国外竹类植物组织培养研究最新进展

组织培养技术已经成为竹类植物快速繁殖的重要手段之一。文章概述了近10年来国外在竹类植物组织培养研究方面的最新进展,包含了基于固体、液体培养基所开展的以芽繁芽、愈伤组织诱导、体细胞胚胎发生等途径的繁殖研究,以及细胞悬浮培养与种质保存、次生代谢物质诱导与分离等应用研究。对外植体的选择与消毒、组培种苗产权保护和试管开花与转基因育种等进行了讨论。     

Induction of flavonoid production by UV-B radiation in Passiflora quadrangularis callus cultures

Callus cultures from several species of Passiflora were initiated in vitro, and their capacity to produce four glycosyl flavonoids (orientin, isoorientin, vitexin, isovitexin) was analysed. The aim of the present work was to examine the possible role of UV-B irradiation and elicitation with methyl jasmonate (MJ) on the production of these compounds in callus cultures. All the species tested (P. incarnata, P. quadrangularis, P. edulis) formed friable callus from leaf explants after 4 weeks on medium supplemented with kinetin and 2,4-dichlorophenoxyacetic acid. Among them, P. quadrangularis turned out to have a faster growth rate and a more friable texture, and was therefore chosen for experiments with elicitors. In callus cultures only small amounts of isoorientin were found, while the concentration of the other flavonoids was below the detection limit. UV-B irradiation of calluses was able to increase the production of all four glycosyl flavonoids. After a 7-day exposure of cultures to UV-B light, the production of isoorientin reached concentrations similar to those found in fresh leaves from glasshouse-grown plants. Elicitation with methyl jasmonate also enhanced orientin, vitexin and isovitexin concentrations, even though the stimulation was about 6-fold weaker for orientin and vitexin and about 40-fold for isovitexin, than that exerted by UV-B treatment. Callus cultures treated with the UV-B dose which most enhanced flavonoid production showed a higher antioxidant activity compared to untreated calluses, with an increase ranging from 28% to 76%. Results show that the secondary metabolite biosynthetic capacity of Passiflora tissue cultures can be enhanced by appropriate forms of elicitation.     

Interactions between callus cultures of Pinus sylvestris and pine fungi with different trophic properties

Fungal virulence may be studied using tissues cultures of host plants in dual cultures in vitro, enabling analyses of interactions with undifferentiated cells of their host plants. Three genotypes of Pinus sylvestris callus, initiated by somatic embryogenesis, were used for establishing dual cultures with fungi pathogenic, endophytic or saprotrophic on pine needles or shoots. Fungal growth towards the plant callus tissue differed, depending on the life strategy of the fungus. The pathogen Gremmeniella abietina proved the slowest colonizer of callus whereas the saprotrophic Phacidium lacerum was the fastest. Gremmeniella abietina partially overgrew the callus, causing extensive necrosis and death within 10 days after inoculation. Anthostomella formosa, an endophyte of pines, did not cause evident symptoms of callus degradation: after 10 days of dual culture, the callus cells remained greenish and at least 50% of cells were alive. In dual cultures Ph. lacerum, callus remained alive until the end of the experiment, maintaining a white‐creamy colour with a loose cell structure. Electrophoresis of protein extracts from the callus showed the presence of additional bands of 25–35 kDa only in host tissues challenged with the pathogen G. abietina, possibly indicating the production of pathogenesis‐related proteins. This work has shown that pine callus does not respond equally to challenge with different fungal isolates. In general, one‐third of the isolates of each fungus examined showed greater virulence compared to other isolates.     

