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。     

根癌农杆菌介导Bt基因转化水稻的研究

为了培育出无筛选标记基因的转基因水稻,试验将loxp-hpt-loxp基因与成基因连锁在-起转化水稻方法,得到loxp-hpt—loxp—Bt转基因水稻植株,再与同质的带有ere基因的水稻杂交,以定向删除潮霉素抗性筛选标记。试验表明以水稻品种“皖粳97”为供试材料,将成熟胚来源的愈伤组织用根癌农杆菌EHA105／pCAMBIA1305．1感染后,筛选出抗性愈伤组织并获得再生植株。经PCR验证,得到20棵转基因水稻植株。     

药用野生稻TAC克隆转化籼稻的体系初探

采用已构建的药用野生稻TAC文库,比较研究4个籼稻品种在不同培养基的诱导率及愈伤组织对潮霉素抗性。通过2种不同诱导方法产生的愈伤组织,将携带药用野生稻大片段DNA的TAC克隆转化到籼稻品种中的结果表明,除了‘粤香占’以外,其它3个品种在心诱导培养基中添加微量B,后,其愈伤诱导率最高。不同品种的潮霉素筛选适合浓度存在差异,其中‘华粳籼74’的适合筛选浓度为50mg．L-1,‘粤香占’为40mg．L-1。用预培养5d的愈伤组织进行遗传转化,在潮霉素筛选之前先以头孢拉定抑菌处理的,容易获得转基因植株。4个品种中以‘粤香占’的抗性愈伤组织筛选率和分化率最高,分别为22．58％和14．86％。分子检测11株转基因水稻的结果表明,其中8株含有抗性标记基因。据此认为,药用野生稻TAC文库在水稻创新育种中可能有一定的利用前景。     

Pi-ta+基因和几丁质酶基因双价基因导入水稻的研究

为了提高‘云资粳41’和‘云资粳43’的抗稻瘟病能力,利用农杆菌介导法将二价表达载体pCAMBIA1300-Pi-ta⁺-Bchi转化到水稻愈伤组织中。经组织培养获得再生苗,再通过氯酚红显色法、PCR检测和抗稻瘟病鉴定法获得抗稻瘟病的转基因植株,为进一步创建持久、广谱抗稻瘟病水稻新材料奠定基础。结果显示:(1)抗性愈伤组织经分化和生根培养后,共获得T⁰代水稻再生苗137株,其中‘云资粳41’14株,‘中花11’82株,‘云资粳43’41株。(2)经氯酚红显色法和PCR对再生苗检测,‘中花11’、‘云资粳41’、‘云资粳43’的转化苗阳性率分别为66%、43%和55%。证明2个外源基因已经整合到水稻基因组中。(3)对转化阳性植株温室接种稻瘟病病菌66b鉴定结果显示,转基因植株较非转基因植株对稻瘟病的抗性明显增强,而且转Pi-ta⁺基因和几丁质酶基因双价的水稻植株比转单价Pi-ta⁺基因或几丁质酶基因的水稻植株抗稻瘟病能力强。（4）氯酚红检测结果存在一定的假阳性,PCR检测结果更真实可靠,但氯酚红显色法方便、快速,结果观察直观,可对大量的转基因植株进行初步筛选。研究表明,转Pi-ta⁺基因和几丁质酶基因双价基因的水稻植株具有更高的抗稻瘟病能力。     

简单高效的根癌农杆菌介导的水稻基因转化方法

本文在研究影响农杆菌介导的水稻转化的主要因素基础上,建立了一套简单、高效的水稻转基因系统。将水稻成熟胚来源的愈伤组织用农杆菌EHA101/pHQ9,EHA 101/pHQ 10,EHA 101/pHQ T3感染后,筛选抗性愈伤,经分化获得转化株。抗性愈伤的平均得率为约100个愈伤/g愈伤外植体,抗性愈伤的分化频率平均高达85%。转基因植株的GUS染色、Southern杂交结果表明,T-DNA上的外源基因已整合进转基因植物的基因组中。转基因植株T1代对潮霉素的抗性表明,多数转基因株系符合孟德尔分离比3∶1。该系统的建立将有助于应用T-DNA标签法和基因打靶法进行水稻功能基因组的研究。     

