In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

Haploid Plantlet Formation from Female Gametophytes of Ephedra foliata Boiss. in vitro

Female gametophytes (at the archegonial stage) excised fromyoung ovules of Ephedra foliata Boiss, were cultured on a basalmedium (Murnshige and Skoog's combinations of major and minorsalts, Iron source, vitamins, myo-inositol along with 2 percent sucrose and 10 per cent coconut milk) under aseptic conditions.Growth and morphogenetic responses of the explants to auxinswere compared at different concentrations and a study of theirinteractions with cytokinins has also been made. At 2 mg 1^–1,2, 4-D induced profuse callusing which subsequently producedroots. NAA at 4 mg 1^–1 was optimal for callus growth androoting. Combinations of 2,4-D and kinetin were more effectivein inducing roots and shoot buds than those of 2,4-D and benzylamino-purine (BAP). Addition of BAP (0.05 mg ^1–1) to themedium containing optimal concentrations of NAA resulted information of a large number of roots. Kinetin induced only rootingin the presence of 4 mg 1^–1 NAA. A high concentrationof BAP (8 mg 1^–1), stimulated shoot bud formation. Forthe further development of shoot buds, neither auxin nor cytokininwas needed. Cytological observations revealed the presence ofhaploid number of chromosomes, i.e. seven. Ephedra foliata, tissue culture, callus, regeneration, 2,4-dichlorophenoxyacetic acid, naphthalene acetic acid, kinetin, benzyl amino-purine     

Plantlet Production from Morphogenetically Competent Cell Suspensions of Daylily

Cell suspensions of the diploid daylily cultivar Autumn Blazewere produced from larger masses of tissue by culture in thebasal medium of Murashige and Skoog supplemented with 10 percent v/v coconut water and 2 mg 1^–12,4–D. By drasticallylowering the level of 2,4–D, followed by transferral toa modified White's or Schenk and Hildebrandt medium, clustersgrow and ultimately give rise to embryonic structures. A finalperiod in a semi-solid medium stimulates shoot and root growthto the point where successful transplanting of plantlets tosoil is assured provided safeguards to prevent ‘dampingoff’ and desiccation are taken. Normal plantlet formationmay be arrested in the formation of neomorphs which do not seemper se to be capable of further development but they can giverise to morphologically normal plantlets after they are stimulatedto form callus which, in turn, is given an appropriate sequenceof stimuli. Hemerocallis, daylily, totipotent cells, micropropagation, tissue culture.     

Some Physiological Responses of Chlorella pyrenoidosa to 2,4-Dichlorophenoxyacetic Acid

An 18-h treatment of synchronously-grown Chlorella pyrenoidosawith 2,4-D did not significantly alter the size, dry weight,degree of synchrony, or pigment content of the cells, nor weredetectable quantities of ethylene produced. When Chlorella pyrenoidosawas treated with 5?10^–4 M 2,4-D, there was a statisticallysignificant stimulation of both net oxygen uptake and productionwhile 5?10 M 2,4-D inhibited both processes. When Chlorellapyrenoidosa was treated with 5?10^–4 M and 5?10^–3M 2,4-D, significantly greater amounts of glycollate were presentin the culture medium, even though an assay for glycollate dehydrogenaseshowed that the activity of this enzyme from 2,4-D-treated Chlorellapyrenoidosa was three times greater than in control cells. Looselybound 2,4-D was partitioned from a nonaqueously isolated chloroplastfraction, while other cell fractions failed to show detectablequantities of 2,4-D. It is postulated that in Chlorella pyrenoidosathe chloroplast is a target for 2,4-D action and that interferencein photorespiratory processes may underlie the observed responses.     

Factors Affecting Growth and Aggregate Dissociation in Batch Suspension Cultures of Datura innoxia (Miller)

Growth kinetics of Datura innoxia batch suspension cultureswen monitored by a Klett-turbidimetric technique. While cultured. wt varied linearly with Klett units, f. wt and packed cellvolume did not. Turbidimetrically determined doubling timeswere highly reproducible. The method proved to be useful inthe determination of acutely lethal conantrations of a seriesof anti-metabolites. In certain circumstances, aggregate dissociation in batch suspensioncultures of D. innoxia was found to be coupled to growth rate.Suspensions maintained with 10^–5 M 2,4-D exhibited a relativelyslow growth rate with a high degree of aggregate dissociation:10^–4 M 2,4-D promoted a maximum growth rate, but dramaticallysuppressed aggregate dissociation. At 10^–5 M 2,4-D, themitotic index of smaller-aggregate fractions was greater thanthe mitotic index of the large-aggregate fraction. At 10^–5M 2,4-D the converse was observed. Supraoptimal 2,4-D concentrationsthus enhanced both aggregate dissociation and the growth ofsmaller aggregates. When present in concentrations promoting optimal growth. malicand succinic acids caused a decrease in aggregate dissociation.Casein hydrolysate dramatically enhanced growth, but did notaffect aggregate dissociation to the same degree as 2,4-D orthe Krebs cycle organic acids. Suggestions are made concerningmedium composition to be used in future mutant selection schemesusing D. innoxia. Datura innoxia (Miller), suspension culture, growth kinetics, mitotic index, 2,4-dichorophenoxy acetic acid     

