Movement to bark and metabolism of xylem cytokinins in stems of Lupinus angustifolius

Following uptake of [(3)H]zeatin riboside and [(3)H]dihydrozeatin riboside by girdled lupin (Lupinus angustifolius L.) stems via the transpiration stream, rapid lateral movement of the radioactivity from xylem to bark was observed. Short-term studies with intact stems, and other studies with excised stem tissues, revealed that the ribosides and/or the corresponding nucleotides were the cytokinin forms which actually moved into the bark tissues. Relative to cytokinin metabolism in xylem plus pith, metabolism in bark was both more rapid and more complex. Riboside cleavage and formation of the O-acetylzeatin and O-acetyldihydrozeatin ribosides and nucleotides were almost completely confined to bark tissues. Exogenous (3)H-labelled O-acetylzeatin riboside was converted to zeatin riboside in bark tissue, but the presence of the acetyl group suppressed degradation to adenine metabolites. The sequestration and modification of xylem cytokinins by stem tissues probably contributes significantly to the cytokinin status of the shoot. New cytokinins identified by mass spectrometry in lupin were: O-acetyldihydrozeatin 9-riboside, a metabolite of exogenous dihydrozeatin riboside in stem bark; O-methylzeatin nucleotide and O-methyldihydrozeatin 9-riboside, metabolites of endogenous cytokinins in stem bark; O-methylzeatin nucleotide and O-methylzeatin 9-riboside, metabolites of exogenous zeatin riboside in excised pod walls.     

Regulators of cell division in plant tissues. XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence

Kinetic studies of formation of glucosides of 6-benzylaminopurine (BAP) in excised radish cotyledons indicated that the 3-, 7-, and 9-glucosides (N-glucosides) were each formed directly from BAP. The 7- and 9-glucosides of BAP and the 7-glucoside of zeatin exhibited great stability in the cotyledons, but the 3-glucoside was converted to free BAP and to the 7- and 9-glucosides of BAP. When³H-labeled zeatin was supplied to developed cotyledons, at high concentrations (100 M), 7-glucosylzeatin was the principal metabolite, but an appreciable proportion of the extracted³H was due to O-glucosylzeatin. In immature cotyledons, as used in the radish cotyledon cytokinin bioassay, this O-glucoside was shown to be converted into zeatin 7-glucoside probably via free zeatin.Metabolism of BAP and zeatin in radish cotyledons was studied in relation to cytokinin-induced cotyledon expansion. Cytokinin N-glucosides were not metabolites responsible for the observed cytokinin-induced expansion, and were not detoxification products, or deactivation products formation of which was coupled with cytokinin action. However, the free base, its riboside, and nucleotide were possible active forms of BAP associated with cotyledon expansion. The possible significance of cytokinin N-glucosides is discussed.Senescent and nonsenescent cotyledons differed in their metabolism of BAP, zeatin, and zeatin riboside. Senescence was associated principally with a reduction in ability to form 7-glucosylzeatin, enhanced metabolism to adenine derivatives, and an inability to form appreciable amounts of 3-glucosyl-BAP.A two-dimensional thin layer chromatography (TLC) system, based on adjoining layers of cellulose and silica gel, for separating zeatin metabolites is described. This does not completely separate zeatin and zeatin riboside from the corresponding dihydro-compounds. A reversed phase TLC method for achieving these separations is also reported.     

Regulators of cell division in plant tissues. XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence

Kinetic studies of formation of glucosides of 6-benzylaminopurine (BAP) in excised radish cotyledons indicated that the 3-, 7-, and 9-glucosides (N-glucosides) were each formed directly from BAP. The 7- and 9-glucosides of BAP and the 7-glucoside of zeatin exhibited great stability in the cotyledons, but the 3-glucoside was converted to free BAP and to the 7- and 9-glucosides of BAP. When³H-labeled zeatin was supplied to developed cotyledons, at high concentrations (100 μM), 7-glucosylzeatin was the principal metabolite, but an appreciable proportion of the extracted³H was due to O-glucosylzeatin. In immature cotyledons, as used in the radish cotyledon cytokinin bioassay, this O-glucoside was shown to be converted into zeatin 7-glucoside probably via free zeatin. Metabolism of BAP and zeatin in radish cotyledons was studied in relation to cytokinin-induced cotyledon expansion. Cytokinin N-glucosides were not metabolites responsible for the observed cytokinin-induced expansion, and were not detoxification products, or deactivation products formation of which was coupled with cytokinin action. However, the free base, its riboside, and nucleotide were possible active forms of BAP associated with cotyledon expansion. The possible significance of cytokinin N-glucosides is discussed. Senescent and nonsenescent cotyledons differed in their metabolism of BAP, zeatin, and zeatin riboside. Senescence was associated principally with a reduction in ability to form 7-glucosylzeatin, enhanced metabolism to adenine derivatives, and an inability to form appreciable amounts of 3-glucosyl-BAP. A two-dimensional thin layer chromatography (TLC) system, based on adjoining layers of cellulose and silica gel, for separating zeatin metabolites is described. This does not completely separate zeatin and zeatin riboside from the corresponding dihydro-compounds. A reversed phase TLC method for achieving these separations is also reported.     

Mass-spectrometric quantification of cytokinin nucleotides and glycosides in tobacco crown-gall tissue

the cytokinins of tobacco crown-gall tissue have been analysed by quantitative mass spectrometry using ²H₂-labelled cytokinin riboside 5-monophosphates and ¹⁵N₄-labelled cytokinin glycosides as internal standards. The principal endogenous cytokinin of this tissue is zeatin riboside 5-monophosphate. The biologically inactive 7-glucoside of zeatin is the most abundant basic cytokinin in the tissue. These findings expose the limitations of previously reported analyses of similar tissues, which were restricted to biologically active basic cytokinins. The present study demonstrates that the endogenous cytokinins of tobacco crowngall tissue show a clear correspondence to the range of metabolites formed when exogenous cytokinins are supplied to nontumorous tobacco cells.Abbreviations DHZ dihydrozeatin - DHZ7G dihydrozeatin 7-glucoside - DHZMP dihydrozeatin 9-riboside 5-monophosphate - DHZR dihydrozeatin 9-riboside - GC-MS coupled gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Z7G zeatin 7-glucoside - Z9G zeatin 9-glucoside - ZOG zeatin O-glucoside - ZMP zeatin 9-riboside 5-monophosphate - ZR zeatin 9-riboside - ZROG zeatin 9-riboside O-glucoside     

外植体与不同理化因子对虎杖愈伤组织诱导及次生代谢产物形成的影响

考察了外植体、培养基及光照条件对中药植物虎杖愈伤组织形成及次生代谢产物生产的影响。总的来看,所有愈伤组织中总酚和总黄酮含量比原植物的含量高2—3倍;而蒽醌的含量比原植物中含量低。外植体对愈伤组织形成及次生代谢产物生产的影响很大,所考察的3种外植体中。叶外植的愈伤组织诱导率最高而源于根外植体的愈伤组织具有最好的次生代谢能力。所考察的6种培养基中,MS＋0．5mg／L 2,4-D＋1．0mg／L 6-BA和N6＋0．5mg／L 2,4-D＋1．0mg／L 6-BA无论对于愈伤组织的产生还是次生代谢产物的累积都有较优表现。光照对愈伤组织诱导及次生代谢产物产生有明显影响。但二者无规律性联系。     

Effects of Explants,Medium Formulations and Light on Callus Induction and Secondary Metabolites Accumulated in the Calli of Polygonum cuspidatum (Polygonaceae)

Callus initiation of a Chinese traditional medical plant , Polygonum cuspidatum Sieb. et Zucc (Polygonaceae) was studied, with a focus on the factors that could influence the callus induction ability and secondary metabolites accumulation. In comparison with the parent explants, the levels of total phenolics and flavones were 2 to 3-fold higher and the levels of anthraquinones (AQ_s) were lower in all calli induced. Different explants exhibited great variability in callus induction rate and secondarymetabolite accumulation. Among the three explants tested, leaf explants were with the best capacity in callus induction, while root explants accumulated the highest levels in secondary metabolites. Two media of six tested, MS and N6 respectively plus 0.5 mg/L 2, 4-dichlorophenoxy acetic acid and 1.0 mg/L 6-benzylaminopurine, showed the best performances in both callus growth and secondary metabolite accumulation. Illumination shower a remarkable effects on both callus induction and secondary metabolite accumulation, but no regular pattern was observed.     