简单高效的根癌农杆菌介导的水稻基因转化方法

本文在研究影响农杆菌介导的水稻转化的主要因素基础上,建立了一套简单、高效的水稻转基因系统。将水稻成熟胚来源的愈伤组织用农杆菌EHA101／pHQ9,EHA101／pHQ10,EHA101／pHQT3感染后,筛选抗性愈伤,经分化获得转化株。抗性愈伤的平均得率为约100个愈伤／g愈伤外植体,抗性愈伤的分化频率平均高达85％。转基因植株的GUS染色、Southern杂交结果表明,T-DNA上的外源基因已整合进转基因植物的基因组中。转基因植株T1代对潮霉素的抗性表明,多数转基因株系符合孟德尔分离比3：1。该系统的建立将有助于应用T—DNA标签法和基因打靶法进行水稻功能基因组的研究。     

干旱响应转录因子DREB1A基因导入水稻光温敏核不育系的研究

以成熟胚诱导的愈伤组织作为农杆菌转化的受体材料,将诱导型启动子rd29A驱动的拟南芥DREB1A基因导入粳型光温敏核不育系水稻4008S,共获得67株再生苗.再生苗经0.75 mg/L除草剂草铵膦涂布筛选,有62株再生苗表现出对草铵膦抗性.PCR检测抗性苗中DREB1A基因,结果全为阳性.挑选部分进行Southem检测.结果表明目的基因已经整合到水稻基因组中.在干旱胁迫下,转基因水稻当代(T1代)植株的电导率显著低于非转基因对照植株(P＜0.05),脯氨酸含量显著高于对照植株(P＜0.05),证明DREBIA基因能提高水稻对干旱胁迫的耐受性.     

多年生黑麦草成熟胚再生体系的建立及基因枪转化

目的:建立以多年生黑麦草成熟胚为起始材料的再生体系,用于基因枪转化。方法:多年生黑麦草成熟种子在附加 5mg L 2,4 D的MS培养基上诱导愈伤组织,转至新继代培养基上产生胚性愈伤组织。分化培养基为无激素MS培养基。再生植株在培养基成分减半的无激素MS培养基生根,之后移栽至土壤。基于这一再生体系,用含有水稻几丁质酶基因RC2 4的质粒pARN6和含有草丁膦乙酰转移酶基因Bar的质粒pDB1,通过基因枪轰击胚性愈伤组织。用附加PPT的继代培养基进行转化植株的抗性筛选。结果:共获得 2 4 3株再生植株。通过PCR进行检测,获得1 8株整合有RC2 4基因的植株,1 5株整合有Bar基因的植株,同时转入 2个基因的植株 2株。     

马齿苋多糖提取物对翅果油树愈伤组织诱导的影响

采用传统热浸提工艺,从马齿苋植株中提取多糖.以B1培养基为基础,通过添加0.15 g/L的抗褐变剂Vc及不同浓度的马齿苋多糖,研究了其对翅果油树愈伤组织培养的抗褐变作用.结果显示:(1)马齿苋多糖对愈伤组织培养中的褐变有一定缓解作用.(2)经筛选,得到0.19 g/L为最适的多糖添加物浓度,子叶愈伤组织产生生物量最多,抗褐变效果与Vc的作用相似,愈伤组织生命周期也明显缩短.     

以耐草甘膦2mG2-epsps基因为选择标记的玉米转化体系的建立

转化细胞的筛选和再生是植物遗传转化体系中重要的组成部分,筛选剂的选择和筛选压力的高低直接影响着外源基因的转化率。以草甘膦异丙胺盐为筛选剂,通过比较在不同的草甘膦浓度及筛选时间的条件下,玉米愈伤组织生长和分化情况,发现经1mM的草甘膦异丙胺盐筛选15d后玉米愈伤组织分化受到明显抑制,故以此作为玉米遗传转化实验中的筛选压力。通过基因枪轰击,将构建好的pMAGUHM载体（其上携带有抗草甘膦基因2mG2-epsps基因）转化到玉米愈伤组织,利用草甘膦筛选得到耐草甘膦植株80株,其中PCR检测阳性植株为36株,转化率为45％。     

