Plant regeneration via adventitious bud formation from cotyledon explants of Panax ginseng C. A. Meyer

Cotyledon explants of ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on medium without growth regulators, with 89% of the explants forming somatic embryos. Cytokinin treatment greatly suppressed somatic embryo formation but stimulated the direct formation of adventitious buds. BAP treatment was more effective than the kinetin treatment for adventitious bud formation. Auxin (0.05 mg/l IBA) in combination with cytokinin enhanced adventitious bud formation, with the highest frequency, 40%, at 0.05 mg/l IBA and 5 mg/l BAP. Adventitious buds were mainly formed near the distal portion of the cotyledons, while somatic embryos were formed near the proximal excised margins. Shoots were developed from adventitious buds after transfer to MS medium with 10 mg/l GA₃. Root formation from the shoots was obtained after the shoots were transferred to half-strength MS medium with auxin (IAA). When the plants derived from adventitious buds were transferred to greenhouse soil, 36% were successfully acclimatized. Received: 7 November 1997 / Revision received: 12 January 1998 / Accepted: 7 February 1998     

禿杉组织培养研究

本文详细报道了从秃杉(Taiwania flousiana Gaussen)离体胚诱导不定芽、不定根及从无菌苗茎端培养再生植株的过程。诱导不定芽要求较低的蔗糖浓度(以3％最好);同时BA是必须的,在附加0.1—3 mg/1 BA的White培养基上,从离体胚的子叶或胚轴上诱导了不定芽的发生(以1 mg/1最好);NAA与BA结合使用,对不定芽诱导无促进作用;适当提高光照有利于不定芽的诱导。在诱导不定芽的同时,在子叶表面还观察到有许多无结构的“不定突起”。不定芽起源于子叶表皮下1—2层细胞。IBA对诱导离体胚上产生不定根效果较好。在有或无生长素的培养基上,从生长1月龄的无菌苗茎端培养获得了不定根的产生,在加有细胞分裂索的培养基上,从无菌苗上产生了腋芽。     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.     

Micropropagation of Sesbania aculeata (Pers.) by adventitious organogenesis

Multiple shoots differentiated from hypocotyl explants of Sesbania aculeata (Pers.) syn S. cannabina (Retz.) Pers., a leguminous woody shrub, when cultured on Murashige and Skoog's basal medium supplemented with auxin (IBA, NAA) or auxin and cytokinin (IBA + BAP, NAA + BAP). Shoot budding occurred directly from the explant as well as from callus. Differentiation of shoot and root occurred in one step in the same concentration of auxin or auxin and cytokinin. Elongation of shoots occurred in the shoot induction medium.     

Influence of galactoglucomannan oligosaccharides on root culture of <Emphasis Type="Italic">Karwinskia humboldtiana</Emphasis>

Galactoglucomannan oligosaccharides (GGMOs) activity in K. humboldtiana root culture has been determined. GGMOs inhibited adventitious root growth and lateral root induction in contrast to IAA, IBA, and NAA stimulating effect in these processes. Similarly, the combination of GGMOs with natural auxins (IAA, IBA) evoked an inhibition of adventitious root growth and lateral root induction that depended on the oligosaccharides concentration and the type of auxin. The growth stimulating effect of the synthetic auxin, NAA, in adventitious roots was negatively affected by GGMOs, but they were without influence on lateral root induction. The presence of oligosaccharides triggered lateral root position on adventitious roots and the anatomy of adventitious roots (diameter, proportion of primary cortex to the central cylinder, number and size of primary cortical cells, intercellular spaces, and the number of starch grains in cells of primary cortex) in dependence on their coactions with auxin.     

