大叶种胡椒实生苗茎尖培养和合子胚培养研究

利用各种表面消毒方法对采自海南岛三个地区的胡椒大田植株的外植体进行消毒试验,由于内源性污染,除胡椒成熟种子外,其它各种大田外植体的表面消毒均未能成功。以胡椒成熟种子无菌萌发的实生苗茎尖作外植体,在1/2MS(MS或B5)+1.5mg/LBA+0～0.2mg/LIAA(或NAA)上可实现丛生芽增殖。茎尖水平或竖直接种方法显著影响茎尖的增殖;水平接种茎尖的生长和增殖效果优于竖直茎尖接种方式。茎尖增殖率随BA浓度的增加而提高,但BA浓度大于2.0mg/L时会使苗芽的质量降低,愈伤组织产生严重,苗芽细小,抽出不明显,颜色发黄甚至变白。附加或不附加100mg/LAdSO4对丛生芽增殖没有明显影响。生根培养基以1/2MS+1.0mg/LIBA+0.5～1.0mg/LIAA为最优,生根率可达100%;在细沙∶土∶椰糠(1∶1∶1)的基质中常规炼苗,成活率可达98%以上。液体纸桥法对胡椒种胚进行培养,在不附加任何生长调节物质的培养基(MS、B5或SH)上只产生单苗,而在附加不同种类和不同浓度的生长调节物质的培养基上则诱导形成愈伤组织,但未能实现分化;以胡椒无菌萌发的实生苗胚轴和叶片切段作外植体进行培养,较易诱导产生愈伤组织,但难以实现分化。     

叶子花的组织培养与微繁技术研究

叶子花茎尖、茎段接种在MS附加不同浓度组合的6-BA、IAA、IBA、NAA培养基上可诱导茎尖及腋芽的生长而获得无性系芽,无性系芽茎尖和茎段在MS附加6-BA0．5mg／L,NAA0．05mg／L进行继代培养,一般不形成丛生芽,以茎尖生长和腋芽萌生增殖,增殖系数为2．73;高度大于3cm的增殖芽在1／2 MS附加IAA lmg／L、IBA lmg／L、NAA0．2mg／L培养基上生根率为80．5％,生根苗用“二步法”移栽成活率在97％以上。无性系芽的幼叶、茎段在MS附加6-BA和NAA、2,4-D培养基上能高频产生愈伤组织,但愈伤组织在所试验的所有培养基上均无不定芽或胚状体的分化。     

香水白掌的组织培养和快速繁殖

1植物名称白鹤芋属品种香水白掌（Spathiphyllum“Xiangshui”）。2材料类别茎段、茎尖。3培养条件以MS为基本培养基。诱导丛生芽培养基：（1）MS＋6－BA4～6mg·L-1（单位下同）＋NAA0．2。丛生芽增殖培养基：（2）MS＋6－BA2～4＋NAA0．2;（3）培养基（2）＋KH2PO485。4生长与分化情况4．1丛生芽的诱导取香水白掌茎段,剥去外层叶片,75％酒精消毒lmin,然后用灭菌净浸泡20～35min,切取茎尖和茎段,置于培养基（1）上培养。7d后开始出现侧芽萌发和茎尖萌发成苗。30d后将萌发苗基部切割,置于培养基（1）继续培养,2周后出现了…     

黄山药丛生芽诱导与植株快速繁殖

采用黄山药的带芽茎段为外植体,试验了不同激素处理对芽诱导的影响,通过继代培养诱导丛生芽使大量增殖,经诱导生根和炼苗移栽而完成植株的快速繁殖再生。结果表明,MS 6-BA2.0mg/L NAA0.5-1.0mg/L为芽诱导的最适培养基,MS 6-BA1.0mg/L NAA0.5-1.0mg/L为继代增殖的最佳培养基,月增殖系数约为3倍,1/2MS 1BA0.5mg/L培养基诱导生根相对较好,诱导率为50%,组培苗经炼苗后,移栽成活率可达80%。     

