根癌农杆菌介导的西瓜遗传转化研究

利用带有内含子的GUS基因的瞬时表达,研究影响根癌农杆菌介导的西瓜遗传转化的若干因素。结果表 明:西瓜子叶块外植体对潮霉素较为敏感,15 mg/L是适宜的筛选浓度,可明显抑制非转化组织的生长;脱菌过程中 采用500 mg/L的头孢霉素,对外植体生长影响较小;农杆菌菌株EHA105对西瓜子叶块的侵染能力较强;预培养有 利于转化;共培养3-4 d有利于提高转化频率并避免了农杆菌的过度生长;OD600值为0.3的菌液侵染10min效果最 佳;共培养培养基中添加乙酰丁香酮可提高转化频率。     

根癌农杆菌介导的白菜类作物转化体系研究进展

白菜类作物是我国乃至世界性的大众蔬菜,利用农杆菌介导技术将目的基因转入蔬菜中,从而选育具有特殊性状的新品种。文章综述了根癌农杆菌介导的白菜类作物高频再生转化体系构建的影响因素,简要介绍了转化植株的分子检测及遗传行为,在此基础上分析了目前遗传转化技术在白菜类作物改良上存在的问题及广阔的应用前景。     

Some factors affecting the in vitro propagation of gladiolus

Callus tissue cultures have been established from the excised segments of the inflorescence, flower stalks, denuded flower, bract, perianth and leaf segments of 2 cultivars of Gladiolus grandiflorus. Of all the explants and the media tested, the best callus was obtained from the segments of the flower stalks, cultured on a basal medium supplemented with naphthalene acetic acid and kinetin. The callus mostly underwent rhizogenesis, and occasionally differentiated shoots. Complete plants were regenerated from the in vitro cultured cormels, cormel tips and the axillary buds, and 6 plants were formed from the segments of 1 cormel, whereas in nature only 1 plant is obtained per cormel. Cultured young anthers callused and developed leaf-like petaloid structures, and occasionally showed multicellular pollen.     

In vitro regeneration and genetic transformation of common bean (Phaseolus vulgaris L.)—Problems and progress

Common bean is one of the most cultivated species in the Leguminosae family. Thus far, progress in bean improvement has been achieved mainly by conventional breeding methods. These methods, however, in addition of being time-consuming and labor-intensive, are met with a wide range of problems including traits associated with low genetic variation, low survivability of interspecific hybrids, specific inheritance of some valuable characteristics such as yield, disease, and pest resistance, as well as harvesting characteristics. Plant biotechnology offers different strategies to overcome these difficulties. With some exceptions, species belonging to the Leguminosae are difficult to regenerate in vitro. In this review, we discuss the potential and limitations of in vitro cultivation of Phaseolus vulgaris L. based on the existing literature data. Different tissue culture methods like somatic regeneration are discussed and evaluated as well as gene transfer and other approaches in bean modification in vitro.     

苦瓜下胚轴的离体再生体系

为了建立高频的苦瓜离体再生体系,为苦瓜遗传转化提供技术基础,筛选了最适消毒方法和外植体类型。以MS培养基为基础培养基,添加不同浓度激素组合,筛选最适于苦瓜不定芽诱导、伸长和生根的培养基。最佳的消毒方法为:用水浸泡去壳的种子15 min,在超净工作台中用75%酒精浸泡30 s后再用4%NaClO浸泡6 min,用无菌水冲洗4～5遍,污染率为0,发芽率为98%;最佳的愈伤组织诱导培养基为MS+2.5 mg·L~(-1)6-BA+0.01 mg·L~(-1)IBA,愈伤组织密度为0.79 g·cm~(-3),不定芽的分化率为39%,最佳外植体为下胚轴(12 d),诱导率为41%;最佳的增殖培养基为MS+1.0 mg·L~(-1)6-BA+0.005 mg·L~(-1)IBA;最佳的生根培养基为MS+0.1 mg·L~(-1)IBA,移栽存活率为74%。初步建立了苦瓜离体再生体系。     