Effects of explant types and media salt strength on growth and secondary metabolite accumulation in adventitious roots of <Emphasis Type="Italic">Periploca sepium</Emphasis> Bunge

In order to study the capabilities of Periploca sepium adventitious root induction in different types of explants, we selected leaves, roots and stems with or without buds. The growth of adventitious roots and periplocin content in these roots were determined. In order to investigate the suitable media salt strength, we cultured the adventitious roots in different salt strength (0.25, 0.50, 1.0, 1.5, 2.0) of Murashige and Skoog (MS) media supplemented with 1 mg/l indole butyric acid (IBA) and 30 g/l sucrose. The results showed that both leaf and root explants were proven suitable for the adventitious root induction; however, the stems could hardly induce adventitious roots no matter whether the stems had buds or not. Further studies reported that adventitious root proliferation and periplocin production derived from root explants were higher than those derived from leaf explants. So the root explants were the optimum explants for adventitious root induction, growth and periplocin production. The salt strength experiment showed that with the increasing salt strength (1.0–2.0 MS), adventitious root growth decreased significantly, as well as periplocin content in comparison with lower (0.25–0.5 MS) salt strength media.     

Plant genotype and growth regulators interaction affecting in vitro morphogenesis of blackberry and raspberry

The morphogenic response of somatic (leaf and petiole) and de-differentiated tissue (callus) of two blackberry (Rubus fruticosus) and one raspberry (Rubus idaeus) cultivars have been studied in vitro. With the aim to induce regeneration the effect of two sets of plant growth regulator (PGR) combinations (high cytokinin/auxin ratios and high auxin/cytokinin ratios) in Murashige and Skoog basal medium, were analysed. The three cultivars were characterised by a qualitatively different morphogenic response to the PGR combinations. Raspberry adventitious shoot regeneration from somatic tissue was improved by the 6-benzylaminopurine (BAP)/indol-3-butyric acid (IBA) combinations. On the contrary, shoot regeneration of both blackberry cultivars was reduced by high concentrations of BAP and completely inhibited by BAP/IBA combination. Media supplemented with high auxin/cytokinin ratios promoted callus production and root differentiation according to genotype and type of auxin. All the genotypes responded to media supplemented with IBA. 2,4-dichlorophenoxyacetic acid induced good callus formation in blackberry, but was toxic to raspberry. Indirect shoot formation was observed only in callus of blackberry cultivar Hull Thornless cultivated on medium with 10 μM BAP, the same concentration able to trigger efficient direct shoot regeneration from leaf explants of the same cultivar. This revised version was published online in July 2006 with corrections to the Cover Date.     

Acides-aminés et amines libres ďexplants foliaires de Nicotiana tabacum cultivés in vitro sur des milieux induisant la rhizogenèse ou la caulogenèse

Free amino acids and amines in leaf explants of Nicotiana tabacum cultivated in vitro on media inducing rhizogenesis or caulogenesis.
Foliar explants of Nicotiana tabacum cv. Xanthi n.c. were cultivated on three different media: (1) a basal medium without hormone, so that no differentiation occurred in the explants; (2) with auxin added; and (3) with auxin plus cytokinin added, where the additions (2) and (3) promote rhizogenesis and caulogenesis, respectively. The content of free amino acids and amines of the three kinds of explants were investigated. In the two media lacking cytokinin, the explants contained great amounts of five amino acids (asparagine, glutamine, proline, glutamic acid and histidine) and of one aromatic amine, tyramine. In the cytokinin containing medium, only two amines accumulated in the explants: one aliphatic polyamine (putrescine) and one aromatic amine (phenethylamine). The increase in amino acids began immediately on the first days of culture. It was related neither to a more active proteolysis nor to the breaking of the correlations from the mother plant. It was induced by the addition of nutritional elements into the medium. On the other hand, the accumulation of aromatic amines occurred after a few days of culture and was transitory. A decrease was observed after the first emergence of new organs. The relation between the accumulations of these aromatic compounds and formation of roots or shoots is discussed.     

In vitro propagation of birch (Betula verrucosa Ehrh)

Shoot t ps, young internodal segments and young developing leaves ofBetula ver rucosa Ehrh. in contact with agar nutrient medium formed tissue with numerous buds if medium contained a low concentration of cytokinin (BAP) and auxin (IBA or NAA). Tissue with induced buds transferred on a fresh nutrient medium continued in a formation of new buds which developed into shoots. Excised shoots were rooted on agar medium with a low concentration of auxin. Regenerated trees showed a genetic uniformity.     

