用组织培养法筛选水稻抗白叶枯病突变体——Ⅰ.水稻愈伤组织抗白叶枯病病原菌的选择及其再生植株的抗病性鉴定

选用感病品种南粳34去壳籽粒的愈伤组织,按种白叶枯病病原菌菌株Ks 8-4,共同培养,以期筛选抗菌愈伤组织。在365块接菌愈伤组织中,有63块局部生长,从中得到抗菌新愈伤组织63块,并分化出再生植株。鉴定再生植株当代R_1 45株,有44株抗病,1株感病。部分株系得到了R_2、R_3代植株,多代鉴定表明,其抗病性是稳定的和可遗传的。     

离体筛选小麦抗赤霉病和根腐病突变体 I．小麦兼抗赤霉病和根腐病愈伤组织的筛选及其抗病性的鉴定

本文选用冬小麦和春小麦品种及其杂种后代的幼穗进行离体培养， 以赤霉病菌及根腐病菌的培养 滤液为选择剂，按不同的筛选方法筛选兼抗赤霉病和根腐病的突变体，用赤霉病菌和根腐病菌分生抱子 直接接种在经筛选的愈伤组织上，进行抗病性鉴定。试验结果表明：不同筛选方法对筛选兼坑赤霉病和 根腐病的愈伤组织的效果有明显差异，二步筛选法比混合筛选法效果好。供鉴定的120块愈伤组织中， 有村块愈伤组织具有兼抗赤霉病菌和根腐菌侵染的能力。     

利用辣椒疫霉培养滤液体外筛选胡椒抗瘟病无性系研究

在胡椒（Piper nigrum Linn．)茎尖丛生增殖技术的基础上,以印尼大叶种“Lampong Type”无菌实生苗作外植体源,利用辣椒疫霉(Phytophthora capsici)培养滤液对胡椒茎尖及其增殖形成的丛生芽进行体外选择。辣椒疫霉培养滤液的不同灭菌方法对辣椒疫霉培养滤液的毒性影响显著,过滤灭菌方式可以保持辣椒疫霉培养滤液的毒性,而高温高压灭菌方式则不能。随着辣椒疫霉培养滤液浓度的增加,茎尖和丛生芽的存活率和增殖率都在下降。在存活的茎尖或丛生芽培养中,一部分可正常增殖,其余的形成愈伤组织,或者保持生长停滞的休眠状态。在选择性培养基上继代培养2次后进行生根和移栽,利用离体叶片针刺接种法对温室条件下生长的移栽植株进行抗瘟病测定。以3次抗病检测均无明显症状的植株作为抗病株。随着辣椒疫霉培养滤液浓度的增加,得到的再生植株数量降低,但其中抗病株的比例提高。利用过滤灭菌方式加入选择性培养基的处理中,25％、50％和75％的辣椒疫霉培养滤液分别获得1株、4株和3株抗病株,分别占各处理再生植株总数的1．54％、20．00％和42．86％,共获得8株,占该组处理再生植株总数的8．70％。     

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。     

离体筛选中国水仙抗叶大褐斑病突变体初步研究

选用中国水仙鳞茎块离体培养,以叶大褐斑病原菌的培养滤液作为选择,结合γ－射线物理诱变,从1420块外植体中得到9个抗病再生种球,抗病性测定表明,经病原菌培养滤液筛选处理得到的再生种球,抗病性比对照明显提高。     

筛选西洋参抗黑斑病胚性愈伤组织无性系的研究

应用黑斑病菌滤液筛选西洋参胚性愈伤组织,获得了抗西洋参黑斑病的胚性愈伤组织无性系及再生植株。试验证明,“浓度梯度筛选－继代培养－再筛选”的方法,对于筛选西洋参抗病胚性愈伤组织无性系是有效的。     

筛选西洋参抗黑斑病胚性愈伤组织无性系的研究

应用黑斑病菌滤液筛选西洋参胚性意伤组织,获得了抗西洋参黑斑病的怀性愈伤组织无性系及再生植株。试验证明,“浓度梯度筛选－继代培养－再筛选”的方法,对于筛选西洋参抗病胚性愈伤组织无性系是有效的。     

