In vitro establishment of Cryptomeria japonica (Taxocidaceae)

In this work, it was achieved to establish in vitro shoots of Cryptomeria japonica from 20 year old trees. The shoots were disinfected and treated with six different concentrations of kinetin and belciladenine in order to induce their development and budding. It was evaluated the effect of quality and lighting intensity on these using for this orange light at 20 microEs-1 m-2 and white light at 30 microEs-1 m-2. For shoots rooting it was used different concentrations of NAA (naftalen acetic acid) and IBA (indolbutiric acid) alone or combined. BA and KIN induced the bud formation in Cryptomeria but it was observed the best budding with BA at 9.1 microM. White light and orange light promoved the growth of explants as well as the growth of new buds but it was higher with orange light. The bud rooting was observed but it was not possible to find the best auxin concentration for rooting because of the plentiful callus formation on the base of explants and the root formation was very sporadic. The rooted shoots were placed on a substrate for their acclimation in greenhouse conditions.     

廊坊杨3号快繁和转基因的再生系统

对杂交杨树新品种廊坊杨3号（Populus langfangensis 3,(P. deltoides (“Shan Hai Guan”)×((P. simonii × pyramidalys)₁₂×Ulmus pumila) 的离体叶片和茎段在附加BA、NAA、IBA和2,4-D的MS培养基上的直接和间接的器官分化、愈伤组织形成和植株再生进行了研究。叶柄和叶片最容易在叶脉处直接诱导生芽。从叶片直接诱导生芽的激素条件为1～2 mg·L^-1 BA和0.5 mg·L^-1 IBA,最高生芽率可达90%。2,4-D促进愈伤组织的形成。由愈伤组织诱导生芽的激素条件为0.3～0.5 mg·L^-1 BA 和 0.02 mg·L^-1 IBA或NAA,生芽率达76%。较好的生根条件为0.1 mg·L^-1 BA和0.2～0.5 mg·L^-1 IBA,生根率可达67％。以上再生条件为廊坊杨3号的转基因育种和无性快繁技术提供了可能。     

In vitro root induction in axillary microshoots of Quercus robur L.

Whilst considerable efforts have been made to optimise shoot multiplication and rooting in oak, little attention has been paid to the impact of conditions used for multiplication on subsequent root formation. An optimised technique for rooting of oak microshoots has been developed to assess the effect of cytokinin treatments applied to shoot multiplication cultures on the subsequent rooting of microshoots. We found IBA to be more effective at inducing root formation in microshoots than NAA. Efficient rooting of oak microshoots (80%) was achieved after 35 days on medium supplemented with 1.0 mg litre^-1 IBA. Lower concentrations of IBA reduced the frequency of root formation and significantly increased the time taken for microshoots to form roots. High concentrations of IBA (3.0 mg litre^-1) produced similar rooting frequencies but with significantly increased numbers of roots formed by each microshoot. However, high concentrations of IBA stimulated the production of basal callus. Rooting of microshoots was unaffected by the concentration of BA used during shoot multiplication, although basal callusing was greater in microshoots taken from multiplication medium supplemented with the highest concentration of BA (1.0 mg litre^-1) and rooted on medium supplemented with 3.0 mg litre IBA. Reducing the period of exposure to auxin to 7 days by transferring microshoots to auxin-free medium increased the frequency of root formation (84%), led to more rapid root formation and a reduction in basal callus formation.     

High frequency axillary bud multiplication and ex vitro rooting of Wedelia chinensis (Osbeck) Merr.--a medicinal plant

An efficient protocol was achieved for rapid propagation of Wedelia chinensis (Osbeck) Merr. through axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium supplemented with benzyladenine (BA; 8.87 microM) and indole-3-butyric acid (IBA; 2.46 microM) was optimal for axillary bud proliferation, which developed a mean of 8.3 shoots/node. Excision and culture of node segments from in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 days. Excision and culture of nodes in succession enhanced the number of shoots. Shoot multiplication did not exhibit decrease in the number of shoots even at 10th subculture. Nevertheless, the shoots exhibited a tendency towards stunted nature. But reduction of BA to 4.44 or 2.22 microM resumed normal growth of shoots. Half strength MS medium fortified with IBA (2.46 microM) induced the highest number of roots. All in vitro rooted shoots survived in field. Dipping of the basal end of shoots collected from multiplication medium in IBA (2.46 microM) solution for 7 days induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100%). Rooting ex vitro by direct transfer of shoots from multiplication medium exhibited 89.2 per cent survival. Use of commercial sugar and tap water and also the omission of in vitro rooting reduce the propagation cost 50-70 per cent. The protocol enables to harvest more than 50,000 plantlets within 150 days starting from a single node explant.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