外殖体与不同理化因子对虎杖愈伤组织诱导及次生代谢产物形成的影响

考察了外植体、培养基及光照条件对中药植物虎杖愈伤组织形成及次生代谢产物生产的影响。总的来看, 所有愈伤组织中总酚和总黄酮含量比原植物的含量高2～3 倍; 而蒽醌的含量比原植物中含量低。外植体对愈伤组织形成及次生代谢产物生产的影响很大, 所考察的3 种外植体中, 叶外植的愈伤组织诱导率最高而源于根外植体的愈伤组织具有最好的次生代谢能力。所考察的6 种培养基中, MS + 0.5 mg/L 2 , 4 -D + 1.0mg/L 6-BA 和N6 + 0.5 mg/L 2 ,4 -D + 1.0 mg/L 6-BA 无论对于愈伤组织的产生还是次生代谢产物的累积都有较优表现。光照对愈伤组织诱导及次生代谢产物产生有明显影响, 但二者无规律性联系。     

In Vitro Shoot Regeneration from Callus, Leaf Axils and Petioles of Sugar Beet (Beta vulgaris L.)

Procedures are described for producing axillary shoots fromseedling apices and adventitious shoots from petioles and leaf-derivedcallus of sugar beet cultivars. The rate of adventitious shootregeneration from petioles was influenced by temperature, BAPconcentration of the medium, and the time in culture of theseedling apices from which the petioles were excised. Petiolesectioning confirmed that adventitious shoots originated inthe sub-epidermal parenchyma. Two distinct types of callus wereproduced from leaf explants, but only white friable callus wascapable of shoot development. This callus developed from browntissue and was composed of thin-walled cells with dense cytoplasmand prominent nuclei. Green compact callus with thick-walledlignified cells developed from green tissue, but did not produceshoots. Successful seed sterilization and shoot regenerationfrom petiole explants and callus was cultivar-dependent. Adventitiousshoots were rooted and successfully transplanted to pottingcompost under glasshouse conditions. Key words: Adventitious shoots, axillary shoots, callus, sugar beet (Beta vulgaris L.)     

In vitro propagation of guayule (Parthenium argentatum) — a rubber yielding shrub

Nodal explants (0.5 to 0.8 cm long) isolated from 2-year old shrubs of guayule, Parthenium argentatum Gray, when cultured on MS medium supplemented with different concentrations of KN, BAP, 2,4-D, 2,4-D + BAP, NAA and NAA + BAP produced callus tissues and shoots simultaneously with varying frequencies. Shoots were regenerated with a high frequency (80–88%) from callus on MS medium containing NAA + BAP with or without glutamine. Addition of glutamine to these media improved considerably the number of shoots formed from a known amount of callus. Shoots could be regenerated from 200 day old callus cultures with a very high frequency but the organogenetic capacity declined thereafter. Increase in the concentration of sucrose (upto 4%) significantly enhanced the shoot forming ability of callus, but higher concentrations (6%) suppressed it. Rooting was induced only in dark when IAA, IBA and NAA were used, but 2,4-D could induce them both in light and dark. The system is suitable for the mass propagation of this important rubber yielding plant.Abbreviations MS Murashige and Skoog (1962) - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - KN Kinetin - BAP 6-Benzylaminopurine     

Adventitious shoot regeneration from in vitro cultured leaves of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd<Emphasis Type="Italic">.</Emphasis>)

Adventitious shoots were successfully regenerated from leaf explants of in vitro cultures of Platanus acerifolia Willd. The leaves of three clones (genotypes), designated as PH1, PH2 and PC, respectively, were wounded by three to four transverse cuts through the midvein and cultured on 26 media based on Murashige and Skoog (MS) basal medium, containing different concentrations of 6-benzylaminopurine (BAP) in combination with different concentrations of indole-3-butyric acid (IBA). The highest regeneration rate (>90%) and the largest number of shoot clumps per regenerating leaf (>4 shoot clumps/explant) were obtained with leaves of genotype PH2 cultured on MS basal medium supplemented with 17.76 microM BAP and 4.92 microM IBA. The other two genotypes, PH1 and PC, showed very low capability of shoot regeneration (<10%) on all the media tested. Shoots on leaf explants originated mainly from callus that developed around the cut end of petioles and along the cuts across the midvein. The regenerated shoots were micropropagated, rooted and transplanted to the soil successfully.     

