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.     

毛白杨植物络合素合酶(PtPCS)基因克隆及其表达研究

植物络合素(PCs)是植物细胞中一类螯合重金属离子的多肽,对细胞解毒和重金属的富集有重要的作用。植物络合素合酶(PCS)是合成PCs途径中的关键酶。本研究克隆了毛白杨的植物络合素合酶基因PtPCS,该基因cDNA序列全长1512bp,可编码503个氨基酸;与其它8种植物同源基因比对显示,它们的氨基酸序列相似性在67%～74.1%之间;构建进化树发现,这9种植物明显的分为两大分支,并且两大分支PCS蛋白的高级结构显示出明显的差异,一种呈月牙状,一种为伞状,重金属超富集植物均为月牙状结构。对毛白杨在不同镉浓度处理下PtPCS基因的表达情况分析发现,毛白杨根、茎、叶中PtPCS基因均表现出先升高后缓慢降低的表达趋势,并且在同一镉浓度处理下,毛白杨不同器官PtPCS基因的表达具有规律性,即根表达量最强,茎次之,叶片最弱。研究结果不仅为植物育种提供了重要的基因资源,也为剖析毛白杨的镉抗性和富集特性的研究奠定了良好的基础。     

Development of molecular markers to assess the level of introgression of Populus tremula into P. alba natural populations

Morphological traits traditionally adopted to discriminate between Populus alba L. and P. tremula L. have frequently led to misclassifi‐cation of their spontaneous hybrid P. × canescens Sm. Moreover, they may not be of any help in cases of spontaneous backcross phenomena. These limitations can be overcome by molecular markers, which are not environmentally influenced nor subjectively assessed. In this study, the effectiveness of amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers in species and hybrid discrimination was evaluated by analysing a set of reference samples of P. alba, P. tremula and P. × canescens. Species‐specific and species‐indicative AFLPs, as well as diagnostic SSR alleles, were recorded in both P. alba and P. tremula reference samples. The results allowed a clear distinction between the two poplar species and their hybrid. Using these diagnostic markers, a natural population of P. alba trees sampled along the Ticino river basin in northern Italy was analysed, and P. × canescens individuals, intermingled with P. alba trees, were detected.     

Establishment of reliable methods of in vitro pollen germination and pollen preservation of Brassica rapa (syn. B. campestris)

A simple and reliable method for evaluating the viability of Brassica pollen was established in which the in vitro germination rate of pollen was adopted as the index of the viability of pollen grains. Pollen grains were preincubated in an atmosphere in which the relative humidity (RH) was fixed to 52% or 66% at 20 °C for 5 hours. They were cultured for 16 hours at 25 °C in a liquid Kwack's medium (1964) supplemented with 20% sucrose, and the pH was adjusted to 8.0. They were then observed under a microscope and the number of germinating and unchanged pollen grains were counted. The germination rate of pollen was improved and stabilized by preincubation and the use of a high pH medium. More than 90% of the freshly harvested pollen grains of Brassica rapa (syn. B. campestris) germinated constantly in these conditions Undehisced anthers were collected from flowers at anthesis and dehydrated by incubation at 20 °C for 16–24 hours in an atmosphere where the RH was fixed to 15% or 32%. They were put into a plastic vial and preserved in a freezer at -20 °C. The germination percentage of the preserved pollen was scored at intervals during preservation. The germination rate of the pollen grains preserved at -20°C for 1 year was higher than 50% and the pollen proved to be efficient for seed set. Most of the seeds germinated normally. This revised version was published online in August 2006 with corrections to the Cover Date.     

