枸杞花药愈伤组织悬浮培养条件下胚状体发生与植株再生

采用枸杞花药进行离体培养,建立细胞系,诱导植株再生,结果在含不同激素的4种培养基上都诱导出了愈伤组织,诱导率为17%～169%。愈伤组织在MS 2,4D05mg/L的固体培养基上,经2～3次培养后,获颗粒状胚性愈伤组织,颗粒状胚性愈伤组织转入含相同成分的液体培养基中进行振荡培养,24h后获得大量单细胞。单细胞液经过多次继代培养,建立起稳定的悬浮系。悬浮细胞在液体培养基中培养8～10d可获得含有大量胚状体的愈伤组织块,收集悬浮培养物转移到MS 6BA02mg/L的固体培养基上,胚状体能够萌发形成大量绿色小芽,小芽转入生根培养基(MS NAA02mg/L)中20d后得到完整植株。植株根尖细胞经细胞学鉴定为单倍体。     

黄瓜胚性细胞悬浮培养及其原生质体的植株再生

黄瓜子叶原生质体来源的胚性愈伤组织,在液体培养中形成胚性悬浮细胞系,已继代两年,仍保持胚胎发生能力,不同的ABA和蔗糖浓度对胚状体的生长发育和同步化有明显影响。1mg/l的ABA和7－9％的蔗糖能显著地减少培养物的愈伤化,并同步地控制胚状体处于球形或球形后期,低浓度的蔗糖（1％）有利于胚状体的早期萌发,继代3－5天的胚性是浮细胞团酶解后,获得大量有活力的原生质体,原生质体在DPDK,液滴或浅层培养,或褐藻酸钠包埋培养中活跃分裂,形成细胞团和球形胚,转至固体培养基上或将包埋培养物直接转入液体振荡培养,胚状体可不断增殖,胚状体在大量元素减半,不加外源激素的MS培养基上发育成小植株。     

黄瓜胚性细胞悬浮培养及其原生质体的植株再生

黄瓜子叶原生质体来源的胚性愈伤组织,在液体培养中形成胚性悬浮细胞系,已继代两年,仍保持胚胎发生能力,不同的ABA和蔗糖浓度对胚状体的生长发育和同步化有明显影响。1mg/l的ABA和7－9％的蔗糖能显著地减少培养物的愈伤化,并同步地控制胚状体处于球形或球形后期,低浓度的蔗糖（1％）有利于胚状体的早期萌发,继代3－5天的胚性是浮细胞团酶解后,获得大量有活力的原生质体,原生质体在DPDK,液滴或浅层培养,或褐藻酸钠包埋培养中活跃分裂,形成细胞团和球形胚,转至固体培养基上或将包埋培养物直接转入液体振荡培养,胚状体可不断增殖,胚状体在大量元素减半,不加外源激素的MS培养基上发育成小植株。     

黄连体细胞胚胎发生的研究

黄连(Coptis chinensis)叶片外植体在 MS 2,4-D 1 ppm 培养基上很容易产生愈伤组织。愈伤组织在转入分化培养基 MS 6-BA 0.5ppm NAA 1ppm 培养基上以后,能产生大量胚状体。胚状体可经过球形、心形、鱼雷形及子叶期等诸阶段发育成小植株。对胚状体用4%的藻酸钠和2%的氯化钙进行人工种皮包埋后,在无菌条件下,胚状体转变成苗。愈伤组织在分化培养基上经几次继代后,整个愈伤组织可转变为胚性愈伤组织并形成一个个胚性细胞团。胚状体可从其表面或愈伤组织内的任一细胞团产生。这一研究结果为获得大量分散的单个胚状体及人工种子的研制提供了良好的实验系统。     

采用低pH值和适量的玉米素、ABA配合恢复褐化的水稻愈伤组织

在建立水稻体细胞悬浮培养体系的实验中,我们观察到当将胚性愈伤组织从固体培养基转入液体分化培养基进行悬浮培养并去掉2.4-D 时,随着培养时间的延长,愈伤组织往往出现褐化现象。与这种情况类似的是,当将悬浮培养的愈伤组织和成熟胚状体转入固体培养基时,也出现严重的褐化现象。这种褐化现象一般认为是不可逆的,伴随着胚性愈伤组织和成熟胚状体的褐化,胚性愈伤组织逐渐丧失分化能力,成熟胚状体则逐渐死亡。     

