Variation of chromosome numbers and essential oil components of plants derived from anther culture of the diploid and the tetraploid in Pelargonium roseum

Summary Anthers of the diploid (2n=77) and the colchi-tetraploid (2n=154) Pelargonium roseum were cultured in vitro. In both ploidy level anthers containing uninucleate or binucleate microspores were incubated on a modified White's medium. Calli formed were then subcultured on Murashige and Skoog's medium for organoid differentiation. Plants developed from organoids were transferred to filter paper bridges and after that transplanted into pots. Plants derived from anthers of the tetraploid had diploid chromosome number. Wide variation of their essential oil components suggested their genetic heterogeneity. Further, high correlations between different seasons in the rate of essential oil components showed that the wide variation was due to genetic differences. Therefore, these plants probably originated from pollen grains. On the other hand, plants derived from anthers of the diploid had diploid chromosome number. Small variation and low correlations between different seasons in essential oil components indicated their genetic homogeneity. Their origin was ascribable to the somatic tissues of the mother plant. It is concluded that in plant species in which usual sexual reproduction is difficult, anther culture of chromosome-doubled plants will give a useful method for obtaining genetic variation.     

Effect of high temperature on the production of 2n pollen grains in diploid roses and obtaining tetraploids via unilateral polyploidization

To integrate the gene pool of a wild species (primarily diploid) into a cultivated pool (primarily tetraploid), a crossing between a dihaploid cultivated rose and a hybrid of Rosa wichurana allowed to obtain interspecific diploid hybrids that produced 2n pollen grains. A return to a tetraploid level sought by breeders can then be considered using sexual polyploidization, obtained by crossing a tetraploid cultivated rose with these hybrids. Application of a high‐temperature regime led to a small but significant increase in the percentage of 2n pollen grains in these hybrids of up to 4.6%. This result was obtained by applying high temperatures close to 32°C during the day to plants cultivated in a glasshouse during recurrent cycles of bloom. Crosses were made between an unreleased tetraploid hybrid tea rose, as a female, and the diploid hybrid that produces the most 2n pollen grains. Tetraploid (42.1%) and triploid (57.9%) offspring were obtained. The use of these 2n pollen grains of the first division restitution type should facilitate the introgression of complex traits of interest.     

Germinal pore number in pollen grains as a selection basis in the production of tetraploid clover

Experiments with the variety Fahl of Berseem clover and with different material of red clover have shown that the occurrence of tetraploid and diploid tissues can be detected in a rapid and reliable way by determining the number and arrangement of germinal pores of the pollen. The advantages over the conventional methods are mentioned and described.The determination of the germinal pore pattern, which is combined with pair crossing of the tetraploid heads, makes it possible to obtain in an efficient way completely tetraploid material with a great genetic variability.Samenvatting Proeven met het ras Fahl van Alexandrijnse klaver en met verschillend materiaal van rode klaver hebben uitgewezen dat het voorkomen van tetraploide en diploide weefsels op een snelle en betrouwbare wijze aangetoond kan worden door middel van bepaling van het kiemporiënbeeld van het stuifmeel.De bepaling van het kiemporiënbeeld gevolgd door paarsgewijze kruising van de tetraploide hoofdjes maakt het mogelijk op een efficiënte wijze te komen tot zuiver tetraploid materiaal met een grote genetische variabiliteit.     

Influence of flower and flowering characteristics on seed yield in diploid and tetraploid red clover

Tetraploid red clover (Trifolium pratense L.) often produces insufficient seed to be economically interesting. Numerous studies have identified poor pollination due to long corolla tubes as its cause, but other studies disagree. Therefore, we investigated seed yield, corolla tube length and flowering traits in 244 plants from five diploid and five tetraploid cultivars during 2 years. Tetraploids produced fewer seeds, fewer flower heads and fewer seeds per head, but an equal number of flowers per head when compared to diploids. Although corolla tubes were longer in tetraploid than in diploid red clover (P < 0.001), no correlation between corolla tube length and seed yield was detected. Therefore, the corolla tube length is likely neither the main nor only cause of low seed yield in tetraploid red clover. Instead, three putative causes for low seed yield were identified: tetraploids produced less inflorescences per plant, possessed a lower degree of determinacy and had markedly less seeds per head when compared to diploids. Possible causes for such differences include lower pollen viability and higher rates of embryo abortion.     

