Correlation of spermine levels with ovary senescence and with fruit set and development inPisum sativum L.

Separation and quantitation of polyamines from unpollinated pea (Pisum sativum L.) ovaries and young fruits induced by application of gibberellic acid to unpollinated ovaries showed, in both cases, a decrease in putrescine and spermidine levels between anthesis and 4 d later. By contrast, spermine levels increased prior to the onset of senescence of the unpollinated ovaries (3 d post anthesis) and decreased during fruit development. Low levels of putrescine, spermidine and spermine were also observed in young fruits obtained by self-pollination and by treatment of unpollinated ovaries with 2,4-dichlorophenoxyacetic acid. In-vitro culture of ovary explants in a medium containing spermine showed that a reduction of the growth of gibberellic acid-treated unpollinated ovaries was associated with a rise in the level of spermine in the fruits. The results obtained indicate that changes in spermine levels are involved in the control of ovary senescence and of fruit set and development.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophen-oxyacetic acid - GA₃ gibberellic acid - HPLC high-performance liquid chromatography     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Induction of pollen callus in anther cultures of Feijoa sellowiana Berg. (Myrtaceae)

Summary Anthers of Feijoa sellowiana Berg. (feijoa) produced pollen callus when cultured in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine or in nurse cultures. Somatic callus was also formed in large amounts from the connective and from the cut end of the filament. Anthers containing microspores at the stage immediately prior to the first pollen mitosis cultured in the presence of 3% sucrose, presented the highest frequencies of induction. Androgenetic divisions were initiated by the formation of two morphologically equal cells, the so-called B-pathway. Attempts to regenerate pollen plants were unsuccessful but leaf-like structures could be obtained in regeneration media containing combinations of gibberellic acid and benzyladenine.Abbreviations 2,4-D 2,4-diclorophenoxyacetic acid - BA benzyladenine - FDA fluorescein diacetate - GA₃ gibberellic acid - Kn kinetin - MS Murashige and Skoog (1962) medium     

Influence of culture conditions on embryo formation and maturation in auxin-induced embryogenic cultures of Digitalis obscura

Somatic embryogenesis was induced in hypocotyls of Digitalis obscura using indoleacetic acid or 2,4-dichlorophenoxyacetic acid with different culture and subculture conditions. Indoleacetic acid-induced embryogenic cultures were used to investigate the effects of amino acids, polyamines and growth regulators on embryo differentiation and maturation. Supplementation of the media with amino acids, polyamines or abscisic acid did not influence or had an adverse effect on embryogenic response. Gibberellic acid at 1.4 M in either culture (30 days) or subculture medium was effective in promoting both differentiation and normal embryo development. The efficiency of somatic embryogenesis was greatly enhanced when isolated indoleacetic acid-induced proembryogenic masses were subcultured in liquid medium with reduced auxin content.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - IAA indoleacetic acid - Ptr putrescine - Spd spermidine - Spn spermine     

Ribulose-1,5-bisphosphate carboxylase and fruit set or degeneration of unpollinated ovaries of Pisum sativum L.

The polypeptide patterns obtained by sodium dodecylsulphate-polyacrylamide gel electrophoresis of undigested and autodigested extracts from pea (Pisum sativum L.) ovaries at the early stages of development or degeneration have been studied. Development of unpollinated ovaries was stimulated by application of different plant growth regulators (gibberellic acid, 2,4-dichlorophenoxyacetic acid, and N⁶-benzyladenine) or by plant topping. Polypeptide bands of similar mobility to ribulose-1,5-bisphosphate carboxylase (RuBPCase) subunits (16 and 55 kDa) could be detected in all types of autodigested extracts from stimulated ovaries. However these bands were absent in electrophoretic patterns of autodigested extracts from unstimulated ovaries after 3 d post anthesis and in patterns of autodigested mixtures of these extracts with either those from stimulated ovaries or those from unstimulated ovaries before day 3. These observations indicate that a proteolytic activity which promotes the hydrolysis of RuBPCase appears in unstimulated ovaries about 3 d after anthesis. This event coincides with the loss of the capacity of unpollinated ovaries to develop in response to gibberellic acid and with the degeneration of the ovary wall.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS-PAGE sodium dodecylsulphate-polyacrylamide gel electrophoresis     