竹子的离体培养研究

近２０ａ来已对２０个属７０余种竹子进行离体培养研究,以侧芽,顶芽,成熟胚作外植体诱导愈伤组织,由愈伤组织制备悬浮细胞进行细胞悬浮培养,由悬浮细胞制备原生质体进行原生质体培养。竹子愈伤组织经不定芽途径或体细胞胚发生途径再生完整植株。通过芽尖培养增殖新生芽进行竹微繁殖,并获得脱病毒种苗。以芽为外植体增殖的新芽或组织再生苗经继代培养诱导竹试管开花结实。     

白刺花胚性愈伤组织诱导及体细胞胚发生

【目的】探讨不同植物生长调节剂对白刺花胚性愈伤组织诱导的作用,以及培养基中氮源和无机盐浓度对白刺花体细胞胚发生和植株再生的影响,以期建立白刺花体细胞胚发生、发育及调控技术体系,为白刺花种苗快速繁殖体系建立及遗传转化研究提供参考。【方法】以白刺花叶片为外植体,研究生长调节剂2,4-D(1.0、2.0、3.0、4.0 mg ·L -1 )、NAA(0、0.5、0.8、1.0 mg ·L -1 )、6-BA(0.2、0.5、1.0、2.0 mg ·L -1 )和TDZ(0、0.2、0.5、1.0 mg ·L -1 )组合对胚性愈伤组织诱导,及NAA(0、0.2、0.5 mg ·L -1 )、6-BA(0、0.5、1.0 mg ·L -1 )和TDZ(0、0.2、0.5 mg ·L -1 )组合对体细胞胚发生的调控作用,筛选最优生长调节剂组合;并研究培养基中KNO 3和NH 4NO 3比例对体细胞胚发生的作用,及MS培养基中无机盐浓度(1/5MS 、1/4MS、1/3MS、1/2MS)对体细胞胚萌发的影响,筛选最佳的体细胞胚发育及成熟萌发条件。【结果】白刺花叶片外植体胚性愈伤组织诱导适宜培养基为MS + 2,4-D 3.0 mg ·L -1 + NAA 0.5 mg ·L -1 + 6-BA 0.2 mg ·L -1 + TDZ 1.0 mg ·L -1 +蔗糖40 g ·L -1 +琼脂7.0 g ·L -1 ,诱导率为42.0%。采用MS基本培养基时,最佳的体细胞胚发生培养基为MS + NAA 0.5 mg ·L -1 + 6-BA 1.0 mg ·L -1 + TDZ 0.5 mg ·L -1 +蔗糖40 g ·L -1 +谷氨酰胺100 mg ·L -1 +琼脂7.0 g ·L -1 ,体细胞胚发生率为78.46%,总胚数为对照的3.6倍;MS培养基中,KNO 3浓度提高1倍、NH 4NO 3降至1/2时,体细胞胚发生率可提高至91.33%,总胚数为采用MS基本培养基时的1.4倍;1/3MS培养基有利于体细胞胚的萌发,萌发率为82.75%,幼苗长势良好,单株平均鲜质量为76 mg,幼苗驯化移栽1个月后成活率达90%以上。【结论】白刺花叶片接种于添加2,4-D、NAA、6-BA和TDZ不同组合的诱导培养基上,可脱分化形成愈伤组织或胚性愈伤组织,2,4-D浓度对愈伤组织形态和质地有较大影响。TDZ有利于体细胞胚的形成,适宜浓度的生长素与细胞分裂素组合及硝态氮和铵态氮的比例对体细胞胚的形成和发育具有调控作用,降低MS无机盐浓度可提高体细胞胚萌发率,本试验体系的再生植株移栽成活率达90%以上。     

Somatic embryogenesis inCephalotaxus harringtonia embryo-megagametophyte co-culture

