The in vitro metabolism of [8-14C]benzyladenine by excised organs of tomato plants

The metabolic fate of [8-¹⁴C]benzyladenine applied to the excised organs of tomato (Lycopersicon esculentum Mill. cv. Heinz 1370) was investigated after 2 and 6 h of feeding. Although the roots were the most effective at uptake of the cytokinin the leaves metabolised it the most efficiently. The predominant metabolite in all of the tissues was an unknown compound which did not have a retention time corresponding with any of the standards used. The roots contained the most extensive range of metabolites which included the unknown metabolite and compounds co-eluting with adenine, and the riboside, nucleotide and 9-glucoside of benzyladenine. The 9-glucoside was detected only in the root material. The stem yielded the highest levels of radioactivity at the retention times of benzyladenosine-5-monophosphate and benzyladenosine. The radioactivity associated with these two cytokinins was transient in the leaf extract. This organ ultimately yielded radioactivity only at the retention times of the unknown metabolite and adenine. Since only the roots and leaves contained relatively large peaks of radioactivity at the elution volume of adenine it seems that degradative metabolism was more predominant in these organs than in the stem.Abbreviations Ade adenine - Ado adenosine - BA benzyladenine - BAR benzyladenosine - BA3G 3-glucosylbenzyladenine - BA9G 9-glucosylbenzyladenine - BARMP benzyladenosine monophosphate - HPLC high performance liquid chromatography - MS mass spectrometry     

Regeneration of plants from achenes and petals of Chrysanthemum coccineum

Achenes and petals of Chrysanthemum coccineum were cultured on MS and White medium supplemented with BA and NAA or 2,4-D. Without being transferred, shoots were formed directly and immediately after callus formation on the surface of the achene walls and from the cut ends of the petals. High concentracions of BA and NAA supported the callus induction and shoot formation, but higher concentrations of 2,4-D inhibited shoot formation. The shoots obtained from both explants formed roots when transferred to hormone-free medium and they could be transplanted to soil for further growth. The regenerated plants contained as much pyrethrins as the original plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthalene acetic acid - BA 6-benzyladenine     

High efficiency shoot regeneration from callus of quaking aspen (Populus tremuloides Michx.)

Callus was induced from leaf segments of aspen (Populus tremuloides Michx.) on modified B5 (mB5) medium with 0.1 mg/1 benzyladenine (BA) and 0.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting callus was either subcultured to solidified Woody Plant Medium (WPM) with 0.5 mg/1 BA directly for shoot regeneration or sieved into liquid mB5 medium for suspension culture. After 3 weeks of suspension culture, when the callus clumps grew to 3–4 mm in diameter, they were transferred onto solidified WPM with 0.5 mg/1 BA for shoot regeneration. Almost 100% of the clumps formed shoots on WPM when subcultured directly from mB5 with an average number of 6 shoots per callus. When transferred from suspension culture in mB5 to WPM, an average of 6 shoots per callus were produced from 51% of calli. These shoots could be easily rooted on either mB5 or WPM with 0.2 mg/1 indole-3-butyric acid (IBA) and transferred to pots. Transplanted plants were kept under intermittent mist for 2–4 weeks before normal growth in the green house.Abbreviations BA 6-Benzyl-adenine - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - mB5 medium modified B5 medium - WPM Woody Plant medium     

In vitro shoot and root organogenesis, plant regeneration and production of tropane alkaloids in some species of Schizanthus

A rapid in vitro propagation system leading to formation of shoots from callus, roots, and plantlets was developed for Schizanthus hookeri Gill. (Solanaceae), an endemic Chilean plant. The genus Schizanthus is of particular interest due to the presence of several tropane alkaloids. So far, in vitro propagation of species of this genus has not been reported. Propagation of S. hookeri consisted of two phases, the first one for callus initiation and shoot formation and the second for rhizogenesis and plantlet regeneration. From a single callus that rapidly increased in cell biomass (from approximately 50 mg to approximately 460 mg/culture tube [25 x 130 mm] in 60 days) in the presence of 2.69 microM NAA and 2.22 microM BA, more than 10 shoots/callus explant were formed. From the latter, approx. twenty plantlets formed after 90-110 days shoot subculture in medium devoid of growth regulators that favored root formation. Ten alkaloids ranging from simple pyrrolidine derivatives to tropane esters derived from angelic, tiglic, senecioic or methylmesaconic acids were obtained from in vitro regenerated plantlets. One of them, 3alpha-methylmesaconyloxytropane, was not previously described. The same growth conditions, as well as other growth regulator levels tested, were required to induce callus and root formation in S. grahamii Gill. Root organogenesis occurred despite a high level of BA vs. NAA used, (i.e., 4.44 microM BA and 0.54 microM NAA); however no shoot formation was achieved. In the case of S. tricolor Grau et Gronbach, only callus formation was obtained in the presence of various growth regulators.     