In vitro Clonal Propagation and Organogenesis in Spilanthes acmella (L) Murray: A Herbal Pesticidal Plant of North-East India

Multiple shoots were regenerated in MS medium using different concentrations of BAP and Kn and different combinations of BAP with IAA, NAA and IBA. Highest multiplication of shoots was obtained with BAP (0.75 mg l^?1) with 28.4 shoots per explant after 60 days of culture. Shoots rooted best on IBA (0.5 mg l^?1), numbering 48.8 per explant. Organogenesis was maximum in callus cultured on MS medium supplemented with BAP (2.0 mg l^?1) and IAA (1.0 mg l^?1).     

Production of haploids of neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss.) by anther culture

Androgenic haploids of the neem tree (Azadirachta indica A. Juss.) were produced by anther culture at the early- to late-uninucleate stage of pollen. Haploid formation occurred via callusing. The best medium for inducing callusing in the anther cultures was Murashige and Skoog's basal medium (MS) (9% sucrose) supplemented with 1 microM 2,4-D, 1 microM NAA and 5 microM BAP, while anther callus multiplied best on MS medium supplemented with 1 microM 2,4-D and 10 microM Kn. These calli differentiated shoots when transferred to a medium containing BAP; 5 microM BAP was optimum for young calli (75% cultures differentiated shoots), but older calli showed the best regeneration with 7.5 microM BAP. Shoots elongated at a lower concentration of BAP-0.5 microM. These shoots were multiplied by forced axillary branching and rooted in vitro. The plants were subsequently established in soil. Of the plants that regenerated from anther callus 60% were haploid, 20% were diploid and 20% were aneuploid.     

An efficient protocol for the production of triploid plants from endosperm callus of neem,Azadirachta indica A. Juss

Triploid plants of neem were obtained by immature endosperm culture. Immature seeds, at the early dicotyledonous stage of embryo development, is the best explant to raise endosperm callus on MS + NAA (5 mumol/L) + BAP (2 mumol/L) + CH (500 mg L-1). Maximum shoot bud differentiation from the endosperm callus occurred on MS + 5 mumol/L BAP. Shoots were multiplied by forced axillary branching and rooted in vitro. The plants were established in soil. Over 66% of the plants were triploid with chromosome number 2n = 3x = 36. A characteristic feature of the shoots of endosperm origin is the presence of a large number of multi-cellular glands.     

In vitro culture ofBellevalia romana (L.) Rchb.

Summary Bellevalia romana (L.) Rchb., a monocotyledonous plant characterized by few (2 n=2 x=8) and very large chromosomes, is a useful subject for studying developmental problemsin vitro. Cytological analysis of callus revealed that the majority of cells were diploid, but the remaining cells had aneuploid nuclei with a wide range of chromosome numbers, tetraploid and haploid nuclei. The frequency of aneuploid and polyploid cells was higher in callus grown in the presence of 2,4-D than in callus grown in NAA plus BAP. These nuclei seemed to increase with the duration of culture. The chromosome number distribution as determined by chromosome counts in calli at different culture times was confirmed by DNA cytophotometry. Chromosome number mosaicism (mixoploidy and aneusomaty) also occurred in all root apices of 9 out of 46 plantlets regenerated from callusvia adventitious shoots.     

Phenylacetic acid induced organogenesis in cultured leaf segments of Dianthus chinensis

Summary Shoot regeneration was achieved from leaf derived callus of Dianthus chinensis using Phenylacetic acid (PAA). Callus from basal leaf segments, raised on Murashige and Skoog's (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 1-Naphthaleneacetic acid (NAA) in combination with 6-benzylamino purine (BAP), was subcultured on medium supplemented with BAP in combination with 2,4-D, NAA or PAA. Shoots were induced only when leaf derived callus was subcultured on medium containing BAP (2.0, 5.0 mg/l) in combination with PAA (0.5, 1.0 mg/l). No shoot regeneration was observed when 2,4-D, NAA or BAP were used in the medium either singly or in different combinations. These results demonstrate that PAA in combination with BAP was essential to trigger shoot regeneration from cultured leaf callus of D. chinensis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DPX dibutylphthalate xylol - MS Murashige and Skoog (1962) basal medium - NAA 1-Naphthaleneacetic acid - PAA Phenylacetic acid     

