Aflp markers tightly linked to the sex locus in Asparagus officinalis L.

Nine AFLP markers linked to the sex locus in Asparagus officinalis L. have been identified by non-radioactive AFLP technique and bulked segregant analysis. A composite map of one F₂ and two F₁ populations identified three very tightly linked markers. These markers did not give recombinants in the three different populations and mapped 0.5, 0.7 and 1 cM to the sex locus. Codominant scoring of the markers in the F₂ population from a selfed andromonoecious plant could distinguish the XX, XY and YY asparagus plants. The AFLP markers were isolated from the gel and cloned into plasmid vectors. The marker E41M50, which is a low-copy sequence and did not give any recombinants in the screened populations, detected polymorphism between female and male plants when used as RFLP probe. The AFLP markers we obtained are important to plant breeding, particularly in the development of sex specific PCR primers that could be used in the screening of different asparagus plants at the seedling stage. They are likewise important in the elucidation of the mechanisms underlying sex determination and differentiation in this species.     

Isozyme gene markers in the dioecious species Asparagus officinalis L.

Summary Extracts from phylloclads of Asparagus officinails were electrophoretically analyzed for isozyme polymorphism. Fourteen enzyme systems were examined using four buffer systems: seven enzymes (acid phosphatase, catalase, glutamate-oxaloacetate transaminase, isocitrate dehydrogenase, malate dehydrogenase, peroxidase, and 6-phosphogluconate dehydrogenase) exhibited clear and consistent banding patterns. Isozyme polymorphism was studied in seven pairs of male and female doubled haploids and in their male F₁s. Segregation of polymorphic loci was examined in the backcross progenies and was found to be consistent with a simple Mendelian inheritance in all cases, except for three anodical peroxidases, where two factors have been hypothesized. No linkage could be found between isozyme markers that were segregating in the same cross, but association was demonstrated between one malate dehydrogenase locus and the sex determining genes. The availability of isozyme markers may be useful in breeding and, in particular, the localization of one malate dehydrogenase locus on the sex chromosomes may be helpful in mapping the sex genes.     

芦笋皂苷的抗肿瘤作用研究进展

芦笋是一种常见蔬菜,富含多种营养物质,在多种疾病的预防和治疗中发挥良好的药理效应。芦笋中的甾体皂苷是其生物活性的主要表现物质,现已从芦笋中分离出的皂苷单体有19种。本文概述了它们的来源及结构,对其中已被报道的几种皂苷单体在肿瘤预防和治疗方面的作用、机理及研究进展加以综述,为进一步分离新的芦笋皂苷单体及其对肿瘤的预防和治疗提供参考。     

A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers

 An integrated genetic map of the dioecious species Asparagus officinalis L. has been constructed on the basis of RFLP, RAPD, AFLP and isoenzyme markers. The segregation analysis of the polymorphic markers was carried out on the progeny of five different crosses between male and female doubled-haploid clones generated by anther culture. A total of 274 markers have been organized to ten linkage groups spanning 721.4 cM. Since the haploid chromosome number of asparagus is ten, the established linkage groups probably represent the different chromosomes; however, the only group associated with a specific chromosome is the one which includes sex, whose determinant genes have been located on chromosome 5. A total of 33 molecular markers (13 RFLPs, 18 AFLPs, 2 RAPDs and 1 isoenzyme) have been located on this chromosome. The closest marker to the sex determinant is the AFLP SV marker at 3.2 cM. Received: 26 March 1998 / Accepted: 30 April 1998     

Change of Superoxide Dismutase Activities During Somatic Embryogenesis in Asparagus officinalis L.

The superoxide dismutase activities in callus and somatic embryoids at different developmental stages of Asparagus officinalis L. were determined by means light of induced oxidation-reduction of nitro blue tetrazolium. It was shown that the superoxide dismutase activity was higher in callus than in somatic embryoids at different developmental stages. The activity increased with growth, differentiation and maturation of somatic embryoids during somatic embryogenesis. The relations between superoxide dismutade activity and somatic embryogenesis were discussed.     