亳芍茎尖组织培养的研究

选取亳芍茎尖为试验材料,探究不同培养条件对亳芍组织培养的影响,结果表明:亳芍茎尖在1/2MS+6-BA 1.0 mg/L培养基上培养39 d后,茎尖分化出芽的同时也形成较多的丛生芽;丛生芽在1/2MS+6-BA1.0mg/L培养基上增殖速度最快;来自不同启动培养基上的丛生芽在相同培养基上,接种15 d后观察,不同来源的丛生芽长势不同,30 d后仍存在一定的差异;幼苗在1/2MS+IBA 0.1生根效果最好。     

大花美人蕉茎尖组织培养技术研究

以大花美人蕉(Canna×generalis)根茎茎尖为外植体进行组织培养技术研究,筛选出芽诱导适宜的培养基为MS+6-BA 8.0mg/L (单位下同)+TDZ 0.03;MS+6-BA 8.0+TDZ 0.03+NAA 0.1培养基能较好地诱导分化出丛生芽,继代增殖培养中与MS+6-BA 3.0+TDZ 0.03+NAA 0.1培养基交替使用可减少畸形芽,增殖系数达1.67;适宜的生根培养基为MS+6-BA 1.0+NAA 0.5,生根率达66.67%,且植株生长健壮,移栽易成活。     

芋茎尖离体培养和快速繁殖

对芋优良品种莱阳毛羊头进行茎尖培养和快速繁殖。试验表明,外植体表面灭菌的最佳方案是乙醇→新洁尔灭→剥幼叶→升汞。在较软的培养基中,茎尖大于0．6mm,成活率为90％以上。不定芽分化的最适培养基为MS＋BA1（mg／L以下单位相同）＋NAA0．2＋Spm20,其分化率为100％。不定芽在MS＋BA2＋NAA0．1＋Spm50的培养基中,可实现继代繁殖和诱导生根一步完成,月增殖系数达7．3,生根率88．3％。芋试管苗移栽于沙壤土基中,成活率为90％以上,且生长健壮,根系发达。     

人参果的脱毒和快速繁殖

用人参果无菌实生苗茎尖进行组织培养,经过5代高温加剥离茎尖培养的方法,可以完全脱除病毒。无菌小苗茎尖接种在MS 6-BA1．0mg／L NAA0．1mg／L 2．4-D0．10mg／L的培养基上能够产生淡黄色愈伤组织,继续培养分化出较多芽点;把带芽点的愈伤组织转移至MS 6-BA1．0mg／L IAA0．05mg／L的培养上可分化出丛生芽且继代增殖效果较好;丛生芽在1／2MS NAA0．1mg／L PP3330．2mg／L的培养基上易生根,可以获得健壮完整的小苗。     

连钱草组培快繁技术研究

用连钱草无菌茎尖为外植体进行快速繁殖,分别诱导、分化、生根形成再生植株进行快速繁殖,并移栽成活。结果表明在MS+6-BA1.5mg/L+NAA0.1mg/L培养基上诱导丛生芽效果最佳。在MS+IBA1.0mg/l+KT1.0mg/L培养基中根的诱导率为100%。     

丹参离体微繁技术研究

以丹参(Salvia miltiorrhiza Bunge)离体幼茎、叶、叶柄为外植体,对其丛生芽、不定芽的诱导和增殖、生根、移栽等方面进行系统研究,探讨了有关丹参的离体快速微繁技术。试验表明：MS 6-BA1．0mg／L是诱导初代培养的芽产生丛生芽的最佳培养基,其诱导生芽率为100％,丛生芽增殖的最佳培养基为MS 6-BA1．0mg／L NAA0．01mg／L;以叶为外植体,用MS 6-BA 0．5～2．0mg／L诱导不定芽可取得较好效果,其诱导生芽率为100％,不定芽增殖的最佳培养基为MS 6-BA1．0mg／L,其增殖倍数达24倍;诱导生根较好的培养基为1／2MS 0．1mg／L IBA,移栽先水培再土培,成活率可达100％。     

In vitro selection of gamma irradiated shoots of ginger (Zingiber officinale Rosc.) against Fusarium oxysporum f.sp. zingiberi and molecular analysis of the resistant plants

