含硫氨基酸基因植物表达载体的构建及对百脉根的转化

在本研究中,用限制性内切酶Nco Ⅰ和Bgl Ⅱ从中间载体pMD18zeolin和pMD18zein 上切下zeolin基因(约1 600bp)和γ-zein基因(约720 bp),将其定向连接在经相同酶切的质粒载体pCAMBIA1302上,构建成植物表达载体pCBzeolin和pCBzein.采用冻融法将pCBzeolin和pCBzein导入根癌农杆菌(Agrobacterium tumefaciens)菌株LBA4404,利用该菌株转化百脉根(Lotus corniculatus L.).经过共培养、筛选分化、再生,得到抗件植株.对抗性植株进行了PCR、RT-PCR检测表明,γ-zein和zeolin基因已经整合到百脉根基因组中,在核酸水平得到了表达.含硫氨基酸数据分析表明,转zeolin基因植株含硫氨基酸含量极显著高于转γ-zein基因植株和对照植株的含量(P＜0.01),但转γ-zein基因植株与对照植株间差异不显著.     

Effect of selection and temperature stress on the production of 2n gametes in Lotus tenuis

Five Lotus tenuis genotypes showing 2n pollen production greater than 5% and producing 4x progenies in 4x(male sterile Lotus corniculatus) × 2x crosses were found in different natural populations. Meiotic analyses of these plants showed some second metaphases with disoriented spindles, i.e. parallel and tripolar spindles which resulted in diads and triads, respectively. Because useful genes for winter growth and resistance to powdery mildew from L. tenuis can be introgressed into L. corniculatus via 2n gametes, a programme of selection was started to increase the occurrence of 2n gamete production in L. tenuis. Controlled crosses were performed between genotypes producing 2n gametes and the progenies were checked for 2n pollen production and meiotic aberration, following the same methodologies as used for the parental plants. Although in some progeny plants the frequency of 2n gamete production increased remarkably, the overall progeny level of 2n gamete production remained unchanged. This suggested that different genes for meiotic aberration exist in natural populations and/or differential seed-setting ability exists between genotypes. Diallel crosses between selected genotypes confinned this hypothesis. Evaluation of 2n pollen production of clones grown under different temperature regimes (warm vs. temperate) showed that, in L. tenuis, the effects of genotype, temperature and genotype × temperature interaction are significant. In particular, high temperature increases the level of 2n gamete production in L. tenuis.     

百脉根高频再生体系的建立及兔出血症病毒衣壳蛋白VP60基因的转化

以MS为基本培养基,通过调整激素种类与浓度等培养条件,按正交试验设计的原则,建立了百脉根高频再生体系.结果表明MS 2,4-D 2.0 mg/L KT 2.0 mg/L 培养基可高效诱导愈伤组织的形成,MS 6-BA 1.0 mg/L NAA 0.1 mg/L 培养基可高效诱导芽的分化,MS NAA 0.1 mg/L 培养基可快速诱导根的生成,形成再生植株.通过构建植物表达载体VP60-pBI121,研究了根癌农杆菌介导的兔出血症病毒(RHDV)衣壳蛋白VP60基因对百脉根遗传转化的影响因素,建立了百脉根快速高效遗传转化体系,结果表明,以农杆菌LBA4404为介导菌株、以下胚轴为外植体,预培养3 d,在OD600为0.6的菌夜中浸染20 min,共培养3 d,以及300 mg/L 羧苄青霉素脱菌浓度和50 mg/L 卡那霉素筛选浓度为最佳转化条件.为利用百脉根生产动物口服型疫苗建立了技术基础.     

The occurrence of 2n-pollen and the origin of sexual polyploids in dihaploid roses (Rosa hybrida L.)

Haploidisation by in situ parthenogenesis of 4x R. hybrida resulted in the production of some dihaploid roses (2n=2x=14) able to produce viable pollen. A cytological study of microsporogenesis revealed that, although the first meiotic reductional division occurred normally, the second (equational) division was characterised by frequent abnormalities which concerned spindle formation and led to unreduced gametes of First Division Restitution (FDR) type. Analysis of the hybrid progeny of a parthenogenetically derived male fertile dihaploid plant revealed a selective advantage of the 2n-pollen, especially in the case of an hybridization with a tetraploid female parent. Moreover, crosses carried out among dihaploid partners always resulted in hybrids with ploidy levels≥ 3x. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance and expression of transgenes in T2 and T3 generations of Lotus corniculatus transformed using Agrobacterium tumefaciens

