大豆体细胞胚胎发生与农杆菌介导的遗传转化

以55个大豆基因型未成熟子叶为外植体,用高浓度2,4-D诱导大豆体细胞胚胎发生与植株再生,并对生产上种植面积大、体细胞胚胎发生率高的大豆基因型用农杆菌介导法进行遗传转化。结果表明,东北地区主栽的大豆基因型中有14个基因型体细胞胚胎发生率超过40%。用含有pGBI121S4ABC质粒的LBA4404农杆菌侵染5个东北地区主栽大豆基因型的2147个未成熟子叶,经卡那霉素抗性筛选得到12株PCR阳性植株。 Abstract：Somatic embryogenesis was induced and the regenerated plants were obtained by higher concentrations of auxins with immature cotyledon of 55 genotypes in soybean. Bivalent insect resistant genes were transformed into immature cotyledon of soybean which have high frequency of somatic embryogenesis via Agrobacterium-mediated. The results showed that 14 genotypes possessed high frequency of somatic embryogenesis (more than 40%) among soybean genotypes from Northeast area. 2147 immature cotyledons of 5 different soybean genotypes cultured in Northeast area were inoculated with LBA4404 (including pGBI121S4ABC plasmid). 12 regenerated plants selected by Kanamicy gave positive PCR reaction.     

In vitro response of encapsulated somatic embryos of camellia

Plant regeneration via somatic embryogenesis was achieved in leafbase and leaf tip explants derived from 10-day-oldin vitro-grown seedlings ofEchinochloa colona. Somatic embryogenesis was induced in the callus on Murashige and Skoog (1962) medium supplemented with 4.44 μM 6-benzyladenine, 4.64 μM kinetin and 8.05 μM 1-naphthaleneacetic acid after 4 weeks of culture incubated for 14 days in continuous dark and subsequently under a 14-h photoperiod. The incidence of somatic embryogenesis was greater in leafbase- than in leaf tip-derived calluses. Histological observations revealed various stages of development of somatic embryogenesis. The embryos matured and germinated on fresh medium lacking growth regulators. The somatic embryo-derived plantlets were established in soil.     

Direct somatic embryogenesis from shoot apical meristems of pea, and thidiazuron-induced high conversion rate of somatic embryos

Direct somatic embryogenesis from shoot apical meristems of pea is described. Somatic embryos were induced directly (without callus intervention) from meristematic tissues grown on a medium supplemented with 2.5 μM picloram. Within 4 to 5 weeks, fully morphologically developed somatic embryos were obtained. Somatic embryos originated from apical as well as from basal parts of meristem explants. The initiation and development of somatic embryos was asynchronous, basal somatic embryos developed more quickly than apical ones. Abundant secondary embryogenesis was observed after isolation of primary somatic embryos and culturing them on media for germination. Morphologically normal somatic embryos germinated on medium without growth regulators; the conversion rate was increased by application of 10 μM thidiazuron (TDZ). TDZ was also able to induce shoot bud regeneration on embryos without differentiated a shoot apex, allowing to germinate up to 78 % of all harvested somatic embryos with various morphology. The protocol was successfully tested in 47 out of 48 P. sativum and P. arvense cultivars as well as in two wild peas (P. elatius, P. jomardi). This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis on Thin Cell Layers of a C4 species,Digitaria sanguinalis (L.) Scop

Somatic embryogenesis was obtained from transverse thin cell layers (tTCLs) of Digitaria sanguinalis. tTCLs (0.2 - 0.4mm thick, 1mm in diameter) were excised from 4-week-old seedlings and placed onto Murashige and Skoog media supplemented with a varying concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (from 1 μM to 100 μM) and sucrose (from 3% to 24%). Somatic embryos were obtained in the dark 7-10 days after inoculation from tTCLs excised at specific levels on the seedling and cultured in the presence of 2,4-D (5 μM to 10 μM) and sucrose (3 to 6%). The exposure of the tTCLs to light decreased the percentage of tTCLs forming somatic embryos. Viable plantlets were obtained 2 weeks after transfer onto a cytokinin-containing medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Embryogenesis induction in petals of Araujia sericifera

