Stimulation of in vitro organogenesis from epicotyl explants and successive micropropagation round in Cassia angustifolia Vahl.: an important source of sennosides

This report describes a protocol for the in vitro shoot induction and plant regeneration from epicotyl explants of Cassia angustifolia on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), Kinetin and 2-iP (0.5–10.0 μM). MS medium supplemented with BA (5.0 μM) was the most effective in inducing adventitious shoots and growth. The highest rate of shoot multiplication was achieved on MS medium supplemented with BA (5.0 μM) and Indole-3-acetic acid (IAA, 1.0 μM). The nodal segments excised from the shoots regenerated from BA (5.0 μM) and IAA (1.0 μM) were used as explants for next three round of micropropagation. The number of shoots significantly increased at successive round of micropropagation. For rooting, MS medium supplemented with 2.0 μM indole-3-butyric acid proved to be better than that supplemented with IAA or α-naphthalene acetic acid. The in vitro raised plantlets with well developed shoot and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse. About 52 plants (85 %) survived out of 60 plants transferred in garden soil.     

Non-aerated liquid culture promotes shoot organogenesis in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill

Eucalyptus is very recalcitrant to in vitro culture. In this research, an efficient shoot organogenesis system was developed using 60-day-old plants of Eucalyptus globulus grown in vitro and non-aerated liquid medium to improve shoot proliferation. Cultures were initiated with hypocotyls and leaf segments from plantlets cultivated on semisolid ½ MS modified medium supplemented with 4.44 µM 6-Benzyladenine (BA) and 16.1 µM 1-Naphthaleneacetic acid (NAA). Calli were transferred to shoot induction medium, with either 0.5 or 2.7 µM NAA. Shoot multiplication was carried out on 4.44 µM BA + 0.5 µM NAA medium, and semisolid and non-aerated liquid systems were compared for improving shoot proliferation. Rooting of adventitious shoots was evaluated on medium containing NAA or Indole-3-butyric acid -IBA (5 and 16 µM). Callogenesis was obtained from both types of explants, although shoot formation was only obtained from leaf-derived calli. Shoot proliferation on 4.44 µM BA + 0.5 µM NAA resulted in the most shoots/callus. Non-aerated liquid medium was more efficient in promoting shoot multiplication (53.5 shoots/callus) than was semisolid medium (28.5 shoots/callus). Levels of phenolic compounds were significantly reduced in the shoots cultivated in liquid medium. Efficient rooting (76%) was obtained using 16 µM IBA.     

Direct shoot organogenesis from leaf explants of Embelia ribes Burm. f.: a vulnerable medicinal plant

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Embelia ribes Burm. f., a vulnerable medicinal woody climber of the Western Ghats of India. The in vitro procedure involved three steps that included induction of shoot initials from leaf tissue, regeneration and elongation of shoots from the shoot initials, and rooting of shoots. The induction of shoot initials was achieved on Murashige and Skoog (MS) solid medium supplemented with different concentrations of thidiazuron (TDZ). The best medium for shoot induction was MS with 0.272 μM TDZ. Numerous shoot primordia developed within 2–3 weeks on the leaf margin as well as on the midrib region, without any callus phase. In the second step, the shoot clumps separated from the leaf explant on transfer to MS basal medium, resulting in the differentiation of 90% of the shoot initials into well-developed shoots. The 2- to 3-cm-long shoots rooted on half-strength MS basal medium supplemented with 4.90 μM indole-3-butyric acid (IBA) and 3% (w/v) sucrose in the third stage. The rooted plants could be established in soil with 70% success. This protocol could be utilized for in vitro propagation and conservation of this important threatened medicinal plant.     

In vitro plant regeneration from cotyledon-derived callus cultures of leguminous tree Gleditsia caspica Desf.

