The Effect of 2,3.5 Triiodobenzoic Acid on Caulogenesis in Callus Cultures of Tomato and Pelargonium

Stem explants from winter grown tomatoes cultured on a cytokinin, auxin-free medium, developed one or two adventitious shoots at the top end of the explant. Addition of the auxin transport inhibitor. 2,3,5-triiodobenzoic acid (TIBA) to the medium stimulated caulogenesis with loss of polarity. Callus, initiation in pelargonium and ‘geranium’ petiole explants requires both auxin and cytokinin. On transfer, after callus induction to an auxin-free medium, rhizogenesis occurs in pelargonium cultures followed by caulogenesis. Few shoots develop and unless these are removed, further caulogenesis is suppressed. Bud-like structures were formed in the callus. Subculture on auxin-free medium containing cytokinin and TIBA resulted in shoot formation from these bud-like organs. An analogy with apical dominance is suggested. In ‘geranium’ callus, shoots developed with a low frequency (c. in 2% of the cultures): caulogenesis was increased to 80% when calli were subcultured from auxin-free, cytokinin medium after green nodule formation to cytokinin-TIBA medium. Histological studies of green nodules in ‘geranium’ callus indicated a variation in morphological development within and between nodules. It is suggested that auxin synthesis may occur at some microscopic stage in morphogenesis in ‘geranium’ cultures which suppresses further caulogenesis. This may be overcome by the addition of TIBA to the medium at the appropriate stage in morphogenesis. The possible interaction of endogenous auxin in morphogenesis is discussed.     

Cytokinesis, cell expansion, and the potential for cytokinin-autonomous growth in tobacco pith

Pith tissue from Nicotiana tabacum L. cv `Maryland Mammoth' or `Wisconsin 38' was isolated, free of vascular tissue, and cultured on a medium containing auxin but no cytokinin. Explants from the apical 1 cm of stem, within the pith rib meristem, initiated callus growth with 100% efficiency. Macroscopically visible callus was evident 5 days after the tissue was isolated, and the cultures grew persistently in the absence of cytokinin. Heat treatment, sometimes used to initiate cytokinin habituation, was not required. Explants from tissue basipetal to the pith rib meristem declined in the frequency of habituation with increasing distance from the shoot apex. Although pith tissue which was growing, in vivo, was more prone than mature tissue to establish cytokinin-habituated callus, the basipetal decline in habituation frequency extended well beyond the zone of cell expansion. Explants from mature pith 40 centimeters or more from the shoot apex grew in the absence of cytokinin with 18% frequency, although the response required at least 2 weeks of culture. Further analysis demonstrated that tissue near the periphery of mature pith was more prone to cytokinin-habituation than tissue from the pith center.     

SOMATIC EMBRYOGENESIS AND ORGANOGENESIS FROM CALLUS CULTURES OF SOLANUM CAROLINENSE

Culture of stem segments of Solanum carolinense L. on medium supplemented with 10 mg/1 2,4-dichlorophenoxyacetic acid and 1 mg/1 kinetin, induced callus formation. When subcultured on medium lacking 2,4-D but containing a cytokinin, the callus regenerated. The mode of regeneration depended on the type and concentration of cytokinin employed; high concentrations of benzyladenine and all concentrations of kinetin promoted organogenesis, while low concentrations of benzyladenine induced somatic embryogenesis in addition to organogenesis. With age and continued subculture on 2,4-D containing medium, callus progressively lost its ability to regenerate when the auxin was replaced by cytokinin. In conjunction with previous studies on regeneration from anther cultures of S. carolinense, it appears that in both cases, 2,4-D is required for callus initiation and proliferation but must be exchanged for a cytokinin before differentiation will occur. However, since it was not possible to induce embryogenesis in pollen-derived callus, developmental potential may be influenced by the ploidy level of responding cells in culture.     

Effects of cytokinin on adventitious root formation in callus cultures ofVigna unguiculata (L.) walp

Summary Yellowish compact callus, induced from cowpea hypocotyls on Murashige and Skoog(MS) medium (1962) containing 0.2 mg/l(0.93 μM) kinetin and 0.4 mg/l (1.81 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), was subcultured on MS medium containing cytokinin alone, auxin alone, or auxins plus cytokinins in order to determine the effect of cytokinins on root organogenesis in callus cultures. The callus actively proliferated on the same medium but did not show any organogenic activity macroscopically as well as microscopically. On medium with N⁶-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA), the yellowish compact callus first changed to pale green compact callus and then many green spots appeared on its surface under light culture. But the yellowsih compact callus remained yellowish and white spots appeared on its surface in dark culture. These spots gradually became white nodular structures. Adventitious root formation from the nodular structures occurred not only on the same medium, but also on medium with either auxin or cytokinin but not both. Yellowish compact callus on medium with auxin alone was transformed to yellowish friable callus, which did not develop adventitious roots. The yellowish friable callus could gain rhizogenic activity only after morphological modification to pale green compact callus on medium with auxin plus cytokinin. The modified callus did not form adventitious roots on medium with auxins but only with cytokinins. Therefore, it is suggested that cytokinins have stimulating effects on root formation from callus that previously did not show rhizogenic activity on medium with auxins alone. In addition, the rhizogenic potential of cowpea callus was discriminated from that of leaf explants, which formed adventitious roots directly on medium with auxin alone.     