秋水仙素诱导美洲商陆四倍体的研究

利用改良L.D.Cua法对美洲商陆进行多倍体诱变,在秋水仙碱浓度为0.2%条件下,设置24h、48h和72h三个诱变时间,并对所得到的变异株和对照株进行形态学、细胞学等方面的比较研究,结果表明：诱变48h效果最好;与对照相比,变异株的叶片厚度增加43.75%;叶形指数减少37.95%;气孔纵横径分别增加52.94%和73.01%;保卫细胞内叶绿体数目增加20.91%;花直径增加22.96%;花粉粒直径增加22.47%;果实直径增加23.66%。对变异株进行花粉母细胞减数分裂染色体鉴定,小孢子细胞的染色体数为n=2x=36,而对照小孢子细胞的染色体数为n=x=18,证明变异株为四倍体。     

Establishment, Development and Yield of Forage Legumes and Grasses as Cover Crops in Grain Corn in Eastern Canada

Interseeded cover crops can minimize soil erosion, increase organic matter and nutrient levels and the yield of subsequent crops. However, their performance is very sensitive to local conditions. Field experiments were conducted at two Quebec locations in 1993 and 1994 in order to evaluate the potential use of forage legumes and grasses as interseeds in corn in eastern Canada. Twelve forage species were evaluated. Fall rye ( Secale cereal L.), hairy vetch ( Vicia villosa Roth), a mixture of red clover ( Trifolium pratense L.) and ryegrass ( Lolium multiflorum Lam), a mixture of white clover ( Trifolium repens L.) and ryegrass, subterranean clover ( Trifolium subterraneum L.), yellow sweet clover ( Melilotus officinalis Lam.), black medic ( Medicago lupulina L.), Persian clover ( Trifolium resupinatum L.), strawberry clover ( Trifolium fragiferum L.), crimson clover ( Trifolium incarnatum L.), annual alfalfa ( Medicago saliva L.) and berseem clover ( Trifolium alexandrinum L.) were seeded at two planting dates (10 and 20 days after corn emergence). The control treatments were: hand weeding, chemical weeding and non-weeded. Early seeded forages established better and had higher biomass accumulation than the late seeded ones. In the presence of larger weed populations, the interseeded forages did not develop well due to competition with the weeds. At Macdonald crimson clover provided good soil cover while Persian clover, fall rye and alfalfa provided relatively little cover. Strawberry clover and hairy vetch did not provide early ground cover due to their late development. Forage mixtures of red or white clover and rye grass established well and achieved high populations at the end of the growing season. Fall rye provided good early ground cover but senesced by the middle of the season. The better establishment and early germination of crimson clover caused a 19% reduction in corn grain yield in 1993. In 1994 none of the cover crops caused a reduction in corn yield.     

Cytological mechanism of 2n pollen formation and unilateral sexual polyploidization in Lolium

Summary Plants of Lolium perenne, L. multiflorum and L. hybridum (331, 51 and 18, respectively) were screened for 2n pollen production. The screening was based on the size differences that are expected to be found between n and 2n pollen of a plant. It was found that 28 plants of perennial ryegrass-belonging to 13 cultivars-produced produced pollen grains of heterogenous size (big and small). The estimated frequency of big pollen grain production was higher than 10% in ten out of 28 plants and in three of them it reached a value of 100%. Eight plants obtained from two 4x × 2x crosses, in which the male parent had been previously identified as producing pollen grains of heterogenous size, was tetraploid. The cytological mechanism of big pollen grain formation observed in three plants consists in the lack of spindle formation and, consequently, of chromosome migration in anaphase II. From these breeding and cytological results, it was concluded that the big pollen grains observed were viable 2n pollen grains. The nuclei of the 2n pollen grains produced by this mechanism are genetically equivalent to those formed by the restitution of the second meiotic division. The value of these gametophytes in a breeding program of Lolium is discussed.     