An in vitro regeneration system was developed using organogenic callus derived from in vitro grown cotyledonary explants of Gleditsia caspica Desf., an important leguminous tree. Murashige and Skoog (MS) basal medium augmented with 0.2 g L^?1 myo-inositol and various concentrations of either 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid, or indole-3-butyric acid (IBA) alone as well as combined with cytokinins was used for callus induction. The highest frequency of organogenic yellowish-white and nodular callus (93 %) was obtained from explants grown on medium supplemented with 13.5 μM 2,4-D and 4.4 μM benzyladenine (BA). The yellowish-white and nodular callus when transferred to MS medium supplemented with BA (2.2–17.7 μM) or kinetin (KT; 2.3–18.8 μM) solely or in combination with 2.3 μM 2,4-D produced several microshoots after 5 weeks culture. The calli cultured on MS medium with 4.4 μM BA singly showed superior growth response and produced both maximum shoot regeneration (94 %) and the highest mean number (4.3) of microshoots per callus. Transfer of regenerated microshoots onto modified MS basal medium fortified with 5.8 μM gibberellic acid and 4.4 μM BA resulted in the maximum number of internodes per shoot and the highest shoot elongation after a period of 6 weeks. Optimum rooting of 90 %, an average 6.1 roots per shoot, and a mean root length of 3.6 cm was observed when half-strength MS medium was supplemented with 9.8 μM IBA and 0.92 μM KT. The regenerated healthy plants with well-developed shoots and roots showed a survival rate of 77 % after acclimatization and transplanting to garden soil for a 10-week hardening period under ex vitro conditions.     

A regeneration system using cotyledons and cotyledonary node explants of Toona ciliata

We used the cotyledons and cotyledonary nodes of Toona ciliata(Chinese mahogany) as explants to examine callus and adventitious shoot induction when exposed to different ratios of hormones. We also investigated the effects of seedling age, inoculation method, and genotype on the efficient regeneration of T. ciliata. The results showed that different genotypes exhibited significantly different callus induction efficiency. The cotyledons and cotyledonary nodes of 20-day seedlings inoculated onto MS medium with 0.5 mg/L 6-benzylaminopurine(6-BA), 0.5 mg/L kinetin(KT) and 0.05 mg/L 1-naphthylacetic acid(NAA) achieved a greater regeneration rate than did other concentrations of cytokinin and auxin. The numbers of shoots per cotyledon and cotyledonary node explant were 7.33 and 6.67. The optimal inoculation method for cotyledons was that the distal end of the explants was placed in contact with the medium. The optimal adventitious shoot differentiation medium for cotyledon explants was MS medium containing 0.3 mg/L 6-BA and 0.2 mg/L NAA, producing a 3.4 cm height of shoot on average. This study established an efficient regeneration system for T. ciliata with cotyledons and cotyledonary nodes as explants.     

影响非洲菊叶柄离体培养再生的因素

以3个非洲菊品种的叶柄为材料,研究了不同激素及其浓度对非洲菊离体培养再生的影响。结果表明:3个品种的叶柄在含单一细胞分裂素6-BA的培养基上培养时都不能被诱导出愈伤组织;而在仅含生长素NAA的培养基上培养时均可被诱导出愈伤组织,并且在其愈伤组织发生部位都有不定根发生,但只有品种6267能从不定根发生部位直接分化出不定芽;当在含NAA的培养基上再附加6-BA时也可被诱导出愈伤组织,但无不定根发生。所产生的愈伤组织在分化培养基上培养时只有由品种Ⅱ叶柄在同时含有NAA和6-BA的诱导培养基上产生的愈伤组织才可以分化出不定芽。表明愈伤组织的诱导与分化、不定芽和不定根的发生与品种及培养基中的激素种类有关。     

Establishment of an in vitro plant regeneration protocol for Casuarina cunninghamiana Miq. via indirect organogenesis

An in vitro plant regeneration protocol via indirect organogenesis from morphogenetic callus was established for Casuarina cunninghamiana Miq. Effects of plant growth regulator NAA (naphthaleneacetic acid) and BAP (6-benzylaminopurine), sucrose and AgNO₃ on callus induction, adventitious bud differentiation and shoot development were examined. Explants used were epicotyl fragments from 45-day-old seedlings. The largest callus (4.29 mm in diameter) was obtained after 1 month on a basic culture medium consisting of Murashige and Skoog ? macro- and full strength micro- elements, Nitsch and Nitsch vitamins, supplemented with 0.54 μM NAA, 3.30 μM BAP, and 30 g L⁻¹ sucrose. The calli were subcultured in the same medium above for 2 months. They were then cultured for another 2 months for adventitious bud differentiation and shoot development. The highest mean adventitious bud differentiation, number of shoots formed per callus and number of shoots ≥2 cm long per callus (47.50%, 27.38 and 4.75, respectively) were achieved on the above medium modified with NAA at 0.27 μM and supplemented with AgNO₃ 1 mg L⁻¹. Shoots were successfully rooted without plant growth regulator and the rooted plantlets survived and grew normally. This protocol for in vitro plant regeneration provides a tool not only for vegetative propagation but also for plant genetic transformation and gene function studies of C. cunninghamiana.     

