Involvement of Cyclic AMP in Adventitious Bud Initiation of Torenia Stem Segments

Adventitous bud initiation in epidermis of Torenia stem segmentscultured in vitro, which is usually induced by cytokinin, couldbe induced by application of dibutyryl cyclic AMP in the absenceof cytokinin. Similar stimulatory effects on bud initiationwere observed when various promoting reagents to accumulateendogenous cyclic AMP were added, such as activators for adenylatecyclase, forskolin and prostaglandin E₁, and inhibitors of cyclicnucleotide phospho-diesterase, theophylline and isobutyl methylxanthine. Endogenous content of cyclic AMP in Torenia stem segmentswere increased by the application of the above chemicals, calciumionophore or cytokinin. These results suggested that endogenousconcentrations of cyclic AMP were involved in adventitious budinitiation of Torenia stem segments. Furthermore, some inhibitorsof protein kinases inhibited bud initiation induced by cyclicAMP-accumulating reagents. (Received August 15, 1989; Accepted November 4, 1989)     

Inhibition of Cytokinin-Induced Adventitious Bud Initiation in Torenia Stem Segments by Inhibitors of Serine Proteases

Application of di-isopropyl fluorophosphate (DFP), a highlysensitive inhibitor for serine enzymes, strongly inhibited cytokinin-inducedadventitious bud initiation in Torenia stem segments culturedin vitro. The inhibitory effect was not evident when DFP wasapplied after 3 days of culture. Amount of DFP-binding proteinsremarkably increased in superficial tissues of explants culturedfor 3 and 4 days on a medium containing benzyladenine. At least14 kinds of DFP-binding polypeptides were detected by SDS-polyacrylamidegel electrophoresis and fluorography. DFP-binding to some ofthese polypeptides was inhibited by a prior treatment with phenylmethylsulfonylfluoride and N-p-tosyl-L-lysine chloromethyl ketone. From theseresults, it was suggested that some serine proteases might berelated with biochemical events occurring during the initialstage of adventitious bud differentiation in Torenia stem segments. (Received May 8, 1984; Accepted July 5, 1984)     

Involvement of the Accumulation of Glutamine in the Initiation of Adventitious Buds in Stem Segments of Torenia

Active meristematic divisions in stem segments of Torenia culturedin vitro can be induced in the epidermis by application of cytokininor the calcium ionophore A23187 [GenBank] , resulting in the differentiationof adventitious buds. Endogenous free glutamine accumulatedat a high concentration in the epidermal tissues during theearly stages of such cultures. The accumulation of glutaminewas caused by an increase in glutamine synthetase (GS) activity,and the increase of GS activity was suppressed by the applicationof some inhibitors of GS activity, mRNA synthesis, protein synthesis,or calmodulin. Incorporation of these inhibitors into the culturemedium also inhibited initiation of adventitious buds. The inhibitoryeffect of an inhibitor of GS, methionine sulfoximine (MSX),was apparent only at the very begining of the culture, and theeffect could be overcome by the simultaneous addition of glutamine.The inhibitory action of MSX on initiation of buds seemed tobe caused by an accumulation of ammonium ions. Reduction inlevels of NH₄NO₃ in or its elimination from the culture mediumstimulated the initiation of adventitious buds. Therefore, boththe accumulation of glutamine and the reduction in levels ofammonium ions seem to play a role in the initiation of adventitiousbuds in stem., segments of Torenia. ¹Present address: Faculty of Agriculture, University of Saga,Honjo-cho, Saga, Saga, 840 Japan. (Received October 3, 1988; Accepted March 9, 1989)     

Effects of Wounding on Adventitious Bud Formation in Torenia Stem Segments Cultured In Vitro

In in vitro cultured stem segments of Torenia fournieri Lind.,the formation of adventitious buds can be induced when the culturemedium contains cytokinin. When long stem segments (2.0 cm ormore) were cultured with cytokinin, a large number of buds wereformed in the marginal regions, namely, within the limits of0.5 cm from the cut ends of explants, while only a few budswere initiated in the middle part of the explants. If a slightinjury was made transversely with a scalpel in the central partof an explant, a significant increase in the number of budswas noted within the limits of 0.5 cm from the wound site. Whena wounding treatment was given lengthwise to an explant, a largenumber of adventitious buds were formed over the entire surfaceof the explant compared to the control. Excision itself of explantsfrom mother plants and the additional wounding given to theexplants seemed to trigger the induction of adventitious buddifferentiation in Torenia stem segments. These wounding treatmentsdid not affect the uptake into explants and/or the distributionpattern of radioactive benzyladenine applied to the culturemedium. Key words: Torenia fournieri, Adventitious bud formation, Cytokinin, Wounding     

