Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour., a rare rhoeophytic woody medicinal plant

Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour. by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog (MS) medium. There were no significant differences with respect to the induction of shoots per node or callus and roots per shoot on media prepared either with tap water and commercial sugar or those prepared with double distilled water and tissue culture-grade sucrose. The most effective medium for axillary bud proliferation was MS medium fortified with 1.0 mg l(-1 )N(6)-benzylaminopurine (BAP) and 0.5 mg l(-1 )indole-3-butyric acid (IBA), on which shoots were induced at the rate of 15 per node. The excision of node segments from the in vitro-derived shoots and their subsequent culture on medium supplemented with same concentrations of BAP and IBA facilitated enhanced axillary bud proliferation. Callus that developed from the lower cut end of the node explants induced shoots during subculture on half-strength MS medium with 1.0 mg l(-1 )BAP and 0.5 mg l(-1 )kinetin. The shoots developed rooted best on half-strength MS medium supplemented with 0.5 mg l(-1 )naphthaleneacetic acid (NAA). Rooted shoots, following acclimation in the greenhouse, were successfully transferred to field conditions, and 80% of the plantlets survived. When the basal ends of shoots harvested from multiplication medium were dipped in an NAA (0.5 mg l(-1)) solution for 25 days, a mean of 5.6 roots per shoot developed; the transfer to small pots facilitated the survival of 75% of the rooted shoots. Ex vitro rooting by direct transfer of the shoots from the multiplication medium to the greenhouse resulted in a 65% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economically advantageous. About 750 plantlets were procured in a 3-month period starting from a single node explant.     

Successful propagation in vitro of apple rootstock MM106 and influence of phloroglucinol.

Successful in vitro propagation of clonal apple rootstock MM106 was achieved by culturing axillary buds on MS basal medium with BAP (1 mg/L), GA3 (0.5 mg/L) and IBA (0.1 mg/L). Use of liquid medium (LM) in initial cultures reduced phenol exudation to a greater extent and gave maximum sprouting percentage when transferred to solid MS medium. Phloroglucinol (PG) did not enhance sprouting of buds but increased the rate of multiplication when added in the medium. Maximum number of shoots were obtained when MS medium was supplemented with BAP (0.5 mg/L), GA3 (1 mg/L), IBA (0.1 mg/L) and PG (100 mg/L). For rooting, in vitro regenerated shoots were placed in IBA (30 mg/L) for 3 hr and transferred to solidified auxin free medium. Rooting was recorded in about 80% of shoots. Inclusion of PG in rooting medium was not beneficial but shoot cultures grown in its presence gave higher rooting percentage. Rooted plantlets showed about 70% survival rate in potting mixture of sand:soil:perlite (1:1:1).     

In vitro clonal propagation of bird eye chilli (Capsicum frutescens Mill.)

An efficient and highly reproducible protocol for micropropagation of bird eye chilli Capsicum frutescens was attempted. Murashige and Skoog (MS) medium containing 0.5-3.0 mgl(-1) of 6-benzyladenine (BA), 2-isopentenyl adenine (2iP), kinetin and 0.5-2.0 mg l(-1) of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) along with 1 gl(-1) activated charcoal (AC) were used for shoot regeneration from both shoot tip and nodal explants. Shoot tip explants (100%) grew well on medium containing 1 mgl(-1) of kinetin and 1 mgl(-1) of IBA. Shoot proliferation (1-3) from nodal explants was effective on this medium. The regenerated shoots with 4-7 nodes had further growth upon sub-culturing onto kinetin (1 mgl(-1)) and IBA (1 mgl(-1)) and rooted simultaneously. The rooted plants were transferred to pots after hardening under controlled conditions. The survival percentage in pots was 80-90%.     

