不同基因型玉米愈伤组织诱导与植株再生研究

以5个玉米品系幼胚为外植体,研究了基因型、2,4-D浓度以及胚龄对愈伤组织诱导的影响;6-BA对愈伤组织分化的影响;以及IBA对再生芽生根的影响。结果表明：除。31外,其他基因型的外植体在相同条件下均可诱导出愈伤组织,但是不同基因型间存在显著差异;2,4-D浓度和胚龄显著影响愈伤组织的诱导,且2,4-D浓度为2．0mg／L,胚龄在11—13d之间时,玉米愈伤组织诱导率较高且质量较好。将愈伤组织转入分化培养基后,6-BA促进了愈伤组织的再分化;在生根培养基中,IBA促进了再生芽生根,经过炼苗后移栽获得再生植株。     

小麦遗传转化受体系统建立的研究

选用‘小偃22’和‘宁春16’小麦品种的成熟胚和幼胚进行培养,研究不同种类的胚和培养因子对愈伤组织诱导和分化的影响。结果表明,幼胚和成熟胚的愈伤组织诱导率无明显差异,但较高浓度的2,4-D有利于成熟胚的诱导,而幼胚培养时2,4-D浓度的影响效果因品种而异;两种外植体分化率的高低与KT/IAA的配比均有密切关系,但高浓度的激素水平不利于成熟胚的分化;诱导培养基中低浓度的2,4-D有利于所诱导的愈伤组织的分化。同时,在诱导培养基中添加低浓度的KT能显著提高两品种成熟胚愈伤组织的分化率;各种培养基处理与品种间都存在显著的互作效应,‘小偃22’成熟胚培养的最佳培养基组合为MSD 3.0 mg/L 2,4-D和MSD 0.5 mg/LIAA 1.0 mg/L KT,幼胚培养为MSD 4.0 mg/L 2,4-D和MSD 0.5 mg/L IAA 1.0 mg/L KT;‘宁春16’成熟胚培养为MSD 4.0 mg/L 2,4-D和MSD 1.0 mg/L IAA 1.0 mg/L KT,幼胚培养时为MSD 1.0 mg/L 2,4-D和MSD 2.0 mg/L IAA 2.0 mg/L KT。     

小麦成熟胚愈伤组织诱导及分化研究

以2个小麦品种成熟胚为外植体进行离体培养,研究了不同预处理、不同2,4-D浓度及与KT组合、不同蔗糖浓度等因素对愈伤组织诱导及分化的影响。结果表明:4℃低温预处理可提高愈伤组织的出愈率及再生苗率,2个材料的出愈率及再生苗率均达到90%和30%以上;在不同预处理条件下,2,4-D浓度对出愈率及再生苗率的影响与基因型有关,2,4-D浓度为1~2 mg/L更有利于愈伤组织诱导及分化;附加KT能缓解高浓度2,4-D对再生苗率的抑制作用,而对于在1、2 mg/L 2,4-D的培养基中附加KT则不表现这种作用;蔗糖浓度则在30 g/L条件下更有利于愈伤组织诱导。因此通过4℃低温预处理,在MS基本培养基中附加1~2mg/L 2,4-D及30 g/L蔗糖亦可促进小麦成熟胚愈伤组织的诱导和分化。     

亚麻品种''''双亚5号''''的胚性愈伤组织诱导和体细胞胚胎发生

亚麻品种'双亚5号'的种子在MS 1.0 mg·L-1 2,4-D 30 g·L-1蔗糖培养基上可诱导出愈伤组织,将其转入MS 0.5mg·L-1KT 0.5mg·L-1NAA培养基上培养10周后诱导出大量胚性愈伤组织,结构较致密,浅黄色,表面有成团的紧密粘附在一起的小颗粒形态.但其继代周期不宜太长,继代次数不宜多,否则易回到非胚性化状态.胚性愈伤组织转A.MS 1.5 mg·L-1(T 1.0 mg·L-1 2,4-D分化培养基上培养40 d后可获得大量的球形胚状体.少量球形体胚可萌发形成正常的子叶胚初期形态,较多的球形体胚形成次生体胚或仅有单极性的畸形胚状体.组织解剖学观察表明,诱导出的是亚麻胚性愈伤组织和胚状体.     