The embryogenic capacity of Araujia sericifera petals and some of the factors involved in the induction of embryos was investigated. The influence of 6-benzyladenine and α-naphthalene acetic acid, light intensity (90 or 5 μmol m^-2 s^-1) and silver thiosulphate (inhibitor of ethylene action) were studied. It was found that petals are an easy system in which to induce somatic embryogenesis. Plants were recovered from somatic embryos. Although 6-benzyladenine is essential for inducing an efficient response, a high dosage increased callogenesis and reduced embryogenesis. The highest rate of embryogenesis is induced with high light intensity (90–100 μmol m^-2 s^-1), even though the presence of silver thiosulphate in the medium markedly reduced embryo induction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis from cultured epicotyls and primary leaves of soybean [Glycine max (L.) Merrill]

Summary Regeneration of several varieties of soybean [Glycine max (L.) Merrill] by somatic embryogenesis from cultured epicotyls and primary leaves has been demonstrated. Somatic embryogenesis was induced from epicotyls and primary leaves when cotyledon halves with the intact zygotic embryo axes were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg 1⁻¹ (45.2 μM) 2,4-D. Stable, continuously proliferating globular embryo cultures (GEC) were established from small groups of somatic embryos on MS medium supplemented with 20 mg 1⁻¹ (90.5 μM) 2,4-dichlorophenoxyacetic acid (2,4-D). Rapid multiplication of shoot tips from germinating somatic embryos was achieved on Cheng’s basal medium (CBO) containing 2.5 mg 1⁻¹ (11.3 μM) 6-benzyladenine. Fertile plants were obtained from individual somatic embryos and in vitro propagated adventitious shoot bud cultures.     

Effect and analysis of phenolic compounds during somatic embryogenesis induction in Feijoa sellowiana Berg

Summary. The effect of phenolic compounds on somatic embryogenesis in Feijoa sellowiana was analysed. The results showed that caffeic acid (140–560 μM) significantly increased somatic embryogenesis induction compared with the control. The presence of phloridzin, even at lower concentrations (11.5 μM), or caffeic acid or phloroglucinol at concentrations greater than 140.0 and 197.5 μM, respectively, inhibited somatic embryo development beyond the globular stage. When somatic embryos were transferred to the germination medium, the highest rates of germination (81.9%) were obtained with embryos induced in the presence of phloroglucinol (79.0 μM). At all concentrations tested, somatic embryos induced in medium containing phloroglucinol germinated at higher rates than those induced in the presence of caffeic acid. Histological and ultrastructural studies showed that somatic embryos were formed in close association with phenolic-rich cells which, in more advanced stages of development, formed a zone isolating the embryo from the maternal tissue. A comparative analysis of total phenolic content indicated that phenolics reached a peak by the third week of culture, independently of the medium used. However, after that period, the amount of phenolic compounds was significantly higher in explants cultured in the presence of phloroglucinol than in those cultured in the control or in caffeic acid-containing medium. Attempts to identify the type of phenolic compounds showed that flavan-3-ols and gallic acid derivatives were mainly produced in phloroglucinol-containing medium, whereas flavanones and dihydroflavonols were also present in medium containing caffeic acid. Flavones were the main phenols detected in the control. The ways in which phenolic compounds may affect somatic embryogenesis are discussed. Correspondence: J. M. Canhoto, Departamento de Botanica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Cal?ada Martim de Freitas, 3001-455 Coimbra, Portugal.     