An efficient protocol for micropropagation of lemon (<Emphasis Type="Italic">Citrus limon</Emphasis>) from mature nodal segments

The influence of the basal medium and different plant growth regulators on micropropagation of nodal explants from mature trees of lemon cultivars was investigated. Although the basal medium did not affect any of the variables, explants on DKW medium were greener. Several combinations of 6-benzyladenine (BA) and gibberellic acid (GA) were used to optimise the proliferation phase. The number of shoots was dependent on the BA and GA concentrations and the best results were obtained with 2 mg l⁻¹ BA and 1 or 2 mg l⁻¹ GA. Explants length was shorter with the higher BA concentrations and, in all genotypes, shoot length was greater with 2 mg l⁻¹ GA. The best results for productivity (number of shoots × the average shoot length) were obtained with 2 mg l⁻¹ BA and 2 mg l⁻¹ GA, although explants with chlorosis and narrow leaves were observed. The presence of BA and GA in the proliferation medium was essential for the explant multiplication but GA had a greater influence. The transfer of in vitro shoots to rooting media, containing different concentrations of indole butyric acid (IBA) and indole acetic acid (IAA) produced complete plantlets. Lemon shoots rooted well in all rooting combinations. The highest rooting percentages were obtained on media containing 3 mg l⁻¹ IBA alone or IBA in combination with 1 mg l⁻¹ IAA and on these media the highest numbers of roots were produced. The average root length was affected significantly by the IBA and IAA concentrations. Root length was greater when only 3 mg l⁻¹ IBA was used, and in this rooting medium explants had a better appearance, with greener and larger leaves. The success during the acclimatisation was close to 100% and the plantlets exhibited normal growth in soil under greenhouse conditions.     

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…     

Exogenous trehalose affects morphogenesis in vitro of jojoba

Trehalose is a stress protecting and reserve carbohydrate in a variety of organisms. The effect of trehalose on micropropagation of jojoba [Simmondsia chinensis (Link) Schneider] was investigated using a modified Murashige–Skoog as the basal medium (BM). Proliferation rate was significantly higher in explants grown on BM + 1 mM trehalose compared to that in the phytohormone control medium. In multiplication stage, shoot proliferation rate of 4.8 was achieved in BM with 4.44 μM BA and 1 mM trehalose. The rooting induction with 14.7 μM IBA had a significant effect on root regeneration; the highest rooting percentage (46.6%) was obtained with 7 days of induction. The incorporation of trehalose to the IBA-rhizogenesis media decreased basal callus but with trehalose alone, root development was scant. In vitro plants showed anatomical features typical for the acclimatization stage.     

Plantlet multiplication from white pine (Pinus strobus L.) embryos in vitro: bud induction and rooting

White pine embryos were grown on 4 different media with 6 different benzyladenine (BA) concentrations. Maximum adventitious shoot initiation and growth were obtained on a modified Lepoivre medium with 20 M BA. Modified Schenk and Hildebrandt, Murashige and Skoog, and Gresshoff and Doy media were also tested. Shoot elongation was achieved on half-strength basal medium lacking growth regulators. Three rooting experiments involving indole-3-butyric acid (IBA), sucrose concentration, shoot orientation, IBA pulse length, and light or dark were carried out. Treatment of shoots in an upright position with 50 M IBA for eight days followed by culture in a medium with 3% sucrose in the light produced the most rooting (50% at 3 months). Rooted shoots were transplanted to the greenhouse for further growth.     