新疆天山雪莲体胚诱导与分化研究

以新疆天山雪莲的叶片为外植体,分别用不同配方培养基诱导愈伤组织,后进行体胚诱导和分化培养形成再生雪莲植株.结果表明,诱导愈伤组织的最适培养基为MS 2,4-D 0.5 mg/L BA 1.5 mg/L,诱导率可达到100%;愈伤组织转移至MS 2,4-D 0.5 mg/L BA 1.5 mg/L培养基进行继代培养,增殖后的愈伤组织转移到MS 2,4-D 0.2 mg/L的液体培养基后成功诱导出雪莲体胚,出胚率达40%;将体胚接至MS ABA 0.5 mg/L培养基后,结果分化生长出大量的再生雪莲幼苗.     

从棒头草(Polypogon fugax Nees ex Steud)幼穗培养获得体细胞胚和再生植株

棒头草幼穗在含2,4-D的MS培养基上诱导出了胚性、非胚性和中间型愈伤组织。根据形态、淀粉粒等指标可将组成这些愈伤组织的细胞分为三类。改变培养基中2,4-D的浓度,能诱导三类愈伤组织相互转变。从胚性愈伤组织中诱导形成了大量体细胞胚;体细胞胚是从单个原胚细胞直接发育而来,它们能正常萌发、再生小植株。这种再生能力现已保持了34个月。小植株移植在土壤中可以正常生长、分蘖、开花和结实。     

大豆未成熟胚培养中高浓度植物生长素的作用及其植株再生

用大豆(Glycine max(L.)Merr.)未成熟胚为外植体,在含高浓度 NAA 或2,4-D 的培养基上可诱导绿色结构体和体细胞胚状体。叶状、花状和喇叭形状的绿色结构体在含30mg/l2,4-D 的 MS-1培养基上可重新愈伤组织化。这种由高浓度2,4-D 诱发并保持的愈伤组织,转到低激素水平的培养基上后,能诱导出大量的同类形状的新结构体。绿色结构体可以进一步发育成叶丛状,但顶端不伸长。使用含2m g/l GA,和0.1mg/l IBA 的培养基,能促进顶端伸长和根的发育,获得正常植株。由未成熟胚的子叶(其时种子长度约4—6mm)诱导的愈伤组织具有再生能力。在含高浓度2,4-D(5-30mg/l)的培养基上诱导出的愈伤组织具有较高的产生绿色结构的潜力。而高浓度 NAA(10mg/l)培养基产生的愈伤组织具有较强的产生根状体的潜力。培养的外植体或愈伤组织在含5mg/l 2,4-D 的培养基上,不需要经常的继代培养,即可长期保存。     

刺葡萄幼胚愈伤组织诱导及其高产原花青素细胞系筛选

以刺葡萄幼胚为材料,研究不同培养方式、培养基配方和培养条件对其愈伤组织诱导的影响,采用正交试验设计法筛选刺葡萄愈伤组织继代增殖的培养基配方,并对继代保持的培养条件和方式进行优化,同时进行了高产原花青素刺葡萄愈伤组织细胞系的筛选研究。结果表明,刺葡萄幼胚以平放的方式接种到MS＋1.0 mg·L^-1 2,4-D或MS＋1.0 mg·L^-1 2,4-D＋0.5mg·L^-1 KT的固体培养基上,在黑暗的条件下,能有效的诱导出愈伤组织,诱导效率为80%;刺葡萄愈伤组织继代增殖以MS＋1.5 mg·L^-1 2,4-D或MS＋1.5 mg·L^-1 2,4-D＋0.5 mg·L^-1 KT的固体培养基为佳,并且采用此两种培养基交替继代培养,在光照条件下能长期保持旺盛且生长一致的刺葡萄愈伤组织;筛选出了紫红色松脆状的高产原花青素的刺葡萄愈伤组织细胞系,培养35 d后每克鲜样的原花青素含量可达1 671.16μg。     