大果沙棘无菌外植体的获取

将大果沙棘的幼嫩茎尖、茎段外植体接种在1/2MS附加不同浓度配比的IAA,IBA,BA,NAA培养基上可诱导茎尖和腋芽生长,同时将小叶片和嫩茎接种于不同浓度配比的1/2MS和2/3MS培养基上诱导产生愈伤组织,愈伤组织继续培养后,表面形成大量的绿色突起,进一步分化成不定芽,将不定芽在不同的培养基中继续培养产生无菌外植体。     

沟叶结缕草的组织培养和无性系的建立

通过研究不同基本培养基及植物生长调节组合对沟叶结缕草丛生芽诱导、增殖和愈伤组织诱导、分化的影响,建立起沟叶结缕草试管无性系。以地下匍匐根状茎的顶芽为外植株,在MS 6-BA 2 mg.L-1 NAA 0.1 mg.L-1培养基上诱导丛生芽形成,以MS KT 3 mg.L-1 NAA 0.1 mg.L-1作为继代增殖培养基,建立起丛生芽苗→不定芽发生→丛生芽苗速生试管无性系;以丛生芽苗基部为外植体,以MS 2.4-D 4 mg.L-1作为愈伤组织诱导培养基,以MS基本培养基作为愈伤组织分化培养基,建立起丛生苗→愈伤组织→愈伤组织分化→丛生苗试管无性系。试管无性系室外移栽成活率达95%以上。沟叶结缕草试管无性系的建立为细胞工程育种和基因转化的研究奠定了基础。     

In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes

Acacia sinuata is a valuable multipurpose tree in Southern India. The tree is over exploited, but its regeneration rate in natural habitat is low. Therefore, it is important to study if it can be regenerated through in vitro micro-propagation. Cotyledonary node and shoot-tip explants excised from 15 day-old in vitro grown seedlings were used to initiate cultures. Maximum number of shoots was induced from cotyledonary node explants on Murashige and Skoog's (MS) medium containing6.66 µM 6-benzylamino purine (BAP) and 4.65µM kinetin (Kn). Subculturing was done in the fresh medium of same composition. The number of shoots formed was comparatively greater in the first subculture. Maximum shoot elongation was achieved (5.5 cm)when subcultured on MS medium supplemented with 1.75 µMgibberellic acid (GA₃). In vitro regenerated shoots produced roots when transferred to half strength MS medium supplemented with 7.36 µM indolebutyric acid (IBA). From each cotyledonarynode 30 shoots were obtained within 90 days after two subcultures. The success rate of establishing the rooted plantlets in the field was 55%.This revised version was published online in November 2005 with corrections to the Cover Date.     

爬行卫矛下胚轴高频离体再生体系的建立(英文)

In this paper,a protocol for efficient shoot regeneration was successfully developed from hypocotyl explants of Euonymus fortunei var.radicans.Some factors that influenced shoot regeneration such as different combinations of plant growth regulators,types of medium and inoculation ways were studied in order to establish an efficient plant regeneration for transformation.The results showed that hypocotyl explants wero horizontally cultured on a basic medium composed of MS medium supplemented with 0.5 mg·L-1 BAP and 0.01 mg·L-1 NAA for induction and development of adventidous shoots.Ninety-four percent of regeneration frequency and 5.1 shoots per explants were obtmned after 30 days of culture.Regenerated shootsproliferated efficiently on a shoot multiplication medium consisting of MS medium containing 1.0 mg·L-1 BAP and 0.1 mg·L-1 NAA.Microshoots were rooted on a rooting medium made up of MS medium enriched with O.5 mg·L-1 IBA and O.5 mg·L-1IAA.After hardening,90% of plants were successfully established under greenhouse conditions.Histological observation revealed that shoot primordium originated from subepidermal cells of hypocotyl explants and directly developed into adventitious shoots without caHus formation.     