In vitro regeneration and conservation of wild species of Arachis

Summary

Wild species of Arachis are restricted to South America and generally occur in regions under intensive environmental disturbance. Both in situ and ex situ conservation strategies are required in order to maintain the availability of these genotypes. This work developed in vitro regeneration systems from seed explants of 17 wild species of Arachis from six Sections (Heteranthae, Caulorrhizae, Triseminatae, Erectoides, Procumbentes and Arachis). After seed disinfection, embryonic axes, leaflets and cotyledons were excised aseptically and cultured on Murashige and Skoog (MS) medium supplemented with 8.8 µM, 22 µM or 110 µM 6-benzylaminopurine (BAP). Cultures were maintained in a growth chamber at 28° ± 2°C with a 16 h photoperiod. Regeneration patterns from seed explants were similar among species from all Sections. Embryonic axes produced plants through meristematic amplification or multiple shoot formation, while cotyledons and embryonic leaflets produced shoots at significantly lower frequencies through direct and indirect organogenesis, respectively. Shoots obtained from all explants were transferred to MS medium without growth regulators to induce root formation.     

Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

Sweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.     

In vitro regeneration of Camellia reticulata by somatic embryogenesis

Camellia reticulata L. plantlets were regenerated by direct and indirect somatic embryogenesis from immature zygotic embryos. Initial explants (cotyledon sections and embryonic axes) produced somatic embryos without intermediate callus tissue when grown on Murashige and Skoog’s basal medium with 30 gl^-1 sucrose and no growth regulators; the somatic embryos completed their development in 4-6 weeks in the same medium. Embryogénie competence was increased by 0.5 and 1 mg l^-1 IBA. Histological observation showed the embryos to originate from epidermal and subepidermal cells of the cotyledon and hypocotyl explants. Secondary somatic embryos developed directly from the cotyledons and hypocotyl region of primary somatic embryos by a process that was morphologically very similar to that occurring on zygotic explants. Direct repetitive embryogenesis was maintained by this system. Up to 40% germination occurred when mature somatic embryos were isolated and incubated in medium supplemented with 1 mgl^-1 GA₃ + 1 mgl^-1 IAA. Indirect somatic embryogenesis was induced in callus differentiated on cotyledon explants after three months’ culture in media containing IBA or NAA and/or BAP, embryogenic capacity being retained by callus subcultured on 0.5 mg l^-1 IBA + 1 mg l^-1 BAP.     

Agrobacterium tumefaciens mediated genetic transformation and regeneration of Morus alba L.

An efficient method was established for genetic transformation of Morus alba clone M5 using Agrobacterium tumefaciens mediated gene transfer. Cotyledon explants from in vitro grown seedlings were co-cultivated with disarmed strain LBA 4404 harbouring the binary vector p^BI121 carrying chimeric β-glucuronidase (GUS) and neomycin phosphotransferase (npt II) genes. Maximum transformation frequency of 18.60% was recorded with 48 h of pre-conditioning followed by co-cultivation for the same duration. Expression and presence of transgene was confirmed by histochemical test and polymerase chain reaction. The transgenic plants were micropropagated and successfully acclimatised.     

杏植株再生与遗传转化研究进展

杏是我国主要果树之一,其育种一直以传统方法为主,一般存在周期长、成本高、可利用资源有限、种间杂交后代成活率低等问题。植物生物技术育种方法可以解决这些问题。再生与转化系统是实现杏基因转化的前提与基础。杏的再生与转化技术还不成熟,目前只限于少数几个品种或种子材料,其再生与转化方法有待进一步研究。结合作者的研究结果,综述了杏...     

In vitro plantlet regeneration of Olea europaea ssp. maderensis

Different media were assayed for Olea europaea L. ssp. maderensis Lowe micropropagation. Shoot elongation and propagation was more efficient on DKW medium supplemented with 4.4 μM BA and 0.4 μM IBA. Higher branching was obtained on OM medium supplemented with 18.2 μM zeatin. Rooting was achieved at higher rates on half strength DKW medium supplemented with 20.7 μM IBA. Plants were acclimated to greenhouse and up to now no morphological changes were observed among the regenerated in vitro plants.     

In vitro regeneration from alginate-encapsulated microcuttings of Quercus sp.