The histological analysis of indirect somatic embryogenesis on <Emphasis Type="Italic">Drosera spathulata</Emphasis> Labill

We studied indirect somatic embryogenesis in the callus tissue of Drosera spathulata Labill. originated from isolated leaves. Callogenesis was induced on MS medium (Murashige and Skoog 1962), supplemented with various concentrations of NAA and BA. Somatic embryos regenerated on half-strength MS medium supplemented with 20 μM of NAA or without growth regulators. The highest efficiency of somatic embryo production was achieved on hormone-free medium. Globular, heart-, torpedo- and cotyledonary-shaped embryos were observed in embryogenic clusters. Histological and scanning electron microscopy analysis verifies somatic embryogenesis. Regenerated plants were transferred to soil and were grown to maturity.     

利用mRNA差别显示技术分析枸杞体细胞胚发生早期基因的差别表达

本研究选用枸杞体细胞胚发生体系中的继代愈伤组织(对照)、胚性愈伤组织和早期胚体为实验材料,提取细胞总RNA,在12种锚定真核生物mRNA3'末端的OligodT12VN中,随机选用OligodT12GA为引物合成了以上三种材料的cDNA第一链,以此cDNA为模板,用随机引物进行PCR扩增,选择差别表达的片段。我们选用了OPA、OPH、OPK和OPB四组的60个随机引物对所得的c DNA进行了PCR扩增,得到了三个在体细胞胚发生早期组织中基因特异表达的片段。结果表明,在体细胞胚发生早期有胚胎发生特异性基因的表达,而且这种特异表达的基因在继代愈伤组织中没有表达,说明植物的体细胞胚发生过程就是细胞内基因差别表达的结果。 Abstract:Embryogenic calli and early embryo can be obtained from both auxin and auxin-free medium.The analysis of differential gene expression in early somatic embryogenesis has been hindered by above-mentioned material.The modifications of the recently described mRNA differential display method were reported and differential gene expression in early slmatic embryogenesis was analyzed.We have obtained three differential bands of cDNA in early somatic embryogenesis.The results indicate that gene expression has temperal and spalil order in early somatic embryogenesis of Lycium barbarum L.Plant somatic embryogenesis is the results of differential gene expression in cell.     

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μm kinetin or zeatin, but was highest on medium containing 4.5 μm 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 μm 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 μm 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse. Received: 14 January 1997 / Revision received: 17 June 1997 / Accepted: 5 July 1997     

Plant regeneration via somatic embryogenesis in pigeonpea (Cajanus cajan L. Millsp)

Efficient plant regeneration via somatic embryogenesis has been developed in pigeonpea. Cotyledon and leaf explants from 10-day-old seedlings produced embryogenic callus and somatic embryos when cultured on Murashige and Skoog (MS) medium supplemented with 10 μm thidiazuron (TDZ). Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal medium. The rooted plantlets were transferred and acclimatized on vermiculite where they showed normal morphological characters. Received: 23 December 1996 / Revision received: 22 July 1997 / Accepted: 2 August 1997     

Regeneration of intergeneric somatic hybrids by protoplast fusion between Onobrychis viciaefolia and Medicago sativa

Protoplast fusion was induced between sainfoin and alfalfa by an improved polyethyleneglycol (PEG) method. The intergeneric somatic calluses were selected based on complementation of hydroxyproline-resistance of sainfoin and hormone autonomy growth of alfalfa transformation cell line. 17 somatic hybrid plantlets were regenerat-ed. PEG could induce the tight agglutination of protoplasts. During diluting and washing process, cyclization of the linked membrane and formation of vesicle-like structures were observed, resulting in protoplast fusion. 5%-10% glycerol supplemented in the fusion inducing solution markedly increased the frequency of heterogeneous fusion. Better fusion results were obtained when mixed protoplast suspension was dripped in petri dishes in which PEG solution was previously placed. Chromosome number of regenerated hybrid buds varied from 30 to 60. The genome of hybrids in-cluded the small chromosome from sainfoin and two chromosomes with two clear constrictions from alfalfa. The hybrid     