药蒲公英再生体系的建立和优化

通过愈伤组织诱导和直接不定芽再生途径 ,建立了药蒲公英的快速高效再生系统。叶片外植体在含0.2mg LIAA和1mg LTDZ的MS培养基中培养 2周后便产生大量的丛生芽 ,在含有 0.5mg/L 2 ,4D和2mg/L 6BA的MS培养基中培养 30d后 ,形成明显的愈伤组织 ,愈伤组织块在含10mg/L 6BA的MS培养基中成功再生。对 9株再生植株进行RAPD检测表明 ,部分植株在DNA水平上发生了变异。与对照相比 ,再生植株的主要抗氧化成分无明显变化 ,保证了有效成分的稳定。     

水稻抗白叶枯病菌毒素细胞无性变异系的初步筛选

应用湖北水稻白叶枯病强毒菌株“江陵６９１”活细菌、细菌培养滤液、病菌粘液多糖和菌体多糖。对水稻粳１０５、粳３０３７和科选１号的幼穗及花药所形成的愈伤组织进行共培养筛选。获得体细胞再生植株４４３株,花粉植株３１株,其中１７株为单倍体（ｎ＝１２）。同工酶谱显示,筛选出的粳１０５愈伤组织的过氧化物酶同工酶活性加强,带数增多。     

大麦转化体系的改进及TrxS基因的转化

以啤酒大麦品种“晋引6号”的幼胚为转化起始材料,用基因枪法将分别携带有目的基因(TrxS)和除草剂基因(筛选基因,Bar)的两个质粒进行了共转化,同时对基因转化的相关技术和植株再生的培养方案进行了优化。结果表明,受体材料宜选用预培养15d的幼胚;在培养前2周添加1mg／L ABA可抑制胚芽萌发而且有助于胚性愈伤组织的形成;1．0mg／L ZT与0．1mg／L IAA激素配比可有效促进愈伤组织的分化。利用优化的培养条件,经在含3～5mg／L筛选剂PPT的培养基上筛选、再生及生根培养。共在178块抗性愈伤组织上获得11株再生植株,再生率达到6．2％,经对T0、T1、T2代PCR、nested PCR和Southern杂交检测表明,TrxS基因已经稳定整合到大麦基因组中且遗传稳定、结构完整。     

利用细胞工程技术筛选小麦抗病新种质的研究

在利用细胞工程技术筛选小麦抗根腐病和赤霉病突变体研究工作的基础上,将已获得的突变株继续进行多年多点的抗病鉴定,对突变株后代的抗病性进行测定,并对农艺性状作详细的观察。结果表明,抗病突变株不论是同一年份在不同鉴定点上,还是在同一鉴定点上多年重复鉴定,都表现有较强的抗根腐病菌和赤霉病菌侵染的能力,突变株的抗病性不因代数的增加而发生变化。已从中选出４个对根腐病和赤霉病抗性强而稳定、农艺性状亦较好的新种质,提供给一些育种单位利用。     

In vitro selection and regeneration of carnation (Dianthus caryophyllus L.) plants resistant to culture filtrate of Fusarium oxysporum f.sp. dianthi

Callus cultures derived from internodal segments of two cultivars of carnation susceptible to Fusarium oxysporum f.sp. dianthi were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant lines were selected by culturing calli on growth medium containing various concentrations of the culture filtrate of F. oxysporum f.sp. dianthi. Resistant calli obtained after two cycles (25 days/cycle) of selection were used for plant regeneration. About 32% of the plants regenerated from the resistant calli had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

In vitro selection of barley and wheat for resistance against Helminthosporium sativum

Summary Calli derived from immature embryos of barley and wheat genotypes were screened for their resistance to purified culture filtrate produced by the fungus Helminthosporium sativum P.K. and B. Two selection methods were used: a continuous method in which four cycles of selection were performed one after another on toxic medium and a discontinuous method in which a pause on non-toxic medium was given after the second or third cycle of selection. The latter was superior as it allowed the calli to regain their regeneration ability. About 3,000 calli from two barley genotypes and 2,000 from two wheat genotypes were used for selection. The selection with the pathotoxins resulted in 6% to 17% surviving calli. Toxin tolerant callus lines of barley were characterised by protein isozymes. Zymograms showed one more isozyme than with the unselected sensitive callus. Barley and wheat plants have been regenerated from callus lines surviving the toxin treatment and in vivo testing against pathogen revealed that the majority of these plants were less sensitive.     

