Mother plant age and seasonal influence on in vitro propagation of <Emphasis Type="Italic">Quercus euboica</Emphasis> Pap., an endemic,rare and endangered oak species of Greece

A micropropagation method for Quercus euboica Pap. was developed. Nodal explants from seedlings gave higher multiplication rates than explants from adult plants. Cultures initiated at the beginning of May produced the highest percentage of shoot forming explants and multiplication rate. Woody Plant Medium (WPM) salts, with 100 mg l⁻¹ myoinositol, 1 mg l⁻¹ thiamine, 0.5 mg l⁻¹ pyridoxine, 0.5 mg l⁻¹ nicotinic acid and 3% sucrose was used as basal medium and several cytokinins at various concentrations were evaluated for their effect on shoot multiplication. The highest shoot multiplication rate was obtained with 4.44 μΜ BA. IBA at 9.84 μΜ in the culture medium during the first week of culture, and if followed by culture in hormone-free medium, gave the best rooting results. Darkness at the beginning of the rooting period did not improve rooting. The use of plastic wrap as a cover material of the culture vessels enhanced rooting percentage and root number. Plantlets acclimatized ex vitro in soil from the natural environment of the species survived at a higher percentage (up to 93%) and had more vigorous growth than plantlets grown in a compost–perlite (2:1 v/v) medium (up to 36%).     

In vitro rooting of almond (Prunus dulcis Mill.)

Summary Shoot cultures of the paper shell almond (Prunus dulcis Mill.) cultivars ‘Ne Plus Ultra’ and ‘Nonpareil’ were subcultured for 4 wk at 4°C on growth regulator-free basal medium under low light conditions. Elongated shoots were excised and their response to a range of rooting treatments determined. Various concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid were compared over a range of incubation periods to determine the optimum auxin for root formation. In addition, the effect of shoot base shading, phloroglucinol (PG), and basal salt composition were examined. The treatment resulting in the best rooting of both cultivars was shoot insertion for 12 h into water-agar (0.6% w/v) with 1.0 mM IBA, followed by 2 wk in basal medium without auxin but with 100.0 μM PG. Explants were maintained under dark conditions for 3 d at the start of the treatment period, then exposed to light. Extending the darkening period did not improve rooting ability. Whilst half-strength Murashige and Skoog basal medium was suitable for rooting “Ne Plus Ultra’ shoots, full-strength Almehdi and Parfitt medium resulted in the best rooting of ‘Nonpareil’. Under these conditions, 60.0% of explants developed adventitious roots.     

火力楠种胚组培快繁研究

为探索火力楠组培快繁技术体系,以其优良家系种子为材料,从外植体选材与消毒、基本培养基、PVP浓度、生根促进剂类型及浓度等方面开展试验研究。结果表明:①应用0.1%的升汞溶液消毒胚6~9 min,无菌萌发率达60%~65%。②LY培养基适宜大部分胚系的增殖生长,35 d转接1次,增殖倍数高达3.50,高3.0 cm以上芽苗比率达50%以上。③应用4~8 g·L^-1浓度的PVP能够显著降低培养基褐化程度,促进芽苗抽高生长。④各胚系之间生根难易程度差异极显著,其生根率为0~93.6%,两种生根促进剂的最佳浓度均为8.5 mg·L^-1,ABT2号生根粉的生根诱导效果极显著(P<0.01)优于IBA。⑤生根瓶苗移植30 d后成活率达90%以上,未生根瓶苗移植60 d后成活率达80%以上。本研究为火力楠优良无性系的组培快繁研究及产业化生产提供理论与技术基础。     

Plant regeneration from seedling explants of Eucalyptus grandis × E. urophylla

Hypocotyls, cotyledons, cotyledonary nodes and primary leaves were used as explants to establish a regeneration protocol for Eucalyptus grandis × E. urophylla. These seedling-derived explants were incubated on a modified MS medium (SP medium), supplemented with 2.0 M TDZ. After 1 month, the calluses obtained were transferred to SP medium containing different concentrations of BA and NAA or zeatin and NAA. Shoots were induced from these calluses at a high frequency. Shoot elongation was then stimulated on SP medium supplemented with BA, NAA and GA₃ for 20 to 30 days. For rooting, 50 mm long shoots were cultivated on root induction medium containing IBA (2.5 M) for different periods and then transferred to the same medium but without auxin, for 30 days. Plantlets were then successfully transplanted to greenhouse conditions.     