‘范妮’亚麻高频率体细胞胚胎发生及组织学观察

以亚麻品种‘范妮’的子叶和下胚轴为外植体,分别接种在不同组合的植物生长调节剂的MB(MS培养基的无机盐加B5培养基的维生素)培养基上培养,结果所有使用的培养基都能诱导出愈伤组织,但只有在附加了0.5~4.0 mg.L-12,4-D培养基上才能诱导出胚性愈伤组织。在胚性愈伤组织和体胚诱导中,源于下胚轴的材料均比源于子叶的材料所需的2,4-D浓度低。在培养基中,单独使用2,4-D诱导体胚的效果明显好于使用其它植物生长调节剂的组合,源于子叶和下胚轴的体胚诱导数分别达到了224个/g和265个/g。组织学观察发现有大量正常的球形胚、心形胚、鱼雷胚和少量子叶胚,还有少量畸形胚。本文为‘范妮’与其它麻类的体细胞杂交育种、‘范妮’变异无性系的筛选及相关的植物基因工程和细胞工程研究奠定了一定的工作基础。     

秦艽(Gentiana macrophylla Pall.)离体培养再生过程中体细胞胚的发生(简报)

本文以秦艽叶片和茎段作为外植体,通过离体培养对秦艽植株再生途径进行研究。愈伤组织在添加2mg/L 2,4-D和0.5mg/L BA的MS培养基上诱导,两周内可出现愈伤组织。愈伤组织在相同激素配比并附加500mg/L LH的MS培养基上继代。愈伤组织的分化在添加有0.1mg/L 2,4-D和0.5mg/L BA的MB培养基上进行。通过显微观测,疑似体细胞胚可以在叶片和茎段的愈伤组织上产生。形态学和组织学的分析进一步证实了秦艽离体再生过程中体细胞胚发生的现象。体细胞胚和合子胚一样,也经历球形、心形、鱼雷和子叶胚等发育时期。相对独立的结构说明秦艽的体细胞胚可能是单细胞来源。体细胞胚在愈伤组织的表面和内部都有出现。在本实验中,体细胞胚发生途径是在秦艽愈伤组织形成后观察到的唯一再生途径。     

Embryo Sacs of Hevea brasiliensis and Carica papaya Isolated with Enzyme-Squash Technique

The enzyme-squash technique had been employed to study the development of megaspores and embryo sacs from tropical plants of Hevea brasiliensis Muel-Arg. and Carica papaya L. Ovules fixed with FAA or FPA were enzymatieally macerated in 3% pectinase cellulase solution or in 3–4% snailase solution for 4–6 h, at 28–30℃ to isolate the embryo sacs after being squashed. The whole structural images of megaspores and embryo sacs at different developmental stages were observed and identified without staining under Olympus BH-2 phase contrast microscope or light microscope after ron-aceto-carmine staining.     

ACC oxidase from Carica papaya: Isolation and characterization

Most of the studies done on 1‐aminocyclopropane 1‐carboxylic acid (ACC) oxidase were done in vivo. It is only recently that in vitro studies have been carried out successfully on the enzyme. Here we report on in vitro studies of the enzyme that was isolated from Carica papaya . The enzyme had a K_m of 37 µ M and was inhibited by n ‐propyl gallate (0.240 m M ), sodium dithionite (0.022 m M ), sodium metabisulphite (0.021 m M ) and cobalt sulphate (0.100 m M ). The activity of the enzyme increased with ripening, the enzyme was somewhat labile and activity was lost after 4 days at 14°C; activity was prolonged when the crude homogenate was kept at −15°C. Isolation and purification were achieved with ammonium sulphate precipitation followed by gel‐filtration (Sephadex G 100‐120) and ion‐exchange chromatography (DEAE‐Sephadex). Gel electrophoresis of the purified enzyme gave a single band which corresponded to a molecular mass of 27.5 kDa. The amino acid content of the enzyme showed a relatively high percentage of valine (10.4%). Enzyme activity was enhanced when dithiothreitol (3 m M ) and bicarbonate ion (30 m M ) were added to the assay medium. The production of ethylene from Carica papaya did not require pretreatment of the fruit with ethylene.     

