长寿花嫩叶片的组织培养研究

以长寿花嫩叶片为外植体,进行了不同灭菌时间对长寿花嫩叶片再生能力的影响实验,不同培养基对长寿花嫩叶片愈伤组织诱导及不定芽生根的影响实验。实验结果表明,长寿花嫩叶片的最佳消毒方法为,75%酒精灭菌30 s,无菌水冲洗4次,再用0.1%升汞消毒12 m in,最后无菌水冲洗7次;长寿花嫩叶片的最佳分化培养基为MS+6-BA3.5 mg/L+NAA0.2 mg/L;长寿花不定芽在1/2MS+IBA 0.15 mg/L+6-BA 0.15 mg/L上生根效果最好。通过实验获得了长寿花嫩叶片的组培技术,为大规模生产长寿花提供了相关技术支撑。     

The origin and chromosome numbers of cultivars of Kalanchoe blossfeldiana Von Poelln.: Their history and evolution

Summary Kalanchoe blossfeldiana was described by Von Poellnitz in 1935. It is an endemic species of Madagascar where it was discovered in 1924 by Perrier de la Bâthie, who described it as the variety coccinea of K. globulifera. By 1930 living plants had been spread throughout Europe and the United States. At the same time commercial growing was initiated by Blossfeld at Potsdam, Germany. Selection within the species and, from 1939, hybridisation with several other Kalanchoe species yielded cultivars from which many have disappeared. K. blossfeldiana, like two selections, has 2n=34 chromosomes. The remaining cultivars, which are likely of hybrid origin, are polyploids. They are usually tetraploids (2n=ca 68), but in some cases numbers of 2n=72, 75, 84, 85 and 96 have been counted. Polyploidy already occurred in 1939, when the first interspecific cross, in this case with K. flammea (2n=34), was recoreed. Other species which have been crossed with K. blossfeldiana are K. pumila (2n=40), K. grandiflora (2n=34), K. schumacheri (2n=?), K. kirkii (2n=?) and K. manginii (2n=34).     

Production and characterization of inter-sectional hybrids between <Emphasis Type="Italic">Kalanchoe spathulata</Emphasis> and <Emphasis Type="Italic">K. laxiflora</Emphasis> ( = <Emphasis Type="Italic">Bryophyllum crenatum</Emphasis>)

The genus Kalanchoe is currently divided into section Kalanchoe and section Bryophyllum, and there has been no successful report on the production of inter-sectional hybrids. Therefore, reciprocal crosses were made between Kalanchoe spathulata (sect. Kalanchoe) and K. laxiflora (sect. Bryophyllum) in order to obtain basic information on the reproductive barriers between these two sections. The seeds were aseptically germinated in vitro and the plants were grown in greenhouse till flowering. When K. spathulata was used as a maternal donor, 39 out of 80 plants showed intermediate characteristics between K. spathulata and K. laxiflora. In contrast, no plants were obtained in the reverse crosses. Hybridity of these plants was confirmed by flow cytometric analysis, chromosome numbers and RAPD analysis. Bulbil formation on the leaf margin as one of the conspicuous characteristics of K. laxiflora was not observed in the hybrids. Some of the hybrid lines showed some pollen fertility, but failed to yield viable seeds by self-pollination or backcross-pollination. Successful production of the inter-sectional hybrid between the two species suggests that they are not so distantly related as considered previously.     