Plant regeneration from leaf protoplasts of apple

Protoplasts were isolated from young leaves or etiolated shoot apices. For initiation of divisions the protoplasts were embedded in sodium alginate and cultivated in MS or MI medium supplemented with 2.2 M BA, 2.6 M NAA and 2.2 M 2,4-dichlorophenoxyacetic acid. The protoplasts of all seven lines tested developed to protocalluses at high frequencies. No genotypic differences were observed. When BA was used in combination with NAA in the regeneration experiments, only a few protocalluses (highest frequency 3%) exhibited shoot organogenesis. When BA was replaced with thidiazuron, the percentage of protocalluses that developed shoots increased in two of three tested lines to 7% and 56%, respectively. Shoot development was achieved under light conditions. The shoots were then rooted and transferred into soil.Abbreviations ABA abscisic acid - BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - IBA indole-3-butyric acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Plant regeneration from cotyledons of niger

Callus initiation from seedling explants of niger (Guizotia abyssinica Cass) cv. Ootacamund was found to be better on LS medium containing kinetin (1.4 M) plus 2,4-dichlorophenoxyacetic acid (9 M) than its analogues. Embryoids were induced directly from cotyledons on LS medium supplemented with 2,4,5-trichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid. When cotyledon-derived callus was subcultured onto medium with 10.7 M naphthalene-acetic acid and 2.3 M kinetin, embryogenesis was observed. Multiple shoots were obtained from cotyledonary explants in presence of MS medium containing 4.4 M benzyladenine and 11.4 M indoleacetic acid. Regenerated plants that were transferred to pots and grown to maturity were morphologically normal and fertile.Abbreviations NAA naphthaleneacetic acid - IAA indoleacetic acid - BA benzyladenine - GA₃ gibberellic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxypropionic acid - 2,4,5-TP 2,4,5-trichlorophenoxypropionic acid - ABA abscisic acid     

Somatic embryogenesis and organogenesis in Encephalartos dyerianus and E. natalensis

Callus cultures were initiated from zygotic embryos of Encephalartos dyerianus and E. natalensis. Callus of both species were transferred onto a modified B5 medium containing different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Somatic embryogenesis and shoot organogenesis occurred in both species. The embryos were dicotyledonary. To date none of the embryos have matured.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

High-frequency induction of adventitious shoots from hypocotyl segments ofLiquidambar styracifiua L. by thidiazuron

The effects of thidiazuron (TDZ) on adventitious bud and shoot formation from hypocotyl segments of sweetgum (Liquidambar styracifiua) were tested alone and in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). The combination of 1 mg/1 TDZ with 0.01 mg/l 2,4-D resulted in the highest frequency of bud production. Lower concentrations of TDZ stimulated shoot production, generating the most shoots at 0.1 mg/1 TDZ with 0.01 mg/1 of 2,4-D. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium lacking TDZ or containing naphthaleneacetic acid and benzyladenine in addition to TDZ. Shoot production in liquid culture was significantly greater than that in solid culture. Comparisons of in vitro and ex vitro rooting of the adventitious shoots demonstrated that ex vitro rooting produced plants with faster growth rates and more extensive root systems.Abbreviations BA Benzyladenine - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyacetic acid     

Somatic embryogenesis and plant regeneration in garden leek

High frequency plant regeneration via somatic embryogenesis has been induced from in vitro shoot-base cultures of seedlings of garden leek (Allium porrum L.). Four main steps are involved in the procedure using BDS medium:

- shoot multiplication with 17.6 mM benzyladenine;

- induction of nodular callus from the in vitro shoot base with 9 mM 2,4-dichlorophenoxyacetic acid;

- initiation of embryogenic callus from nodular callus with 9 mM 2,4-dichlorophenoxyacetic acid +7.6 mM abscisic acid;

- plant regeneration from embryogenic callus with 9.8 mM N⁶-(2-isopentenyl)adenine.

The presence of 2,4-dichlorophenoxyacetic acid in the medium and light conditions were shown to be essential for nodular callus induction and somatic embryogenesis. Abscisic acid was not a prerequiste for somatic embryogenesis, but it significantly increased the frequency.     