Occurrence and relative incidence of viruses infecting papaya in Venezuela

A survey of the main papaya (Carica papaya L.) production fields in Venezuela during 1997, indicated that crops were heavily affected with various virus‐like symptoms. A total of 745 samples from papaya plants showing symptoms suggestive of virus infection were collected and analysed using electron microscopy and enzyme‐linked immunosorbent assay (ELISA). Papaya ringspot virus (PRSV) and Papaya mild yellowing virus (PMYV) were the most frequently found viruses, which also occurred, in mixed infections. Rhabdovirus‐like particles were found only in samples collected in Distrito Federal (D.F). Papaya mosaic virus (papMV) and Tomato spotted wild virus (TSW V) were not detected during the survey.     

Regeneration of transgenic papaya plants via somatic embryogenesis induced byAgrobacterium rhizogenes

AnAgrobacterium rhizogenes-mediated procedure for transformation of papaya (Carica papaya) was developed. Transgenic plants were obtained from somatic embryos that spontaneously formed at the base of transformed roots, induced from leaf discs infected withA. rhizogenes. Transformation was monitored by autonomous growth of roots and somatic embryos, resistance to kanamycin, β-glucuronidase activity (GUS), and Southern hybridization analysis. Over one-third of the infected leaf explants produced transformed roots with GUS activity, from which 10% spontaneously produced somatic embryos. Histological analysis ofA. rhizogenes-transformed embryos showed that they have an altered symmetry between the shoot apex and the root meristem when compared to somatic embryos induced with hormone treatment from control explants. Transgenic papaya plants containingA. rhizogenes rol genes were more sensitive to auxins, developed wrinkled leaves, and grew slower than nontransformed plants.     

番木瓜果实内生细菌MGP3 菌株的鉴定及拮抗作用

【目的】从番木瓜果皮内筛选具有较强拮抗活性的内生细菌防治番木瓜采后炭疽病和疫霉病,以减少果实采后病害带来的损失。【方法】采用稀释分离和平板抑菌圈法进行内生细菌的分离筛选,结合形态特征、生理生化特性及16S rDNA部分序列同源性分析对菌株进行鉴定,菌株经利福平诱抗处理后田间接种到果树树干上,测定内生菌的定殖动态,采用采前和采后生防试验测定菌株对番木瓜炭疽病和疫霉病的生防效果。【结果】从番木瓜果皮中分离筛选到一株具有拮抗活性的内生细菌MGP3,对10种病原菌有较强的拮抗作用,鉴定该细菌为铜绿假单胞菌(Pseudomonas aeruginosa,登录号JF708186),MGP3可进入番木瓜叶片、叶柄和果皮中定殖。MGP3对采后番木瓜炭疽病和疫霉病的防治效果分别达到50%和71%;除苗期外,采前4个不同时期经MGP3菌液处理可以显著降低采收后果实炭疽菌的潜伏侵染率和炭疽病的病情指数。【结论】番木瓜内生拮抗细菌MGP3具有潜在的生防应用价值。     

Novel method for cell immobilization and its application for production of organic acid

