of the chloroplast number in guard cells as a measure for ploidy level was evaluated. Haploids of tomato were very instable, which instability was observed already in somatic root tip and leaf cells. The number of regenerated plants that retained the original ploidy level differed significantly between the tested haploids. The plants that were regenerated from leaf explants of diploids were predominantly diploid in contrast to the plants regenerated from established callus cultures and protoplast where the majority was tetraploid.     

The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum

Summary To produce hexaploid (or other polyploid) hybrids, diploid or tetraploid Hordeum vulgare was crossed with hexaploid or octoploid H. bulbosum, and perennial triploid hybrids between the two species were treated with colchicine. The crosses did not yield viable plants: seedset was low, the seed aborted and embryo culture was unsuccessful. The colchicine treatments geve rise to plants in which hexaploid chromosome numbers were observed. At the hexaploid level chromosomal instability occurred, resulting in chromosome elimination.The colchicine-treated triploid hybrids showed in the first years after the treatment better fertility after open flowering than untreated plants, but the level of fertility remained very low. The offspring consisted of haploid, diploid and approximately triploid plants like H. vulgare, tetraploid and approximately tetraploid plants like H. bulbosum, and plants with hybrid morphology and unstable chromosome number, which were highly sterile. Thus the crossing barrier between H. vulgare and H. bulbosum could not be broken down at higher ploidy level.