Occurrence of 2n and jumbo pollen in the Brazilian ginseng (Pfaffia glomerata and P. tuberosa)

An analysis of microsporogenesis was carried out in four populations of Pfaffia tuberosa (2n=4x=68) and in ten populations of P. glomerata(2n=2x=34). Monads, dyads and triads were reported at high frequency in all populations of P. tuberosa. In P. glomerata, on the other hand, only four populations presented monads, and at low frequency. Dyads and triads were observed in both species, albeit at a higher frequency in P. tuberosa populations. Certain abnormalities of spindles in the second division, such as sequential and tripolar spindles, led to triad formation by the rejoining of two chromosome sets. The precise causes of monad and dyad formation have not been found during meiosis, although evidence exists that dyads originated by omission of second meiosis and monads by omission of both meiotic divisions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetics of isozyme variants in Populus tremula,P. tremuloides and their hybrids

Summary Eight Populus tremula and six P. tremuloides clones as well as 49 full-sib families were studied in GOT, LAP, 6-PGDH, and SKDH by horizontal starch gel electrophoresis. For GOT one polymorphic zone was found and segregation of seven full-sib families suggests Mendelian inheritance. For LAP and 6-PGDH two zones each were clearly scored. For LAP two polymorphic loci were proposed based on the phenotypic segregation of isozyme variants in six and 34 full-sib families, respectively. In 24 full-sibs families the presence of null alleles was inferred for both loci. The genetic control of the upper zone of 6-PGDH was demonstrated by a segregation analysis of 17 full-sib families. SKDH also demonstrated a Mendelian inheritance pattern in 12 of the full-sib families analysed. The electrophoretic patterns of pollen were similar to those of buds, but migration rates of the supposed corresponding isozymes were slightly modified (Lap-B, Skdh, 6Pgdh-A). Lap-A was not present in pollen extracts and hybrid bands were not found when gels were stained for dimeric enzymes (6-PGDH, GOT).     

Induction of 2n pollen in tulips by arresting the meiotic process with nitrous oxide gas

Triploid tulips have agronomically desirable traits such as vigorous growth and large flower size, but only a portion of all cultivated tulips is triploid. To apply 2n pollen to polyploid breeding of tulips, the polyploidizing agent, nitrous oxide gas (N₂O), was applied to bulbs. In tulips, meiosis in anthers occurs inside the bulbs from mid- to late-October. When meiosis in anthers (excised from bulbs) reached metaphase I, we treated other bulbs of the same clones with N₂O for 24–48 h. Most of the treated plants produced pollen grains with a wide-ranging or bimodal size distribution, indicating a mixture of n, 2n and aneuploid pollen grains. The use of pollen containing a relatively high proportion of giant pollen grains tended to yield larger numbers of triploids in the progeny. The number of giant pollen grains could be increased when N₂O-treated pollen grains were suspended in 10% sucrose and then sieved through a nylon mesh. Very few polyploids were observed in some cross combinations, even those involving pollen with a relatively high proportion of giant grains. Even so, this low polyploid yield most likely is due to a triploid block, because the capsules obtained in the crosses of the diploid×N₂O-treated plants contained some abnormal seeds, which were mostly triploid. Embryo culture was useful in rescuing abnormal embryos. The present study reveals that 2n pollen can be produced at high frequency using N₂O during tulip breeding.     

Pollen and pollination experiments. V. An empirical basis for a mentor pollen effect observed on the growth of incompatible pollen tubes in pear

Summary The self-incompatible pear cultivar Doyenne du Comice was selfed with the aid of the mentor pollen technique (self pollen mixed 1:1 with compatible pollen) and the pioneer pollen method (compatible pollen applied 14 h in advance of the self pollen). Observations on tube growth in the style showed that inviable methylated pollen was ineffective either as mentor or pioncer pollen, having no effect on the performance of the self pollen which stopped growing at about one quarter of the style from the stigma. Calculations made on the basis of the obtained data indicated that the viable untreated or irradiated pioneer and mentor pollen, the former somewhat better than the latter, aided the self pollen tubes to reach the base of the style.     

Identification and cytological analysis of 2n-pollen producers in Lotus tennis Wald. et Kit.

Two meiotic mutants of L. tennis (2n = 2x = 12) producing unreduced pollen are described. When crossed to male sterile L. corniculatus (2n = 4x = 24) plants, all progeny plants were morphologically similar to L. corniculatus, had 2n = 24 chromosomes, and in the cross, were fully compatible with L. corniculatus, indicating that the male parent plants were 2n-pollen producers. One of them also had ‘giant’ pollen grains. In metaphase II of both genotypes, there were parallel and tripolar spindles leading to dyad and triad formation, the latter being found most frequently. Since both the above-mentioned mechanisms result in first-division restitution-type microspores, the genotypes examined could be useful in breeding Lotus.     