In the present investigation, an attempt was made to develop Fusarium yellows resistant plants of ginger (Zingiber officinale Rosc.) var. Himgiri using in vitro mutagenesis and selection technique. Axenic in vitro cultures were subjected to gamma irradiation (10–100 Gy) for mutation induction. LD₅₀ was calculated after every 4 weeks, and was observed to be 15 Gy after 16 weeks of irradiation. Surviving 10 Gy irradiated shoots were cultured on selective medium containing different concentrations (0–20%) of fungal culture filtrate (FCF) obtained from Fusarium oxysporum f.sp. zingiberi for in vitro selection. FCF concentration of 17.5% in the selective medium was found to be the highest on which 5% shoots survived after first selection cycle, which further reduced to 1.1% after third continuous cycle of selection. It was followed by 4.3% shoot survival after third selection cycle on 15% FCF concentration. Surviving, selected shoots from 15 and 17.5% FCF were multiplied on multiplication medium and rooted plantlets were hardened with 100% survival. On in vivo evaluation 46.4 and 52% plants selected at 15 and 17.5% FCF were found to be highly resistant. Molecular analysis with disease specific SSR primers differentiated between FCF selected, tissue culture propagated and gamma irradiated plants. Two unique bands were obtained in 15 and 17.5% FCF selected plantlets with GIN 6 and GIN 9 primers, sequencing, of which showed 98 and 97% homology with disease resistance protein-like gene CC-NBS-LRR from clones ZwP627 and ZoP620 of Z. officinale. After in vivo bioassay, SSR analysis of selected highly resistant plants again confirmed the unique bands with both the primers. The DNA sequences obtained from these primers have been published in GenBank under accession number MN497252 and MN497253.

     

通过组织培养筛选小麦抗赤霉病突变体的研究

选用中抗赤霉病的春小麦品种和品系的花药进行离体培养,以小麦赤霉病29号菌株产生的致病培养滤液为选择剂,结合物理诱变处理,进行抗病鉴定,用赤霉病菌分生孢子直接接种在愈伤组织和再生植株筛选抗病突变体。在83块愈伤中有53块抗病。在11株的再生植株中有9株均比未经培养滤液处理的对照提高了抗病性,从中选出4株抗病性接近或超过“苏麦3号”品种。     

In vitro selection of yellow passion fruit genotypes for resistance to <Emphasis Type="Italic">Fusarium</Emphasis> vascular wilt

Fusarium vascular wilt (caused by Fusarium oxysporum f. sp. passiflorae) is a limiting factor in the cultivation of yellow passion fruit (Passiflora edulis). Since there is no effective and economically viable control available, development of resistant or at least tolerant cultivars are in demand. A number of procedures have been used for the initial selection of plant genotypes resistant to various fungal pathogens by means of a fungal culture filtrate or purified toxin. In this study, seeds and in vitro-grown plantlets of passion fruit were screened with different concentrations of either Fusarium oxysporum f. sp. passiflorae (FOP) culture filtrate (0, 20, 30, 40 or 50%, v/v) or fusaric acid (0.10, 0.20, 0.30 or 0.40 mM) supplemented in Murashige and Skoog (MS) basal media. Subsequently, selected plants were inoculated with a conidial suspension of FOP to assess correlation between in vivo and in vitro responses. In vitro sensitivity to the selective agents and the resistance response to the pathogen were also compared. Root growth was markedly influenced by FA, culture filtrate, and conidial suspension culture treatments. Observations indicated that roots were primary targets for attack by F. oxysporum. Successful in vitro selection of resistant genotypes by both FA and culture filtrate treatments suggested that this strategy was viable for accelerating breeding of passion fruit for resistance to the Fusarium vascular wilt.     