Effect of BA,NAA, and 2,4-D on red maple callus growth

Established red maple (Acer rubrum L.) callus was cultured on media varying in auxin (NAA or 2,4-D) and cytokinin (BA) concentrations. Callus growth was positively affected by the presence of both an auxin and cytokinin in the medium. Optimal growth depended on the ratio of cytokinin/auxin as well as the total amount of plant growth regulators in the medium.Abbreviations (NAA) naphthaleneacetic acid - (2,4-D) 2,4-dichlorophenoxyacetic acid - (BA) and 6-benzylaminopurine     

Kinetics of Determination in the Differentiation of Isolated Mesophyll Cells of Zinnia elegans to Tracheary Elements

Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing 0.5 micromolar α-naphthaleneacetic acid and 0.5 micromolar benzyladenine. The cells do not differentiate when cultured in medium in which the concentration of auxin and/or cytokinin has been reduced to 0.005 micromolar. Cells require an initial 24-hour exposure to inductive cytokinin and 56-hour exposure to inductive auxin for differentiation at 72 hours of culture. Freshly isolated Zinnia cells can be maintained in medium having low concentrations of both auxin and cytokinin for only 1 day without significant loss of potential to differentiate upon transfer to inductive medium. Initial culture for up to 2 days in medium having high auxin and low cytokinin, or low auxin and high cytokinin, allows full differentiation on the third day after transfer to inductive medium and potentiates the early differentiation of some cells.     

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.     

Interspecific hybridization of red clover (Trifolium pratense L.) with T. sarosiense Hazsl. Using in vitro embryo rescue

Summary Interspecific hybrid clover plants from the cross Trifolium sarosiense Hazsl. X T. pratense L. were obtained in the present investigation. Immature hybrid embryos were excised aseptically from the pistillate parent, T. sarosiense (2 n = 48), and cultured in vitro prior to in situ abortion. Agar-solidified nutrient media modified from that developed previously for tissue and cell cultures of red clover (2 n = 14) were used for embryo rescue.The heart shaped embryos obtained were cultured for 8 to 14 days on a medium containing a high level of sucrose, a moderate level of auxin, and low cytokinin activity. Viable embryos were then transferred to a standard medium with low auxin and moderate cytokinin levels for the direct germination of shoots. Some embryos produced only callus. Plants were regenerated from callus using an alternate culture scheme. Hybrid shoot numbers were increased on a low auxin, high cytokinin medium and subsequently rooted before transfer to soil in the greenhouse.About 10% of the hybrid embryos were rescued using the optimal culture sequence. Five full-sib families of the F₁ hybrid were successfully grown to maturity. Root-tip cells of hybrid plants possessed the expected somatic chromosome number of 31. The genetically determined leaf-mark trait carried by the staminate parent and the rhizomatous root habit of the pistillate parent were expressed in hybrid plants.The investigations reported in this paper (No. 81-3-151) were performed in connection with projects of the Kentucky Agricultural Experiment Station and the paper is published with the approval of the Director     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

Differential callus type formation by auxins and cytokinin in in vitro cultures of pepper (Capsicum annuum L.)

ABSTRACT

Two types of callus were produced by pepper explants cultured in various media containing auxins, the cytokinin 6-benzylaminopurine (BAP) and the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA). Callus produced on media containing auxins alone was friable, grey-green or green-orange in colour and more compact, whereas when BAP was added to culture media with a low concentration of auxin or when the medium contained TIBA alone, the callus produced was white and very hard. This type of callus was also produced in cultures of older tissues and of young tissues cultured on hormonefree medium. Results are discussed in relation to the role of cytokinins in wounding, phenylpropanoid metabolism and lignin biosynthesis.     