猕猴桃胚乳培养中的胚胎发生

中华猕猴桃及硬毛猕猴桃的胚乳培养在MS+Zeatin 3 ppm+2,4-D0.5ppm+CH 400ppm的培养基上诱导产生愈伤组织,在MS+Zeatin 1 ppm+CH 400ppm的分化培养基上产生胚状体和长成完整小植株。细胞组织学观察表明,胚状体起源于愈伤组织内单个细胞,经原胚、球形胚、心形胚等阶段发育成苗。猕猴桃胚乳属于细胞型胚乳,它的这种胚状体起源于愈伤组织内单个细胞的发生方式,也不同于常见的胚状体发生于愈伤组织表面的方式。     

防风悬浮细胞的原生质体再生植株

防风(Saposhnikovia divaricata(Turcz.)Schischk)试管苗的根尖,下胚轴或叶柄切段在含有1mg/l 2,4-D 的 MS 固体培养基上,形成含有胚性细胞团的愈伤组织。愈伤组织经液体振荡培养,形成含有大量胚性细胞团的悬浮培养物。用含有 Onozuka R-10 1.5%、Mace-rozyme R-10 0.3%、蜗牛酶0.5%、CaCl_2 5mmol/l 和甘露醇0.6 mol/l(pH=5.8)的酶液从胚性细胞团游离得到原生质体。原生质体在培养的第4天出现第一次分裂,50天左右形成的细胞团大小为1—2mm。这些细胞团在含有0.5 mg/l 2,4-D 的 MS 固体培养基上形成愈伤组织。在含有0.1 mg/l 6-BA 或0.1 mg/l 2,4-D+0.5mg/l 6-BA 的 MS 固体培养基上,原生质体再生的愈伤组织分化出胚状体。胚状体在不含任何生长调节剂的 MS 固体培养基上发育成完整的原生质体再生植株。     

石刁柏胚乳愈伤组织内胚胎发生的组织细胞学观察

石刁柏(Asparagus officinalis L.)的幼嫩胚乳接种于MS+NAA 5ppm+6-BA 1ppm和MS+NAA 1ppm+6-BA 0.5ppm的培养基上诱导产生愈伤组织。将愈伤组织转移到MS+6-BA 1ppm+NAA 0.5ppm的分化培养基上,培养30天后即可形成胚状体,组织细胞学观察表明:胚状体起源于愈伤组织内或近表层的单个胚性细胞。在胚状体发生的早期阶段,观察到与柳叶菜型和藜型胚胎发育大致相似的细胞分裂方式,从而出现了T型或线型的四个细胞的原胚。在多细胞原胚及球形胚期具有单列或不规则排列的多细胞胚柄。石刁柏胚乳愈伤组织中的胚胎发生是不同步的,在同一块愈伤组织的切片中可以观察到不同发育阶段的胚状体。在外形上还可以观察到一个子叶、两个子叶或四个子叶等多种不同形态的胚状体。一部份胚状体能发育成完整小植株。     

从未授粉烟草子房诱导单倍体植株的细胞学和胚胎学研究

由未授粉的子房培养出的单培体植株来自两条途径:1.来自大孢子,直接进行多次分裂,经胚状体各阶段,发育形成;2.来自成熟卵细胞,经胚状体各阶段发育而成。此外,从上述途径产生的芽的基部和顶端形成愈伤组织,在同样培养基上分化形成胚状体,最后发育成植株。外源激素对未授粉子房高频率地诱导形成胚状体足必需的,而对诱导胚性愈伤组织形成胚状体以及进一步分化成植株并非必需。培养基中增加维生素 B_1,B_6,烟酸,叶酸和肌醇等的使用量时,未授粉离体子房和胚性愈伤组织诱导胚状体的频率显著提高。     

苦荞胚性愈伤组织诱导与植株再生研究

以苦荞子叶和下胚轴为外植体,进行了不同浓度激素组合的MS和SH固体培养基对胚性愈伤组织诱导及植株再生的研究。结果发现,MS培养基比SH培养基更有利于胚性愈伤组织诱导;2,4-D是诱导愈伤组织的有效激素,KT能有效促进胚状体的形成;下胚轴和子叶都能有效诱导出胚性愈伤组织和再生植株。下胚轴在MS 1.5mg·L-12,4-D 1.5mg·L-1BA培养基,子叶在MS 2mg·L-12,4-D 0.5~1.5mg·L-1BA上能高效诱导出愈伤组织;愈伤组织在MS 2mg·L-12,4-D 0.1mg·L-1KT培养基中继代,能有效诱导胚性愈伤组织;来自下胚轴的胚性愈伤组织在1/2MS 2.0mg·L-1BA 0.5mg·L-1KT 0.1mg·L-1NAA培养基上能够高频再生出芽,来自子叶的胚性愈伤组织在1/2MS 1.0mg·L-1BA 0.1mg·L-1KT 0.1mg·L-1NAA培养基上芽诱导率较高;MS 1mg·L-1NAA是适宜的再生苗生根培养基。     