In-vitro selection of Vitis vinifera `Chardonnay' with Elsinoe ampelina culture filtrate is accompanied by fungal resistance and enhanced secretion of chitinase

 Proembryogenic masses of grapevine (Vitis vinifera L.) `Chardonnay' (clone 02Ch) were exposed to the culture filtrate of Elsinoe ampelina (deBary) Shear, the causal agent of anthracnose disease. After four or five cycles of recurrent in-vitro selection with medium containing 40% fungal culture filtrate, putative resistant lines RC 1 and RC 2 respectively, were established. The selected lines inhibited the growth of E. ampelina and Fusarium oxysporium (Schlecht.) (isolated from watermelon) in a dual-culture assay and reduced the growth of mycelium on a conditioned-medium test, thus suggesting the involvement of extracellular compounds in resistance. Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis of extracellular proteins from spent suspension-culture medium showed enhanced secretion of new proteins by selected lines. A 36-kDa protein was immunodetected by a chitinase antiserum. This chitinase continued to express constitutively in differentiated somatic embryos and also in the intercellular fluids of plants regenerated from the selected lines. Somatic embryos from selected lines grew uninhibitedly in a medium containing 40% fungal culture filtrate, whereas non-selected (control) somatic embryos became necrotic and died within a few days. Plants regenerated from selected lines exhibited resistance to infection by E. ampelina in both greenhouse tests and detached leaf bioassays. Results suggest that embryogenic cells can be selected for resistance following in-vitro selection, resulting in resistant plants. Whether or not resistant cells pre-existed in the original embryogenic culture or were induced by the selection pressure could not be determined. Received: 12 November 1999 / Accepted: 3 December 1999     

Evaluation of soybean resistance to Phialophora gregata culture filtrate in tissue culture

Summary Resistance to the fungal pathogen, Phialophora gregata (Allington and Chamberlain) W. Gams, the cause of brown stem rot (BSR) in soybean [Glycine max (L.) Merr.], is an important trait for cultivars grown in the northern USA. A novel tissue culture method was developed where ten soybean cultivars were differentiated on the ability of their excised cotyledons to remain green and initiate callus in a tissue culture medium containing P. gregata culture filtrate. Cultivar BSR classifications by the cotyledon method corresponded to greenhouse root-dip assay classifications in 80%, 100%, and 90% of the three P. gregata isolate treatments. Another method, employing pieces of somatic callus exposed to the culture filtrate, had a 70% average correspondence to the greenhouse results. Physiologic specialization was demonstrated in parallel in vivo/in vitro assays for the first time. These data suggest that the cotyledon method would accurately identify soybean lines resistant to certain aberrant or wild-type P. gregata isolates.     

A physiological study on the toxicity of culture filtrate of helminthosporium sativum P.K. & P.

Summary When symptoms of the leaf-spot disease of barley caused byHelminthosporium sativum could be reproduced by dipping host plants in fungal filtrate, it was thought to trace any correlation that may exist between nitrogenous constituents of the filtrate and its wilt-inducing capacity to test plants (lupin seedlings). The fungus was grown on a modified Fries' No 3 medium with sodium nitrate or ammonium chloride or an equivalent mixture of both as nitrogen source. The nitrogen content of the culture medium and its wilt-inducing capacity were traced over 34 days. The nitrogen content, after reaching a minimum, was found to rise again; the rise being more rapid and bigger in nitrate and ammonium-nitrate media than in ammonium media. Only a partial apparent correlation could be noted between the trend in the wilt-inducing capacity of filtrates obtained at different periods of incubation, and the trend in the change in their nitrogen content. It is suggested that the toxicity of the culture filtrate may be attributed to more than one substance, one of them being a nitrogenous product (possibly of peptide nature). The observed degree of toxicity would therefore depend on the relative amounts of the nitrogenous product and other substances.     

In vitro selection at cellular level for red rot resistance in sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> sp.)