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

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

Somatic embryogenesis and plant regeneration from immature embryos of western larch

Somatic embryogenesis was initiated from immature embryos of western larch (Larix occidentalis Nutt.) on media containing 2,4-dichlorophenoxyacetic acid and N6- benzyladenine. The effects of explant type and ammonium nitrate and glutamine concentrations on initiation were tested. Although 21–93% of explants rendered cultures in various experiments, only 3% yielded sustainable embryogenic lines. Excised embryos at the early cotyledonary stage were optimal for initiation. Maturation of somatic embryos was promoted by abscisic acid. Response to abscisic acid concentrations and duration of exposure to abscisic acid varied with genotype. Maximal results were obtained with 0.025 M abscisic acid for 1 to 2 weeks followed by individual culture on medium without growth regulators. Mature somatic embryos developed into shoots with roots. Plantlets have been established in peat.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - IBA indole-3-butyric acid - ABA abscisic acid     

Tissue cultures of Artemisia pallens: Organogenesis,terpenoid production

Germinated seedlings of Artemisia pallens gave three types of cultures on MS medium supplemented with different plant growth hormones. Medium containing BA+2,4-D stimulated unorganized callus; BA+IAA medium, semi-organized tissues interspersed with shoot buds; and BA+NAA+IAA medium, multiple shoot cultures. The in vitro shoots developed roots in medium devoid of growth hormones. TLC and GLC analysis of the tissue extracts showed that linalool was present in the cultured tissues, with maximum concentration in the unorganized tissue. Although the TLC profiles of the three culture extracts were similar, the extracts did not contain the major polar compounds of the plant. The plant extracts contained more polar compounds and gave the characteristic fragrance of davana.Abbreviations MS Murashige & Skoog's basal medium - BA benzyladenine - Kn kinetin - NAA naphthaleneacetic acid - IAA indoleacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume     

Effect of 2,4-dichlorophenoxyacetic acid on the dark- and light-induced pulvinar movements in Cassia fasciculata Michx

2,4-dichlorophenoxyacetic (2,4-D) applied to excised leaves of Cassia fasciculata modified the dark-induced (scotonasty) and light-induced (photonasty) leaflet movements, showing that this compound acts on rapid turgor variation and the concomitant ion migrations, in particular K⁺. 2,4-D inhibited the scotonastic closure in a dose-dependent manner from 10^–8 M to 10^–5 M and promoted the photonastic opening in the same range of concentrations. The compound acted rapidly since a treatment as short as 5 min gave an obvious effect on the motile reaction; however, a lag period of 45–60 min was needed to observe its effect. Although 2,4-D is a weak acid, its greatest physiological efficiency was obtained with pH values close to neutrality. The physiological results are discussed in relation to the chemical properties and the characteristics of transport of the molecule.Abbreviations ABA abscisic acid - 6-BAP 6-benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - GA₃ gibberellic acid - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid] - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - MES 2-(N-morpholino)-ethanesulphonic acid     

Roots as an enhancing factor for the production of artemisinin in shoot cultures of Artemisia annua

Artemisinin was produced in differentiated shoot cultures of Artemisia annua L. but was undetected in callus or cell cultures. The growth regulators benzyladenine, kinetin, chlormequat, and daminozide, at concentrations which severely reduced rooting, reduced artemisinin production. A highly significant correlation (1% level) was observed between shoot artemisinin content and number of roots (r=0.775^**), but shoot number and artemisinin content were unrelated (r=-0.198). Benzyladenine increased shoot proliferation at 0.5 and 5.0 M, but decreased root production at 0.5, 5.0, and 50 M. The highest levels of artemisinin production (0.287% DW) were obtained in hormone-free medium when root production was maximized. Removal of roots from shoots cultured in hormone-free liquid medium reduced shoot artemisinin by 53% and shoot arteannuin B by 60%. Neither artemisinin, arteannuin B, or artemisinic acid were detected from roots developed in semi-solid or liquid medium.Abbreviations BA benzyladenine - CCC chlormequat - DW dry weight - FW fresh weight - GA₃ gibberellic acid - GC/MS gas chromatography/mass spectrometry - HPLC-EC high-performance liquid chromatography with electrochemical detection - MS Murashige & Skoog basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid Journal paper no. 14558 of Purdue Agricultural Research Progress     

In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil)

A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA₃) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

Plant regeneration from protoplasts of Felicia and Brachycome

Protoplasts were isolated from leaves of axenic shoot cultures of Felicia bergeriana (Kingfisher Daisy) and Brachycome iberidifolia (Swan River Daisy) and from callus cultures of Felicia. Plants were regenerated from all three sources and since both species are of ornamental value (blue flowered) the establishment of plant regeneration provides a basis for their incorporation in somatic hybridisation programmes involving important ornamentals such as Chrysanthemum.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - KIN 6-furfurylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - FDA fluorescein diacetate - f. wt. fresh weight