长寿花叶盘离体培养及植株高效再生研究

以长寿花叶盘做为外植体进行再生体系的建立,通过一定的激素组合诱导,结果表明MS+6-BA2.0 mg/L +NAA0.5 mg/L组合愈伤组织诱导率可达86.6%,再生率达83.3%,外植体叶盘的摆放方式也对再生有显著影响,叶盘叶面向上放置,有利于其再生,反之不利于再生。加入AgNO3（4.0mg/L）溶液,对芽苗的分化有影响,再生苗开瓶练苗一周后容易生根,移苗入土后生长正常。     

大叶落地生根SAHH基因功能鉴定

S-腺苷-L-高半胱氨酸水解酶(S-adenosyl-L-homocysteine hydrolase,SAHH)是已知的唯一能够水解S-腺苷高半胱氨酸(S-adenosylhomocysteine,SAH)成半胱氨酸和腺苷的酶.SAH作为转甲基反应的抑制剂,与S-腺苷甲硫氨酸(S-adenosyl-L-methionine,SAM,体内甲基供体)的比值常用来作为衡量植物整体甲基化水平的重要指标.为鉴定大叶落地生根(Kalanchoe daigremontiana) SAHH的功能,本研究根据已知的大叶落地生根SAHH基因(KdSAHH)序列(GenBank登录号:KF953475)设计引物,对其进行干旱胁迫下的表达分析、亚细胞定位分析以及烟草(Nicotiana tobacum)转化,并对T1代苗用20％ PEG6000模拟干旱处理,测定0、12和24 h的相对含水量(relative water content,RWC)、叶绿素(chlorophyll,Chl)含量、丙二醛(malondialdehyde,MDA)含量、过氧化物酶(peroxidase,POD)含量、超氧化物歧化酶(superoxide dismutase,SOD)含量等与抗旱相关的生理指标.结果表明,KdSAHH基因在轻度(20 d)、中度(35 d)、重度(50 d)干旱胁迫下表达量依次升高,表明该基因与抗旱性相关;亚细胞定位结果显示,该蛋白定位于细胞质与细胞核;对转基因烟草进行PCR检测,获得9株阳性植株;测定干旱胁迫下的相关生理指标,结果显示,与野生型烟草相比,转KdSAHH烟草在干旱胁迫下能够保持相对稳定的RWC、Chl含量以及相对较低的MDA、POD含量.与野生型相比,转KdSAHH烟草受伤害轻,抗旱性增强,表明KdSAHH基因能够提高烟草植株的抗旱性.研究结果丰富了KdSAHH基因的功能,为后续研究该基因提供了借鉴和参考.     

长寿花离体快繁研究

[目的]研究长寿花离体快繁的培养基配方和培养条件。[方法]以长寿花的幼嫩叶片作为外植体,以MS为基本培养基,诱导出不定芽进行快速繁殖,并比较添加4种浓度的BA和NAA对长寿花不定芽的诱导和生根效果的影响。[结果]长寿花幼嫩叶片的诱导和生长与生长素、细胞分裂素2种激素有关系,两者的配比直接影响了叶片的再生频率。长寿花叶片诱导和不定芽增殖的最适培养基为MS+6-BA1.0 mg/L+NAA0.5 mg/L,最适生根培养基为1/2 MS+NAA0.5 mg/L,不定芽在此培养基上的生根率最高,移栽成活率为95%。通过直接诱导可获得较高的不定芽诱导率,同时增殖培养的增殖系数达到10,大大缩短了常规育苗时间。[结论]该研究建立了长寿花组织培养的再生体系,为其快速繁殖及大规模的工厂化育苗提供科学依据。     

应用SMARTer cDNA合成技术扩增落地生根叶片的总RNA

以落地生根(Kalanchoe daigremontiana)未长芽和长芽的叶片为材料,提取落地生根的总RNA,采用SMAR-Ter cDNA合成技术合成cDNA第1链,优化扩增第2链,通过设置梯度PCR扩增循环数,电泳分析后鉴定cDNA质量。结果表明:未长芽和长芽的材料各1μg总RNA分别成功扩增质量较高的双链cDNA。此方法的应用解决了研究材料有限的问题,可由微量的总RNA扩增出足够多的高质量双链cDNA,为进一步从基因水平研究落地生根奠定基础。     

Isolation and Characterization of a New Virulence Gene (abvA) of Agrobacterium tumefaciens