The inheritance and expression of the reporter gene uidA, encoding β-glucuronidase (GUS), was previously analysed in the T₁ generation of 25 independent transformed lines of Lotus corniculatus cv. Leo. In the work reported here, GUS activity in various tissues of seven of these lines was tested in the T₂ generation. Four representative lines were chosen for more detailed study in the T₃ generation. Lines 25 and 38 had multiple, independently segregating transgene inserts; lines 24 and 39 appeared to transmit one segregating transgene insert to their T₁ progeny, although transgene expression was low and was detected in fewer seedlings than expected in line 39. The uidA gene was inherited and expressed in seedlings of T₁, T₂ and T₃ generations of all four lines. In all lines, transgene expression varied between tissues, with more embryos than seedlings having detectable GUS activity. Studies in the T₂ generation showed that use of transgenic plants as female or male parents altered the frequency of expression of the transgene in progeny. By contrast, in the T₃ generation the use of transgenic plants as female or male parents did not effect either frequency of transmission, or expression of the transgene, in any of the four lines. Transgene inheritance was also similar among individual pods within flower heads and between individual flower heads. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Mitosis Observation on the Tetraploid Lilium oriental and 2n Gamete Hybrid Offspring of Lilium oriental

对东方百合四倍体正常植株,变异植株,2n配子杂种后代进行有丝分裂观察,结果表明四倍体正常植株的有丝分裂均正常而变异植株出现染色体桥现象;2n配子杂种后代多数植株有丝分裂正常,少数植株的有丝分裂极不正常出现了染色体桥和染色体落后,双核及多极分裂现象,从而产生染色体数目的多样性。     

山东莲藕(Nelumbo nucifera Gaertn.)种质资源的RAPD鉴定和遗传多样性分析

山东莲藕(Nelumbo nucifera Gaertn.)栽培历史悠久,在中国北方具有重要影响。但是,山东莲藕种质资源研究一直没有开展。为收集和保存莲藕种质资源,本研究从山东省8个种植面积较大的地区采集了76份莲藕材料,并利用RAPD分子标记对这些材料进行了遗传相似性分析,共鉴定出38份种质资源。遗传多样性指数分析显示,38份种质资源的Nei's多样性指数(H)平均值为0.26,Shannon信息指数(I)平均值为0.41。研究数据表明,山东莲藕种质资源相对丰富,但要建立高质量的种质资源圃,仍需要加强种质资源的引进。     

绿豆雄性不育突变体msm2015-1的遗传学与细胞学分析

单产水平低一直是我国绿豆存在的产业问题。绿豆杂种优势显著是提高产量的有效途径,而雄性不育材料是实现杂种利用的宝贵资源。本研究利用~(60)Co-γ辐射诱变技术首次在绿豆中获得雄性不育突变体,并命名为‘不育绿豆2015-1’(male sterile mungbean2015-1,简写为msm2015-1)。msm2015-1营养生长期表型与‘苏绿1号’没有明显差异。花器官形态正常,但花药发白且不能正常开裂散粉,成熟期座荚率几乎为0。遗传分析显示,育性分离群体中可育与不育分离比符合3∶1,表明不育性状由单个隐性核基因控制。花粉发育的细胞学分析发现, msm2015-1的盛开花朵中花粉粒几乎未在柱头附着,体外亦未萌发。花粉细胞质未被Alexander染液正常着色, I_2-KI染色出现典败、圆败和染败3种类型。DAPI染色显示, msm2015-1花粉细胞核发育异常。醋酸洋红染色表明,败育发生在花粉发育早期,花粉母细胞在四分体时期减数分裂的不对称和异常多分裂是导致花粉败育的主要原因。     

The implications of 2n egg gametes in nobilization and breeding of sugarcane

Summary Nobilization in sugarcane is viewed from a genetic angle considering the significance of 2n gametes. The inbreeding coefficients resulting from 2n gametes, for first and second nobilizations are worked out. The implication of 2n gametes to sugarcane breeding are discussed.     

Pollen viability and meiotic behaviour in intraspecific hybrids of Hydrangea aspera subsp. aspera Kawakami group x subsp. sargentiana

Pollen viability and male meiosis in intraspecific hybrids of Hydrangea aspera subsp. aspera Kawakami group (2n = 2x = 36) and subsp. sargentiana (2n = 2x = 34) were investigated. Although it had been assumed that they were sterile, pollen viability was observed; it varied from 2.5% to 12.1%. The production of gametes with different chromosome numbers was implied by the analysis of the dispersion of the diameter distribution of pollen grains. Analysis of male meiosis made it possible to identify the origins with two major categories of meiotic aberrations: abnormal chromosome distribution (early chromosome migration at metaphase, lagging chromosomes at anaphase, micronuclei at telophase), leading to the formation of unbalanced tetrads and/or ones with supernumerary microspores; and abnormal spindle orientation in metaphase II (tripolar, fused and parallel spindles), leading to the formation of dyads or triads. The mode of 2n pollen formation is of the First Division Restitution type. The high level of parental heterozygosity that is normally associated with them should facilitate the transfer of a polygenic trait in breeding programme.     