Ethylene and Ethane Production in 2,4-D Treated and Salt Treated Tobacco Tissue Cultures

Gas chromatography was used to measure ethylene (ethene) andethane production by tobacco (Nicotiana tabacum cv. Wisconsinno. 38) callus tissues grown on media containing inorganic saltsaccording to Murashige and Skoog (1962), sucrose, myo-inositol,thiamine-HCl kinetic according to Linsmaier and Skoog (1965),and either 2,4-dichiorophenoxyacetic acid (2,4-D) in the range0–100 mgl^–1 or 2 mgl^–1 indoi-3-ylacetic acidplus NaCl in the range 0–200 Meq l^–1. Ethylene productionrates were high (> 500 nl h^–1 g^1– fresh weight)initially in all treatments. Subsequently, ethylene productiondeclined in rapidly growing cultures but remained high in moderatelyand severely 2,4-D (> 0·5 mgl^–1) stressed andin severely NaCl (150 Meql^–1) stressed cultures. Highinitial rates of ethane production (> 200 nl h^–1 g^–1fresh weight) were obtained under conditions of severe stresscaused by 2,4-D or NaCl but not in control or moderately inhibitedcultures. With further incubation ethane production declinedin the severely stressed cultures. It is concluded that ethyleneproduction can be used as an index of moderate 2,4-D stressand severe NaCl stress by virtue of the high persisting ratesof ethylene production in stressed cultures. Ethane productioncan be used as an early index of severe stress caused by either2,4-D or NaCl in vitro. Nicotiana tabacum L., tobacco, ethylene, ethenen, ethane, 2,4-dichlorophenoxyacetic acid, auxin, stress, callus tissue     

Callus Growth and Plantlet Regeneration in Taro, Colocasia esculenta var. esculenta (L) Schott (Araceae)

Explants of taro cultivars belonging to Colocasia esculentavar. esculenta have been nearly impossible to culture untilrecently. Here, we describe a method which induces callus formationfrom bud explants of Colocasia esculenta var. esculenta cv.Akalomamale, brings about shoot and root production, and leadsto plantlet regeneration. The medium used is half-strength Murashige-Skoog(HMS) solution containing 25 ml taro tuber extract (TE), 2 mg2, 4, 5-trichlorophenoxyacetic acid and 200 mg glutamine 1^–1.TE is an important requirement for bud explants and callus tissues.Root induction on callus-derived shoots (i.e. plantlet formation)occurs on HMS containing only 25 ml TE and 100 ml coconut water1^–1. Taro, Colocasia esculenta var. esculenta (L) Schott (Araceae), coconut water, micropropagation, plantlet regeneration, root formation, taro extract, tissue culture     

The Localization in Cultured Carrot Cells of Factors that Induce their Growth

Various previously recognized parts of the complex of growthfactors present in the liquid endosperm of the coconut or inimmature fruits of Aesculus woerlitzensis were generally tritiated.The labeled growth factors were applied singly to culture mediawhich contained balanced requirements that had caused carrotexplants to proliferate and grow in accordance with combinationsof growth factors supplied. By the usc of electron microscopyand autoradiography, the radioactivity from each source wasdetected in the cells and its density and distribution, in theform of developed grains over different cellular compartmentsand organelles, was determined. The tabulated data relate tofour labeled sources as observed over seven cellular compartmentsunder six experimental treatments. Electron micrographs alsoshow how the radioactivity from the various sources relatedto organization of the cells. The distribution of radioactivity within the cells varied withthe source. Both ³H-myo-inositol and the tritiated growth factorsfrom Aesculus (³H-AF_1Aesc) with which it interacts (as in so-calledGrowth Promoting System I) contributed radioactivity, preferentially,to cell walls and sites of their formation in culturcd carrotcells. Both ³H-IAA and ³H-zatin (as in so-called Growth PromotingSystem II) contributed their radioactivity preferentially tothe nucleoli of the cultured cells. Some other conspicuous distributionsof radioactivity (e.g. from ³H-AF_1Aesc to plastids and from³H-IAA to the interstitial substance, i.e. middle lamella, whereenlarging cells separate) involved these tritiated moietieswithout regard to their counterparts in Growth Promoting SystemsI and II, respectively. The problems raised by such multiple effects due to differentgrowth factors acting singly and in combinations at differentcell sites are both recognized and discussed. growth factors, Aesculus woerlitzeensis, autoradiography, tritiation, cell sites, carrot, Daucus carota, coconut, electron microscopy     