Somatic embryogenesis was initiated fromCephalotaxus harringtonia (Forbes) K. Koch embryo culture. Explants consisted of embryo and megagametophyte halves both cut longitudinally. They were removed aseptically from mature seeds and grown together on a solid Murashige and Skoog modified medium supplemented with 5 mg·l ⁻¹ 2,4-dichlorophenoxyacetic acid. Embryogenic cultures started from callus after three or more months on the primary medium. The embryogenic callus originated from the suspensor region of the embryo. All chromosome counts made in the cells of the embryonic structures demonstrated a diploid stage, which suggest that they originated from zygotic embryo tissue. The early stages of somatic embryogenic development were achieved,i.e., formation of small clusters consisting of an embryonal region made up of isodiametric meristematic cells. A more advanced stage was reached in some cultures in which the distal embryonal end of the embryo appeared smooth and opaque. The ultrastructural characteristics of the embryos, the two types of embryo cells, embryonal and suspensor cells, as well as their contents were similar to those already reported in the case of somatic embryogenesis of other conifers.     

刺五加体细胞胚状体发生及成苗的研究

以刺五加试管苗叶片为试材,将叶片接种于含有不同种类和浓度激素的培养基上进行培养,研究不同激素对刺五加胚性愈伤组织诱导、胚状体发生及生根的影响。结果表明：诱导叶片胚性愈伤组织发生的最适培养基为MS＋6-BA 1.5 mg.L-1＋2,4-D 1.0 mg.L-1,胚状体生长发育的最适培养基为MS＋6-BA1.0 mg.L-1＋IBA0.5 mg.L-1,生根的最适培养基为1/2MS＋NAA0.4 mg.L-1。     

我国木本植物体细胞胚胎发生研究进展

综述了我国木本植物组织培养体细胞胚胎发生研究领域的研究现状，分别从影响木本植物体细胞胚胎发生的主要因子、生理生化基础、遗传变异性等几个方面进行了评述，对我国木本植物体细胞胚胎发生研究领域提出了一些建议。     

Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis

The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature embryos of Korean pine(Pinus koraiensis Sieb.et Zucc.;Pinaceae) as explants,but there are problems,such as low proliferation efficiency,loss of embryogenicity,poor vigor;thus,best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear.To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation,we varied hormone concentration,subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages,then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design.The results showed that the family origin and collection date of explants significantly affected callus induction(induction rate reached1.67%).Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L~(-1)6-benzyl adenine,1 mg L~(-1) naphthaleneacetic acid,30 g L~(-1)sucrose,500 mg L~(-1),L-glutamine,500 mg L~(-1) casein hydrolysis and 6.5 g L~(-1) agar.In addition,the combination of 2,4-dichlorophenoxyacetic acid+6-benzyl adenine also had a better proliferative effect on callus.The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different.Transfer to new medium every 13-15 days not only maintained robust callus vigor,but also yielded a larger proliferation coefficient.The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.     

Micropropagation and tissue culture of Eucalyptus-a review

Micropropagation has the potential to provide very high multiplication rates of selected tree genotypes, with resulting short-term silvicultural gains. Aseptic cultures have been established from seeds, seedlings, shoots, flowers and lignotubers. Callus cultures have been established from a wide range of tissue sources for at least 30 species of Eucalyptus. Plant regeneration from callus was successful for 12 of these species. Micropropagation through axillary proliferation, or adventitious shoot proliferation on nodal explants, or both, has been successful. An agar-based medium of Murashige and Skoog with a low auxin/cytokinin ratio is most commonly used for shoot multiplication. Vitrification and shoot senescence remain problems. Gibberellic acid was added in some media to stimulate shoot elongation. Various media are used for in vitro root initiation. Suspension and protoplast cultures have been achieved and plants have been regenerated from protoplasts. In vitro techniques are presently being applied to Eucalyptus to achieve genetic transformations.     