Meiotic restitution mechanisms and 2n pollen formation in a Solanum tuberosum dihaploid and in dihaploid × wild species hybrids*

A Solatium tuberosum dihaploid that proved to be a 2n pollen producer, exhibited two types of meiotic abnormality involved in 2n pollen formation: premature cytokinesis at prophase II and parallel/tripolar spindles in meiosis II. For this reason such a dihaploid was preferentially used as a seed parent in crosses with the diploid species S. tarijense, S. sanctae-rosae, and S. phureja. Diploid interspecific hybrids were evaluated for male fertility, for 2n pollen production and stability over three years and for meiotic nuclear restitution mechanisms. Pollen stainability ranged from 6 to 85% and 2n pollen from 5 to 35% among hybrids. The meiotic mechanism generating 2n pollen was that of spindle abnormality in meiosis II and consequent dyad/triad formation. High correlation was found between fused/parallel spindles and dyads.     

The site of action of the crossability genes (Kr 1, Kr 2) between Triticum and Secale. I. Pollen germination,pollen tube growth,and number of pollen tubes

Summary Seven genotypes of common wheat (Triticum aestivum L.) were crossed with rye (Secale cereale L.) in order to find the site or sites of action of the crossability genes, Kr ₁ and Kr ₂, of wheat. The data obtained, by fluorescence microscopy, were compared to the controls (wheat x wheat). The results indicate that the crossability genes have little effect on pollen germination and on the time taken for the pollen tubes to reach the microphyle, irrespective of their crossabilities with rye. The number of pollen tubes reaching the microphyle is, however, affected by the Kr-genes, as high crossable genotypes have more pollen tubes than the low crossable ones. There was a high correlation between the mean number of pollen tubes at the micropyle with seed set, which also reflects the crossability. The Kr-genes seem to manifest themselves in the retardation and inhibition of pollen tube growth between the style base and the top of the embryo sac, where the effect is most distinct in the low crossable genotypes.     

Carbamate-induced flowering in asparagus (Asparagus officinalis L.) seedlings: optimization of treatment and cultivar variation in flowering response and pollen germination

Optimal carbamate treatment conditions were studied for flower induction in seedlings of an asparagus cultivar ‘Mary Washington 500 W’. Flower induction was most accelerated by soaking seeds in 50 mg l⁻¹ carbamate solution for 12 days at 25 ^°C under the fluorescent light. Longer exposure to carbamate over a 12 day period induced a higher percentage of seedlings to flower. A higher percentage of flowering seedlings were male. Flower induction frequency among seven cultivars through carbamate treatment widely ranged from 13 to 67%. ‘Geynlim’, ‘Mary Washington 500 W’ and ‘Welcome’ exhibited a high percentage of flowering seedlings, while ‘Larac’ and ‘Vulkan’ showed low values. Only male flowers were induced in all-male cultivars. Variation in pollen germination was found within all cultivars. ‘Geynlim’, ‘Cito’ and ‘Mary Washington 500 W’ showed high values of average pollen germination. Application of carbamate compound to induce flower production can rapidly produce homogenic cultivars which include both sexes. This is necessary for genetic studies and breeding purposes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of molecular markers linked with crown rust (Puccinia coronata f. sp. lolii) resistance in perennial ryegrass (Lolium perenne) using AFLP markers and a bulked segregant approach

Crown rust resistance is an important selection criterion in ryegrass breeding. The fungal disease caused by P. coronata causes yield loss and a reduced quality of the fodder crop. Molecular markers were used to unravel the genomic organization of crown rust resistance in a segregating L. perenne population. Two genomic regions involved in crown rust resistance were identified that together explained 35% of the phenotypic variance present. Bulked segregant analysis in combination with AFLP markers was a suitable method to identify DNA markers associated with genomic regions of major effect. One cluster of AFLP markers explained 6.1% of the variance and mapped to linkage group 2, a genomic region known to contain crown rust resistance genes. A second cluster of AFLP markers detected a novel genomic region of major effect that explained 27.7% of the phenotypic variance in crown rust resistance. This cluster was unlinked to the cluster on linkage group 2. Divergent selections performed within the segregating F₁ population on the basis of genotype and phenotype revealed that the markers associated with crown rust resistance identified in this study have potential for marker assisted selection. Selection of plants on the basis of markers was more straightforward than the selection on the basis of phenotype.     