Significance of Spermidine in the Initiation of Adventitious Buds in Stem Segments of Torenia

When stem segments of Torenia fournieri Lind. were culturedin vitro, the initiation of adventitious buds, which is usuallyinduced by cytokinin, was induced by application of polyamines,such as putrescine and spermidine. Addition of arginine hada slight inductive effect. When cyclohexylamine, an inhibitorof spermidine synthase, was added simultaneously with putrescine,induction of the initiation of buds by putrescine was stronglyinhibited. However, application of the inhibitor together withspermidine had no effect on the spermidine-induced initiationof buds. The induction of initiation of buds by a cytokinin,by a calcium ionophore, by cyclic AMP, and by a phorbol ester,which was accompanied in each case by elevation of the levelsof endogenous spermidine, was also inhibited by cyclohexylamine.These results suggest the involvement of spermidine in the initiationof adventitious buds in stem segments of Torenia. ²Present address: Radiation Effects Research Foundation, Hijiyamakouen5-2, Minami-ku, Hiroshima, 732 Japan.     

Effects of Cytokinin and Anticytokinin on the Initial Stage of Adventitious Bud Differentiation in the Epidermis of Torenia Stem Segments

In the epidermis of Torenia stem segments cultured in vitro,meristematic zones (MZ) were initiated prior to adventitiousbud differentiation. Application of benzyladenine (BA) stimulatedMZ and bud formation, and the average number of MZ per epidermalstrip increased linearly with rising concentrations of BA. Thepresence of naphthaleneacetic acid together with BA in the mediumsuppressed MZ formation. Some derivatives of 4-substituted-2-methylpyrrolo[2,3-d]pyrimidinewhich have anticytokinin activity inhibited BA-promoted MZ formation.Interactions of various cytokinins and anticytokinins in MZand adventitious bud formation were also examined. The numberof MZ formed by treatment with 5 µM BA was reduced 50%by the simultaneous application of one of the anticytokininsat the same concentration. (Received June 19, 1982; Accepted September 27, 1982)     

Chemical Factors Controlling Floral Bud Formation of Torenia Stem Segments Cultured in Vitro II. Effects of Growth Regulators

Correlative effects between growth regulators added to a mediumand different physiological states of explants on adventitiousbud formation and flowering were investigated using Toreniastem segments cultured in vitro. Indoleacetic acid stimulatedfloral bud formation and its development in explants taken fromreproductive plants. These stimulative effects were clearlyseen in explants taken from plants in which flower abscissionwas taking place, but insignificant when explants were preparedfrom younger materials. Abscisic acid acted in a reverse wayto auxin, greatly promoting floral bud initiation and floweringof originally vegetative explnts. Zeatin at a concentrationof 1 mg/liter inhibited floral bud formation, and at a low concentrationsit was generally ineffective. However, floral bud formationand flowering of explants taken either from basal parts of stemsor from 18- to 20-week-old plants were promoted by zeatin treatment.The action of gibberellic acid seemed rather indirect: at aconcentration of 0.01 mg/liter, it generally stimulated floralbud formation but at a concentration of 1 mg/liter, it was ofteninhibitory. (Received January 17, 1981; Accepted February 21, 1981)     

Chemical Factors Controlling Floral Bud Formation of Torenia Stem Segments Cultured in Vitro I. Effects of Mineral Nutrients and Sugars