蛇床幼茎离体培养中体细胞胚胎形成的观察

蛇床幼茎外植体经诱导产生了愈伤组织。在ＭＳ＋２,４－Ｄ,０．２ｍｇ／Ｌ＋ＺＴ０．４ｍｇ／Ｌ培养基中,愈伤组织转变成胚性愈伤组织。转入ＭＳ＋ＮＡＡ０．２ｍｇ／Ｌ＋ＺＴ０．８ｍｇ／Ｌ培养基以后,胚性愈伤组织分化出体细胞胚胎。体细胞胚胎在ＭＳ＋ＮＡＡ０．５ｍｇ／Ｌ培养基中可直接发育成为完整植析。显微观察表明,体细胞胚胎产生于愈伤组织的表层细胞或内部细胞。在鱼雷胚期已有螺纹导管的分化。子叶期的维管组织从两     

Callus and embryoid formation from protoplasts of Helianthus annuus

Sunflower hypocotyl protoplasts have been isolated and cultured. Optimum plating density for cell division and colony formation was in the range of 5 to 7×10⁴ cells/mi in an agarose medium supplemented with BAP (1 mg/l) and NAA (1 mg/l). Plating efficiency was 60% after 21 days of culture. In the resultant culture a mixed population of calli and embryoids was observed. Thirty seven percent of the cell clusters exhibited a developmental pattern similar to an embryoid. Many stages of embryogenesis were observed in the same cultures.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - NAA 1-naphtaleneacetic acid - IAA Indole-3-acetic - BAP 6-benzylamino purine - GA₃ Gibberellic acid     

从石刁柏原生质体培养再生植株

石刁柏,又名芦笋(Asparagus officinalisL.)是百合科天门冬属植物。其栽培品种含有丰富的维生素类及蛋白质。同时,石刁柏对于某些疾病有一定的药效,因此它已成为人们所喜爱的一种高级营养蔬菜。国外已有不少关于石刁柏试管苗繁殖的报告,但至今只有Bui Dang Ha等从石刁柏枝状叶分离的原生质体得到愈伤组织,并由此愈伤组织诱导获得了再生植株。此后,未见在石刁柏的原生质体培养方面再有新的工作。在本文中,我们利     

Studies on Somatic Embryogenesis and Histological Observation in Gossypium hirsutum L. cv. Coker 312

A reproducible 3-step procedure of somatic embryogenesis of Gossypium hirsutum L. cv. Coker 312 has been developed. Calli were initiated on LSC medium containing 0.1mg/L 2,4-D plus 0.1 mg/L KT from cotyledon tissue of 5-day-old-seedlings, and subcultured on the same medium with 4 mg/L NAA and 1 mg/L KT. Embryogenic calli and few globular embryos developed at a frequency of 67.5% after 55 days’ culture in the latter medium. When the embryogenic calli were transferred to growth regulator-free medium, embryogenesis occured and all stages of normal zygotic embryos, globular-, heart- and arrowhead- or torpedo-shaped embryo, ,were developed. Cyto-histological study showed that embryogenic calli were very easily distinguished from non-embryogenic calli. Embryoids were mainly initiated from the cells in the peripheral area of embryogenic calli. At the early stage the development of embryoid was limited in a boundary of thicken cell wail. There were 2 peaks of starch accumulation in the process of embryogenesis, one was at the early globular stage, and the other at the later torpedo-shaped stage.     

Study on Tissue and Protoplast Culture of Wild Cotton (Gossypium davidsonii)

This paper deals with the study on the condition of callus formation, embryogenesis, organogenesis, plant regeneration and protoplast culture of wild cotton (G. davidsonii) Callus cultures derived from several organs such as root, stem, leaf, cotyledon and hypocotyl. The results obtained in these cultures showed that the modified MS medium containing 2,4-D 1.0+KT 0.1; 2,4-D 0.1+KT 0.01; NAA (IAA) 2.0+KT 0.1 and NAA (IAA) 1.0+KT 0.1 mg/L were favorable to callus formation. Modified MS medium containing 2,4-D was suitable for initiated callus of G. davidsonii Besides, suspension cultures from callus of G. davidsonii were saccessfully initiated. Optimum concentration of 6BA (or ZT, or 2ip) and NAA (IAA) was for shooting, somatic embryo or leaf formation. Plantlets regenerated from somatic embryo at lower concentration of 6BA, or ZT, or 2ip. As to protoplast culture of this species, the age and physiological condition of callus or suspension cells and concentration of enzymes used for protoplast isolation affected the yield and survival of protoplasts. Protoplast of this species cultured in modified MS medium containing 2,4-D 0.5+NAA 0.5+ZT 0.1–0.2 mg/L. and divied after 3–4 days. The rate of division was 3--4% and cell cluster formed after 14 days, then these cells died.     