STUDIES IN THE BACTERIAL DISEASES OF SUDAN CROPS

The atypical symptoms first described by Bryan (1932) of the angular leaf spot disease of cotton caused by Xanthomonas malvacearum (E. F. Sm.) Dowson were reproduced by inoculation into seeds, stem apices or buds. The lesions that developed on the veins of the newly produced leaves were elongated and water-soaked, becoming dark brown. The leaf tissue dependent upon infected veins became yellow, flaccid and withered. The development of these symptoms was enhanced when inoculations were made into opening buds or germinating seeds as compared with inoculations into closed buds or dormant seeds.
In other bacterial diseases caused by Xanthomonas spp., somewhat atypical symptoms could also be produced by bud inoculation into the appropriate host. Those produced by X. ricini (Yoshi & Takimato) Dowson on castor, closely resembled the vein lesions described above on cotton but resulted only from bud inoculations; inoculations into stem apices and seeds failed to produce them. In dolichos bean inoculated with X. phaseoli (Smith) Dowson, atypical symptoms were produced only by seed inoculations and were confined to the first simple leaves (prophylls).
The differences in the production of atypical symptoms on the three hosts are correlated with differences in host structure and with the degree of virulence of the pathogen. The leaf parasite X. ricini , for example, which cannot infect castor bean stems, does not produce atypical symptoms when inoculated below the stem apex.
From the data discussed below, the incidence of atypical symptoms is attributed to infection either of an actively growing tissue or of a telescoped structure which subsequently completes its development.
The atypical symptoms of the cotton disease are not caused by a special strain of X. malvacearum. Further, they are not a peculiarity of this disease but may also develop in other necrotic diseases under similar conditions.     

STUDIES IN THE BACTERIAL DISEASES OF SUDAN CROPS

The bacterial leaf spot disease of jute ( Corchorus olitorius ) is characterized by the development of brown circular spots, about 2–5 mm. in diameter, on the leaves, surrounded, sometimes, by a yellow halo. The disease on the stems causes elongated lesions, and on the capsules produces small sunken spots. Defoliation and death of the stems may result in severe infections. The disease is ascribed to a new variety of Xanthomonas nakatae (Okabe) Dowson, the cause of a somewhat similar disease of Corchorus capsularis in Formosa (Elliott, 1951), on the ground that: (i) the minor differences between the two pathogens in biochemical characters are considered well within strain differences, and (ii) the Sudan pathogen does not infect C. capsularis , and Xanthomonas nakatae has not been reported to infect Corchorus olitorius . These differences do not justify the creation of a new species. The name Xanthomonas nakatae v. olitorii is suggested for the cause of the disease under consideration.     

STUDIES IN THE BACTERIAL DISEASES OF SUDAN CROPS: II. BACTERIAL LEAF BLIGHT DISEASE OF CASTOR (RICINUS COMMUNIS L.)

The bacterial disease of castor in the Sudan has been reinvestigated. Typically the disease symptoms are brown or black, round or angular lesions on leaves; occasionally the thin succulent branches are attacked, but not the vascular tissue.
The bacterium responsible for the disease was previously thought to be Pseudomonas solanacearum E.F.Sm. but comparative studies show that the Sudan species differs in pathogenicity and physiology from P. solanacearum. It closely resembles Xanthomonas ricinicola (Elliott) Dowson (= Bacterium ricini Yoshi and Takimoto) in characters, host range and pathogenic effects. In culture on solid media the growth of X. ricinicola is typically yellow, while that of the Sudan species is dirty white; but this difference is not considered sufficient to justify the establishment of a new species.
The author proposes the specific name ricini originally adopted by Archibald (1927) and Yoshi & Takimoto (1928), for the species of Xanthomonas causing the leaf blight disease of castor, and considers that the causal organism in the Sudan is an atypical strain of this species.     