Internodal segments of Torenia fournieri Lind. were culturedon various media to investigate chemical factors influencingin vitro flowering. The elimination or dilution of ammoniumnitrate from Murashige and Skoog's medium increased the formationof adventitious buds which subsequently differentiated floralbuds. The dilution of mineral salts in Murashige and Skoog'smedium enhanced adventitious bud formation, but did not influencethe ratio of cultures with floral buds to those with adventitiousbuds. Among various media tested, in vitro floral bud formationand development of Torenia was best on a medium having 1/5 ofthe mineral salts and no NH₄NO₃. Eighty-seven percent of thecultures produced floral buds on this medium. Using this medium,the effects of various sugars were also examined. Increasingthe concentration of sucrose in the medium (up to 60 g/liter)increased the rate of cultures with floral buds, and stimulatedthe development of floral buds led to anthesis. (Received January 17, 1981; Accepted February 21, 1981)     

Distribution Pattern of Cell Division Centers on the Epidermis of Stem Segments of Torenia fournieri during de Novo Bud Formation

The stem epidermis in Torenia fournieri, which has budding potentialialities, is composed of one cell layer which can be easily separated from the rest of the stem segment at different stages of bud formation. As the buds are formed directly from the epidermis, without intermediate callus formation, it is possible to observe simultaneously the cell division centers over the entire excised epidermal surface. The quantitative analysis at the 6-day stage of bud formation showed that the cell division centers do not have a random distribution on the epidermal surface. With respect to the length of the stem segment, the frequency of cell division centers increases toward the base which is also the direction of auxin transport. With respect to the width, the maximum number of division centers is observed on either side of the median zone. The median zone and the lateral zones have few division centers. An anatomical study showed that the zones with few division centers are the closest to underlying vascular tissue. A more uniform distribution of division centers can be obtained by addition of auxin to the medium.     

Regulation by Abscisic Acid of In Vitro Flower Formation in Torenia Stem Segments

In Torenia stem segments cultured on a defined medium withoutphytohormones, in vitro flower formation was influenced by thephysiological states of the explants. Endogenous contents ofABA, but not those of IAA, were closely correlated with thephysiological states of the explants. Application of ABA (100ng/ml) to the culture medium stimulated flower formation inthe originally vegetative explants which otherwise had littleflower-forming capacity. Thus, endogenous ABA seems to be oneof the factors controlling the flower-forming capacity of Toreniastem segments. The highest rate of flower formation in the stemsegments was obtained when endogenous contents of ABA (whichresulted from both endogenously present and externally appliedABA) in the stem tissues was between 16 and 20 ng/g fresh weight. ¹ Present address: Bioscience Research Center, Mitsui PetrochemicalIndustries Ltd., Waki-cho, Kuga-gun, Yamaguchi 740, Japan. (Received November 22, 1984; Accepted March 1, 1985)     

Effects of IAA, Zeatin, Ammonium Nitrate and Sucrose on the Initiation and Development of Floral Buds in Torenia Stem Segments Cultured In Vitro

In Torenia stem segments cultured on a defined medium from whichammonium nitrate and growth regulators were omitted, adventitiousbuds were readily formed from epidermal tissue, with subsequentdifferentiation of floral buds. Using this plant material, thecorrelation between the time of application of various chemicalsand the time-course of floral bud differentiation was investigated.Histological examination showed that adventitious buds werevegetative during the first two weeks of the culture, and floralprimordia appeared after about three to four weeks of culture.We divided the flowering process in Torenia stem segments intothe following 3 phases: the first phase (first 2 weeks) duringwhich adventitious buds are formed, the second phase (3rd and4th weeks) during which floral buds are initiated and the thirdphase (5th to 12th weeks) during which floral buds develop.Then we added IAA, zeatin, ammonium nitrate or a high concentrationof sucrose to the medium during one, two or three of these phases.Ammonium nitrate added during the third phase suppressed floralbud development, but the high concentration of sucrose givenduring this phase stimulated it. These two chemicals influencedonly the development of floral buds previously initiated. Theapplication of IAA during the first phase promoted both theinitiation and development of floral buds. However, its applicationafter 2 weeks of culture failed to promote floral bud formation.Zeatin inhibited floral bud formation in a manner similar toammonium nitrate, but if it was added to the medium only duringthe first phase, it slightly promoted the initiation and developmentof floral buds. (Received July 7, 1981; Accepted October 12, 1981)     

Involvement of the Cotyledon in Adventitious Root Initiation in Impatiens balsamina L. Seedlings