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…     

Factors affecting the establishment and maintenance of ‘Titan’ red raspberry root organ cultures

Root organ cultures of red raspberry (Rubus ideaus cv. Titan) were established from two cm long terminal segments of adventitious roots induced in vitro on micropropagated shoot cultures. Growth of root organ cultures was almost entirely by initiation and elongation of lateral roots rather than by terminal elongation from the apical meristem of the original root explants. Indolebutyric acid (IBA) was required for lateral root initiation and elongation. The optimal IBA concentration for lateral root initiation decreased from 0.5 to 0.1 mg/L from the first to the fifth passage in culture. Two cm root explants initiated more lateral roots per unit length than four cm explants. Liquid Anderson's medium was superior to other basal nutrient and vitamin formulations tested. Root growth in liquid culture was stimulated by aeration. Root organ cultures also grew on media solidified with gelrite or agar. A rapid transfer technique was developed for subculturing these which involved cutting and transferring 1 cm discs of roots and underlying medium. Adventitious bud formation occurred spontaneously, but sporadically in liquid cultures, and was not influenced by cytokinin, auxin (spat) light, or chilling.     

Isolation of <Emphasis Type="Italic">HAG1</Emphasis> and its regulation by plant hormones during in vitro floral organogenesis in <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L.

Floral organs have been successfully induced from the regenerated floral buds of Hyacinthus orientalis L. by precisely controlling exogenous hormones in the medium. Under high concentrations of cytokinin and auxin, the regenerated floral bud produces only tepals. However, at reduced levels of the hormones, the regenerated floral bud can produce stamens and/or carpels with ovules. To understand the molecular mechanism of hormone-regulated flower development, a MADS-box gene, HAG1, which is homologous to AGAMOUS (AG) in Arabidopsis, was isolated from the floral tissues of Hyacinthus. Overexpression of HAG1 in Arabidopsis created flower phenotypes resembling those of the apetala2 mutant and AG transgenic Arabidopsis plants. Furthermore, the HAG1 expression pattern was similar to that of AG, confirming that HAG1 is the ortholog of AG in Hyacinthus. HAG1 mRNA was first detected in cultured explants at day 5 in the medium containing high levels of cytokinin and auxin, which could induce floral regeneration in vitro. However, no HAG1 mRNA was detected in the cultured explants until day 10 in media with low or no hormones. Further, HAG1 mRNA was detected in the stamens and carpels of regenerated floral buds, but not in the tepals. Our data support the hypothesis that hormone-regulated HAG1 activity is required for the induction of floral buds and the determination of floral organ types during the regeneration of floral buds.     

植物生长调节剂对白头翁叶片培养的影响

以白头翁试管苗叶片为外植体,以MS为基础培养基,探讨不同细胞分裂素和生长素对叶片不定芽诱导、愈伤组织的诱导、增殖、再分化的影响。实验结果表明：细胞分裂素TDZ适合于白头翁叶片培养,与生长素搭配使用使叶片发生分化;2,4-D对愈伤组织的诱导能力相对较强。叶片直接诱导不定芽的激素组合为TDZ0．3mg／L＋NAA0．1mg／L;愈伤组织增殖的激素组合为TDZ0．2mg／L＋2,4-D0．2mg／L,将愈伤组织转接到TDZ和NAA组合的培养基上时会再分化。     