Shoot proliferation and HPLC-determination of iridoid glycosides in clones of <Emphasis Type="Italic">Gentiana cruciata</Emphasis> L

Gentiana cruciata L. (Cross gentian) is a medicinal and ornamental plant, threatened in its natural habitats. The wild root extracts of this species are known to exhibit many curative properties. In the present study, an efficient protocol for in vitro propagation of G. cruciata L. was developed from node culture. A semi-solidified Murashige and Skoog (MS) basal medium supplemented with 2.22 μM 6-benzyladenine (BA), 2.46 μM indole-3-butyric acid (IBA) and sucrose (3% w/v) improved the production of multiple shoots directly from nodal segments, providing 3.9 shoots per explants on average. The highest rooting (81.7%) was observed with half-strength MS medium supplemented with 2.46 μM IBA. Plants with well-developed roots were transferred to pots containing turf/vermiculite mixtures and acclimatized in plant growth chamber conditions. Acclimatized plants showed 100% survival and remained healthy. The content of secondary metabolites in the clones was determined by HPLC, and the presence of gentiopicroside, loganic acid, swertiamarin, and sweroside in the samples was confirmed. Gentiopicroside was found to be the major compound.     

长叶皇冠的组织培养与快速繁殖技术研究

以长叶皇冠短缩茎为外植体进行离体培养,对外植体灭菌方法以及培养基中不同种类和浓度生长调节剂对增殖、生根的影响等进行研究。结果表明:以0.1%的HgCl2为灭菌剂,采用8min+(6～8)min、间歇时间为24h的间歇灭菌法,可获得20%以上的成活无菌外植体;试管苗茎纵切成4份后,接种在1/2MS+6-BA6.0mg·L-1增殖培养基上培养42d,增殖系数可达9.3;在1/2MS+NAA0.2mg·L-1培养基上培养28d后,生根率达100%,平均每株约生根18条;炼苗后移植成活率100%。     

Facile transformation of Arabidopsis

Summary A protocol is described for the simple, rapid and efficient production of transgenic Arabidopsis plants. The procedure was developed using growth regulator regimes that promote adventitious embryogenesis during or immediately following Agrobacterium mediated transformation. Both the RLD and Columbia genotypes of Arabidopsis were transformed using slightly different growth regulator regimes. For the Columbia genotype two modifications of the protocol were identified which substantially improved regeneration. Cold treatment of the plants used as a source of root explants resulted in a three-fold increase in the number of morphogenic sectors produced. A more important modification was the inclusion of 25 mg/l silver nitrate (an inhibitor of ethylene action) to the medium used for shoot regeneration. This provided a ten-fold increase in the number of shoots produced. These procedures made it possible to obtain over 100 putative transformants of RLD or Columbia from a single 10 cm petri dish, within 2 or 4 weeks after exposure of root explants to the bacteria. When these were transferred to rooting media containing antibiotics, approximately 20% were able to root after kanamycin selection and 80% after hygromycin selection. All the rooted plantlets tested were shown to contain integrated donor DNA as determined by Southern blot analysis.     

红厚壳茎段丛生芽诱导与植株再生

红厚壳(Calophyllum inophyllum)为藤黄科红厚壳属多年生木本植物,有很高的药用价值。该研究以红厚壳带节茎段为外植体,探讨生长调节剂对腋芽萌发及丛生芽诱导、伸长和试管苗生根的影响。研究结果表明,外植体腋芽萌发和丛生芽诱导效果最好的培养基是MS+NAA1.0+TDZ0.5,在此条件下培养21天后,转入添加0.5 g·L–1活性炭且无生长调节剂的MS培养基,可有效促进不定芽的伸长。将带不定芽的外植体先在附加1.0 mg·L–1NAA的1/2MS培养基上进行生根诱导4周,之后转入附加1.0 g·L–1活性炭的无激素培养基进行根的伸长培养,这样的两步生根法能有效促进红厚壳生根。     