AFLP and SCAR markers linked to the suppressor gene (Rf) of a dominant genetic male sterility in rapeseed (Brassica napus L.)

Rs1046AB is a line which is true breeding for a dominant genetic male sterility gene (Ms) but which is a mixture of male fertile and sterile individuals (a two-type line) because it is segregating for a dominant suppressor gene (Rf). This system provides a promising alternative to the CMS system for hybrid breeding in Brassica napus. In order to identify molecular markers linked to the rf gene, a near-isogenic line (NIL) population from the cross between a sterile individual (MsMsrfrf) and a fertile individual (MsMsRfrf) in Rs1046AB was subjected to amplified fragment length polymorphism (AFLP) analysis, with a combination of comparing near isogenic lines (NILs) and bulked segregant analysis (BSA). From 2,816 pairs of AFLP primers, six fragments showing polymorphism between the fertile and sterile bulks as well as the individuals of the bulks were identified. Linkage analysis indicated that the six AFLP markers are tightly linked to the Rf gene and all are distributed on the same side. The minimum genetic distance between the Rf gene and a marker was 0.7 cM. Since the AFLP markers are not suitable for large-scale application in MAS (marker-assisted selection), our objective was to develop a fast, cheap and reliable PCR-based assay. Consequently, three of the four closest AFLP markers were converted directly to sequence characterized amplified region (SCAR) markers. For the other marker a corresponding SCAR marker was successfully obtained after isolating the adjacent sequences by PCR Walking. The available SCAR markers of the Rf gene will greatly facilitate future breeding programs using dominant GMS to produce hybrid varieties.     

Crop breeding for salt tolerance in the era of molecular markers and marker‐assisted selection

Crop salt tolerance (ST) is a complex trait affected by numerous genetic and non‐genetic factors, and its improvement via conventional breeding has been slow. Recent advancements in biotechnology have led to the development of more efficient selection tools to substitute phenotype‐based selection systems. Molecular markers associated with genes or quantitative trait loci (QTLs) affecting important traits are identified, which could be used as indirect selection criteria to improve breeding efficiency via marker‐assisted selection (MAS). While the use of MAS for manipulating simple traits has been streamlined in many plant breeding programmes, MAS for improving complex traits seems to be at infancy stage. Numerous QTLs have been reported for ST in different crop species; however, few commercial cultivars or breeding lines with improved ST have been developed via MAS. We review genes and QTLs identified with positive effects on ST in different plant species and discuss the prospects for developing crop ST via MAS. With the current advances in marker technology and a better handling of genotype by environment interaction effects, the utility of MAS for breeding for ST will gain momentum.     

Transmission of bacterial wilt resistance by First Division Restitution (FDR) 2n pollen via 4x×2x crosses in potatoes

Summary Resistance to bacterial wilt (Pseudomonas solanacearum) found in tuber-bearing Solanum species was transferred into a diploid potato breeding population. Simultaneous selections were made for agronomic characters, production of first division restitution (FDR) 2n pollen, and resistance. Diploid resistant genotypes were identified via inoculation with a virulent isolate (CIP-204) of race 3 of Pseudomonas solanacearum. These resistant diploid genotypes were crossed to susceptible tetraploid potatoes. An investigation was made to assess whether resistant diploid genotypes transmit resistance to bacterial wilt, which is a quantitatively inherited trait, to tetraploid potatoes via FDR 2n pollen. Tetraploid seedlings from 4x×2x crosses were inoculated with the same isolate CIP-204, and the percentage of surviving seedlings was scored. Some 4x×2x families from resistant diploid genotypes demonstrated a high level of survival rate. The transmission of bacterial wilt resistance was achieved by the use of FDR 2n pollen. It was speculated that a female x male interaction effect on the survival rate of the evaluated progeny may exist. Selecting proper 4x and 2x parents would be important for obtaining a higher frequency of transmission of resistance to bacterial wilt in the progeny.     