Regeneration of Lasiurus scindicus from Tissue Culture

Embryogenic callus cultures were initiated from mature embryosof Lasiurus scindicus on Murashige and Skoog's medium supplementedwith 6 mg l^–1 2,4-Dichlorophenoxyacetic acid (2,4-D).These cultures were maintained on 2 mg l^–1 2,4-D. Plantletswere regenerated via somatic embryogenesis when the calli weretransferred onto hormone-free MS basal medium. Young plantswere successfully transplanted to pots and grown to maturityin a greenhouse. Grass, Lasiurus scindicus, Thar Desert, drought tolerant, somatic embryogenesis, plant regeneration     

Purification and Properties of an Auxin-Binding Protein from the Shoot Apex of Peach Tree

A soluble auxin-binding protein was purified from the shootapices of peach trees by chromatography on columns of CM-Toyopearl,Sephacryl S-200, 2,4-D-linked-Sepharose 4B and ConA-Sepharose.The molecular mass of the purified protein was estimated tobe about 100 kDa. After electrophoresis on a denaturing gel,the protein gave a single band with a molecular mass of 20 kDa.From Scatchard analyses, the dissociation constant for 2,4-Dwas calculated to be 4.1 10^–5 M and the specific bindingof 2,4-D at saturating concentration was 42 nmol (mg protein)^–1.The binding of [¹⁴C]-2,4-D to the protein was reversible andwas inhibited by IAA, 1-naphthylacetic acid and p-chlorophenoxyisobutyricacid. (Received June 25, 1992; Accepted October 20, 1992)     

Somatic Embryogenesis from Clonal Leaf Tissues of Cassava

Leaf lobes were isolated from palmate leaves of clonal cassava(Manihot esculenta Crantz) material growing in vitro or in glasshouseconditions and subjected to a two-stage culture procedure involvingincubation on Murashige and Skoog (MS2) basal medium supplementedwith 2–12 mg l^–1 2,4-D for 20 d (Stage I) beforetransfer to MS2 basal medium supplemented with 0.01 mg l^–12,4-D and 0.1 mg l^–1 6-benzylamino purine (BAP) (StageII medium). Embryogenetic tissues, foliose structures and somatic embryosdeveloped from leaf lobes at all Stage I 2,4-D concentrations,except on those explants isolated from shoot-tip cultures incubatedon MS2 basal medium supplemented with 0.1 mg l^–1 NAA and1.0 mg l^–1 BAP. Leaf lobes isolated directly from glasshouse plants showed optimalembryogenetic competence when subjected to a Stage I cultureperiod of 17 d, although foliose structure initiation was optimalwith shorter Stage I durations. Leaf lobes of 2–4 mm lengthand those isolated from phyllotaxic leaf numbers 4 and 5 showedthe greatest embryogenetic competence. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, morphogenetic competence     

Effects of Growth Regulators on the Induction of Anthocyanin Synthesis in Carrot Suspension Cultures

The effects of plant growth regulators were investigated onanthocyanin synthesis induced by removing auxin from carrotsuspension cultures. Of the auxins tested, 2,4-D showed thestrongest inhibiting effect on anthocyanin synthesis and hadthe strongest promoting effect on undifferentiated growth. When2,4-D was added to anthocyanin synthesizing cells, in whichcell division had ceased, anthocyanin synthesis was repressedimmediately, accumulated anthocyanin disappeared and cell divisionresumed. All cytokinins examined promoted anthocyanin synthesisin the absence of auxin. Both gibberellic acid (GA₃) and abscisicacid inhibited anthocyanin synthesis in media lacking 2,4-D,though GA³ showed no effect on cell division. These effectsof growth regulators on anthocyanin synthesis are similar tothose reported for their effects on embryogenesis [Fujimuraand Komamine (1975) Plant Sci. Lett. 5: 359, (1979) Z. Pflanzenphysiol.95: 13, (1980) Z. PJlanzenphysiol. 99: 1]. The relationshipbetween the induction of anthocyanin synthesis, metabolic differentiation,and embryogenesis are discussed. ¹ Present address: Department of Biology, College of Arts andSciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo153, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted July 23, 1986)     

Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells

A calmodulin like domain protein kinase (CPK) homologue wasidentified in alfalfa and termed MsCPK3. The full-length sequenceof cDNA encoded a 535 amino acid polypeptide with a molecularweight of 60.2 kDa. The deduced amino acid sequence showed allthe conserved motifs that define other members of this kinasefamily, such as serine-threonine kinase domain, a junction regionand four potential Ca²⁺-binding EF sites. The recombinant MsCPK3protein purified from E. coli was activated by Ca²⁺and inhibitedby calmodulin antagonist (W-7) in in vitro phosphorylation assays.The expression of MsCPK3 gene increased in the early phase ofthe 2,4-D induced alfalfa somatic embryogenesis. Heat shockalso activated this gene while kinetin, ABA and NaCl treatmentdid not result in MsCPK3 mRNA accumulation. The data presentedsuggest that the new alfalfa CPK differs in stress responsesfrom the previously described homologues and in its potentialinvolvement in hormone and stress-activated reprogramming ofdevelopmental pathways during somatic embryogenesis. Key words: Medicago sativa, CPK, stress, 2,4-D, phosphorylation, somatic embryogenesis.     

Auxin-Binding Protein in Etiolated Mung Bean Seedlings: Purification and Properties of Auxin-Bindmg Protein-II

An auxin-binding protein (ABP-II) was purified from the extractof etiolated mung bean seedlings by affinity chromatographyon 2,4-D-linked Sepharose 4B and by gel filtration on Sepharose4B and Sephacryl S-200. The molecular weight was estimated tobe about 190,000 by gel filtration on Sephacryl S-200. ABP-IIgave a single band corresponding to a molecular weight of about48,000 on SDS-polyacrylamide gel electrophoresis. The dissociationconstants of ABP-II for 2,4-D determined by amrnonium sulfateprecipitation and equilibrium dialysis were 9.5?10^–6 Mand 1.1?10^–5 M, respectively. ¹⁴C-2,4-D-binding to ABP-IIwas reversible and inhibited by addition of IAA, naphthalene-1-aceticacid, 2,4,5-trichlorophenoxyacetic acid or p-chlorophenoxyisobutylicacid to the assay mixture. (Received September 5, 1984; Accepted November 5, 1984)     

In Vitro Embryo Culture and Induction of Multiple Shoots in Lychee (Litchi chinensis Sonn.)

Immature embryos of different sizes and ages from commercialvarieties of lychee (Litchi chinensis Sonn.) were cultured ina range of different media. Embryos as small as 3 mm could becultured using in vitro techniques and subsequently grown intoplants. MS solid medium with 2% sucrose supplemented with 150ml l^–1 coconut water was most effective in stimulatingthe germination of immature lychee embryos. Embryos of lycheewere treated to induce adventitious buds from embryonic shootsas a means of achieving multiplication. The different varietiesexhibited differences in response, with Bengal embryonic shootsproducing 15 adventitious buds after pretreatment with 100 mgl^–1 BAP for 3 h. Root formation was achieved in 65% ofadventitious shoots using MS medium supplemented with 0.5 mgl^–1 NAA and activated charcoal. These plants were successfullydeflasked and grown on in the glasshouse. This technique providesof means of producing some multiple shoots from lychee embryosand has value for multiplication in a breeding program wherea method of micropropagation is unavailable. Litchi chinensis Sonn., lychee, embryo culture, multiple shoots, in vitro     

Somatic Embryogenesis and Plant Regeneration from Cultured Leaf Explants of Zea mays

Young leaf segments of Zea mays L. seedlings were cultured onMurashige and Skoog's basal nutrient medium supplemented with2 mg l^–1 2, 4-D and sub-cultured on medium containing8 mg l^–1 2,4-D. Two types of callus tissues appeared—embryogenicand non-embryogenic. The embryogenic callus tissue producednumerous somatic embryos which on transfer to media containinglow amounts of 2,4-D or ABA produced plantlets. Callus tissuesexhibited embryogenic potential for more than 1 year. Zea mays L. cv. Ageti-76, Zea mays L. cv. N-L-D-Comp., maize, leaf, callus, somatic embryogenesis, regeneration     

Growth Stimulation by Conditioned Medium and Spermidine in Low-Density Suspension Cultures of Rice

Suspension cultures of Oryza sativa L. var IR 20 grew in Murashigeand Skoog medium (MS) supplemented with 2,4-D and kinetin ina density-dependant manner with a critical minimum inoculation-densityof 10,000 cells ml^–1. Conditioned medium obtained fromthese cultures and added to MS+2,4-D+kinetin induced the growthof cultures at 1,000 cells ml^–1. Growth stimulation byconditioned medium was mimicked by spermidine but not by otherpolyamines viz. putrescine and spermine. This is the first reportof a polyamine substituting for conditioned medium in cultures. ² Present address: Vice-Chancellor, Pondicherry University,Pondicherry 605 014, India.     