Incidence and molecular markers of 2n pollen in Populus tomentosa Carr.

Microscopic examination, amplified fragment length polymorphism (AFLP) and SCAR (sequence-characterized amplified region) molecular markers were employed to determine the incidence of 2n pollen (unreduced pollen) in Chinese white poplar (Populus tomentosa Carr.) and to identify related molecular markers. The presence of a parallel and tripolar spindle at metaphase II and the absence of cytokinesis at telophase II were found to be determining factors in 2n pollen formation. A group of 298 clones that originated from their indigenous areas were investigated for the production of 2n pollen based on pollen size differences, both within a clone and between n and 2n pollen. Pollen grains were collected from 224 of the clones, six of which were subsequently determined to produce only normal pollen; the remainder produced 2n pollen at different frequencies (0.6–21.9%). The frequency at which 2n pollen was produced was significantly and highly significantly different among and within indigenous populations, respectively. Clones produced by the six normal and twenty-two 2n pollen clones were selected for AFLP analysis. Following an initial screening with 55 primer combinations, the E50-M38 (CAT/ACT) primer was identified: it generated a PCR fragment (246 bp) from the normal clones, but not from the 2n pollen producers. In addition, the E31-M50 (AAA/CAT)-amplified DNA fragment (204 bp) was present in 2n pollen producers, and absent in normal clones. These two discriminating AFLP markers were developed into easily detectable SCAR (sequence characterized amplified region) markers which can be used in combination with previously developed AFLP markers to distinguish between normal and 2n pollen clones.     

Reproductive behaviour of tetraploid alfalfa plants obtained by unilateral and bilateral sexual polyploidization

The objective of the present study was to evaluate the fertility of tetraploid plants obtained from diploid mutants that produced 2n gametes via bilateral (BSP) and unilateral sexual polyploidization (USP) schemes. Controlled matings between selected plants from BSP and USP were carried out according to a complete diallel. The level of male and female fertility of each plant was estimated within full-sib, half-sib and non-inbred crosses. Crosses with unrelated self-fertile and male-sterile testers were also performed. Pollen size and stainability were determined by acetocarmine staining. The number of ovules per ovary was counted and the callose deposition within ovules examined to evaluate embryo sac fecundity. Cross-fertility was generally much higher for USP than BSP plants (on average, 0.3 vs. 0.03 seeds per flower pollinated). Both male and female fertility were inversely related to the inbreeding level of cross combinations. The in vitro pollen germinability was positively correlated to male fertility of BSP plants. The level of in vivo embryo sac sterility was not correlated to female fertility in either BSP or USP plants. Female fertility was restored in both BSP and USP groups when plants were crossed with unrelated tetraploid testers. Male fertility also increased in USP plants but remained rather low in the BSP plants. Scaling up the ploidy level by means of 2n gamete union resulted in tetraploid plants with low fertility. In particular, the BSP process yielded virtually self-sterile and highly cross-sterile plants. The well documented positive effect of sexual tetraploidization on forage yield was accompanied by worsening of fertility traits. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tracing of 2n egg occurrence in perennial ryegrass (Lolium perenne L.) using interploidy crosses

Several genotypes of Lolium perenne (2n = 2x = 14) were screenedfor the production of 2n eggs. In each cross a diploid genotype, used asfemale parent, was pollinated with a tetraploid genotype. All interploidycrosses were made in isolation cages. Flow cytometrical analysis of theprogeny of 154 diploid plants revealed 28 tetraploid descendants. Theseoffspring plants were harvested on 15 different diploid mother plants.Amplified Fragment Length Polymorphism (AFLP) markers were used tocheck the parenthood of these tetraploids. Two seedlings died, theremaining plants could, after AFLP fingerprinting, be identified as meiotictetraploids. The results obtained allowed to conclude that the tetraploidprogeny plants harvested on 15 diploid mother plants were the result ofthe fertilisation of an unreduced egg by a reduced pollen grain of thetetraploid plant.     