Micropropagation of Pinus peuce

In Pinus peuce zygotic embryo culture grown on Gresshoff and Doy (1972; GD) basal medium, 2.22 μM benzyladenine (BA) was superior in promoting adventitious bud induction during 4 weeks comparing to kinetin or BA + kinetin. Shoot elongation was achieved on half-strength GD medium devoid of plant growth regulators and containing activated charcoal. Pulse treatment with 1 mM indole-3-butyric acid (IBA) for 2 h, followed by transfer to half-strength GD medium, produced the most efficient rooting. Rooted shoots were transplanted to the greenhouse and plantlets continued to grow and developed into phenotypically normal plants. Up to 10 plants per explant can be obtained within 36 weeks from culture initiation.     

影响四季桔器官发生的因素

对四季桔胚轴直接出芽、愈伤组织诱导及植株再生能力的研究表明：上、下胚轴在含有BA的MT培养基上,均能直接出芽,但上胚轴出芽能力明显高于下胚轴,最高出芽率为96．9％。外植体在培养基上的接种方式,对出芽也有一定影响,上胚轴切段以形态学下端垂直插入,出芽率高,且在培养前期表现尤为突出。胚轴愈伤诱导的适宜培养基为MT＋IBA0．5mg-1＋BAgmg-1。愈伤组织分化时,上、下胚轴愈伤组织所要求的BA浓度分别为4mgL-1及2mgL-1。在根的诱导中,附加的生长素种类,不仅影响生根率的高低,而且影响上部茎叶的生长,其中以加入lmgL-1IBA的效果最好,生根率达84．4％。     

Micropropagation of a <Emphasis Type="Italic">Casuarina</Emphasis> hybrid (<Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> L. × <Emphasis Type="Italic">Casuarina glauca</Emphasis> Sieber ex Spreng) following facilitated seed germination

A suitable protocol for micropropagation of Casuarina hybrid, Casuarina equisetifolia L. × Casuarina glauca Sieber ex Spreng (C. e. × C. g.), was developed. When seeds without seed coats were cultured on 4 germination media, the optimal seed germination percentage (91%) was obtained on 0.8% agar solidified water medium. Shoot multiplication was achieved by culturing 2-cm long epicotyls, excised from germinated seedlings, on MS (Murashige and Skoog 1962) basal medium supplemented with BA (6-benzylaminopurine) at 4.4, 8.8, 17.8 and 35.6 μM. The greatest percentage of axillary bud sproutings (87.5%), mean number of sprouts per explant (3.8), and shoot length (3.2 cm) were achieved on MS medium supplemented with 17.8 μM BA. MS medium supplemented with 4 different concentrations of IBA (indole-3-butyric acid) (4.3, 8.7, 13.0 and 17.4 μM) were used for rooting of in vitro grown shoots. The highest rooting percentage (65.6%), mean number of roots per explant (2.5) and mean length of roots per explant (1.6 cm) was achieved at 13.0 μM IBA. Rooted shoots grew well after transfer to a substrate of peat and pinebark (7:3) in the greenhouse.     

Factors affecting induction and development of in vitro rooting in apple rootstocks

Shoots of apple rootstocks raised in vitro were transferred to various rooting media to study the effect of different factors on root initiation and development. Various concentrations of indole-3-butyric acid (IBA) initiated rooting but maximum rooting percentage was found with 2.0 and 2.5 mg l(-1) of IBA in M7 and with 1.0 mg l(-1) of IBA in MM106. The drawback was that the roots were thick, short and with profuse callus. The presence of activated charcoal (AC) in the rooting medium improved the rooting quality but reduced the rooting percentage in both the rootstocks. In high auxin dip of 70, 80 and 90 mg l(-1) IBA for 2, 2 and 1 hr showed 75-85 per cent rooting in M7, but lacked reproducibility of the results. Whereas in MM106, 66 - 70 % rooting was achieved with 70 mg l(-1) of IBA dip for 3 h. Root induction in shoots in IBA containing liquid medium (LM) in dark for few days and root elongation in IBA--free medium in light proved most effective. On the other hand, continuous light treatment showed reduced rooting. Reduction of MS salts and sucrose in root elongation medium showed decreased rooting. Plantlets from two--stage rooting procedure showed more rapid growth and satisfactory survival during hardening of plants and on transfer to field.     