几种因素对白芦笋试管苗生根的影响

白芦笋的不同筛选株系之间生根能力存有差异;培养温度是影响生根率的关键因素,高温[≥(25±1)℃]不利生根,发根的适宜培养温度为(20±1)℃;低光照强度(≤25mmol·m-2·s-1)对生根不利,适宜的光合光量子通量密度(PPFD)为40μmol·m-2·s-1;PP333、嘧啶醇不能提高试管苗生根率,且浓度过高还会降低生根率和抑制植株生长。     

Cytohistological Study on Somatic Embryogenesis in Asparagus officinalis L.

The single cell was the first cell of somatic embryoid in Asparagus officinalis L.. Electron microscopic observations revealed that early embryogenic cells and meristematic cells had the same characteristics in cell shape and structure. The embryogenic cell was small in size, with large nucleus, dense cytoplasm, thin wall, lots of small vacuoles, and rich in organelles. At later stage, the polarity of embryogenic cell appeared, i.e. nucleus situated at one end of the cell, while the other end was occupied by a large vacuole. The polar type of the embryc)genic cell was similar to that of a zygote. Light microscopic observations revealed :hat ihe embryoids were sequentially differentiated through 2-cells, 4-cells, 8-cells, multicellular proembryo, globularshaped, pear-shaped, rod-shaped, cotyledonary-differentiated and mature embryo stages. The heart-shaped and torpedo-shaped stages were observed during the early stage of embryogenesis. In addition, a typical embryoids were also found in vitro.     

芦笋性别决定与性别分化研究进展

从芦笋性别表现及其决定的遗传基础、性别分化途径,性别决定基因的定位以及性别分化特异表达基因的分离与分析等方面来综述芦笋性别决定与性别分化最新研究进展。目前,已构建了围绕芦笋性别决定基因M比较精细的遗传图谱,将M定位在L5染色体着丝点附近的0.63 cM区域内,并构建了含有8个跨叠克隆群的物理图谱,但由于大量重复序列的存在,跨叠克隆之间的空隙不能闭合;同时先后分离得到11个芦笋花器官发育特异表达基因,并通过序列分析和原位杂交等技术对这些基因的功能进行了分析。最后,对今后进一步研究提出了建议。     

RP-HPLC法测定芦笋中黄酮类化合物芦丁的含量

以芦丁标准品为对照,利用反相高效液相色谱法对芦笋中黄酮类化合物芦丁的含量进行定量测定。采用Agilent Eclipse XDB-C18色谱柱,柱温25℃,流动相由甲醇-水-磷酸(55∶44.5∶0.5)组成,流速为0.7 mL/min,检测波长390 nm。结果表明黄酮类化合物中各组分基线分离良好;进样量在0.07~0.7μg/20μL范围内,峰面积A与进样浓度C呈良好的线性关系,回归方程为A=1 5176C-10.388,相关系数R2=0.999 4;加样回收率为101.051%,RSD=3.306%;以保留时间和峰面积作精密度试验,RSD分别为0.199%和1.24%。该方法样品处理简单,准确度高,精密度好,适合于芦笋中芦丁含量的测定。     

芦笋皂苷的抗肿瘤作用研究进展

芦笋是一种常见蔬菜,富含多种营养物质,在多种疾病的预防和治疗中发挥良好的药理效应。芦笋中的甾体皂苷是其生物活性的主要表现物质,现已从芦笋中分离出的皂苷单体有19种。本文概述了它们的来源及结构,对其中已被报道的几种皂苷单体在肿瘤预防和治疗方面的作用、机理及研究进展加以综述,为进一步分离新的芦笋皂苷单体及其对肿瘤的预防和治疗提供参考。     

芦笋茎叶游离氨基酸的提取及含量测定

通过对芦笋茎叶游离氨基酸提取工艺中温度、提取时间、料液比、乙酸浓度等影响因素的试验分析,确定芦笋茎叶游离氨基酸测定过程中的最佳提取条件为：提取温度60℃;料液比为1：35;提取时间为2．5h;并对芦笋茎叶游离氨基酸含量进行了测定,确定芦笋茎叶游离氨基酸含量为76．54mg／100g。     