桉树体细胞培养研究进展

体细胞培养能获得稳定的遗传材料,还能通过变异获得抗性的育种材料。在现代科学技术的支撑下,通过体细胞培养来获得次生代谢物和进行细胞育种的技术不断完善。本文从桉树愈伤组织培养、原生质体培养、悬浮培养、体细胞胚胎发生和人工种子等五个方面对二十世纪八十年代以来桉树体细胞培养的研究进展进行了综述。     

Efficient embryogenic callus induction and plant regeneration from embryonic axis explants inQuercus acutissima

Embryogenic callus ofQuercus acutissima was successfully induced from embryogenic cultures, and plants were regenerated from the callus. The development of the techniques involved will allow mass propagation and gene transformation in this species. Embryogenic cultures were formed from embryonic axis explants (i.e., embryos without cotyledons) excised from immature embryos, after culture on Murashige and Skoog (MS) medium containing indolebutyric acid and benzyladenine. Attempts to induce embryogenic cultures from cotyledon explants were unsuccessful. Embryogenic calli were induced at high frequency from embryogenic cultures on MS medium containing 2,4-dichlorophenoxyacetic acid. However, benzyladenine inhibited embryogenic callus formation. Somatic embryo development from embryogenic calli occurred on MS medium in all of the seven cell lines tested. Germination of somatic embryos was induced on half strength MS medium without plant growth regulators. Finally, acclimated plants growing in soil were obtained.     

Decreased morphogenetic potential in peach palm stem-like cells in long-term in vitro conditions

Peach palm(Bactris gasipaes Kunth)has been micropropagated from pre-procambial cells that provide stem-like cell niches,(i.e.,pre-procambial cells),multipotent,pluripotent and totipotent for direct vascularization,adventitious buds and somatic embryogenesis,respectively.The direct induction of adventitious buds and somatic embryogenesis reduces the frequency of mutations when compared to indirect morphogenesis.Long-term in vitro cultivation of perennial species such as peach palm cause the clones to age and deteriorate;however,the consequences for morphogenesis potential are not fully clear.The morphogenic potential of peach palm clones established and in vitro cultivated for 8 years(regeneration of adventitious buds without callus formation)was investigated in leaves,roots and stem bases using histological and histochemical analyses.Data from long-term cultures(8-years-old)was compared to data from short-term cultures(1-year-old).Morphogenic pathways monitoring for direct induction of somatic embryos and adventitious buds revealed a strong morphogenic reduction potential in the pre-procambial cells,parenchyma cells in the proximal region of stem bases,and external cells of leaf sheaths.Initial cells of shoot apical meristems and pre-procambial cells commit cell reprogramming to the undifferentiated state and subsequent acquisition of cellular competence.These results are applicable in the micropropagation of peach palm,with consideration to obtaining clones and their long-term in vitro culture.     

桉树组织培养研究进展

桉树组织培养在理论和实践中都具有非常重要的意义。迄今为止,全世界约有60种桉树进行过组织培养技术研究,包括胚培养、芽培养及嫩茎培养、花药培养、悬浮培养、原生质体培养、体细胞胚培养等技术,绝大部分成功获得完整植株。     

利用悬浮培养和培养基选择改善火炬松的体细胞胚胎发生和植株再生(英文)

三个基固型的火炬松成熟合子胚被培养在附加 8mg·L-12 ,4 D ,4mg·L-1BA ,4mg·L-1KT ,5 0 0mg·L-1水解酪蛋白和 5 0 0mg·L-1谷氨酰胺的愈伤组织诱导培养基上诱导愈伤组织 .来自于子叶、胚轴和胚根的愈伤组织在附加 1 6mg·L-12 ,4 D ,0 8mg·L-1BA和 0 8mg·L-1KT的愈伤组织增殖培养基上培养 9周后 ,可获得 16 9%的胚性愈伤组织 .通过建立胚性细胞悬浮系和研究ABA、PEG和活性炭对体细胞胚成熟的促进作用 ,优化的体细胞胚胎发生体系被建立 .71棵再生小苗被用于移栽试验 ,2 3棵小苗在田间移栽成活     

Early selection improves clonal performance and reduces intraclonal variation of Norway spruce plants propagated by somatic embryogenesis