AIMS: The aim was to develop a novel and simple technique for the entrapment of fungal hyphae. METHODS AND RESULTS: A novel immobilization technique was developed by using a structural fibrous network (SFN) of papaya wood as an immobilizing matrix. The technique is simple and a stable entrapment was achieved simply by inoculating the Aspergillus terreus hyphae within culture medium containing SFN pieces for 3 days, without any prior chemical treatment. Results show that SFN has no detrimental effect both on growth and bioactivity of fungi. A 23.5% increase in the itaconic acid production by SFN-immobilized A. terreus was noted when compared with free biomass. SFN-immobilized fungal biomass retained 95% itaconic acid productivity for five repeated batch cycles, 7 days each, without any disintegration/release of hyphae in the production medium. CONCLUSIONS: This is the first report on the use of SFN, a structural material, as an immobilizing matrix for the entrapment of any kind of microbial biomass and its application in organic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: The low cost of SFN and simplicity of the technique applied for immobilization of fungal hyphae within/onto SFN make its use ideal for the immobilization of fungal biomass to produce commercially valuable products.     

Transmission of the Phytoplasma Associated with Bunchy Top Symptom of Papaya by Empoasca papayae Oman

Transmission tests were conducted with field‐collected Bunchy Top Symptoms (BTS) phytoplasma‐infected specimens of Empoasca papayae. BTS developed in all eight inoculated papayas 3 months later. The BTS phytoplasma was identified in six of eight inoculated papayas, whose partial 16S rRNA sequence (GenBank Accession no. FJ6492000 ) was 99.9% identical with those from the collected papayas (GenBank Accession no FJ649198 ) and E. papayae (GenBank Accession no. FJ649199 ), all of which are members of group 16SrII, ‘Candidatus Phytoplasma aurantifolia’. Results confirmed the ability of E. papayae to transmit the BTS phytoplasma.     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

T4转基因番木瓜遗传性和果实品质分析

对T 4代转基因番木瓜进行了分子生物学和果实品质分析,结果表明,筛选获得的转基因番木瓜均为转番木瓜环斑病毒(PRV)复制酶突变体基因(RP),且对PRV抗性达到了高抗或免疫,RP基因在转基因植物中能稳定遗传至后代并在RNA水平上表达。在田间种植时,转基因木瓜的生长状况普遍好于普通番木瓜,尤其在生长后期(10月以后),普通番木瓜100%发病(大规模种植时),而大部分(约91.8%)转基因植株生长良好,果实较多且表面光洁、基本上无环斑。与非转基因亲本相比,T 4代转基因番木瓜的果实长度增加2.6%~5%,果实直径变小0.6%~1.5%,果肉厚度增加了12%~15%,因而果实形状与亲本相近或更好,且信用价值更高。转基因番木瓜果实中水分、蛋白质、氮、脂肪、还原性糖、维生素A、维生素C和类胡萝卜素的含量与对照都无显著性差异,即转基因番木瓜与亲本具有实质等同性,这表明转入的外源基因对番木瓜果实品质没有不良影响。     

CO₂ Assimilation Rate in Production Systems for Papaya Crops

The aim of this study was to evaluate some physiological aspects of papaya crops in semi conventional and organic production systems. The following factors assessed in this experiment were: 1. Production systems (organic and semi conventional); 2. Genotypes (Maradol and Maradona F1), and 3. Cover crop plants (Canavalia, vegetative cover and no cover). Twelve treatments were obtained -product of factors’ combination- and distributed under a threerepetition experimental design of subdivided parcels. The factors examined in this study, that changed the CO₂ assimilation rate, were production system and genotype. It was determined that the greatest gas exchange in papaya crops happened at 13:40 h but achieving the highest CO₂ assimilation was also affected by the production system and genotype. Similarly, they showed some effects in CO₂ assimilation, transpiration, stomatal conductance, intercellular CO₂, leaf temperature, chlorophyll, and temperature. In general, the combination of factors that accentuated in this experiment were the semi conventional-Maradona-Canavalia with a crop yield of 53.5 t ha^-1, followed by treatments organic-Maradona-no cover and semi conventional-Maradona-vegetative cover.