Development of sex-linked PCR markers for gender identification in Actinidia

 Two sex-linked random amplified polymorphic DNA (RAPD) markers identified from Actinidia chinensis were converted into sequence-characterised amplified regions (SCARs) for the large-scale screening of Actinidia breeding populations. Initial SCAR primers converted one RAPD (SmX) into a dominant marker, but the other (SmY), which was potentially more useful because of its linkage to the male determining ‘Y’ locus, failed to retain polymorphism. This difficulty was overcome by cloning and sequencing the alternate ‘allele’ from female plants, and then designing ‘allele’-specific primers that utilised nucleotide differences between the sexes. Using a quick squash-blot method of DNA extraction, the SCAR primers were tested in 120 A. chinensis plants to determine their gender. The system is now in use for large-scale screening of seedling populations in the Actinidia breeding programme. The sex-linked SCAR primers also functioned with plants from some other geographically separate accessions of A. chinensis and with plants in the closely related polyploid species A. deliciosa, but did not amplify a sex-linked band in more distantly related species of Actinidia. Received: 27 December 1997 / Accepted: 5 March 1998     

石刁柏胚性细胞诱导过程中的内源激素和多胺含量变化

用高效液相色谱法分析石刁柏愈伤组织胚性细胞诱导过程中不同时期内源激素和多胺含量的结果表明,在胚性细胞诱导过程中,Put和GA3一直呈上升趋势,胚性细胞出现时,IAA、Put和GA3含量都达到最高水平,显示高含量的IAA以及高比例的Pu“（Spd＋Spm）可能有利于胚性细胞的形成。     

Identification of two markers linked to the sex locus in dioecious <Emphasis Type="Italic">Asparagus officinalis</Emphasis> plants

One hundred decamer primers of random-amplified polymorphic DNA were tested on dioecious Asparagus officinalis plants to identify sex-linked molecular markers. One primer (S368) produced two markers (S368-928 and S368-1178) in female plants. These two DNA markers were identified in 30 male and female plants, respectively, and a S368-928 marker was proved to be linked to the female sex locus. The female-linked S368-928 marker was sequenced and specific primers were synthesized to generate a 928 bp marker of sequence characterized amplified regions (SCAR) in female plants, SCAR₉₂₈. SCAR₉₂₈ could be used to correctly screen homozygous mm female plants of A. officinalis. However, results of Southern blot analysis suggest that the hybridization pattern of S368-928 was presented in both sex plants. This text was submitted by the authors in English.     

The MADS box gene AOM1 is expressed in reproductive meristems and flowers of the dioecious species Asparagus officinalis

MADS box genes are implicated in different steps of plant development. Some of them are expressed in vegetative organs. Most of them, however, are expressed in flower tissues and are involved in different phases of flower development. Here we describe the isolation and characterization of an Asparagus officinalis MADS box gene, AOM1. The deduced AOM1 protein shows the highest degree of similarity with FBP2 of Petunia hybrida and AGL9 (SEP3), AGL2 (SEP1) and AGL4 (SEP2) of Arabidopsis thaliana. In situ hybridization analyses, however, show that the expression profile of AOM1 is different from that of these genes: AOM1 is expressed not only in flower organs but also in inflorescence and flower meristems. These data indicate a possible function of AOM1 during flower development as well as in earlier stages of the flowering process. Asparagus officinalis is a dioecious species which bears male and female flowers on different individuals. AOM1, which is expressed very early during the process of flowering and has a similar expression profile in male and female flowers, does not seems to be involved in asparagus sex differentiation. Received: 3 July 2000 / Revision accepted: 4 August 2000     

正交试验优选超声提取芦笋总皂苷的工艺

目的:优选超声提取芦笋总皂苷的最佳工艺。方法:以高氯酸作为显色剂,用紫外分光光度法测定芦笋中总皂苷的含量,并以提取率为评价指标,采用单因素实验和正交试验优选最佳提取工艺。结果:芦笋总皂苷超声提取的最佳工艺为乙醇浓度70%,料液比1:15(W/V),超声时间50 min,超声温度40℃。结论:该提取工艺可行,为芦笋总皂苷的进一步研究提供了依据。     

Improved callus formation and plant regeneration for shed microspore culture in asparagus (Asparagus officinalis L.)