In Vitro Culture of Orobanche aegyptiaca

Calli derived from seedlings of the parasitic plant Orobancheaegyptiaca were grown on enriched nutrient agar. Growth wasenhanced by the addition of 0.1 mg 1^–1 NAA and 5 % coconutmilk. The calli developed two kinds of elongated protrusions:thin ones which resemble the young root radicle and thick oneswhich resemble the adventitious roots of Orobanche plants. Bothformed pad-like structures when touching the substrate. In liquidculture, some of the calli were observed to surround isolatedcultured tomato roots. Orobanche aegyptiaca Pers., in vitro culture, germination, coconut milk, root parasite     

The importance of linoleic acid and linolenic acid as precursors of hexanal and trans-2-hexenal in black tea

During tea fermentation, linoleic acid in the neutral fat fraction,and linolenic acid in both the neutral fat and phospholipidfractions from leaves decreased. The addition of linoleic orlinolenic acid to leaf macerates during fermentation resultedin an increase in hexanal or trans-2-hexenal in the volatilefraction. Tracer experiments showed the direct conversion oflinoleic-U-¹⁴C and linolenic-U-¹⁴C acids to labeled hexanaland trans-2-hexenal, respectively, which were identified as2,4-DNPH derivatives. Further conversion of hexanal and trans-2-hexenal into hexanoicand trans-2-hexenoic acids during tea fermentation was suggestedby the increases in these compounds after the addition of hexanaland trans-2-hexenal to leaf macerates. (Received December 21, 1971; )     

Transport and Accumulation of IAA-14C in Tumour-forming Nicotiana Hybrids

The polar transport of indol-3yl-acetic acid (IAA-2-¹⁴C) instem explants and decapitated shoots of tumour-prone Nicotianahybrids (2n, 3n, and 4n) was compared with that in the normal,non-tumorous parent species N. glauca and N. langsdorffii. Thetotal uptake of the auxin from donor blocks was greatest inthe hybrids and N. glauca. The velocity of the basipetal movementof IAA-¹⁴C was the same in all species tested, i.e. 8 mm/h.The transport capacity for the hormone, however, was decreasedin the three tumour-prone hybrids. Gas chromatography showedthat between 70 and 90 per cent of the transported auxin waspresent in the form of IAA, between 10 and 30 per cent in theform of indol-3yl-aldehyde (IAld). The basipetal transport exceeded the acropetal transport inyoung (third) intemodes of all plants studied, whereas in olderstem segments (tenth intenodes) the reverse was found. The polarity of auxin transport was less well expressed in thetumorous hybrids. Blocking the active transport by pre-treatment of stem cuttingswith 2,4-dinitrophenol (2,4-DNP) caused a drastic reductionin the polar IAA-¹⁴C movement; in all plants tested the auxintransport was reduced to the same low level. The accumulation of auxin at the base of cuttings was higherin N. glauca and the 2n hybrid than in N. langsdorffii, i.e.about seven times higher after 1-h and three times higher after12-h transport experiments. The release of ¹⁴C from the cuttinginto an agar receiver block, however, was markedly reduced inthe 2n hybrid, whereas in N. glauca the labelled substancesmoved more freely into the receiver blocks. Differences in the capacity for the accumulation and the releaseof IAA-¹⁴C in hybrid and N. glauca stem tissues were studiedusing decapitated greenhouse plants wounded by incision abovethe fourth internode. Accumulation of the auxin occurred onlyabove the wound-cut in hybrid plants. This observation is consistentwith the view that tumour formation on hybrid stems occurs atsites of wounding. Our data suggest an elevated auxin levelto be present during tumour initiation at these sites. These results on polar transport and accumulation of IAA-¹⁴Cin tumorous Nicotiana plants together with our previous dataon various endogenous auxins suggest that the induction of neoplasticgrowth in tobacco plants is correlated with increased auxinlevels and an accumulation of the hormone at sites of wounding.