Studies on Induction of Pollen Plantlets from the Anther Cultures of Lily

Anthers with the filament of lily (Lilium davidii var. Willmottiae (Wilson) Roffill) were cultured on modified MS medium. Supplemented with different concentrations and compatible ratios of growth hormones (Z 2 mg/L,or 2,4-D 2 mg/L + KT 2mg/L, or 2,4-D 4mg/L+ 6 BA 2 mg/L). At this time the pollen grains in the anthers were at the late uninueleate stage. Anther cultures were incubated at 25—27 ℃, and illuminated with daylight of about 800–1200 lx. After 30 days, the calli or embryoids were produced from anthers. The frequency of the calli or embryoids induction was 8.89%. After transfer eventually to the differentiation medium, these calli or embryoids developed into plantlets in 70 days. Among the root tips of regenerated plantlets haploid, diploid and aneuploid cells were found, but the haploid cells were produced in about 86.4% of the root tips. It is quite evident that haploid plantlets are derived from the pollen grains.     

Correlation of cotyledonary node shoot proliferation and somatic embryoid development in suspension cultures of soybean (Glycine max L. Merr.)

Suspension cultures of soybean were initiated from hypocotyl or cotyledon callus tissue of several soybean genotypes. When these were grown on L2 medium with 0.4 mg/liter 2,4-D several genotypes produced numerous embryoids while others produced only a few such structures. Due to internal anatomy, no embryoid developed into a complete plant. A genotype's propensity to form normal appearing embryoids was correlated with the ability to proliferate shoots at the cotyledonary node on a medium with benzylaminopurine as determined in previous testing.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP Benzylaminopurine - L2 Phillips and Collins (1979) legume medium     

太白米组织培养的研究

太白米地下鳞茎在MS附加不同浓度KT、BA、IAA、NAA、2,4－D的培养基上,可诱导产生愈伤组织,其中在MS附加NAA 0.5mg/L,KT0.1mg/L的培养基上愈伤组织诱导率最高,可达65％,且生长快;培养在MS附加2,4－D 1.0mg/L,KT 0.1mg/L培养基上的鳞茎可经不定根直接发育成新的鳞茎,由此建立了太白米的鳞茎再生体系,鳞茎再生率达2．17倍。     

Somatic embryogenesis and plant regeneration in cultured immature inflorescences of Setaria italica

Young inflorescence explants of Setaria italica in culture showed high capacity for regenerating plantlets through somatic embryogenesis. Embryogenic callus formation was initiated from the explants cultured on Murashige and Skoog's medium with 2 mg/l 2,4-D and 0.2–0.5 mg/l KT or BAP, but it was better for the maintenance of embryogenic growth to subculture the calli on the medium with 2,4-D and KT/BAP and on the medium with 2 mg/l 2iPA and 0.2 mg/l NAA alternately. A number of plantlets were regenerated when embryogenic calli were transferred onto the same basic medium but with 2 mg/l BAP and 0.5 mg/l NAA. Plant regeneration capacity has been maintained in some embryogenic calli during fourteen months of subculture.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA 3-indoleacetic acid - 2iPA N⁶-(²-isopentenyl) adenosine - BAP 6-benzylaminopurine - KT kinetin - CH casein hydrolysate     

Plant regeneration from protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of six cultivars of Glycine max L. and cultured in the KP8 liquid medium supplemented with 0.2 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L ZT. The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and small calli in 6 weeks. The calli further grew to 2–3 mm on the gelritesolidified K8 medium and were transferred onto the MSB medium with 1 mg/L 2,4-D and 0.25 mg/L BA, to obtain compact and nodular calli. Shoot formation was initiated on MSB medium with 0.15 mg/L NAA, and BA, KT and ZT, 0.5 mg/L of each, with or without 500 mg/L CH. It was followed by plant regeneration. So far, 87 plants have been regenerated from 4 cultivars, and normal seeds were obtained from them after transplanting into pots.Abbreviation IAA indol-3-acetic acid - NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - KT kinetin - BA 6-benzyladenine - ZT zeatin - CH casein hydrolysate