 To establish an efficient asparagus microspore culture system, experiments were conducted to investigate the effects of medium components, period of cold pretreatment for flower buds, and period of anther co-culture on culture response. All factors affected the frequency of asparagus microspore division and the yields of microspore-derived calli. The best results were obtained by pretreating genotype G459 flower buds at 4  °C for 7–9 days, co-culturing anthers with shed microspores for 14 days, and including 6% sucrose, 2 mg l^–1α-naphthaleneacetic acid and 1 mg l^–1 N⁶-benzylaminopurine in the culture medium. After 4 days of culture, most shed microspores contained starch-like bodies and died. The 2% of shed microspores lacking these structures divided to produce microcalli. For the best treatments in the different experiments, about 140 calli per 100 anthers were recovered. Cultured on four different regeneration media, 19.6–21% and 3.9–8.0% of microspore-derived calli produced shoots and embryos, respectively, and ultimately plantlets, among which 49% were haploid, 34% diploid, 4% triploid and 11% tetraploid. Received: 3 September 1998 / Revision received: 25 November 1998 / Accepted: 5 December 1998     

Optimization of the choice of molecular markers for varietal identification in Vitis vinifera L.

 The aim of this study was to develop a cultivar identification tool based on molecular analysis and a statistical approach. From the PIC parameter we defined the D parameter, which evaluates the efficiency of a primer for the purpose of identification of varieties; i.e. the probability that two randomly chosen individuals have different patterns. D can be used to compare different types of markers even if only the allelic frequencies are known. We used this parameter to develop an algorithm for selecting the optimal combination of primers necessary to identify a set of varieties. The optimal combination of primers determined for a small elite group of varieties applied on a larger set induces a risk of confusion involving 1 of the elite varieties. We estimated the risk of confusion using the D value of each primer of the combination. We applied this methodology on a set of 224 varieties of Vitis vinifera screened with 21 RAPD primers and two microsatellite loci. The discriminating power of the primers did not only depend on the number of patterns it generates but also on the frequencies of the different patterns. A combination of 8 primers (6 RAPD and two microsatellite) was found to be optimum for the discrimination of these 224 varieties. A subset of 38 elite varieties was also investigated. The determined optimal combination of 4 primers (3 RAPD and one microsatellite) applied on the 224 varieties gave 9 risks of confusion involving 1 of the elite varieties. Confusion can happen between varieties with the same origin as well as between varieties of very diverse geographical origins. Received: 22 April 1998 / Accepted: 9 June 1998     

Interactions of ancymidol with sucrose and α-naphthaleneacetic acid in promoting asparagus (Asparagus officinalis L.) somatic embryogenesis

Interactions of varying ancymidol concentrations with those of α-naphthaleneacetic acid (NAA) or sucrose in embryo induction medium were related to the production and development of asparagus (Asparagus officinalis L.) somatic embryos, and to the ability of these embryos to germinate. A significant sucrose×ancymidol interaction was observed only for the production of bipolar embryos; 4% sucrose with 0.75 mg l^–1 ancymidol gave the best result, 78 g^–1 callus. The frequency of globular embryos decreased as sucrose or ancymidol concentrations increased. Sucrose concentration affected embryo germination; 3% and 4% sucrose were optimal with approximately 60% and 40% of bipolar and globular embryos germinating, respectively. Significant ancymidol×NAA interactions were observed for the production of bipolar and globular embryos and their germination. Varying ancymidol concentrations affected embryo production and germination in combination with 0.1 mg l^–1 NAA, but not with 1.0 mg l^–1 NAA. The treatment combination of 0.1 mg l^–1 NAA with 0.75 mg l^–1 ancymidol produced the most bipolar embryos, 64 g^–1 callus, and the greatest percentages of bipolar and globular embryos germinated, 63% and 42%, respectively. Received: 19 August 1996 / Revision received: 24 April 1997 / Accepted: 22 May 1997     

外源腐胺对石刁柏愈伤组织胚性能力的影响

石刁柏胚性愈伤组织继代过程中,添加浓度为10 mg·L^-1的外源腐胺能有效地保持愈伤组织的胚胎发生能力,并减少愈伤组织的褐化,但不能提高愈伤组织的体细胞胚的诱导率.腐胺处理过的胚性愈伤组织的内源腐胺含量明显提高.这可能是外源腐胺保持细胞胚性、降低褐化程度的原因.