Height growth during the first and second growth periods (i.e., the June-September period in consecutive years) and intraclonal variation were assessed in 13 Norway spruce (Picea abies (L.) Karst.) clones propagated by somatic embryogenesis. The plants were acclimatized and grown in a greenhouse until mid-July and then transferred outdoors. The clonal mean heights after the first and second growth periods were lower for somatic embryo plants than for seedlings from corresponding families sown at the time of somatic embryo plant ex vitro transfer, because a large proportion of somatic embryo plants were small. We determined whether certain selection criteria at ex vitro transfer can be used to identify somatic embryo plants with height growth characteristics comparable with those of seedlings. Epicotyl length and presence of lateral roots proved to be important parameters for selection, whereas main root length was less useful. A combined selection for somatic embryo plants with lateral roots and with an epicotyl length exceeding 8 mm resulted in taller plants and reduced intraclonal variation after the first and second growth periods. The growth of somatic embryo plants selected in this way was similar to that of seedlings from the corresponding families. We conclude that selection according to these criteria at ex vitro transfer can result in improved performance of clonal stock propagated by somatic embryogenesis.     

Callus induction and plant regeneration from mature bluegrass (Poa pratensis L.) seeds

A protocol was discussed for high efficient plant regeneration from seven bluegrass (Poa pratensis L.) cultivars via an indirect callus induction and somatic embryogenesis method. Mature seeds were used as explants for callus initiation. Callus induction and proliferation efficiencies were investigated on NB, modified MS (MMS) and MS media, supplemented with 2.0 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D). The MMS medium performed best. Based on the MMS medium, direct and indirect callus induction effects of bluegrass from mature seeds were compared at the range of 1-5 mg·L-1 2,4-D contained in the medium. Under the direct callus induction method, the most suitable 2,4-D concentrations varied among cultivars. Under the indirect callus induction method, a significantly high callus induction frequency (93.33%-98.33%) was obtained and there were barely any statistically significant differences among the tested genetically diverse cultivars. Somatic embryos were promoted on the MMS medium supplemented with 3 mg·L-1 2,4-D, 0.1 mg·L-1 kinetin and 0.8 mg·L-1 CuSO4. Embryogenetic calli developed into plantlets on the MMS medium containing different concentrations of thidiazuron (TDZ), and the differentiation frequencies varied in the range from 20.15% to 77.65%. The 0.25 mg·L-1 TDZ was generally the most suitable concentration for the tested cultivars.     

核桃体细胞胚发生与转基因研究进展

总结了核桃体细胞胚发生的研究进展,列表统计已报道的核桃５个种和３个杂种体细胞胚发生的外植体与诱导条件,重点论述了影响核桃体细胞胚发生与次生胚发生的因素,介绍了核桃体细胞胚萌发与转化的方法。还总结核桃转基因研究的进展,提出了用核桃体细胞胚发生系统进行外源基因转移的操作模式。     

Establishment of cryopreserved gene banks of European chestnut and cork oak

Cryopreservation of selected genotypes of European chestnut and cork oak was carried out in two laboratories in a project involving conservation of field collections. Plant material was selected on the basis of disease resistance (chestnut), growth habit, phytosanitary performance and cork quality (cork oak). The cryopreservation technique comprised of vitrification of shoot apices isolated from in vitro stock shoot cultures (chestnut) and somatic embryos (cork oak). Forty-three out of 46 chestnut genotypes assayed survived the freezing process, but only 63% recovered their capacity to produce new shoots. After completion of multiplication and rooting steps, the surviving shoots produced plants that were morphologically identical to those derived from non-supercooled material. All 51 cork oak genotypes withstood freezing and were able to produce new somatic embryos through a process of secondary embryogenesis. Multiplication and germination of the recovered embryos enabled production of plants that were morphologically identical to those derived from non-supercooled material. In light of the results obtained, long-term cryopreservation of these species is feasible, thereby ensuring conservation of valuable genotypes during field evaluation.