In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

甘薯叶柄原生质体有效植株再生

将甘薯(Ipomoea batatas (L．)Lam．)‘元气’和‘白星’(‘White Star’)的叶柄原生质体培养在含有0.05 mg·L-1 2,4-D和0.5 mg·L-1 KT的改良MS液体培养基中,3～4 d后细胞开始分裂。培养8～9周后,将直径达1～2 mm的愈伤组织转移到添加0.05～0.2 mg· L-1 2,4-D和0～0.5 mg·L-1 KT或添加0.5～2.0 mg ·L-1 NAA和1.0～3.0 mg·L-1 BAP 的MS固体增殖培养基上使其增殖。转移3～5周后,将愈伤组织再转移到MS基本培养基或转移到添加2.0～3.0 mg·L-1 BAP的MS培养基上。当进一步转移到MS基本培养基上后,从愈伤组织或从愈伤组织形成的不定根上再生出植株。‘元气’植株再生率高达60.0 ％,White Star高达43.4％。     

Uptake and translocation of [14C]-labelled benzylaminopurine in apple shoots grown in vitro in relation to shoot development

Shoots of apple (rootstock A2, Malus sylvestris (L.) Mill var. domestica Borkh.) were grown in vitro on agar media containing 4.4 μ M benzylaminopurine (BAP) during the whole of a 36-day culture period or during the first 6 days of the period. Development of lateral shoots and an appreciable increase in fresh weight were obtained only when BAP was present during the whole period. Treatment for 6 days only was not sufficient for shoot proliferation although the transferred shoot continued its growth on fresh medium without BAP. Uptake of the cytokinin was studied by adding [8–¹⁴C]-benzylaminopurine to the medium and then following the distribution of the isotope in the shoots during the 36-day culture period. There was a steady, almost linear, uptake of ¹⁴C during the whole period. Most of the activity accumulated in the lower part of the shoot. It is concluded that BAP, due to slow translocation, is active mainly in the basal parts of the shoot where it stimulates formation and growth of buds. Its action is dependent on continuous supply of new BAP from the medium, indicating metabolism of BAP in the tissue.     

红豆草耐盐愈伤组织的筛选及植株再生

将红豆草种子在含１．２％ＮａＣｌ的ＭＳ培养基上萌发以消除盐敏感的幼苗,把存活的幼苗下胚轴切段在含１ｍｇ／Ｌ２,４－Ｄ、０．５ｍｇ／Ｌ６－ＢＡ及１．２％ＮａＣｌ的ＭＳ培养基上诱导愈伤组织,通过连续筛选得到可耐受１．８％ＮａＣｌ的愈伤组织,在有０．２ｍｇ／Ｌ ＮＡＡ和１ｍｇ／Ｌ ＩＡＡ存在下该愈伤组织分化出芽,待幼,待幼苗长至３ｃｍ左右时转至含２ｍｇ／ＬＮＡＡ和或ＩＢＡ的１／２ＭＳ培养基上生根。对对照     

Influence of benzyladenine on condensed tannin formation and callus growth in cultures from sainfoin (Onobrychis viccifolia Scop.) cotyledons

Cotyledons from aseptically grown seedlings of sainfoin (Onobrychis viciifolia Scop.) were used as explant material to grow callus tissue for periods of 21 and 31 days. The formation of cells containing condensed tannins was induced by adding a range of BAP concentrations to an mB5 culture medium containing 2,4-D. After 21 days the fresh weight of calli treated with BAP was much greater than the control and appeared highest at the 0.6 mg/L level. Fresh weight was reduced at high BAP levels (2 to 8 mg/L) but still remained well above the control. The formation of tannin-filled cells was genotype-specific but occurred in all treatments with BAP. After 21 days in culture, fresh weight and tannin formation increased with the BAP level at the lower BAP concentrations. After 31 days, the growth rate slowed in the control and the lower BAP concentration, but continued at a high rate in the remaining treatments; the number of tannin-filled cells appeared to decline. The results show that in sainfoin callus culture, BAP induces the formation of condensed tannin, a secondary metabolite, and concomitantly produces a high growth rate.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - PAR photosynthetically active radiation Contribution No. 974 of Agriculture Canada, Research Station, 107 Science Cres., Saskatoon, Saskatchewan, S7N 0X2 Canada