In vitro morphogenetic studies on three apomictic species of Garcinia

Comparison of morphogenetic potential of three important Indian species of Garcinia??G. indica, G. cambogia and G. xanthochymus has been reported. Apomictic seeds of G. indica were found to be morphogenetically most potential followed by G. cambogia. The explants of G. xanthochymus were highly recalcitrant towards in vitro conditions and failed to induce adventitious buds on any of the media tested. High frequency direct shoot bud differentiation was induced in aseptic seed cultures of G. indica and G. cambogia on MS medium supplemented with cytokinins (BAP, kinetin or TDZ). Amongst the three cytokinins tested, TDZ (0.1?C0.5???M) was most effective for adventitious bud differentiation in both G. indica and G. cambogia, however, the proliferating buds failed to elongate. Substantial number of buds induced on BAP supplemented media elongated into shoots after subculture on elongation medium. Addition of NAA along with cytokinins in the induction medium enhanced callusing without improvement in bud induction response. The induced adventitious buds were elongated on MS basal medium containing 0.2% activated charcoal. Direct rooting was achieved in both G. indica and G. cambogia on auxin supplemented media with best response at 10???M IBA concentration in both the species. The in vitro raised plantlets showed 90% survival in the field when transferred after hardening and acclimatization.     

Influence of growth regulators on plant regeneration from epicotyl and hypocotyl cultures of two groundnut (Arachis hypogaea L.) cultivars

This study was designed to evaluate the effect of phytohormones on plant regeneration from epicotyl and hypocotyl explants of two groundnut (Arachis hypogaea) cultivars. Explants cultured on media with auxins and in combination with cytokinin produced high frequency of callus. After four weeks, callus from these cultures was transferred to medium with cytokinin and reduced auxin, shoot buds regenerated from the cultures. A high rate of shoot bud regeneration was observed on medium supplemented with 2.0 mg/L BAP and 0.5 mg/L NAA. Among the different auxins tested, NAA was found to be most effective, producing the highest frequency of shoot buds per responding cultures. Of the two explants tested, epicotyl was found to be best for high frequency shoot bud regeneration. Multiple shoots arose on MS medium supplemented with BAP or kinetin (1.0–5.0 mg/L) plus IBA (1.0 mg/L), with maximum production occurring at 5.0 mg/L. The elongated shoots developed rootsin vitro upon transfer to MS medium supplemented with NAA or IBA (0.5–2.0 mg/L) and kinetin (0.5 mg/L) for 15 days.In vitro produced plantlets, were transferred to soil and placed in a glasshouse developed successfully, matured, and set seeds.     

The dynamic distribution of NO and NADPH–diaphorase activity during IBA-induced adventitious root formation

Nitric oxide (NO) is a multifunctional molecule involved in numerous physiological processes in plants. In this study, we investigate the spatiotemporal changes in NO levels and endogenous NO‐generating system in auxin‐induced adventitious root formation. We demonstrate that NO mediates the auxin response, leading to adventitious root formation. Treatment of explants with the auxin indole‐3‐butyric acid (IBA) plus the NO donor sodium nitroprusside (SNP) together resulted in an increased number of adventitious roots compared with explants treated with SNP or IBA alone. The action of IBA was significantly reduced by the specific NO scavenger, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (c‐PTIO), and the nitric oxide synthase (NOS, enzyme commission 1.14.13.39) inhibitor, N^G‐nitro‐l ‐arg‐methyl ester (l ‐NAME). Detection of endogenous NO by the specific probe 4,5‐diaminofluorescein diacetate and survey of NADPH–diaphorase activity (commonly employed as a marker for NOS activity) by histochemical staining revealed that during adventitious root formation, NO and NADPH–diaphorase signals were specifically located in the adventitious root primordia in the basal 2‐mm region (as zone I) of both control and IBA‐treated explants. With the development of root primordia, NO and NADPH–diaphorase signals increased gradually and were mainly distributed in the root meristem. Endogenous NO and NADPH–diaphorase activity showed overall similarities in their tissue localization. Distribution of NO and NADPH–diaphorase activity similar to that in zone I were also observed in the basal 2–4‐mm region (zone II) of IBA‐treated explants, but neither NO nor NADPH–diaphorase signals were detected in this region of the control explants. l ‐NAME and c‐PTIO inhibited the formation of adventitious roots induced by IBA and reduced both NADPH–diaphorase staining and NO fluorescence. These results show the dynamic distribution of endogenous NO in the developing root primordia and demonstrate that NO plays a vital role in IBA‐induced adventitious rooting. Also, the production of NO in this process may be catalyzed by a NOS‐like enzyme.     