Production of glycyrrhizin in callus cultures of licorice

Licorice plants, Glycyrrhiza glabra, G. uralensis, and G. inflata, were investigated for callus induction using Murashige and Skoog (MS) medium combined with auxins and cytokinins. After 4 weeks of culture, 33-100% of leaf or stem explants formed calli. Maximum of shoot induction from callus cultures was achieved by G. inflata stem explants cultured on MS medium supplemented with 1 mg/l alpha-naphthaleneacetic acid (NAA) and 0.5 mg/l 6-benzyladenine (BA) (67%) which also gave maximum shoot formation per explant (two shoots per explant). These results indicated that all three Glycyrrhiza species regenerated shoots from callus cultures on MS medium combined with NAA and BA or only thidiazuron (TDZ; 0.1 and 0.5 mg/l). Glycyrrhizin contents of G. uralensis calli induced using MS medium in combination with NAA and BA [(27.60 +/- 8.47) microg/g DW] or TDZ alone [(36.52 +/- 2.45) microg/ g DW] were higher than those found in other combinations.     

亚洲百合花丝离体培养器官形成的细胞形态学研究

对亚洲百合的花丝进行离体培养,并利用常规石蜡制片技术对诱导效果最好的材料进行细胞形态学观察,研究花丝在离体培养过程中器官形成的细胞形态学变化。结果表明:花丝在MS+BA0.5 mg/L+NAA0.5 mg/L的培养基上诱导效果最好。离体培养后其形态学下端切口内方的1～3层细胞首先启动脱分化,然后是内方的10～12层细胞,而其他部位的细胞自始至终未启动脱分化。亚洲百合的再生方式为器官发生型,器官通过胚性愈伤组织间接产生,在胚性愈伤组织团表面附近形成芽原基,或在胚性愈伤组织团内部形成根原基,有时同时分别在内、外形成根原基和芽原基后再通过维管组织连接成完整的植株。本研究为亚洲百合的人工调控提供基础理论依据。     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

In vitro induction of multiple shoots and plant regeneration from shoot tips of mung bean (Vigna radiata (L.) Wilczek)

Conditions for plant regeneration from excised shoot tips of Vigna radiata were studied. Complete plants were regenerated directly without an intervening callus phase from shoot tips on basal medium (MS salts+B₅vitamins). Regeneration frequency varied with genotype, explant size and growth regulator combinations in the medium. Addition of cytokinins induced a variable amount of callus at the base of the shoot tip, followed by multiple shoot formation. Benzyladenine (BA), kinetin and zeatin at 5×10^-6 M each induced multiple shoots in 100% of the explants but the highest number of regenerants per explant (9) was produced with BA. The efficacy of BA for shoot multiplication was not improved when it was supplemented with naphthaleneacetic acid (NAA) or indoleacetic acid (IAA). NAA or adenine sulphate, when applied alone, induced complete plantlets. The growth regulator requirement of explants for the induction of multiple shoots varied with explant size. The shoot tip explants maintained proliferation ability on subculture. None of the treatments was effective in inducing shoot bud differentiation from callus. Regenerated shoots were rooted on MS basal medium and MS supplemented with either IAA or indolebutyric acid. The rooted plants were transferred to the field; 60% subsequently survived and grew.Abbreviations BM basal medium [MS (Murashige & Skoog 1962) salts+B₅ (Gamborg et al. 1968) vitamins] - BA 6-benzyladenine - AdS adenine sulphate - IAA indole-3-acetic acid - NAA-1 naphthaleneacetic acid - IBA indolebutyric acid     

The metabolism of 6-(2,3,4-trihydroxy-3-methylbutylamino) purine by soybean callus

6-(2,3,4-trihydroxy-3-methylbutylamino) purine (trihydroxyzeatin) applied to soybean callus is metabolised slowly. After 48 h only one peak of biological activity which co-eluted with the applied cytokinin was detected. When the callus was incubated on a medium which contained 10^–5 M trihydroxyzeatin, spiked with 8 {¹⁴C} trihydroxyzeatin, for 28 days, three peaks of biological activity and three peaks of radioactivity were detected. One of the biologically active and radioactive peaks co-eluted with zeatin. Another of the radioactive peaks co-eluted with N-(purin-6-yl) glycine. From the data obtained it apears that trihydroxyzeatin can be both oxidized and reduced by soybean callus. The potential to be converted to zeatin may explain why trihydroxyzeatin and its parent compound, which is usually rapidly metabolised by living material, are equally active in the soybean callus bioassay. From the radioactive data obtained it appears that trihydroxyzeatin is susceptible to oxidation to form N-(purin-6-yl) glycine.     