用基因枪法将人工雄性不育基因导入小麦的研究初报

利用ＰＤＳ１０００／氦气基因枪将人工构建的雄性不育基因（ＴＡ２９－Ｂａｒｎａｓｅ基因）导入小麦栽培品种豫责１８号的幼胚细胞。然后在含有１０～２０ｍｇ／Ｌ除草剂Ｂａｓｔａ的培养基础上筛选与分化。从１７０个幼胚中获得６株绿苗,对照的７０个幼胚中未得到绿苗。对其中３株已生根且长势好的绿苗进行Ｓｏｕｔｈｅｍ杂交分析,结果表明,这３株绿苗皆为转基因植株,转化效率达１．８％。     

Transgenic expression of polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the flower pathogen Fusarium graminearum

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important diseases of wheat worldwide, resulting in yield losses and mycotoxin contamination. The molecular mechanisms regulating Fusarium penetration and infection are poorly understood. Beside mycotoxin production, cell wall degradation may play a role in the development of FHB. Many fungal pathogens secrete polygalacturonases (PGs) during the early stages of infection, and plants have evolved polygalacturonase-inhibiting proteins (PGIPs) to restrict pectin degradation during fungal infection. To investigate the role of plant PGIPs in restricting the development of FHB symptoms, we first used Arabidopsis thaliana, whose genome encodes two PGIPs (AtPGIP1 and AtPGIP2). Arabidopsis transgenic plants expressing either of these PGIPs under control of the CaMV 35S promoter accumulate inhibitory activity against F.?graminearum PG in their inflorescences, and show increased resistance to FHB. Second, transgenic wheat plants expressing the bean PvPGIP2 in their flowers also had a significant reduction of symptoms when infected with F.?graminearum. Our data suggest that PGs likely play a role in F.?graminearum infection of floral tissues, and that PGIPs incorporated into wheat may be important for increased resistance to FHB.     

Use of cell culture to screen sunflower germplasm for resistance to Phomopsis brown/gray stem spot

Resistance to Phomopsis sp. (Diaporthe sp.) brown/gray stem spot was confirmed by screening sunflower calli on shoot induction media amended with fungal filtrate. Calli of sunflower genotypes OCMS 74 and NS-H-45, which show resistance to the disease in field trials, remained viable on media with 15% (v/v) fungal filtrate, while calli of field susceptible genotypes RHA 273 and PAG/SF 103 were killed on media with as little as 7.5% fungal filtrate. Fresh weight of calli of all genotypes was significantly reduced by 2.5% fungal filtrate, and calli of all genotypes were killed by filtrate concentrations of 20%. These in vitro results corroborate prior field observations for disease reaction of these genotypes.     

Stably transformed callus of wheat by electroporation-induced direct gene transfer

Fertile plants of wheat have been regenerated from protoplasts in several laboratories. The objective of this study was to develop a transformation system using protoplasts as target cells. Protoplasts were isolated from cell suspensions initiated from an anther-derived callus. The protoplasts were transformed by electroporation using pBARGUS or pBAS, both carrying the Basta resistance (BAR) gene. A total of 2,761 calli were produced from electroporation transformed protoplasts in 3 independent experiments. Six calli survived selective culture on 10 mg/l phosphinothricin (PPT), a concentration that completely inhibited the growth of non-transformed wheat callus. Five PPT resistant calli showed phosphinothricin acetyltransferase (PAT) activity, whereas the sixth probably was a mutant. The transformed wheat calli could tolerate PPT concentrations up to 2,560 mg/l. Southern blot analyses confirmed the integration of the BAR gene in wheat genomes. The integrated DNA sequence may have partially methylated and tandemly repeated at least once. These results demonstrate the production of stably transformed wheat calli by electroporation-mediated direct gene transfer into protoplasts.     

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.