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.     

Identification of AFLP and SSR markers linked with the male fertility restorer gene of CMS 06J45 in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

In this study, AFLP and SSR techniques were combined with the bulk segregant analysis (BSA) method to map the restorer gene BrRfp using an F₂‐segregating population comprising 258 individuals developed by crossing the polima (pol)‐like cytoplasmic male sterility (CMS) line 06J45 and the restorer line 01S325 of heading Chinese cabbage. A survey of 2048 AFLP primer pairs identified 21 polymorphic fragments, approximately half of which exhibited high similarity with the A09 chromosome sequence of Brassica rapa in the Brassica database (BRAD). Based on the genome sequence, three specific AFLP fragments linked with BrRfp were successfully converted into sequence‐characterized amplified region (SCAR) markers, named SC1233, SC2673 and SC2141. Subsequently, 178 pairs of SSR primers were redesigned for further screening, with five producing polymorphic amplification patterns. Linkage analysis showed that these markers were distributed along both sides of the BrRfp gene, with two markers, SSR03 and SSR2528, co‐segregating with the BrRfp locus in the F₂ population. These results may be valuable for marker‐assisted selection and map‐based cloning in heading Chinese cabbage.     

利用大白菜抗感干烧心病F2群体构建AFLP遗传连锁图

大白菜(BrassicacampestrisL.ssp.pekinensis)原产于中国,是我国蔬菜栽培中分布最广、种植面积最大的蔬菜作物之一,也是我国的主要出口创汇蔬菜之一。但是近年来大白菜干烧心病却严重影响了其内部质量和出口创汇。此病是由于缺钙引起的生理性病害,不同品种间在抗干烧心病特性上表现出明显的差异,选育抗干烧心病品种值得引起关注。本实验利用大白菜离体叶片扦插方法,结合多年青岛农科院研究人员的田间观察对大量的大白菜品种进行广泛筛选,得到了抗病品种和感病品种。以大白菜抗干烧心病品种F1-6-5-2-2-2-3和感干烧心病品种P14-2-1-15杂交的F2代115个单株作为构建遗传图谱的群体,通过对192对引物组合的筛选,利用32对引物得到了280个AFLP多态性位点,经Jionmap3.0软件处理,得到了一张含105个标记位点、11个连锁群、覆盖长度为669.7cM的连锁图。每个连锁群上的标记数在4￣27之间,平均图距在3.3￣12.5cM之间,连锁群长度在23.4￣98.9cM之间。105个AFLP位点中偏离孟德尔遗传规律的比率为39.0%。为大白菜干烧心QTL定位和分析奠定了基础。     

Factors affecting haploid production in wheat using the Hordeum bulbosum system. 1. Genotypic and environmental effects on pollen grain germination,pollen tube growth and the frequency of fertilization

Summary A quantitative examination of pollen grain germination and pollen tube growth within the overy wall was made in selected wheat × tetraploid H. bulbosum hybridizations, to investigate the effect of the wheat and the H. bulbosum genotype on these characters. As expected, variation at the known crossability loci had no effect on pollen grain germination. The frequency of pollen tube penetration of the ovary wall was, however, severely reduced when the dominant alleles were present. Pollen tube penetration was nevertheless observed in 3 of the 110 ovaries examined of the non-crossable cultivar Highbury. The H. bulbosum genotype had a much smaller effect on these characters, but significant differences between the clones were observed in the numbers of pollen tubes initially penetrating the ovary wall. Although two H. bulbosum genotypes showed no significant differences in the number of ovaries with pollen tubes at the base of the ovule, significant differences in the frequency of fertilization were observed. The possible cause of this discrepancy is discussed.The frequency of fertilization in crossable wheat × H. bulbosum hybridizations was improved by the application of gibberellic acid within 10 minutes of pollination, and reduced by an increase in the ambient temperature from 20°C to 26°C. Fertilization following the pollination of non-crossable wheat genotypes was not affected by either of these factors.