The histogenesis of somaclones from tomato (Lycopersicon esculentum Mill.) cotyledons

Summary A histological study ofin vitro cultured cotyledonary expiants of tomato (Lycopersicon esculentum) was performed in order to determine the site (differentiated tissue or developing callus) and the mode of plant regeneration.Results have shown that callus develops at the excision sites of cotyledonary expiants and that shoots are formed exclusively within the unorganized callus: excision areas are the only morphogenetic sites and the proximal excision is the preferred site for plant regeneration.Shoots differentiate by organogenesis within the superficial region of the callus. Few neocambial cells cooperate in the neoformation. Origin from a single cell is highly unlikely since rarely observed single activated cells never developed into shoots.Regenerated plants may be chimeras if invitro culture induces genetic diversity in the initial cells.Abbreviations IAA Indole-3-acetic acid - c callus - d vegetative dome - s shoot - ad adaxial - ab abaxial - t tracheid - p parenchyma - S sieve tube     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

毛刺槐花药培养及再生植株的获得

以毛刺槐的花药为材料,开展其组织培养和植株再生系统的研究。结果显示：将毛刺槐的花药接种在MS附加2,4—D0．1mg／L和BA3．0mg／L的培养基上,20d时花药愈伤组织诱导率可达41．5％。花药愈伤组织在MS附加BA5．0mg／L的分化培养基上继代培养2个月后,可分化出许多绿色的芽点,待不定芽长至2—3cm高时将其切下,转入MS附加IBA1．0mg／L的生根培养基上,2周后即可得到完整的再生植株。同时,研究就4℃低温预处理和蔗糖浓度对毛刺槐花药培养的影响进行了研究和讨论。     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

Cytokinin-like activities of nucleocyclitols

Although the 9-substituted adenines are commonly inactive as cytokinins, the nucleocyclitol 3-(-adenin-9-yl)-3-deoxy-1,5,6-tri-0-(methylsulfonyl)-muco-inositol (NI) proved to be active in the following bioassays: cell proliferation in soybean cotyledon callus tissue, cell expansion in excised radish cotyledons, and delay of senescence in detached leaves. In these assays, the effect of the compound, applied at the same molar concentration as benzyl adenine, was lower or less uniform than BA. NI completely failed to promote germination of lettuce seeds in conditions of secondary dormancy or thermodormancy, whre BA is effective. NI can substitute for BA in some though not all of the numerous responses evoked by cytokinins.     

草木樨状黄芪高频离体再生体系的建立

以草木樨状黄芪无菌苗茎切段为材料,在含１－２mg/L2,4-D和０－０．５mg/L６ＢＡ的MS培养基上培养获得大量愈伤组织,愈伤组织诱导率在９５％以上,愈伤组织在附加０．２mg/LＫＴ,１mg/L６ＢＡ,０或０．５mg/LＮＡＡ,５００mg/LＣＨ 和２００mg/L ＹＥ的ＭＳ培养基上诱导丛生芽,并进而发育成苗。苗的分化频率为１００％。分化苗或其茎的切段在不国源植物激素的１／２ＭＳ培养基上可出现根的分化,分化频率达９０％以上,再生植株经炼苗后移栽成活率达８０％以上。     

Plant regeneration from seedling cotyledons, leaves and petioles of Glycine clandestina

A B5-based culture medium containing 4.4 μ M N⁶-benzyladenine (BA) and 0.025 μ M indole-3-butyric acid (IBA) induced callus from seedling cotyledons, leaves and petioles of Glycine clandestina Wendl. Only hard, green, nodular callus tissues were capable of producing shoot buds and of five accessions examined, only two (G1231 and G1145) were morphogenetically competent. Callus that did not regenerate could often be induced to produce shoot buds after subculture to fresh regeneration medium. Buds developed into shoots following transfer of callus to a medium containing 0.9 μ M BA and 0.025 μ M IBA. Shoots were rooted in hormone-free, half-strength B5 medium supplemented with 0.2% activated charcoal. The application of these results is discussed in relation to somatic hybridisation between the cultivated soybean and wild Glycine species.     

Plant regeneration from isolated cells ofLavandula latifolia medicus

Summary Single cells were obtained from hypocotyl-derived callus ofLavandula latifolia Medicus. Cells were plated in Murashige and Skoog medium supplemented with indoleacetic acid (IAA), benzyladenine (BA), and several IAA-BA combinations. Cell division required the simultaneous presence of IAA and BA in the culture medium, but callus formation was only achieved with 0.1 or 1 mg/liter IAA and 2 mg/liter BA. To induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on the composition of both the callus induction and the shoot regeneration media, best results being obtained when calli grown in 1 mg/liter IAA and 2 mg/liter BA were subcultured to media containing 2 mg/liter BA and 15% coconut milk. Under these conditions, up to 75% of calli formed shoots that subsequently were rooted and established in soil.     

Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1⁻¹ BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1⁻¹ zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1⁻¹ IBA, and rooted plantlets survived and flowered normally after transference to compost.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.