In the present study, in vitro selection technique using pathogen culture filtrate of Colletotrichum falcatum Went was employed with the aim to identify associations (if any), between selection at the cellular and plant level for red rot resistance in sugarcane (Saccharum sp.). Five to eight months old sugarcane calli of genotypes CoJ 88 and CoJ 64 were screened in vitro against pathogen culture filtrate for two selection cycles. Effect of pathogen culture filtrate on callus survival and/or proliferation was observed to be directly related to its concentration in the selection media. Calli survived and exhibited further proliferation at 5, 10 and 15% v/v pathogen culture filtrate concentrations whereas, at higher concentrations (20 and 25% v/v) proliferation was completely inhibited. Shoot regeneration percent was higher in calli selected on 5% pathogen culture filtrate concentration than those selected on 10 and 15% concentrations. In vivo screening of field transferred somaclones against two pathtypes (Cf 03 and Cf 08) showed considerable variation for red rot resistance. Somaclones regenerated from resistant and/or tolerant calli exhibited better resistance than the parental genotypes. The results indicated that in vitro selection for red rot resistance was effective and expressed when somaclones were screened in the field. This indicated a positive association between in vitro and in vivo methods of selection for disease resistance in sugarcane.     

Capsidiol: Its role in the resistance of Capsicum annuum to Phytophthora capsici

Inoculation of the stems of three Capsicum annuum L. cultivars showing different degrees of sensitivity to the fungal pathogen Phytophthora capsici , resulted in a hypersensitive reaction being expressed along the stems. One of the peppers (cv. Smith-5) showed resistance by total inhibition of fungal growth. Capsidiol, a phytoalexin, which accumulates in the area of necrosis appears to be involved in this resistance. Capsidiol accumulation was analyzed by gas chromatography and was correlated with the restricted growth of P. capsici , in vivo and in vitro, confirming the former's fungistatic and fungitoxic properties. The capacity to inhibit pathogenic growth was evident only when capsidiol production exceeded 1 204 μg ml^-1, a level reached in the resistant variety after 6 days of incubation. Experiments on induced resistance showed that a second inoculation of the stems of the three cultivars also resulted in necrosis and in an accumulation of capsidiol, although to a lesser extent than in the first inoculation. The greater accumulation of capsidiol in the stems of cv. Smith-5 is in accordance with the resistance shown by this cultivar to P. capsici , and confirms the implication of capsidiol in the disease resistance of this cultivar to fungal pathogens. Capsidiol has a fungistatic character at a mean concentration of 3.75 mM, and is fungitoxic at levels above 5 mM. This level must be exceeded and all the growing hyphae must be affected for capsidiol to qualify from being fungistatic to being fungitoxic.     

利用细胞工程技术筛选小麦抗根腐病突变体的研究

以小麦根腐病菌002号菌株产生的致病培养滤液为选择剂,选用春、冬小麦品种、品系以及杂种的花药和幼德进行离体培养,并结合物理诱变技术,已获得抗根腐病的植株,用根腐病菌分生孢子接种鉴定,再生植株50株中有12株抗病。     

In vitro selection of resistant rice plants against rice blast caused by Pyricularia oryzae via tissue culture technique

Abstract

A step by step protocol for resistant calli selection via a tissue culture technique under stress of Pyricularia oryzae culture filtrates was followed. Rice embryos dissected apart from the endosperm of susceptible rice seeds (Giza 176 and Riho) to P. oryzae produced embryonic calli on media containing various growth regulators of 2,4-D at concentrations of 0, 1, 1.5 and 2 mg/L and/or benzyl amino purine (BAP) at 0, 0.5, 1 and 1.5 mg/L when incubated under complete dark conditions for three weeks. Embryonic explants only produced shoots on media containing BAP. Selection of resistant calli was carried out in vitro under the challenging stress of increasing concentration of the pathogen P. oryzae culture filtrate (CF) from “0” up to 100%. The selection protocol has two directions. The first is step-by-step selection from lower to higher selective (CF) concentrations. The second is the exchangeable continuous cycles with and without the same selective (CF) concentration until the end of the selection regime to avoid calli adaptation to (CF). The regenerated calli to plantlets occurred under (CF) stress showed resistance and susceptibility when exposed to the pathogen infection under greenhouse conditions. The results reveal that the resistance in regenerated rice plantlets to P. oryzae pathogen segregated as 1 resistant: 2 moderate resistant: 1 susceptible giving the predication that the resistance in rice to P. oryzae may be controlled by one pair of genes. The in vitro selective regime via tissue cultures is advisable for the selection of novel disease resistant plants because of its time saving, space, money, it is easily applied and has a bio-safe approach.