植物生长调节剂克服甘薯近缘三倍体杂种2n花粉系与甘薯杂交低结实性的研究

甘薯品种与三倍体种间杂种2n花粉系杂交,用4种不同的植物生长调节剂配方克服其低结实性,首次获得有生活力的后代植株.观察上述杂交的花粉粒萌发,花粉管生长,卵细胞受精,胚发育及种子形成的过程,探讨了植物生长调节剂在其中的作用。结果表明,各种不同的植物生长调节剂配方都可以在一定程度上提高结实率,其中以配方100     

Cytogenetic studies and the occurrence of triploidy in the wild potato species Solanum commersonii Dun.

Summary The South American wild potato species Solanum commersonii Dun. exists as two subspecies, subsp. commersonii and subsp. malmeanum (Bitter) Hawkes and Hjerting, and as both diploid and triploid cytotypes. The two subspecies were completely cross fertile but there were reciprocal differences in the number of seeds set per berry and the mean seed weight. Crosses between diploids and colchicine-induced autotetraploids yielded only a few seeds per berry, but 16 of the 18 germinated seeds were triploid or nearly so. Such a high proportion of triploids is unusual for such crosses in potatoes. Some triploids has a high percentage of stainable pollen. The origin of naturally occurring triploids is discussed in the context of 2n gametes, a triploid block, and endosperm balance numbers.     

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.     

Analysis of quantitative variability for seed yield and related characters in Lotus corniculatus L. cv. Leo

Summary Quantitative variability for seed yield and six other characters was analysed in Lotus corniculatus L. cv. Leo. The material consisted of 144 polycross progenies and 100 parents.Wide variability existed for all characters. The characters with the greatest variability were seed yield, forage grading and plant height. The polycross progeny test was employed to study the general combining ability of the parents. Highly significant differences existed for all seven characters under study.Parent-offspring genotypic and phenotypic correlations were high and significant for all characters except genotypic correlations for seed yield and seeds per pod. High h² values (broad sense) were obtained for seed size and days to flowering. Traits with moderate to high h² were seed yield (71% in parents, 64% in progenies), plant height, forage grading, and seeds per pod. The character pods per inflorescence had the lowest h².Positive estimates of % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabiGaaiaacaqabeaadaqaaqaaaOqaaiqabo8agaqcaK% aaavaabeqaceaaaeaacaqGYaaabaGaaeiraaaaaaa!3A89!\[{\text{\hat \sigma }}\begin{array}{*{20}c} {\text{2}} \\ {\text{D}} \\ \end{array} \] were obtained only for seed size. The ratio of dominance variance to additive variance indicated partial dominance for this character. Except for seed yield, in all other cases these estimates had very high sampling errors. In all cases except pods per inflorescence and seeds per pod high positive estimates of % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabiGaaiaacaqabeaadaqaaqaaaOqaaiqbeo8aZzaaja% qbaeqabiqaaaqcaauaaiaaikdaaKaaGeaacaqGbbaaaaaa!3B30!\[\hat \sigma \begin{array}{*{20}c} 2 \\ {\text{A}} \\ \end{array} \] were obtained.The data indicated that it may be possible to simultaneously improve seed yield and maintain forage yield. Seed yield had positive and significant associations with seed size, seeds per pod and pods per inflorescence. The associations of days to flowering with forage grading (negative) and with pods per inflorescence (positive) were also significant.     

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.     

马铃薯2x-2x,4x-2x和2x-4x杂交产生的四倍体杂种块茎产量和特性的优势表现及遗传分析

利用能产生2n配子的二倍体杂种与四倍体普通栽培种(S.tuberosum L.)和四倍体新型栽培种(Neo—tuberosum)进行了2x-2x,4x-2x和2x-4x间的杂交,获得了18个杂交组合的4x杂种家系,将这些家系与其10个4x亲本品种(品系)及对照品种在同一地点进行了连续两年的比较试验。结果表明:4x杂种家系单株平均块茎产量比亲本两年分别高16％和19％;其中最好的6个杂种家系比较好的4个品种(品系)分别高20％和43％;杂种产量的优势主要表现在单株块茎数目的显著增多,但在块茎商品率方面则与4x亲本差异不显著,而且杂种块茎的表现还不如4x亲本及对照品种,这是由于具2n配子特性的2x亲本这些性状较差所致。试验结果还表明杂种的遗传背景愈复杂其杂种优势也愈强,进一步说明马铃薯的产量与遗传异质性(多样性)的强弱可能有明显关系,利用2n配子特性可获得较高的遗传异质性。