A mutant (M-1) was isolated by transposon (Tn5) insertion mutagenesis of Agrobacterium tumefaciens (strain A-208, C58 chromosome, nopaline type T37 pTi, virulent). The M-1 mutant exhibited a complete avirulent phenotype on Kalanchoe daigremontiana leaf and Kalanchoe pinnata stem but a very attenuated virulent phenotype on root of Daucus carota. The mutant had one insertion of Tn5 in pTi. A wild-type target segment (2.3 kb) that included the site of Tn5 insertion in M-1 mutant was cloned. Introducing the 2.3 kb segment into M-1 complemented completely the avirulent phenotype, producing galls as big as strain A-208. The 2.3 kb segment was sequenced, identifying three open reading frames, ORF 1 (354 bp), ORF 2 (261 bp) and ORF 3 (801 bp) in the segment. A Tn5 was inserted between the third and fourth nucleotide of ORF 1 in M-1. The ORF 1 had no homology to any reported genes and thus was named the abvA gene. The ORF 3 had the high homology (identities 44%, positive 68%) to the gene of the sarcosine oxidase β subunit (accession no. sp/P40875). Introduction of the DNA segment (743 bp) containing the abvA gene and its promoter region into M-1 partially complemented the avirulent phenotype of the mutant, producing galls smaller than strain A-208. The abvA gene was distributed not only on nopaline-type pTi (T37) but also on octopine-type pTi (A6NC) and chromosome (C58) of A. tumefaciens. M-1, being avirulent on K. daigremontiana and K. pinnata, had a Tn5 insertion only in the abvA gene on pTi but not in the abvA gene on the chromosome, implying that the abvA gene on the chromosome in strain A-208 is not functional. A binary vector, pIG121-Hm, containing the β -glucuronidase (GUS) gene with an intron was introduced into M-1, which was then applied to leaves of K. daigremontiana to assay GUS activity for monitoring T-DNA transfer to the host nucleus. High GUS activity comparable to that in strain A-208 was detected in M-1 in spite of its inability to induce galls, suggesting that M-1 can transfer T-DNA into the host nucleus, but cannot integrate it into the chromosome. Received 25 October 2000/ Accepted in revised form 28 December 2000     

Phytophthora nicotianae transmission and growth of potted kalanchoe in two recirculating subirrigation systems

Recirculating subirrigation systems are frequently exposed to the risk of plant pathogens transmission, which may deteriorate the growth and quality of the plants. The transmission of Phytophthora nicotianae was examined using Kalanchoe blossfeldiana cv. New Alter in two recirculating subirrigation systems, a nutrient-flow wick culture (NFW) system and an ebb and flow (EBB) system. When the nutrient solution was infested, the pathogen was recovered from roots in both subirrigation systems. However, foliar blights and browning of roots appeared 4 and 7 weeks, respectively, after inoculation in the EBB system. Only a little discoloration appeared in the NFW system. The fresh and dry weights were lower in the EBB system than in the NFW system. When growing medium was inoculated, the pathogen was unable to be isolated from the plants in the NFW system. However, disease symptoms appeared in the EBB system 4 weeks after inoculation, and the pathogen was observed in the basal leaves and roots. Similar to the infested nutrient solution, the plant growth in the EBB system was inhibited. These results suggested that when the nutrient solution was infested, pathogen transmission could occur in plants in both systems, although differences existed with regard to disease symptoms and the time it took for symptoms to appear. However, we observed that when growing medium was inoculated the pathogen was not transmitted to adjacent plants in the NFW system using wick.     

土壤逐渐干旱过程中长寿花叶片生理指标的动态变化

为探讨在土壤逐渐干旱胁迫过程中,长寿花生理生化特性与抗旱性的关系,选取相对含水量、膜透性、可溶性糖和脯氨酸4项指标,研究了其动态变化规律.结果表明土壤干旱胁迫下,与对照相比,相对含水量下降,膜透性、可溶性糖和脯氨酸上升.