The influence of genomes on autonomous growth of pith cultures of Nicotiana glauca-Langsdorffii hybrids

Summary A differential influence of the two parental genomes on cell proliferation and morphogenesis in pith tissue explants can be observed among the various tumorous hybrid combinations between Nicotiana glauca Grah. and N. langsdorffii Weinm.: the F₁ hybrid (GL), its amphiploid (GGLL), and two different triploids (GGL and GLL). This influence was evident when the explants were cultured in the presence of exogenous auxin (indole-3-acetic acid, 2.5 M), supplied either continuously or for a brief period of time. Compared with the F₁ and the amphiploid, the higher proportion of N. glauca genomes in GGL cells resulted in greater growth, the higher proportion of N. langsdorffii genomes in GLL cells in lesser growth. In addition, shoots are produced on the GGL callus, while only roots are formed on calli of the other types in the same medium. When, in addition to auxin, a cytokinin [6-(3-methyl-2-butenyl-amino)purine] was added to the culture medium, the differential growth of the different tissue types was less pronounced; at 1.0 M of the cytokinin, all tissues grew at about the same rate and remained undifferentiated, regardless of their genomic composition.     

The Role of Auxins in Root-Knot Nematode-Induced Growth on Excised Tobacco Stem Segments

Root-knot nematodes, Meloidogyne incognita, induced lumps of callus tissue on the cambial surfaces of peeled tobacco stem segments cultured in vitro. Except for a layer 1 to 3 cells thick, callus was limited to the basal ends of control segments. Indole-3-acetic acid (IAA) applied in agar blocks to the centers of stem segments, when it had any effect on the cambial surface, induced streaks of callus extending from the blocks toward the basal ends of the segments. IAA in agar blocks also increased callus growth at the basal ends of the segments, increased the growth of pith on the undersides of the segments, promoted root initiation, but inhibited bud initiation. Nematodes produced none of these effects, nor did they change the type of organs induced by various concentrations of IAA in the medium. Callus tissue did grow on the cambial surface of stem segments surrounding agar blocks containing 2,3,5-triiodobenzoic acid, an inhibitor of polar auxin transport. Paraffin sections showed that the nematodes were confined to the callus tissue on the cambial surfaces of the segments. Except for occasional syncytia and areas of cell division, nematode-induced callus was composed of thin-walled, irregularly shaped cells arising from the cambium. Differences between the responses of tobacco stem segments to root-knot nematodes and IAA-agar blocks indicate that auxins were not freed from the plant tissue nor secreted by the nematodes. Instead, it is suggested that nematodes enabled the tissue to retain and use endogenous auxins that otherwise would have been transported to the basal ends of the segments.     

Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> and <Emphasis Type="Italic">Morinda citrifolia</Emphasis>

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.     

A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts

Summary Hypocotyl protoplasts of four different Helianthus annuus genotypes were cultivated for 22–28 days in agarose droplets covered with liquid medium. In the first week, supplementation of the medium with plant growth regulators was at a 0.8/1 ratio of cytokinin and auxin followed by a high auxin concentration in the second week and a cytokinin to auxin ratio of 8/1 in the third and fourth week. Following transfer onto solid medium containing cytokinin and auxin in a proportion of 40/1 morphogenic callus started to form globular structures that developed into leaf primordia. Subsequent shoot elongation and rooting were obtained on hormone free medium after dipping the cut shoots into high auxin solution. Thirteen weeks after protoplast isolation, plantlets could be transferred to the greenhouse. Shoot regeneration was obtained for all four cultivars (Florom-328, Cerflor, Euroflor, Frankasol) at different rates reflecting their regenerative potential.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FeNaEDTA ethylenediamine tetraacetic acid ferric sodium salt - IAA indole acetic acid - MES morpholinoethane sulfonic acid - NAA 1-naphtalene acetic acid     

CELL DIVISION IN RELATION TO CYTODIFFERENTIATION IN CULTURED PEA ROOT SEGMENTS

One mm-thick segments cut 10–11 mm proximal to the root tip of germinating seeds of garden pea Pisum sativum were cultured in sterile nutrient medium containing auxin in the presence and absence of kinetin. In the absence of added cytokinin, pericyclic proliferation occurred, the cortical tissues showed no proliferation and were sloughed off, and a callus tissue of diploid cells was formed. In the presence of kinetin concentrations from 0.1–1.0 ppm cortical cells of the segments were induced to divide, beginning at the third day. From experiments with ³H-thymidine incorporation at different times of culture, from cytological squash preparations and from histological sections it was shown that the cortical cells stimulated to divide by cytokinin underwent DNA synthesis prior to division, were polyploid, and following cell division rapidly underwent cytodifferentiation at 5–7 days to form mature tracheary elements. At 10 days, when over 300,000 new cells had been formed per segment about 16% of these cells had formed tracheary elements. It was concluded that cytokinin, together with auxin, was essential for the initiation of DNA synthesis in the cortical cells, for their subsequent division, and finally for their specific cytodifferentiation.     

Pollen plant formation from anther cultures of Digitalis obscura L.

Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration from cultured anthers of Digitalis obscura L. were determined. The presence of auxins was essential for cell proliferation and morphogenesis, and incubation in darkness singificantlyincreased these responses. Callus proliferation usually preceded embryo development, although sometimes direct embryogenesis was observed. On the other hand, bud differentiation was achieved only when callus was transferred to media containing cytokinin or several auxin/cytokinin combinations. Different ploidy levels] were observed in the regenerated plants, with approximately 50% being haploid.     

Embryogenic responses of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. callus induced from transgenic hairy roots

Agrobacterium rhizogenes A4M70GUS-mediated transformation of two local breeding lines of sugar beet was obtained using 4-week-old seedlings. Root formation efficiency was 61.54% for SBa genotype and 36.36% for SBb genotype. Five highly proliferated hairy root lines have been established in liquid hormone-free MS medium. Transgenic nature of the hairy root clones was evaluated by GUS assay, PCR and RT-PCR analyses. Hairy root-derived calli were induced using different plant growth regulators (PGRs): auxin, auxin/cytokinin and cytokinin. The best callus induction response was achieved on MS medium containing both 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg/l thidiazuron (TDZ). Globular embryo-like structures were observed in friable callus after its prolonged cultivation on MS medium supplemented with TDZ and giberellic acid (GA₃) at 1 mg/l each, followed by growth on MS medium containing 1% glucose and 0.5 mg/l 2,3,5-triiodobenzoic acid (TIBA). Histological analysis revealed somatic embryos at different stages of development in hairy root-derived callus of sugar beet.     

Rol genes alter hormonal requirements for protoplast growth and modify the expression of an auxin responsive promoter

Summary Growth characteristics of tobacco protoplasts containing rolA linked to its own promoter, or the rolB, or rolC genes of Agrobacterium rhizogenes linked to the Cauliflower Mosaic Virus 35S RNA promoter were compared with those from untransformed plants. RolA protoplasts require auxin and cytokinin for callus formation. Protoplasts overexpressing rolB and C form callus in the absence of exogenously applied auxin and cytokinin, respectively. Long term callus growth requires auxin, but the requirement for cytokinin is not critical. Optimal transient expression of an auxin responsive promoter element occurred at lower external levels of auxin in rolB and rolC protoplasts compared with untransformed protoplasts. Addition of putrescine was required for auxin responsive transient gene expression in rolA protoplasts suggesting that polyamines, or their products affect gene expression in rolA plants.Abbreviations T-DNA transferred DNA - TL-DNA left transferred DNA - NAA naphthalene acetic acid - PEG polyethylene glycol - GUS glucuronidase - CaMV cauliflower mosaic virus     

Hormone-like effect of vascular tissue on starch accumulation in stem explants of kale, Brassica oleracea

Explants of stem pith of kale ( Brassica oleracea L. var. medullosa cv. Krasa), cultured for several days on agar medium containing sucrose, accumulate starch. Application of streptomycin, 5-fluorouracil and other inhibitors indicates that starch accumulation depends on protein synthesis on 80 S ribosomes. If explants derived from plants grown under natural long-day conditions contained vascular tissue, including cambium, in addition to pith parenchyma, the amount of starch formed in the pith tissue increased up to seven fold when compared with explants without vascular tissue. Similar increase of starch content as caused by vascular tissue was achieved by the addition of kinetin or trans -zeatin (10 μ) in the presence of 5 μ indole-3-acetic acid or 1-naphthaleneacetic acid. A further three-fold increase in starch accumulation could be achieved by application of cytokinin and auxin to explants containing vascular tissue. When explants were derived from plants grown under natural short-day conditions cytokinins and auxins had little or no effect, but vascular tissue enhanced starch formation significantly. The spreading of starch inducing stimulus from vascular tissue (probably from its meristematic region) to the pith parenchyma up to a distance of at least 20 mm was demonstrated. It was concluded that a hormone-like factor other than cytokinin and auxin was involved in the stimulatory action of vascular tissue. The effects of this factor on protein accumulation and growth in the explants and its possible production by meristematic tissues in vivo are discussed.     

Regeneration of Kosteletzkya virginica (L.) Presl. (Seashore Mallow) from callus cultures

Organogenic callus cultures of seashore mallow, Kosteletzkya virginica (L.) Presl., originated from excised mature embryos or stem sections of aseptically germinated plants initially cultured on Murashige & Skoog minimal organics medium containing 30000 mg l^-1 glucose, 2.0 mg l^-1 indoleacetic acid and 1.0 mg l^-1 kinetin. Plants were regenerated via shoots and roots from callus cultures following transfer through a series of media with different cytokinin/auxin ratios and changes in carbohydrate source. Meristematic regions, shoot and root primordia were observed during histological examination of the tissues. Somatic embryos were not found.