In vitro rooting of micropropagated shoots from juvenile and mature Pinus pinaster explants: influence of activated charcoal

The ability for adventitious rooting of micropropagated shoots from juvenile and mature Pinus pinaster Ait. explants was assessed in vitro on a rooting expression medium. The different rooting traits observed, namely the rooting rate, the number and the length of the adventitious roots, and the root score, were greatly influenced by the age of the donor plant: 98% of juvenile explants rooted, while only 49% of mature explants did. Addition of activated charcoal in the rooting expression medium improved the overall rooting capacity of the mature explants to an average of 78%. Whatever the plant material, the number and the length of the adventitious roots, as well as the root score, fluctuated according to the sampling date.Abbreviations BA 6-benzyladenine - NAA naphthaleneacetic acid - REM rooting expression medium - RIM rooting induction medium     

大花银莲花愈伤组织诱导及再生体系的建立

为建立野生大花银莲花(Anemone silvestris)组培再生体系,分别以无菌苗上、下胚轴、叶片和叶柄为外植体,探讨不同浓度植物生长调节剂对不同外植体的愈伤组织诱导、不定芽分化、增殖与生根的影响。结果表明, 4种外植体均可诱导出不定芽,其中上胚轴诱导效果最佳,其在1/2MS+2.0mg·L^–1 6-BA+0.1mg·L^–1 NAA培养基中诱导率最高,为86.67%;最适增殖培养基为1/2MS+1.0mg·L^–1 6-BA+0.05mg·L^–1 NAA,增殖系数为3.67;最佳生根培养基为1/2MS+0.3mg·L^–1 IBA,生根率为100%;在草炭:蛭石=2:1(v/v)的栽培基质中,组培苗的移栽成活率最高,为98.33%。该研究有效解决了野生大花银莲花在园林及药用生产上的种质资源紧缺难题,为工厂化育苗提供了技术支撑。     

High frequency shoot regeneration from nodal and shoot tip explants in Holarrhena antidysenterica L

Shoot tip and nodal segment explants of Holarrhena antidysenterica when cultured on MS medium containing BAP (1.0-3.0 mg/l) with NAA (0.2-1.0 mg/l) and BAP (1.0-3.0 mg/l) with Kn. (0.2-1.0 mg/l) produced multiple shoots. Maximum multiple shoots was found in MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l). Subculture on the same medium resulted in rapid shoot multiplication at an average rate of 16 new shoots per subculture. Addition of urea (100 mg/l) in the medium increased the number of shoots up to 22 per culture. For best rooting, the shoots were excised from the culture flask and implanted individually on half strength MS medium with 0.5 mg/l each of IBA, IAA and NAA. After 20 days of transfer on root induction medium 95% rooting was achieved. Regenerated plantlets were successfully acclimatized and established in soil. About 90% of plantlets survived under open field conditions.     

A Novel Method Based on Etiolation for Increasing Efficiency of In Vitro Propagation of Populus tomentosa

This paper describes a novel method based on etiolation treatment for micropropagation of Populus tomentosa. It includes: (1) Antibiotic “cefotaxime” (2.5 ppm in nutrient medium) is used to inhibit bacteria growth and the cultures which have been contaminated after long period subculture are recovered by sterilization with 1/10000 HgCl2(w/v). (2)Low concentration of TDZ (thidiazuron, 0.005 ppm) replaces expensive zeatin for enhancing bud differentiation and multiplication of leaf explants. (3) Shoot elongation is promoted after etiolation treatment, which will increase the number of shoots suitable for rooting, about 50 etiolated shoots are obtained in a 100 ml flask, 3—5 times more than those produced in traditional method. (4) Etiolated shoots or after their greening are used as cuttings for rooting in vivo and over 90% survival rate could be achieved when the medium is sterilized. This method is labour and money saving and high efficient.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated transformation of <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> and production of flavonoids in hairy roots

Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A₄GUS, R1000 LBA 9402 and ATCC11325. The A₄GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15–20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B₅ medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A₄GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1–30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).     