山茱英的组织培养与植株再生

目的：建立山茱萸的组织培养及植株再生体系。方法：分别以山茱萸的叶片、花柄和花托为材料,进行山茱萸不同外植体的离体培养研究,筛选最佳培养基组成。结果：适宜山茱萸叶片愈伤组织诱导的培养基组合为1／2MS,附加BA2．0mg/L、IBA0,5—1．0mg／L;适宜山茱萸花柄、花托愈伤组织诱导的培养基组合为1／2MS,附加BA1．0mg／L、2,4-D0．5mg／L;在1／2MS附加BA2．0mg／L、IBA0．05mg／L的培养基上,可诱导不定芽的产生;1／2MS附加IBA2．0mg／L的培养基有利于山茱萸试管苗生根。讨论：山茱萸的花托是进行组织培养的最适外植体,白色或翠绿色、结构致密的愈伤组织较易分化产生不定芽。     

云南山楂茎尖的离体培养及其无性系快速繁殖

用云南山楂(Crataegus scabrifolia(Franch.)Rehd.)成年树茎尖和实生芽两种不同发育阶段的材料为外殖体,诱导它们休眠芽萌动,丛生芽条并诱导芽条生根。实验结果如下:1.以成年态的云南山楂侧芽为外植体,培养在附加IAA 0.1—0.5mg/l+6-BA 1-2mg/l的MS培养基上可诱导芽的萌发;将芽继代培养在附加0.5—1mg/l 6-BA的SH或MS培养基上,40天后芽数增殖4—6倍;将芽条截下置于1/2MS培养基上,附加不同浓度的IAA或IBA,可得到50—80％的生根率。2.以实生芽为外殖体,在相同条件下,则20天后芽数增殖便可获4—6倍;98％以上生根。结果表明:云南山楂的幼年态要比成年态易脱分化和再分化。     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

转抗虫基因欧美黑杨离体快繁技术研究

以抗虫欧美黑杨的叶,带腋芽茎段为外植体进行离体快繁技术研究。最佳接种时间为8月份,新芽生长迅速。基本培养基为MS,较适初培养基为MS＋6－BA0.5mg/L（以下单位同）＋NAA0.01mg/,附加30g/L,蔗糖,7g/L琼脂。愈伤组织诱导并同时分化出新芽培养基为MS＋6－BA1.5 NAA0.3,附加40g/L蔗糖,6g/L琼脂。继代增殖培养基为MS 6-BA1.0 NAA0.1 GA2.0,附加30g/L蔗糖,5g/L琼脂。生根培养基为MS＋IBA2．0。     

Effect of plant growth regulators on regeneration of plantlets from bud cultures ofCymbopogon nardus L. and the detection of essential oils from thein vitro plantlets

Cymbopogon nardus L. could be propagated via tissue culture using axillary buds as explants. The aseptic bud explants obtained using double sterilization methods produced stunted abnormal multiple shoots when they were cultured on Murashige and Skoog medium (MS) supplemented with 1.0 mg L^-1 or 2.0 mg L^-1 benzyladenine (BA). Stunted shoots that cultured on MS + 1.0 mg L^-1 BA + 1.0 mg L^-1 N⁶-isopentenyl-adenine (2iP) could induce elongation of shoots from about 60% of the stunted shoots. Normal multiple shoots could be induced at the highest (19.7 shoots per bud) from the bud explants within six weeks when cultured on proliferation medium consisted of MS supplemented with 0.3 mg L^-1 BA and 0.1 mg L^-1 indole-3-butyric acid (IBA). The separated individual shoot produced roots when transferred to basic MS solid medium. The essential oils that were contained in the mature plants namely citronellal, geraniol and citronellol were also found in thein vitro C. nardus plantlets. Citronellal was the main essential oil component in the matured plants while geraniol was the main component in thein vitro plantlets.     

Plantlet regeneration from fascicular buds of seedling shoot apices of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> Sarg