Diphenylurea Derivatives Induce Somatic Embryogenesis in Citrus

The present research investigates the possibility that three diphenylurea (DPU) derivatives, N-phenyl-N′-benzothiazol-6-ylurea (PBU), N,N′-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methilendioxyphenyl)urea (3,4-MDPU), stimulate the induction of somatic embryogenesis in three Citrus species. The hypothetical embryogenic activity was assessed using stigma and styles of Citrus myrtifolia Raf., Citrus madurensis Lour. and Citrus limon (L.) Burm. The three compounds influenced the production of somatic embryos differently as regards the concentrations tested and the citrus species. PBU was able to induce somatic embryogenesis at all the concentrations tested and in all the three species with percentages that ranged from 44 (C. limon) to 85% (C. myrtifolia). 2,3-MDPU and 3,4-MDPU were completely unable to induce the production of somatic embryos in C. myrtifolia while both the compounds at the higher concentration (12 μM) acted positively in both C. madurensis and C. limon (68% of embryogenic explants). The phenylurea derivatives, used for the first time in this study to induce somatic embryogenesis in plant, showed a higher embriogenic performance when compared with 6-benzylaminopurine (BAP), a classical adenine-cytokinin, and with N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), a classical DPU derivative.     

Somatic embryogenesis and plant regeneration of neem (Azadirachta indica A. Juss.)

Somatic embryos were initiated with mature seeds of neem (Azadirachta indica A. Juss.) when cultured on Murashige and Skoog's medium supplemented with thidiazuron (TDZ). Regeneration occurred via somatic embryogenesis: direct embryo formation and through an intermediary callus phase. TDZ was very effective and induced somatic embryogenesis across a wide range of concentrations (1–50 μm). However, somatic embryogenesis was accompanied by callus formation at concentrations of 20 μm and above. Cell suspension cultures were established with the TDZ-induced callus and groups of large cell clumps were formed within 2–3 weeks. Plants were regenerated from both directly formed somatic embryos and somatic embryos derived from cell suspensions plated on semisolid medium devoid of growth regulators. Regenerated plantlets continued to grow after transfer to a greenhouse environment and were similar phenotypically to zygotic seedlings. This simple regeneration system may be beneficial for mass propagation of selected elite clones of neem. Received: 13 May 1997 / Revision received: 13 November 1997 / Accepted: 2 December 1997     

植物激素对体细胞胚胎发生的诱导与调节

以作者自己的工作为背景,结合国内外近几年的有关报道,综述了几种外源和内源激素对植物体细胞胚胎发生的诱导与调节作用。外源生长素和细胞分裂素是诱导离体培养细胞分化与增殖所必需的,2,4-D是诱导胚性愈伤组织的重要激素。在体细胞胚胎发生中内源激素含量和代谢的平衡起着关键的作用,而且外源和内源激素对诱导体细胞胚胎发生起相互调节作用。ABA在提高体细胞胚胎发生频率和质量上具有重要作用,同时,外源与内源ABA对体细胞胚胎发生起相互促进作用。本文还较为深入地讨论了这些激素诱导体细胞胚胎发生的可能作用机制。 Abstract:The paper summarizes the induced and regulatory effects of a few exogenous and endogenous hormones in plant somatic embryogenesis by our studies and related international reports.The exogenous auxin and cytokinin are necessary to induced differentiation and proliferation of cells of culture in vitro.2,4-D is an important hormone of induced embryogenic calluses.The contents and the metabolic balances of endogenous hormones have key effects for somatic embryogenesis.In addition,the exogenous and endogenous hormones have mutual regulatory effects for somatic embryogenesis.ABA has an important effect to improving the frequency and quality of somatic embryogenesis.Meanwhile,the exogenous and endogenous ABA have mutual promoted effects for somatic embryogenesis.The paper discusses possible mechanism of hormones-induced somatic embryogenesis in a deep-going way.     