Efficient in vitro regeneration of fireweed,a medicinal plant

Epilobium angustifolium L. (fireweed) is a medicinal plant that has been used to treat diarrhea, mucous colitis, irritable-bowel syndrome, skin problems, prostate problems, menstrual disorders, asthma, whooping cough, and hiccups. A highly efficient and rapid regeneration system via multiple shoot formation was developed for fireweed. Explants (leaf, petiole, root, and stem segments) excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. Explant browning, a major problem for regeneration, was overcome by adding 100 mg/l ascorbic acid to all prepared media containing growth regulator combinations. Root explants formed more shoots than other explants. Best shoot proliferation was obtained from root explants cultured on media with 0.1 mg/l BA and 0.5 mg/l IAA. Regenerated shoots were transferred to rooting media containing different concentrations of IAA, IBA, NAA or 2,4-D. Most shoots developed roots on medium with 0.5 mg/l IAA. Rooted explants were transferred to vermiculate in Magenta containers for acclimatization and after 3 weeks they were planted in to plastic pots containing potting soil and maintained in the plant growth room.     

High frequency of plant regeneration in sunflower from cotyledons via somatic embryogenesis

Summary A plant regeneration methodvia somatic embryogenesis of severalHelianthus annuus L. genotypes was developed. Starting from cotyledonary explants high frequency embryo induction was obtained following several subcultures on defined media. An appropriate cotyledon developmental stage was identified. Etiolated explants and darkness treatment were necessary to obtain somatic embryos in all tested genotypes. After 20–25 days on somatic induction medium containing an auxin:cytokinin ratio of 1:1, the germination of embryos was induced by a reduction of the hormonal ratio (1:2). Shoots were excised from callus and transferred onto a medium containing various vitamins. The range of embryogenesis frequency was 33–72%, depending on the genotype. High frequency of rooting (49–82%) was obtained using a medium supplemented with 0.5 mg/L of ancymidol and by a reduction of photoperiod. A large percentage of somatic embryos developed into normal regenerated plants producing viable seeds.Abbreviations MS Murashige and Skoog (1962) - NAA naphthaleneacetic acid - BAP benzylaminopurine - GA₃ gibberellic acid - EIM embryo induction medium - GM germination medium - VM vitamins medium - RA2 ancymidol rooting medium - EtOH ethanol     

Micropropagation of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L. using somatic embryo-derived plants

Summary A micropropagation protocol was developed using cacao somatic embryo-derived plant as a source for nodal and apical stem explants, and apical microcuttings. Microcuttings were efficiently rooted and developed into plantlets. Axillary meristems within the remaining decapitated plantlets subsequently developed and were used for production of additional microcuttings, with an average 2.4 growing shoots per decapitated stem. The remaining plantelts were maintained as microcutting stock plants. When nodal stem explants were cultured on thidiazuron medium, axillary buds proliferated and developed into shoots, which were excised and rooted. However, the efficiency of this method is lower than rooting of apical microcuttings harvested directly from stock plants. During root induction, short treatment with indole-3-butyric acid (IBA) increased the total percentage of rooted microcuttings up to 89%. Longer exposures to IBA increased the average number of roots per microcutting (from 1.7 to 5.2). Plant acclimatization after rooting was achieved with an average success of 87%. During several months of growth in the greenhouse, the micropropagated plants developed functional taproots. Currently, cocoa plants produced by this micropropagation method have been successfully acclimated to field conditions in Ivory Coast, Ghana, and Saint Lucia.     