Adventitious root primordia are found in the pre-hypocotyl tissueof developing seeds of Impatiens balsamina L. by the third weekafter petal drop, and are present in the mature seed. Aftergermination, the adventitious roots emerge from a collet swellingon the hypocotyl of the young seedlings. Removal of the colletduring the first five days results in the formation of anotherat the base of the remaining hypocotyl. Older seedlings respondto the excision of the collet by producing one or more rootsnear the cut end, unless the cut is made close to the cotyledon,when, even in nine-day seedlings, a reduced collet is formedassociated with four or fewer roots. The influence of the cotyledonon collet/root regeneration diminishes in older seedlings andin these is manifested only in hypocotyl tissue adjacent tothat organ. Impatiens balsamina, balsam, cotyledon, adventitious roots, collet     

Involvement of Polyamines with Adventitious Root Development in Stem Cuttings of Mung Bean

Spermine enhances the number of adventitious roots developingon stem cuttings of Phaseolus aureus Roxb. This effect is observedwhen spermine is supplied alone to cuttings or in the presenceof indolebutyric acid (IBA). That concentration most effectivein inducing the rooting response also enhances root growth.Other concentrations tested were without effect on growth. Spermidinedoes not influence root number or growth except at high concentration,when it is inhibitory to number only. Methylglyoxal bis(guanylhydrazone)(MGBG) inhibits rooting and root growth in the presence or absenceof IBA. Treatment of stem-cuttings with IBA leads to enhancedlevels of spermine, spermidine and putrescine in the hypocotylprior to development of any root primordia. MGBG reduces thelevels of spermine and spermidine whilst increasing the levelof putrescine. Furthermore, MGBG prevents the IBA-induced increasein spermine and markedly inhibits that in spermidine. Theseresults are consistent with an essential role for polyaminesand their metabolism in the early events which lead to adventitiousroot development. (Received January 10, 1983; Accepted March 17, 1983)     

Inter-Tissue Correlations in Organ Fragments: Organogenetic Capacity of Tissues Excised from Stem Segments of Torenia fournieri Lind Cultured Separately in Vitro

In order to study the effect of inter-tissue correlations on the organogenetic capacities of various tissues of stem segments of Torenia fournieri Lind, different types of explants were excised and grown separately: epidermis, subepidermal parenchyma, epidermis plus subepidermal parenchyma but devoid of vascular tissue and stem segments devoid of epidermis.     

Calcium Antagonists Inhibit Sustained Gibberellic Acid-Induced Growth of Avena (Oat) Stem Segments

The elongation response of Avena sativa (oat) stem segments to gibberellic acid (GA3) is of large magnitude, with high hormonal sensitivity and specificity, but without cell division activity. This system is therefore an excellent model for mechanistic studies on higher plant cell elongation and the action of gibberellin. At millimolar concentrations, the calcium antagonists verapamil, D-600, nicardipine, diltiazem, bepridil, 8-(N,N,-diethylamino)-octyl-3,4,5-trimethoxybenzoate HCl, and lanthanum substantially inhibited the growth of GA3-treated segments but had no effect on the elongation of nonhormone-treated segments. Although verapamil reduced the maximum growth rate and caused premature cessation of growth, even preincubation of the segments with the drug prior to treatment with GA3 failed to inhibit the earliest measured stimulation of growth by the hormone. Inhibition by verapamil was not reversed by increased concentrations of GA3 or calcium. Neither the calcium ionophore A23187 nor agonist BAY K 8644 had any effect on growth. Light microscopic examination of epidermal peels from antagonist-treated internodal tissue revealed no obvious differences from the control except that the cells were not as elongated. Although these results may support a role for calcium ion movement in maintaining the GA3-induced growth of Avena stem segments, they do not support the involvement of calcium ion movement in the hormone-mediated initiation of growth.     