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。     

葡萄柚品种哈路比叶片离体再生体系的优化

为给葡萄柚的大规模育苗提供方法,以葡萄柚品种哈路比的叶片为外植体,在MS培养基中添加不同浓度配比的激素,探索其离体再生的优化体系。试验结果如下:培养基为MS+0.6 mg/L 6-BA+1.0 mg/L 2,4-D愈伤组织诱导率最高为78.57%,愈伤组织呈黄绿色,结构疏松,长势良好;MS+3.0 mg/L 6-BA+1.5 mg/L NAA是诱导愈伤组织分化不定芽的最佳培养基激素浓度配比,分化率最高为48%,不定芽分化快,长势良好;培养基MS+2.0 mg/L IBA有利于诱导生根,生根率达60.74%。初步建立了葡萄柚哈路比的离体再生体系。     

Regeneration and multiplication of Albizia procera Benth. through organogenesis

Vegetative propagation techniques are recognized as indispensable tools for mass multiplication of important multipurpose trees adopted in different agroforestry systems. Albizia procera, one among important species, is difficult to propagate commercially either by stem / root cuttings or layering. A study was undertaken to develop procedure for its in vitro regeneration through organogenesis. Explants collected from 15±2 yr-old mature plus trees and from 15 days old juvenile seedlings were regenerated with exogenous application of different hormones. Epicotyl and hypocotyl explants excised from juvenile seedlings showed higher callusing than axillary bud and shoot tip explants derived from mature trees. Benzylaminopurine (BA) at 3 μg/l was most effective, which induced hundred percent callusing in epicotyl and hypocotyl explants in 1/2 Murashige and Skoog (MS) medium. Callus originated from axillary buds and apical shoot tips of mature trees failed to form organs, however callus derived from epicotyl and hypocotyl explants proliferated and formed de novo shoots and leaflets. A concentration of 3 μg/l of BA was found effective for shoot proliferation. Shoots grew vigorously in 2 μg/l gibberellic acid (GA₃) treatment and rooted in 1/2 MS medium supplemented with indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). Rooting was most successful on medium supplemented with 6 μg/l IBA alone on which 93.3% of the shoots formed roots. Sand or vermiculite supplemented with 4 ml of yoshida solution proved as best hardening media, which recorded 70-80% survival of plantlets. One year old tissue culture raised plants had comparatively more height, collar diameter, biomass, and root shoot ratio than plants raised from cuttings and seeds of the same age. The procedures enumerated provide a basis for the development of in vitro techniques for rapid multiplication of A. procera. This revised version was published online in June 2006 with corrections to the Cover Date.     

弗吉尼亚栎不定芽增殖及试管苗再生影响因子研究

[目的]探析弗吉尼亚栎组织培养中不定芽增殖和生根的关键影响因子,建立弗吉尼亚栎试管苗再生体系,为弗吉尼亚栎无性系选育提供技术支撑。[方法]以弗吉尼亚栎带芽茎段为外植体,采用不定芽增殖途径,研究弗吉尼亚栎组织培养过程中外植体选择、褐化控制以及培养基选择、激素配比对不定芽增殖和生根的影响。[结果]表明:向培养基中添加抗坏血酸、硫代硫酸钠和活性炭均显著降低外植体的褐化半径(p<0.05),其中,外植体在含有3.00、5.00 g·L^-1活性碳的培养基中褐化半径最小。基本培养基筛选试验表明:以1/4MS或WPM为基本培养基时,平均芽长和芽数明显优于MS培养基。不定芽增殖最佳培养基为1/4MS+1.20 mg·L^-1 6-BA,培养周期40 d,增殖倍数可达6.6。最佳生根培养基为1/4MS+0.50 mg·L^-1 IBA+0.50 mg·L^-1NAA,生根率达53.33%,且根粗壮,木质化程度较高。组培苗移植到灭菌河沙中,平均成活率达57.78%。[结论]培养基成分和激素配比是影响弗吉尼亚栎不定芽增殖和试管苗再生的主要因子,低盐培养基(1/4MS或WPM)不仅可以促进不定芽增殖,且能够减轻外植体褐化;还发现野外取材的外植体极易褐化,而来源于无菌苗的外植体,褐化程度明显低于野外取材的外植体。     