香根草体细胞胚胎发生的细胞学特点与形成条件

香根草是一种优良的生态环境治理植物,但也存在着一些局限性。为了对香根草进行遗传改良,选育出性状更优、抗性更强的新品种,开展了香根草离体培养研究。离体培养采用了两种外植体,一是带腋芽的节,二是由器官发生方式所产生的无菌不定芽。基本培养基为MS,根据不同的目的附加不同种类或配比的生长素与细胞分裂素。观察到香根草的外植体的离体发育途径,有器官发生和体细胞胚胎发生两种,依培养基中所含细胞分裂素或生长素的种类和用量不同而异。结果表明,香根草的这两种离体发育途径的植株再生能力均可以长期保持。细胞学的研究显示,香根草离体发育的启动可在外植体的表皮细胞或薄壁细胞中进行,这些细胞逐渐发育成为胚性细胞。胚性细胞分裂活跃,经二细胞、四细胞而发育成为多细胞的胚性细胞团。由显微观察可知,香根草的体细胞胚胎发生是单细胞起源的,成熟的体细胞胚具有单子叶植物典型的胚胎结构。在分化培养基的作用下,体细胞胚组织上所有的胚状体可以出芽而形成再生植株。所建立的香根草体细胞胚胎发生的植株再生体系,完全适用于遗传转化等生物工程方法对离体培养要求。此外,还观察到一些一般只有在双子叶植物才出现的鱼雷形体细胞胚,这是体细胞胚胎发生中的异常现象。认为这种异常胚是离体培养所引起的。     

Micropropagation of wild beet (Beta maritima) from inflorescence pieces

In order to investigate the regeneration of wild beet (Beta maritima) from inflorescence pieces, the effects of growth regulator, genotype, explant source and stage of plant development on adventitious shoot formation and rooting in vitro and subsequent transplanting in the glasshouse were tested. Inflorescence tips produced more adventitious shoots than sub-apical segments and the best micropropagation was achieved on a Murashige and Skoog (MS) medium supplemented with 1.0 mg l^–1 BAP. Addition of auxin was not beneficial. The induction rate of adventitious shoots was genotype-dependent and influenced by the stage of plant development. Adventitious shoots were produced from the base of the flower buds, i.e. from the receptacle, not from axils or stalks and only a few buds on inflorescence tip explants produced adventitious shoots. Rooting was increased by using a MS medium with 3% sucrose supplemented with 1.0 mg l^–1 NAA. There was no variation in leaf morphology of the transplants. This work shows that inflorescence tips can be used successfully as explants for in vitro multiplication of sugar beet and wild beet.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

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。     

Direct shoot regeneration from leaf explants of Jatropha curcas in response to thidiazuron and high copper contents in the medium

Leaf explants of Jatropha curcas cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ; 0.90 μM) in combination with indole-3-butyric acid (IBA; 0.98μM) produced adventitious shoot buds directly on the surface of the explants without formation of intervening callus while shoot bud formation was accompanied with callus formation on medium supplemented with 6-benzylaminopurine (BAP; 13.3 μM) and IBA (2.46 μM). TDZ treatment resulted in more than twice higher rate of shoot bud induction than BAP. Shoot buds were multiplied and elongated following repeated transfers to medium containing BAP (2.22 μM) and gibberellic acid (GA₃; 1.44 μM). The effect of copper sulphate on differentiation of shoot buds from leaf segments was also investigated. Both shoot induction and multiplication media were supplemented with different levels of CuSO₄ (0–5 μM). Significant improvement in shoot bud induction was observed when the concentration of CuSO₄ was increased to 10 times the normal MS level. Healthy elongated shoots were rooted on half strength MS medium supplemented with IBA (2.46 μM). Rooted plantlets were transferred to field and survived. Histological analysis revealed direct formation of shoot buds from leaf explants.