Shoot apices of Pinus roxburghii Sarg were cultured on Murashige and Skoog’s medium (MS) supplemented with cytokinins [6-benzyladenine (BA), kinetin and N-benzyl-9-(2-tetrahydropyranyl) adenine (BPA)] alone and in combination with auxin, α-napthaleneacetic acid (NAA). Of the three cytokinins tested at varying concentrations, medium supplemented with 10 μM BA was found optimal in respect of explant responsiveness (97.22 %) and average number of buds induced per explant (7.42). The concentration of cytokinins in the induction medium had a profound effect on rate of elongation of induced buds on MS basal medium containing 0.5 % activated charcoal. Further, shoots induced on lower concentrations of BA increased up to 2.4 times in length in 4 weeks. Decapitation of the explant enhanced the rate of axillary bud elongation. Proliferating shoot cultures were established by sub-culturing the axillary shoots on MS supplemented with 10 μM BA. Shoots 2–3 cm in length were suitable for culturing as more buds were induced on them compared to longer or shorter shoots. Root primordia were induced on 70.83 % shoots when transferred to 1/2 MS medium supplemented with 5.0 μM NAA. Elongation of root primordia (60 %) was achieved in liquid 1/2 MS basal medium. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 20–22 weeks.     

马尾松高效再生体系的建立(简报)

利用组织培养技术快速繁殖针叶树,不仅为植树造林所需大量苗木开辟了一条快速高效的新途径,是生产优良无性系的捷径,而且也是植物基因工程技术应用于针叶树品种改良的前提条件。它将在林木种苗产业化和林木良种化进程中发挥重要作用。针叶树的离体培养和植株再生一直是植物组织培养研究中难度较大的一个领域。近几十年来,随着世界各国对发展无性系育林业的日益重视,针叶树组织培养研究取得了很大的进展[1-3]。马尾松(Pinus massoniana Lamb.)是我国南方主要的造林及绿化树种,其木材和松脂具有重要的经济价值。然而在生产实践中,种子园种子…     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.     

Rapid In Vitro Propagation of East Indian Rosewood (Dalbergia latifolia Roxb.) through High Frequency Shoot Proliferation from Cotyledonary Nodes

An efficient protocol is described for large scale in vitro propagation of east Indian rosewood (Dalbergia latifolia Roxb.) using cotyledonary nodes derived from axenic seedlings. Of the four different cytokinins tested, BA was most effective in inducing multiple shoot buds in the explants. High frequency shoot proliferation (93%) coupled with maximum number of shoot formation (10-12 shoots/explant) was recorded on Murashige and Skoog’s medium supplemented with 2.0 mg/l BA. The frequency of shoot proliferation declined at higher levels of cytokinins. Shoot culture was multiplied by subculturing the original cotyledonary node on a fresh shoot multiplication medium each time aHer excising the newly formed shoots. Shoots obtained from each passage were multiplied further as nodal explants and each node produced 3-4 shoots. In two months from a single cotyledonary node, about 90 shoots were obtained. Rooting was induced in 72% of the regenerated shoots on half-strength MS containing IAA, IBA and IPA each at 1.0 mg/l. Rooted plantlets were hardened off and eventually established in soil.     

In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil)

A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA₃) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Differential cytokinin effects on the stimulation of in vitro shoot proliferation from meristematic explants of castor (Ricinus communis L.)

A highly efficient and reproducible method of in vitro propagation using meristematic explants has been developed for castor. Embryo axes and shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 0.5–10.0 mg/l of adenine, N⁶-benzyladenine (BA), kinetin (Kn), thiadiazuron (TDZ) and zeatin. TDZ (1.0–10.0 mg/l) gave the maximum number of shoots (37.8–40.0) from embryo axes, while BA (2.0 mg/l) was found superior to other cytokinins for obtaining the highest number of shoots (46.7) from the shoot apex. Adenine and Kn at all of the tested concentrations resulted in low proliferation rates from embryo axes. The carryover effect of the cytokinins was tested by subculturing proliferating shoot cultures from various media onto the medium fortified with 0.5 mg/l BA. There was no significant influence of the cytokinins on subsequent proliferation from the two explant types except for TDZ with embryo axes. The number of shoots from TDZ-habituated embryo axes ranged between 36.0 and 81.7, while it varied from 5.7 to 22.0 and 3.7 to 28.3 in axillary buds and embryo axes, respectively, on the other media. For elongation of shoots, gibberellic acid (GA₃) (0.1–1.0 mg/l) was added to the medium supplemented with 0.2–0.5 mg/l BA. Incorporation of GA₃ (0.1 mg/l) significantly enhanced the frequency of elongated shoots but drastically reduced the multiplication ability. Hence, proliferating shoot clusters were periodically transferred to the medium supplemented with 0.5 and 0.2 mg/l BA for further multiplication and elongation. Well-developed shoots were rooted on half-strength MS medium supplemented with 1.0 mg/l indole-3-butyric acid. The rooted plantlets were acclimatized with more than 60% success. Received: 17 June 1997 / Revision received: 3 September 1997 / Accepted: 20 September 1997     

Direct plant regeneration from cucumber embryonal axis

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 μM BA and 1.59 μM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 μM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 μM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.     