微型月季愈伤组织诱导及植株再生

选取微型月季(Rosa hybrida)幼嫩离体叶片(叶龄12天)为外植体, 以MS为基本培养基, 研究不同配比的植物生长调节剂对愈伤组织诱导、不定芽诱导和生根培养的影响。结果表明: (1) 诱导愈伤组织的最佳培养基配比为1.0 mg·L^–16-BA+3.0 mg·L^–12,4-D, 诱导率为100%; (2) 6-BA在微型月季不定芽诱导中起着关键作用, 诱导不定芽产生的植物生长调节剂配比为1.0 mg·L^–16-BA +(0.05–0.5) mg·L^–1NAA+(0.02–0.2) mg·L^–1TDZ, 其中最适配比1.0 mg·L^–16-BA+0.1 mg·L^–1NAA+0.02 mg·L^–1TDZ的愈伤组织再分化率达到92.6%; (3) 生长素对微型月季的生根有重要影响, 以1/4MS+0.1 mg·L^–1NAA为生根培养基可获得100%的生根率。实验最终建立了微型月季组织培养和高频离体再生体系。     

Relations between auxin and cytokinin contents and in vitro rooting of tree Peony (Paeonia suffruticosa Andr.)

In this work, a combined HPLC-ELISA technique was used to associate in vitro rooting capacity of tree peony micro-cuttings with contents of cytokinin and auxin; the cytokinin mainly detected corresponded to the N⁶-benzyladenine which had been added to the multiplication medium. Rooting capacity of explants was favoured by a preliminary accumulation of endogenous IAA only when levels of the BA absorbed from the multiplication medium had decreased. Main shoots coming from a 5-weeks subculture fulfilled these hormonal conditions and were the best microcuttings for rooting (87% rooting). Main shoots coming from shorter cycles or axillary shoots coming from a 5-weeks cycle always contained high benzyladenine levels and had a low rooting capacity (25–55% rooting). Root induction was associated with an early peak of indole-3-acetic acid followed by a 10-fold lower peak of endogenous ribofuranosyl-isopentenyladenine. Only a low and transitory accumulation of isopentenyladenine occurred during root development, and this could explain the lack of shoot development. Root development was efficient, especially in a medium containing activated charcoal, which led to an almost 3-fold decrease of IAA contents in roots.Abbreviations AC activated charcoal - BA N⁶-benzyladenine - ELISA enzyme linked immunosorbent assay - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - IBA indole-3-butyric acid - iP N⁶-(²-isopentenyl)adenine - RDM root development medium - RIM root induction medium - 9RIP 9--d-ribofuranosyl-iP - 9RZ 9--d ribofuranosyl-zeatin - Z zeatin     

In vitro protocols and field performance of elites of an important bamboo Dendrocalamus hamiltonii Nees et Arn. Ex Munro*

Seedling raised elites of Dendrocalamus hamiltoniiNees et Arn. Ex Munro were chosen as the source of nodal explants from precocious branches. While axillary bud break was accomplished in hormone free 1/2MS medium containing sucrose (3%, w/v), BA supplementation was required for shoot proliferation. A variety of hormonal combinations induced rooting in clumps of shoots. Somatic embryogenesis was also obtained in callus cultures raised in 2,4-D supplemented MS medium and plantlets derived from somatic embryos were hardened for field transfer. Comparative growth performances of plants raised from nodal cuttings of field-grown plants, those from single node cuttings of precocious branches and from somatic embryos indicated that growth performance of the tissue culture raised plants was relatively better than those from nodal cuttings. Improved protocols for efficient micropropagation are visualized to provide an impetus to raising of bamboo nurseries of elite genotypes in bamboo growing areas of western Himalayas.     

High-frequency shoot multiplication in<Emphasis Type="Italic"> Curcuma longa</Emphasis> L. using thidiazuron

The effects of plant growth regulators, explant types, and culture regimens were investigated on in vitro shoot proliferation from terminal bud explants of Curcuma longa. Each bud was longitudinally divided into four equal pieces, each 1 cm in length, and used as explants. These were then cultured on MS medium supplemented with 18.17 microM thidiazuron for 4 weeks prior to transfer to MS medium without growth regulator for 8 weeks. Under these conditions, a shoot induction rate of 18.22+/-0.62 shoots/explant was obtained after 12 weeks of cultures. Spontaneous rooting was achieved. The regenerated plants were transferred to soil under greenhouse conditions and subsequently grown successfully in the field.