Development of breeding populations from interspecific hybrids between Trifolium repens L. and T. occidentale Coombe

Trifolium occidentale is a diploid wild relative of T. repens (white clover) with adaptation to dry, saline coastal habitats. Transfer of drought and salt‐tolerant adaptive traits to white clover could be potentially valuable if interspecific hybridization can be achieved efficiently and leads to fertile hybrid populations. To achieve hybridization, 4x plants of T. occidentale were generated. Efficient techniques for generation of 4x plants and their identification using dry pollen shape are described. Interspecific 4x F₁ plants were achieved without embryo rescue. F₂ populations and first backcross hybrids to white clover were also efficiently achieved. Although male and female fertility were lower than in white clover, they were adequate to produce large amounts of seed from small numbers of inflorescences. Thus, early generation interspecific hybrid prebreeding populations can be readily developed, opening the way for transfer of traits from T. occidentale to white clover.     

秋水仙素对辣椒生长的影响及多倍体诱导效应研究

以秋水仙素为诱变剂,比较了不同浓度和不同处理时长组合下辣椒(Capsicum annuum L.)的生长差异和多倍体诱导效应.结果表明:和对照植株相比,所有处理变异植株在形态上表现为叶片宽大,叶色较深,茎变粗且节间距长,气孔增大;相关指标都表现出显著或极显著的差异,而且随着处理强度的增加差异更为明显.秋水仙素诱导产生的变异植株经染色体倍性鉴定植株表征为嵌合体(2 n=24或2 n=48).相同处理时长下,不同浓度秋水仙素溶液对辣椒多倍体诱导效应差异显著,而对相同浓度下不同处理时长对植株叶片数和气孔面积影响差异不显著(P>0.05),其它指标差异极显著(P<0.01).秋水仙素浓度与处理时长之间存在交互作用,不同处理之间除气孔面积差异不显著之外,其它指标间差异显著或极显著.结合加倍率和植株死亡率综合分析后认为,用0.05%的秋水仙素溶液处理6天诱导效果最佳,诱导诱导加倍率可达26.1%.     

Significance of Pollination in Seed Setting Efficiency of Berseem (Trifolium alexandrinum L.)

The Egyptian clover ( Trifolium alexandrinum L.) commonly known as berseem in India, is a predominantly self-pollinated autogamous fodder legume. It has a simple valvular type mechanism for pollination wherein tripping agencies like bees are needed for better seed setting. The seed setting efficiency and seed yield of berseem (g. m⁻²) under natural tripping or open tripping condition was extremely high (22.38) as compared to completely caged (0.52) which clearly indicates the significance of pollinators/tripping agencies in seed yield of berseem crop. Further, the influence of pollinators was well marked with diploid strains of berseem as compared to tetraploid because of difference in fertility levels (tetraploids are generally less fertile).     

Male fertility and cytology of triploid hybrids between tetraploidSolanum commersonii (2n=4x=48, 2EBN) and Phureja-Tuberosum haploid hybrids (2n=2x=24, 2EBN)

Summary Solanum commersonii Dun. is a diploid (2n=2x=24, 1EBN) wild species of potential value for potato breeding. It is a reproductively isolated species and cannot be crossed with Tuberosum haploids (2n=2x=24, 2EBN) or other diploid 2EBNSolanum species. In order to overcome the EBN barriers, triploid hybrids were produced between Phureja-Tuberosum haploid hybrids, which form 2n pollen grains by parallel spindles, and tetraploidS. commersonii. Microsporogenesis analysis of the triploids indicated a trend towards low values of chromosome distribution at Anaphase I; lagging chromosomes were often observed as well. Despite these abnormalities, the percentage of stainable pollen was very high, ranging from 5.0% to 74.3%. A high variation in pollen grain diameter was also evident. Parallel and tripolar orientation of spindles at Metaphase II of microsporogenesis was a common feature of all the triploids analyzed, but dyads and triads were observed at a very low frequency. Therefore, also the frequency of 2n pollen was very low; the different size of stainable pollen appears to represent the ploidy levels which are possible according to the distribution of chromosomes in Anaphase I. The results obtained also suggest thatS. commersonii could have minor genes acting at the end of meiosis in such a way that, despite the presence of parallel/tripolar spindles, dyads/triads are not formed.Contribution no. 124 from the Research Center for Vegetable Breeding.     