Somatic embryogenesis from chickpea (Cicer arietinum L.) inmature cotyledons: The effect of zeatin, gibberellic acid and indole-3-butyric acid

Studies were conduced to test the effects of various cytokinins on somatic embryogenesis from chickpea (Cicer arietinum L.) immature cotyledons. Zeatin (13.7 μmol) added, to B5 basal medium, supplemented with 1.5 % sucrose and 0.2 μmol indole-3-acetic acid, was the most effective cytokinin. Lobular structures obtained from cotyledons cultures were transferred to B5 basal medium supplemented with gibberellic acid and indole-3-butyric acid at different concentrations. The most effective treatment was B5 medium containing 14.4 μmol gibberellic acid plus 1.0 μmol indole-3-butyric acid in which 42.8 % of lobular structures cultured formed normal somatic embryos. High conversion of embryos into plantlets (61.0–65.2 % embryos regenerated plants) was observed when germinated embryos were placed on plant development medium.     

Induction of somatic embryogenesis and plantlets in tendrils of Vitis vinifera L.

Somatic embryogenesis was observed in callus initiated from tendril explants of Vitis vinifera L. cvs. Thompson, Sonaka and Tas-e-Ganesh on Emershad and Ramming medium supplemented with 1 μm 6-benzylaminopurine. Low-frequency conversion to shoots was obtained in the third and fourth subculture on the same medium. Emerging shoots subsequently formed complete plantlets on liquid rooting medium containing 1 μm indole-3-acetic acid. The possible use of tendrils as a novel explant for somatic embryogenesis in grape is discussed. Received: 3 March 1997 / Revision received: 21 May 1997 / Accepted: 25 June 1997     

Light quality influences germination, root growth and hypocotyl elongation in somatic embryos but not in seedlings of norway spruce

Summary We studied how light from different light sources influences germination and postgerminative growth of plants from somatic embryos and seeds of Norway spruce (Picea abies [L.] Karst). Somatic embryos of three spruce genotypes and seeds were subjected to light from commercially available light sources: Philips TLD Blue 18W/18 (BL), Osram Fluora (FL), Philips Cool White TL 50W/33 (CW), Osram Warm White 18W/30 (WW), Philips Yellow 36W/16 (YE) and Philips TLD Red 36W/15 (RE), 18 h a day, with a photon flux (PAR) at 30 μmol m⁻² s⁻¹. After 6 wk the germination frequencies of the somatic embryo-derived plantlets were 50% under BL and 98% under RE. The corresponding mean root lengths were 6.7 and 15.4 mm. In somatic embryo-derived plantlets cultured under BL, FL, CW and WW, both roots and hypocotyls turned brown, presumably due to production of phenolic substances. Browning was less severe in somatic embryo-derived plantlets cultured under RE and YE. Under RE, the epicotyl elongated in 37% of the plantlets after 6 wk, compared with 70% under the other light sources. Seed germination and postgerminative seedling growth was modestly influenced by light from these light sources. RE and WW initially delayed germination as compared with BL, FL and CW, but after 2 wk, more than 90% of the seeds had germinated under all light sources. In conclusion, germination and postgerminative growth of somatic embryos of spruce is sensitive to differences in light quality, whereas seed germination and seedling growth is not.     

猪圆环病毒2型及猪繁殖与呼吸综合症病毒的快速检测

According to the published genome sequences of Porcine circovirus type 2(PCV2)and Porcine reproductive and respiratory syndrome virus(PRRSV),primers were designed and PCR,RT-PCR were set up for the detection of PCV2 and PRRSV,respectively,With the established methods,38 clinical samples from the respiratory disease pigs were detected for the presence of PCV2 and PRRSV. The results demonstrated that 22 samples were positive for PCV2,27 samples were positive for PRRSV and among the above positive samples,18 samples were positive for both viruses,The data obtained in the present study indicated that PCV2 and PRRSV maybe play an important role in the course of the development of respiratiory diseases.