STEM INFECTION OF COTTON BY XANTHOMONAS MALVACEARUM (E. F. SM.) DOWSON

Varying the position of stem inoculation, the concentration of inoculum and the age of plant affected the reaction of cotton, Gossypium sp., to infection with Xanthomonas malvacearum (E. F. Sm.) Dowson.
The extent of stem discoloration, internal and external, and the probability of disease ocurring in leaves by bacteria moving within the plant increased ( a ) the nearer the point of stem inoculation was to the apex, and ( b ) the higher the concentration of inoculum. The leaf symptoms were not the angular spots typical of primary leaf infection. Instead, bacteria seemed to lodge in, discolour and blacken sections of leaf veins. Then tissue next to the affected veins became water-soaked and leaf sectors dependent upon these veins died and dried. These symptoms usually developed 14 to 55 days after inoculation in the expanding leaves.
The amounts of stem discoloration and the probabilities of leaf symptoms developing were less when hypocotyls of old plants were inoculated than when hypocotyls of young plants were inoculated. The probabilities of leaf symptoms developing were similar, however, when young tissues in young and old plants were inoculated.
American cotton, Gossypium hirsutum , was less affected by stem inoculation than Egyptian cotton, G. barbadense. Of the resistance factors against primary leaf infection only B_6m gave appreciable stem resistance.     

Screening,Identification and Distribution of Endophytic Associative Diazotrophs Isolated from Rice Plants

29 strains of endophyte associated diazotrophs isolated from rice ( Oryza sutiva L. ) "Yuefu" plant were selected by in vitro acetylene reduced activity and 15 N2-flxing activity determination. They were identificated into 14 species of 9 genera: Agrobacterium tumefaciens (Smith et Townsend) Conn, A. radiobacter (Beijerinck et van Delden) Conn; Alcaligenes piechaudii Kiredjian et al., Al. denitrificans (Leifson et Hagh) Ruger et Tan; Bacillus sphaericus Meyer et Neide, B. licheniforrnis Weigmann Chester, B. cereus Frankland et Frankland; Chryseomonas luteola (Kodama, Kinnura et Komagata) Holmes et al.; Enterobacter cloacae ( Jordan ) Honnaeche et Edwards, E. sakazakii Famer et al.; E. agglomeraus (Beijerinck) Ewing et Fife; Pseudomonas pseudoalcaligenes Stanier, P. alcaligeaes Monias, P. putida Biotype A Evans and Aeromonas Kluyver et van Niel, Serratia Bizio, Staphyloccocus Rosenbach, Xanthomonas Dowson. Among them C. luteola, E. sakazakii, E. agglomeraus, P. pseudoalcaligenes have not been previously reported as diazotrophs. Studying on the distribution of endophytic dizoatrophs in rice seeds and rice plants has demonstrated that the diversity of endophytic associated diazotrophs in roots was more than that in other organs of rice plant. Endophytic associative diazotroph E. cloacae not only occurred on the surface but also occurred intercellularly in R48 rice roots as observed with scanning electron microscopy and transmission microscopy.     

Syntaxin of plants31 (SYP31) and SYP32 is essential for Golgi morphology maintenance and pollen development

Pollen development is a key process for the sexual reproduction of angiosperms. The Golgi plays a critical role in pollen development via the synthesis and transport of cell wall materials. However, little is known about the molecular mechanisms underlying the maintenance of Golgi integrity in plants. In Arabidopsis thaliana, syntaxin of plants (SYP) 3 family proteins SYP31 and SYP32 are the only two Golgi-localized Qa-soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) with unknown endogenous functions. Here, we demonstrate the roles of SYP31 and SYP32 in modulating Golgi morphology and pollen development. Two independent lines of syp31/+ syp32/+ double mutants were male gametophytic lethal; the zero transmission rate of syp31 syp32 mutations was restored to largely normal levels by pSYP32:SYP32 but not pSYP32:SYP31 transgenes, indicating their functional differences in pollen development. The initial arrest of syp31 syp32 pollen occurred during the transition from the microspore to the bicellular stage, where cell plate formation in pollen mitosis I (PMI) and deposition of intine were abnormal. In syp31 syp32 pollen, the number and length of Golgi cisterna were significantly reduced, accompanied by many surrounding vesicles, which could be largely attributed to defects in anterograde and retrograde trafficking routes. SYP31 and SYP32 directly interacted with COG3, a subunit of the conserved oligomeric Golgi (COG) complex and were responsible for its Golgi localization, providing an underlying mechanism for SYP31/32 function in intra-Golgi trafficking. We propose that SYP31 and SYP32 play partially redundant roles in pollen development by modulating protein trafficking and Golgi structure.     