Fertility and pollen tube growth in polyploid melons (Cucumis melo L.)

The high sterility of tetraploid melon (Cucumis melo L.) plants is a serious inconvenient to obtain economically useful polyploid lines. In order to study the alterations on diploid pollen tube growth and their influence on sterility, in vitro and in vivo pollen tube growth from three commercial varieties and its tetraploids was observed. Polyploid pollen grains show a lower germination percentage in vitro and a slower germination rate than haploid pollen. Furthermore, diploid pollen grains producing two or three tubes and tube branchings were observed on diploid pollen; this seems to indicate a decrease in its functionality. In vivo and in vitro pollen behaviours are quite similar, but an interaction between the pollen and the pistillar tissues was detected in vivo. In the diploid × tetraploid cross, where fruits were not obtained, pollen tubes showed a slow growth rate at the base of the pistil. The possible effects of these alterations in sterility are discussed in terms of the factors that could affect fecundation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is discussed.     

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of colchicine treatment on Solanum acaule and S. bulbocastanum; A complete analysis of ploidy chimeras in S. bulbocastanum

Summary Seeds of tetraploid Solanum acaule (2n=48) and diploid S. bulbocastanum (2n=24) were germinated in petri-dishes on filter paper soaked in 0.3% colchicine. An additional treatment with 0.3% colchicine was applied one month after sowing at four successive days in the axils of the cotyledons of the seedlings. S. acaule appeared much more sensitive to colchicine (14 surviving seedlings from 500 seeds) than S. bulbocastanum (109 surviving seedlings from 450 seeds). Six S. acaule plants with 2n=96 chromosomes were obtained against 38 S. bulbocastanum plants with 2n=48 chromosomes.The ploidy level in each of the three germ layers L₁, L₂, L₃ was determined in 113 plants of S. bulbocastanum and the following results were obtained. Four of the eight possible ploidy types were detected, viz 2x-2x-2x (72 plants), 4x-2x-2x (3 plants), 2x-4x-4x (9 plants) and 4x-4x-4x (29 plants). Doubling the number of chromosomes resulted in a highly significant increase of the number of chloroplasts in the guard cells of stomata and a greatly significant decrease in the proportion of trimerous pollen, male fertility and leaf index. The variability for all characters studied, except for leaf index, was clearly lowest in the 2x-2x-2x group. All plants with a 2x-L₂ were highly male fertile and self-incompatible, also in the three bud stages tested. Male fertility of the plants with 4x-L₂ varied greatly: 12 plants had more than 90% stainability, 5 plants must be considered male sterile. All non-sterile plants with 4x-L₂ were found to be self-compatible, pointing to a gametophytic system of incompatibility in S. bulbocastanum.     

Production of 2n pollen of Asiatic hybrid lilies by nitrous oxide treatment

Tetraploid varieties of lilies have superior agronomic traits such as large flowers and resistance to physiological disorders. In the present study, we attempted to induce 2n pollen of Asiatic hybrid lilies by arresting the meiotic process with nitrous oxide (N₂O) gas. To determine which meiotic stage is optimal for induction of 2n pollen, plants with attached buds at different meiotic stages were treated with N₂O for 24 h in a pressure-tolerant cylinder. A few 2n pollen grains were induced using plants with anthers in prophase I, whereas mixed pollen grains of differing size were produced using plants undergoing meiotic metaphase predominantly in anthers. Although normal lily pollen grains are elliptical, nitrous oxide exposure induced giant pollen grains that appeared spherical. Flow cytometry analysis showed that the giant pollen grains were diploid. When mixed pollen that included normal and giant pollen was crossed to tetraploid cultivars, the resulting seedlings were tetraploid and aneuploid, indicating that the giant pollen grains were diploids that could generate tetraploid seedlings through fusion to diploid eggs supplied from a tetraploid female parent. Thus, treatment with N₂O is useful for the production of 2n lily pollen and may provide a new approach for tetraploid lily breeding.     