喜树不定芽的诱导及植株再生

1植物名称喜树(Camptotheca acuminate),别名旱莲木. 2材料类别一年生喜树带芽茎段. 3培养条件芽诱导培养基:(1)MS NAA 0.05mg·L-1(单位下同) 6-BA 1.0.芽增殖培养基:(2)MS NAA 0.1 6-BA 0.5;(3)MS NAA 0.1 6-BA 1.0;(4)MS NAA 0.1 6-BA 2.0;(5)MS NAA0.5 6-BA 0.5;(6)MS NAA 0.5 6-BA 1.0;(7)MS NAA 0.5 6-BA 2.0.壮苗培养基:(8)MS NAA 0.05 6-BA 0.1～0.5.生根培养基:(9)1/2MS NAA 0.1 IBA 1.0;(10)1/2MS NAA 0.5 IBA1.0;(11)1/2MS NAA 1.0 IBA 1.0.以上所有培养基均附加6.5 g·L-1琼脂、30 g·L-1蔗糖,pH值为5.9.培养温度为(25±2)℃,光照度为1000～2000lx,光照时间14 h·d-1.     

钾离子对长柄扁桃不定芽诱导的影响

研究了钾离子对长柄扁桃不定芽诱导的影响,结果表明,在MS基本培养基中添加800～1200mg·L-1钾离子有利于长柄扁桃不定芽的形成和生长,不定芽的诱导率和数量分别比对照提高了17％和84％,不定芽的平均高度提高了64％;高浓度钾离子（〉1600mg·L。）可导致长柄扁桃不定芽严重褐化。生理指标测定结果表明,适当浓度的钾离子提高了抗氧化酶（SODPOD）的活性和不定芽的组织细胞活力;高剂量的钾离子（〉1600mg·L-1）显著增加了不定芽中MDA的含量。     

芒果子叶切段不定根形成的影响因素分析

分别用不同成熟时间、不同取材部位、不同品种、不同大小的芒果子叶切段为外植体进行不定根的诱导,以探讨影响芒果子叶切段不定根形成能力的原因.结果表明,芒果子叶切段的生根能力随着芒果成熟度的增加而逐渐提高,花后50和60 d的2.0 cm长子叶切段都无不定根形成,从花后70 d开始有不定根形成,此时生根率为28.6%,之后其生根能力迅速提高,在花后90 d生根率达到76.7%,之后生根率稳定保持直到110 d果实成熟.成熟芒果的子叶切段长度(2.0、1.0、0.5和0.2 cm)对不定根的形成有显著影响,0.2 cm的子叶切段上无不定根形成,另外3个长度的切段都有不定根形成,且生根能力随着长度的增大而逐渐提高.取材位置(靠近或远离胚轴)则对生根影响不大,且紫花芒、红芒、青皮芒等几种常见芒果品种的不定根形成能力基本相同.     

Rapid Initiation of Thymidine Incorporation into Deoxyribonucleic Acid in Vegetative Tobacco Stem Segments Treated with Indole-3-acetic Acid

The short term effect of 11.4 mum indoleacetic acid on the incorporation of (methyl-(3)H)thymidine into DNA in vegetative tobacco (Nicotiana tabacum cv. Wis. 38) stem segments has been investigated. In segments that are defoliated, inverted, and kept in the dark for 7 hours, indoleacetic acid very rapidly (about 60 minutes) and strikingly initiates thymidine incorporation into DNA. The time required before enough indoleacetic acid (2.8 mum) to enhance thymidine incorporation moves into a segment has been found to be about 35 minutes. The initiation response time for segment tissue that already contains 2.8 mum indoleacetic acid should be no more than about 25 minutes. The rate of labeled thymidine incorporation into DNA is affected by physiological treatments of segments. Moving segments from the light into the dark or defoliating segments or inverting defoliated segments decreases the rate of thymidine incorporation. For segments given all three treatments, indoleacetic acid restores the rate of thymidine incorporation as compared to controls. Darkness, or defoliation or inversion of segments, therefore, may decrease thymidine incorporation into DNA by effecting reduced auxin levels in stem segments.     

白桦愈伤组织的高效诱导和不定芽分化

选出白桦愈伤组织诱导的最适培养基为 :IS 6 BA 5 .0mg·L-1 KT 0 .5mg·L -1(茎段 ,叶柄 ) ;IS 6 BA 2 .0mg·L-1 NAA 0 .2mg·L-1(叶片 )。分化培养基为 :IS 6 BA 0 .4～ 0 .5mg·L-1,蔗糖浓度 2 0 g·L-1。愈伤组织诱导率和不定芽分化率分别达到 73 %和 90 %以上。