巨桉无性系EG5叶片高效再生体系的建立

以巨桉栽培无性系EG5无菌苗的叶片为外植体进行愈伤组织诱导与植株再生研究。结果表明:愈伤组织高效诱导和不定芽分化的最适培养基为改良MS+0.12 mg·L-1TDZ+0.25 mg·L-1NAA;硝酸铵对EG5叶片愈伤组织生长及植株分化的影响较大,最适宜质量浓度为0.412 5 g·L-1;最佳伸长培养基配方为改良MS+0.3 mg·L-16BA+0.05 mg·L-1IBA+0.3 mg·L-1IAA;暗培养10 d能促进不定芽分化,延缓愈伤组织老化和褐变速度。生根培养基为改良MS+0.4 mg·L-1NAA时生根率最高,为65.47%,移植成活率为90%以上。     

Changes in enzyme activities,soluble sugars and phenols during in vitro adventitious shoot regeneration from leaflet explants of <Emphasis Type="Italic">Albizia procera</Emphasis> (Roxb.) Benth

Changes in nitrate reductase, i.e. NR (E.C. 1.7.1.1) activity, peroxidase (E.C. 1.11.1.14) activity, soluble sugars and phenols were monitored at various time intervals from day 0 to 60 during in vitro adventitious shoot regeneration from leaflet explants of Albizia procera (Roxb.) Benth. The explants were incubated on semi-solid Murashige and Skoog medium supplemented with 2.6 g l^?1 Phytagel^®, 2.5% sucrose, 10 μM BAP, 1 μM NAA and 15 μM AgNO₃. NR activity, soluble sugars and phenols exhibited initial sharp rise on around day 20 followed by steep decline on day 25, whereas peroxidase activity peaked on day 50, highlighting significance of early input of nitrogen and energy and late emergence of lignification process for cellular differentiation and organization into adventitious shoot primordia. Morpho-anatomical changes in leaflets at various stages of in vitro adventitious shoot formation also followed the endogenous biochemical pattern.     

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.     

卷荚相思组织培养育苗技术的研究

卷荚相思是从澳洲新引种的树种，具有生长迅速，干形通直，木材质量好的特点，试验采用ＭＳ基本培养基，芽增殖培养基附加ＢＡ０．５ｍｇ／Ｌ和ＫＴ０．５ｍｇ／Ｌ，芽增殖率可达４倍以上，壮芽培养基用改良ＭＳ，加活性炭２ｇ／Ｌ，芽可抽长２～３ｃｍ，生根培养基为１／２，ＭＳ，附加ＩＢＡ２ｍｇ／Ｌ，生根率达８５％以上，卷荚相思组织培养为解决种源不足和快速繁殖优良无性素开辟新途径。     

Effect ofl-phenylalanine on rooting of shoots from axillary buds of adultLarix leptolepis

Rooting of shoots derived from axillary buds was examined to establish an efficient shoot culture system of clonal micropropagation in adult tree ofLarix leptolepis Gord. (Japanese larch). Nine out of ten shoots induced calli (90%) on their shoot bases, and the two of them formed root primordia with a red pigment (20%) on the calli surface within 5 weeks after culturing on modified Murashige and Skoog (MS) medium supplemented with 1.5 μM of indolebutyric acid (IBA) and 1.5 μM of naphthaleneacetic acid (NAA). However, the primordia did not elongate actively. The addition of 10 mMl-phenylalanine in the MS medium with the auxins resulted in the formation of roots at high frequency, about 80%, and they elongated actively. Although callus was formed in all the shoots cultured on the medium withl-phenylalanine, it appeared that the callus development was less as compared to the medium withoutl-phenylalanine. Consequently, the rooting might be associated with the suppression of the induced callus.