Aneuploids of perennial ryegrass (Lolium perenne)

Summary Aneuploid plants of perennial ryegrass (Lolium perenne L.) with 2n=15 to 30 chromosomes were obtained by crossing a near-triploid (2n=3x+1=22) with a diploid or on open-pollination with diploids and tetraploids. Aneuploids occurred with a frequency of 83% in near triploid × diploid progeny and 92% on open-pollination with diploid and tetraploid plants. Aneuploid plants with 15 to 18 chromosomes resembled diploids in morphology and those with 19 to 30 chromosomes were akin to tetraploids. Meiotic studies suggested that most aneuploid plants resulted from transmission of aneuploid egg cells (n=8 to 23). Aneuploid plants with 2n=27 to 30 chromosomes in the progeny of 22×14 cross originated from unreduced egg cells. Plants with 19 to 21 chromosomes were recovered only by immature seed culture. Aneuploid plants with 26 to 30 chromosomes and triploids (2n=21) had higher pollen fertility and bigger seeds than plants with 15 to 22 chromosomes.     

芥菜型油菜与诸葛菜属间杂种F1的可育性研究

通过胚胎救的快速繁殖，首次成功地获得了芥菜型油菜与诸葛菜的远缘杂种。杂种植株具有期望的染色体数２ｎ＝３０，具有较好的结实性，花粉萌发率为４２．８％。对杂种Ｆ１的细胞学观察表明：杂种细胞内存在染色体组分割机制，可形成ｎ＝１８和ｎ＝１２的大小配子，从而导致杂种可育的结果。     

Rapid estimation of ploidy levels in in vitro-regenerated interspecific Arachis hybrids and fertile triploids

Summary A significant correlation among chromosome number, chloroplast number and pollen grain size was observed using interspecific hybrids (2n=30,60), Arachis hypogaea (2n=40), A. stenosperma (PI 338280) (2n=20), A. batizocoi (PI 468329) (2n=20) and A. villosulicarpa Hoehne (PI 336984) (2n=20), representing four ploidy groups. Both chloroplast number in guard cells and pollen grain size were found to be positively correlated with ploidy, and provided a reliable method to distinguish diploid, triploid, tetraploid and hexaploid plants from each other regardless of their taxonomic backgrounds. These methods in combination with root-tip chromosome counts enabled us to confirm ploidy determinations on all three dissue layers, L₁, L₂ and L₃ (guard cells, microsporocytes and root meristematic cells, respectively), and to detect the chimeric nature of some colchicine-treated tissue culture-derived plants. Screening pollen grains by size and the detection of highly stainable and viable, 30-chromosome pollen grains have enhanced the use of triploid hybrids in peanut breeding programs.     

Crosses between Beta intermedia bunge and B. vulgaris L.

Summary A tetraploid annual male sterile form of Beta vulgaris L. (2n=4x=36) was crossed with the wild beet species Beta intermedia Bunge (2n=36). The resulting F₁-plants were male sterile annuals being two or three times back-crossed to diploid and tetraploid sugar and fodder beets in the next years. Apart from tetraploid material (36 chromosomes) hexaploid (54 chromosomes) and a number of aneuploid plants developed.The results obtained justify the conclusion that, at a tetraploid level the material mostly propagates apomictically after the F₁ generation. The presence of penta-, hexa-, septa-and even octaploid plants might be explained by assuming that no meiosis has taken place in the crossing partners. Triploid plants are sometimes found in the progeny of hexaploid material and may presumably be considered haploids. Moreover some pentaploid plants were found in the progeny of the open pollinated F₁ which after two generations of bagging are still pentaploids although they produce no pollen. This is another clear indication of apomictic reproduction.The tetraploid generation from the cross between the hexaploid material and diploid sugar beets probably contains the best prospects for breeding.