Anther culture and androgenetic plant regeneration in buckwheat (Fagopyrum esculentum Moench)

Anther culture for haploid induction of buckwheat was studied over a period of five years. Approximately 24,000 anthers were isolated and cultured on different culture media. The regeneration capacity was generally very low. Data are presented for experiments that included 7278 anthers on which 99 calluses were formed and 20 buds regenerations were noted. Regeneration occurred most readily on gellan-gum solidified media, with 90 g l^-1 maltose, 2.5 mg l^-1 BA, 0.5 mg l^-1 IAA, and preferably in darkness. Haploid cells, as established by chromosome counts, were observed in eight regenerants. Several abnormalities of pollen development in vitro were detected. Starch presence in pollen as a possible sign of androgenic capacity was studied. Microspores in uninucleate and early binucleate stages contained only proplastids, while in adult pollen grains a number of amyloplasts were present.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - 2,4 D-2,4-dichloro-phenoxyacetic acid - IBA indole-3-butyric acid - 2iP 6- c,c-dimethylallylaminol-purine - NAA -naphthalene acetic acid     

Gap regeneration of major tree species in different forest types of Japan

Gap regeneration of major tree species was examined, based on the pattern of gap phase replacement, in primary old-growth stands of warm-temperate, cool-temperate and subalpine forests, Japan. Using principal component analysis, the gap-regeneration behavior of major tree species could be divided into three guilds and that of Fagus crenata (monodominant species of cool-temperate forests). The criteria used for this division were total abundance of canopy trees and regenerations and relative abundance of regenerations to canopy trees. The gap-regeneration behavior of species in the first guild was that canopy trees regenerate in gaps from seedlings or saplings recruited before gap formation; they had higher total abundance and more abundant regenerations relative to their canopy trees. The gap-regeneration behavior of F. crenata was same as species in the first guild, but F. crenata had less abundant regenerations relative to its canopy trees. Species in the second guild had lower total abundance and less abundant regenerations to their canopy trees. The guild contained species whose canopy trees regenerate in gaps from seedlings or saplings recruited after gap formation or regenerate following largescale disturbance. The third guild consisted of species with lower total aboundance and more abundant regenerations relative to their canopy trees. The gap-regeneration behavior of some species in this guild was that trees regenerate in gaps from seedlings or saplings recruited before gap formation, and grow, mature, and die without reaching the canopy layer, while the gap-regeneration behavior of other species was same as that of species in the first guild or F. crenata. Major tree species of subalpine forests were not present in the third guild.     

The Role of Arabidopsis ABCG9 and ABCG31 ATP Binding Cassette Transporters in Pollen Fitness and the Deposition of Steryl Glycosides on the Pollen Coat

The pollen coat protects pollen grains from harmful environmental stresses such as drought and cold. Many compounds in the pollen coat are synthesized in the tapetum. However, the pathway by which they are transferred to the pollen surface remains obscure. We found that two Arabidopsis thaliana ATP binding cassette transporters, ABCG9 and ABCG31, were highly expressed in the tapetum and are involved in pollen coat deposition. Upon exposure to dry air, many abcg9 abcg31 pollen grains shriveled up and collapsed, and this phenotype was restored by complementation with ABCG9_pro:GFP:ABCG9. GFP-tagged ABCG9 or ABCG31 localized to the plasma membrane. Electron microscopy revealed that the mutant pollen coat resembled the immature coat of the wild type, which contained many electron-lucent structures. Steryl glycosides were reduced to about half of wild-type levels in the abcg9 abcg31 pollen, but no differences in free sterols or steryl esters were observed. A mutant deficient in steryl glycoside biosynthesis, ugt80A2 ugt80B1, exhibited a similar phenotype. Together, these results indicate that steryl glycosides are critical for pollen fitness, by supporting pollen coat maturation, and that ABCG9 and ABCG31 contribute to the accumulation of this sterol on the surface of pollen.