从麻疯树上胚轴外植体再生植株

以麻疯树上胚轴为实验材料在MS添加IBA和BA的培养基上进行离体培养实验.结果表明,在IBA O.1 mg/L与BA 0.2～0.7 mg/L组合的条件下,不定芽从上胚轴外植体的表面直接被诱导分化,其中以在MS IBA 0.1 mg/L BA 0.5 mg/L上的诱导率最高.从愈伤组织来源的植株再生需要IBA 0.5 mg/L与BA 0.1 mg/L、IBA 0.5 mg/L与BA0.2mg/L以及IBA1.0mg/L与BA0.5mg/L的激素组合,其分化的最佳培养基是MS IBA1.0 mg/L BA0.5 mg/L.生长健壮的不定芽和再生植株能在无激素的MS基本培养基上生根.发育良好的再生苗可成功地转移到温室栽培而没有可见的变异.     

Efficient regeneration of sorghum, Sorghum bicolor (L.) Moench, from shoot-tip explant

Novel protocols for production of multiple shoot-tip clumps and somatic embryos of Sorghum bicolor (L.) Moench were developed with long-term goal of crop improvement through genetic transformation. Multiple shoot-tip clumps were developed in vitro from shoot-tip explant of one-week old seedling, cultured on MS medium containing only BA (0.5, 1 or 2 mg/l) or both BA (1 or 2 mg/l) and 2,4-D (0.5 mg/l) with bi-weekly subculture. Somatic embryos were directly produced on the enlarged dome shaped growing structures that developed from the shoot-tips of one-week old seedling explants (without any callus formation) when cultured on MS medium supplemented with both 2,4-D (0.5 mg/l) and BA (0.5 mg/l). However, the supplementation of MS medium with only 2,4-D (0.5 mg/l) induced compact callus without any plantlet regeneration. Each multiple shoot-clump was capable of regenerating more than 80 shoots via an intensive differentiation of both axillary and adventitious shoot buds, the somatic embryos were capable of 90% germination, plant conversion and regeneration. The regenerated shoots could be efficiently rooted on MS medium containing indole-3-butyric acid (IBA 1 mg/l). The plants were successfully transplanted to glasshouse and grown to maturity with a survival rate of 98%. Morphogenetic response of the explants was found to be genotypically independent.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

Quantitative determination of secoiridoid and xanthone glycosides of Gentiana dinarica Beck cultured in vitro

Gentiana dinarica Beck, native to the Balkan Dinaric Mountains, was established in vitro from axillary shoot buds. It was maintained in the form of shoot cultures on MS medium supplemented with 1.0 mg l^?1 6-benzyladenine (BA) and 0.1 mg l^?1 α-naphthaleneacetic acid and excised root cultures were maintained on ½ MS medium with 0.5 mg l^?1 indole-3-butyric acid (IBA). Shoot cultures, adventitious roots and excised root cultures were analysed by HPLC techniques for the presence of secoiridoids and xanthones. Gentiopicrin and swertiamarin, the dominant components of shoot cultures, could not be detected in root cultures. Xanthones were present in both shoot and root cultures with norswertianin-1-O-primeveroside as the dominant metabolite. The secoiridoid and xanthone content, although characteristic for certain plant organs, was dependent on the concentration of plant growth regulators (BA and IBA) added to the medium. BA in the shoot multiplication stage strongly increased the secondary metabolite (SEM) content of shoot cultures. IBA had little effect on SEM accumulation in shoots during rooting, while it moderately stimulated SEM accumulation in excised root cultures.