Microcuttings with apical and nodal buds from common oak (Quercus robur L.) and Turkey oak (Quercus cerris L.) in vitro clones were encapsulated into an alginate matrix. Apical segments of common oak showed significantly greater in vitro regeneration potential as compared with the nodal ones. Encapsulated Turkey oak nodal segments demonstrated successful regeneration after different periods (2–6 weeks) of cold storage (4 °C), showing no significant deviation from the regenerative frequency of non-cold-treated segments.     

Improved protocol for Agrobacterium mediated transformation of tomato and production of transgenic plants containing carotenoid biosynthetic gene CsZCD

Improved protocol for Agrobacterium mediated transformation of tomato (Lycopersicon esculentum) Micro-Tom was developed to use in corporation of the carotenoid biosynthetic genes CsZCD (Crocus zeaxanthin 7,8-cleavage dioxygenase). From these experiment, a transformation methodology using explants from cotyledons cultured for 1 day on the medium with zeatin 2 mg/L, IAA 0.1 mg/L, carefully submerged in the Agrobacterium inoculum for 20 min, then concultured with the agrobacterium for 3 days on the same medium, followed by a transfer to the same medium with 500 mg/L cefotaxin for 3 days and then by a transfer to the same medium with 100 mg/L kanamycin and 500 mg/L carabenillin for 6–8 weeks and resulted in a greater than 20% transformation efficiency in the concentration of Agrobacterium OD600 = 0.2 tested. In this transformation method, no feeder layers were used and the subculture media was minimal. Among the Agrobacterium concentrations of OD600 = 0.2, 0.5 and 1.0, the best transformation efficiency, 20.87%, was obtained by using OD600 = 0.2, which was significantly higher than that of OD600 = 1.0. The presence of the inserted target genes was checked using a rapid and efficient PCR test. The protocol was successfully employed in the production of transgenic Micro-Tom tomato containing the carotenoid biosynthesis CsZCD under constructive promoter. This procedure represents a simple, efficient and general means of transforming tomato.     

香榧茎段离体培养再生植株的研究

对香榧茎段不定芽诱导研究的结果表明,不定芽诱导的最佳培养条件为:培养基为B5+KT0.1mg/L+IBA0.5mg/L+0.08%活性炭+2%葡萄糖,培养温度为20℃,光强不高于400lx,该条件下的不定芽诱导率可达55%。将不定芽培养在1/2B5+IBA0.1mg/L+0.08%活性炭+2%葡萄糖的生根培养基上,生根率最高达到40%。通过器官发生途径国内外首次建立了香榧离体再生体系和快繁体系,并获得了完整的再生植株,为今后开展香榧的遗传转化和品种改良奠定了基础。     

余甘子下胚轴离体培养中的器官形成与植株再生(英文)

以余甘子(phyllanthusemblicaL.)下胚轴为外植体,通过器官发生途径诱导形成不定芽,探讨不同激素组合对下胚轴器官发生和植株再生的影响,以期建立有效的再生体系,为以后的遗传转化研究奠定基础。结果表明,在附加0.1μmol/LNAA、5μmol/LBA和30g/L蔗糖MS培养基上培养,5周后不定芽再生频率达87.5%,平均每个外植体再生芽数为9.7个,为最佳组合。将分化的不定芽转移至含有10μmol/LIBA的MS培养基上,5周后生根,发育成健康的植株,生根率在29.2%。     

阔叶猕猴桃叶片离体器官发生和植株再生(英文)

以阔叶猕猴桃(A ctinidia latifolia M err.)叶片为外植体,通过器官发生途径诱导形成不定芽,探讨了不同激素组合、暗培养时间以及不同浓度的蔗糖对叶片器官发生和植株再生的影响。结果表明,BW +0.1μm ol/L N AA +5μm ol/L Zeatin 附加20g/L 蔗糖,先进行21d 暗培养然后转到光下培养效果最好,6周后不定芽再生频率达91.67%,平均每个外植体再生芽数6.87个。再生芽生根良好,生根率可达100%。首次报道了阔叶猕猴桃的器官发生和植株再生,建立了叶片的高效再生体系,为今后的遗传转化研究奠定了基础。     

Optimizing shoot regeneration and transient expression factors for Agrobacterium tumefaciens transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency

An efficient, adventitious shoot regeneration protocol was devised, and transient expression studies were carried out to enable Agrobacterium-mediated stable transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency. Leaves, from in vitro stock cultures, with the petiole removed and four partial cuts made transversely and equidistant through the midrib area were found to be the optimum explant type. A 24 h liquid TDZ-pretreatment (0.05, 0.10 or 0.25 mg/l) in MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497.] of leaf explants stimulated shoot formation upon subsequent culture on QL medium [Quoirin, M., Lepoivre, P., 1977. Improved media for in vitro culture of Prunus sp. Acta Hort. 78, 437–442.] supplemented with 3.0 mg/l BAP and 0.5 mg/l NAA. A frequency of 38.9–54.4% of the explants produced at least one shoot with the maximum mean number of shoots, 4.5 per explant with the 0.10 mg/l TDZ pretreatment. The shoot regeneration scheme was subsequently linked with inoculation with Agrobacterium tumefaciens strains EHA105, GV3101 or LBA4404, each harboring the binary plasmid pBISN1. PBISN1 contains an intron interrupted ß-glucuronidase (GUS) gene (gusA) under control of the chimeric super promoter (Aocs)₃AmasPmas. Blue stained leaf cells were observed after co-cultivation with all three strains. Co-cultivation for 4 days with 19.6 mg/l acetosyringone (AS) and assay by GUS indicated over 90% of the leaf explants were infected with an average 7.5–8.8 blue foci per explant. No differences were observed in regard to A. tumefaciens strain used.     

In vitro shoot multiplication and plant regeneration in four Capsicum species using thidiazuron

A procedure of in vitro plant propagation using shoot meristem explants (∼0.5 cm) has been developed for Capsicum annuum cv CA960, C. baccatum, C. frutescens and C. praetermissum on Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497] medium containing various cytokinins. Among various concentrations of cytokinins tested; adenine (Ad), N⁶-benzyladenine (BA), kinetin (Kn), zeatin and thidiazuron (TDZ) individually. TDZ regenerated maximum number (4.2–22.4) of shoots in all the Capsicum species tested. Multiple shoot elongation occurred upon transfer to BA (0.22 μM l⁻¹) + IAA (0.48 μM l⁻¹). Rooting of regenerated shoots was achieved on medium supplemented with 5.71 μM l⁻¹ indole-acetic acid (IAA). Rooting was observed in 72–94% of shoots obtained from TDZ-containing regeneration medium followed by elongation treatment in contrast to 8–22% of shoots without elongation treatment. Plantlets obtained from TDZ-containing media were normal diploid (2n = 24) and could readily be established in the soil under green house conditions with a survival frequency of 68–84%. Regenerated plants were developed into morphologically normal, fertile plants and able to set viable seeds.     

黑莓试管苗叶片植株的再生

以黑莓带芽茎段试管苗叶片为外植体,诱导形成不定芽并进一步形成再生植株。在MS+TDZ2.0mg/L的培养基中叶片不定芽最高分化频率达93.7%(6.52不定芽/外植体),在不定芽伸长培养基BA0.5mg/L上,分化完全的不定芽能够长大伸长,当其高度达到4cm时,切下转接于IBA为0.3mg/L培养基上诱导生根,生根率100%。     

寒富苹果叶片离体再生及四倍体诱导

为建立寒富苹果高效的离体再生体系和多倍体诱导体系,以试管苗叶片为外植体,研究了培养基中激素对寒富叶片再生芽的影响及适宜的四倍体诱导方法。结果表明,当培养基中BA质量浓度为0.5mg/L时,再生频率最高达35.7%;当培养基中BA质量浓度为2.5mg/L时,再生频率超过90%,平均再生芽数在4以上。在培养基中附加1.0mg/LTDZ,再生频率达100%,平均再生芽数达19.47。以附加15、30、60、120mg/L秋水仙素的液体再生培养基处理叶片5d,各个质量浓度处理均诱导出四倍体植株,诱变率在5.3%～22.2%之间;寒富苹果叶片在附加50mg/L秋水仙素固体再生培养基上处理5d亦获得了四倍体植株。研究结果表明寒富苹果叶片具有极强的不定芽再生能力,叶片再生过程中进行秋水仙素处理是获得其四倍体的一个有效途径。