Somatic embryogenesis and plant regeneration from fragments of immature inflorescences and coleoptiles of durum wheat

Use of Hypericum perforatum L. has increased in the past few years due to the antidepressant and antiviral activities found in extracts of this plant. As a result of its potential as a pharmaceutical, a new system was developed for in vitro culture of this species. Leaf explants were inoculated onto MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.45 or 4.5 μM) and 6-benzyladenine (BA, 0.44 or 4.4 μM) or kinetin (0.46 or 4.6 μM). Explants were cultivated under dark or light conditions to induce callus formation. Callus initiation was observed in all media evaluated and the highest cell proliferation was obtained from explants cultivated in the presence of 4.4 μM BA and 4.5 μM 2,4-D in the dark. Shoot induction was obtained from callus induced on 4.6 μM kinetin and 0.45 μM 2,4-D 6 weeks after transferring the callus to a MS medium supplemented with 4.4 μM BA. Roots were induced from shoots on full and half-strength MS media with or without indolebutyric acid (IBA, 4.9 μM) and the highest rooting frequencies were obtained on half-strength MS medium, regardless of the presence of IBA. Regenerated plants were easily acclimated in greenhouse conditions. The procedure reported here allows the micropropagation of H. perforatum in five months of culture and the proliferation of cell masses which could be used for studies on organic compounds of pharmaceutical interest. This revised version was published online in June 2006 with corrections to the Cover Date.     

GIS-facilitated in vitro propagation and ex situ conservation of Achillea occulta

A Geographical Information Systems (GIS)-facilitated approach for the in vitro propagation and ex situ conservation of the conservation priority species Achillea occulta is presented. To realize the species?? ecological requirements, the coordinates of the original habitat were linked with thematic layers derived from digital databases in a GIS environment. From wild plants, shoot tips were established in vitro in a basal medium with 4???M 6-benzyladenine (BA) and 0.5???M indole-3-butyric acid (IBA). A modified basal MS medium (modMS, double amount of Fe) proved to be the most effective for in vitro adventitious shoot production. The effect of BA in combination with ??-naphthaleneacetic acid (NAA) or IBA on shoot proliferation was also tested. The highest number of new microshoots/explant (3.5), with 0.93?cm shoot height was obtained when the modMS was supplemented with 5???M BA and 2.5???M IBA. To evaluate the root induction ability, microshoots produced were transferred to modMS media supplemented with 0?C20???M IBA and 0?C20???M NAA. Rooting proved to be very difficult and only by adding 20???M IBA, a 12.5% rooting percentage was achieved. Acclimatization succeeded only during early spring. Young plants transplanted at the Balkan Botanic Garden of Kroussia produced flowers and seeds in the first year. This GIS-approach provided useful guidelines for A. occulta??s (a) effective propagation (selection of greenhouse temperatures, temperatures during in vitro culture, suitable period for cuttings and acclimatization of plantlets), and (b) ex situ cultivation (selection of watering regime, temperatures, locations and exposures for growing sites).     

巴戟天组织培养和快速繁殖研究

以巴戟天顶芽及嫩茎节段为外植体,以MS为基本培养基,通过不同的激素种类和浓度配比,建立巴戟天组培快繁体系。结果表明,外植体表面消毒以70%酒精预处理60s,再用0.1%HgCl2浸泡10min,效果较好,茎节为外植体优于顶芽。培养基MS+BA1.0mg/L+IBA0.05mg/L利于诱导出芽,可用于初代培养;MS+BA1.0mg/L+IBA0.2mg/L利于形成丛生芽,用于继代增殖,繁殖系数6.0/50d;1/2MS+IBA0.4~0.8mg/L适宜诱导生根获得再生植株,生根率100%;生根苗移栽于排水良好的火土或砂土中,成活率90%。     

金边阔叶麦冬(Liriope platyphylla Wang et Tang var.variegata Hort.)的组织培养

将金边阔叶麦冬（Liriope platyphylla Wang et Tang var.variegata Hort.)不同部位外植体块,接种于附加不同激素配比的MS培养基上,实验结果表明,取自根状茎芽点,培养基为MS BA 1.5mg/L NAA 0.5mg/L的不定芽诱导率最高（100%）,长势较旺,不定芽和愈伤组织均较多,但极易造成性状分离。用MS BA 3.0mg/L NAA 0.5mg/L液体培养可以直接“芽生芽”,避免了脱分化过程,从而稳定保持嵌合性。不定芽每月可转接1次,适宜的生根培养基为1/2MS大量 IBA 0.25mg/L。试管苗在蛭石中炼苗后即可移栽。     

铁核桃离体培养与快速繁殖

以优良单株‘纳雍-1’的单芽茎段为外植体,建立了铁核桃（Juglanssigillata）离体培养与快速繁殖的体系。结果表明,附加6-BA1．0mg·L-1 ＋活·IgK（AC）3．0g·L-1的DKw培养基适宜铁核桃腋芽诱导;适宜铁核桃芽增殖的培养基为DKW＋6-BA1．0mg·L-1 ＋IBA0．02mg·L-1,40d后增殖系数可达7．33;试管苗的茎尖和茎段均可用于增殖培养;一步生根法（低浓度的生长素IBA持续诱导）不利于铁核桃试管苗嫩茎生根;采用二步生根法,生根率最高可达71．73％,其中,不同IBA浓度、暗培养时间、蔗糖浓度和AC含量对试管苗嫩茎生根影响显著,铁核桃试管苗在附]sulBA5,0mg·L-1的1／4DKW培养基中暗培养12d,再转移到不含IBA的1／4DKW培养基（附加AC 3g-L-1和蔗糖20g·L-1）中生根效果最好;生根试管苗采用珍珠岩和营养土两步炼苗,60d后成活率达到87．50％。     

In vitro plantlet regeneration from seedling nodal explants of Acacia catechu

Multiple shoots were initiated after 20 days in stem nodes excised from in vitro grown seedlings of Acacia catechu, on Murashige and Skoog's medium adjuvanted with 1 to 100 microM of N6-benzyladenine (BA). Explants were subcultured on the same medium augmented with 1.5 g l(-1) of polyvinylpyrrolidone (PVP) after 30 days. In the second subculture, after 30 days, the explants were transferred to a medium lacking PVP, but containing 10 microM of BA, where nine or ten shoots differentiated per explant within next 30 days. If individual shoots along with some callus were subcultured on BA (10 microM), nearly 15 shoots per explant regenerated in 90 days. Thus, the average number of shoots obtained from each node was 142 after 180 days. Since a seedling develops four nodes after 20 days, theoretically an average of 568 shoots can be obtained from a single seed. If shoots were individually subcultured on 1/2-strength MS medium with 14.7 microM of indole-3-butyric acid (IBA), roots developed in 20 days. Addition of 40 mg l(-1) of glutamic acid to the rooting medium prevented leaf senescence. These plantlets thrived well in garden soil, sand and silica (1:1:1).     

In vitro propagation of three commercial cut flower cultivars of Anthurium andraeanum Hort

In vitro propagation of Anthurium andraeanum Hort. cut flower cultivars viz. Lima White, Tropical White and Tropical Red through organogenesis using mature plant derived leaf explants was established on Murashige and Skoog (MS) medium fortified with different growth regulators. Cultivar, stage and different regions of the source leaf, and type of growth regulators significantly influenced callus induction. Explants from folded brown leaves were superior in induction of callus. Half strength MS medium fortified with 0.88 microM of benzyiadenine (BA), 0.9 microM of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.46 microM of kinetin (Kn) at pH 5.5 was most effective for callus induction. Transfer of callus to medium with 0.54 microM of NAA in place of 2,4-D induced higher number of shoots. Subsequent cultures displayed enhanced rate of shoot initiation and multiplication. Transfer of shoots onto half strength MS medium supplemented with 0.54 microM of NAA favoured rooting of shoots. Cultivar Tropical White was superior in callus, shoot and root induction compared to Lima White and Tropical Red. Plantlets after acclimation in greenhouse were transferred to net-house, that exhibited ninety seven per cent survival. Plants flowered normally between 12 and 15 months and were morphologically similar to that of the mother plants.