In vitro propagation of Dampiera diversifolia and Prostanthera rotundifolia

Shoot multiplication and root formation was induced in vitro from mature shoot explants of the woody ornamental plant species Dampiera diversifolia and Prostanthera rotundifolia. 0.5 M BAP+0.5 M kinetin in the absence of auxins gave the most prolific shoot multiplication in both species but there were qualitative and quantitative differences between species in rooting responses to auxin applications. A high percentage of rooted plants were successfully grown-on in pot culture.     

Rapid in vitro adventitious shoot propagation of <Emphasis Type="Italic">Scopolia parviflora</Emphasis> through rhizome cultures for enhanced production of tropane alkaloids

A rapid micropropagation system for Scopolia parviflora Nakai (Solanaceae), a rare medicinal plant native to Korea, was established using rhizome cultures. Shoots that originated from adventitious shoots of the rhizome were multiplied when the rhizomes were cultured on half-strength B5 liquid medium supplemented with various growth regulators. Optimum shoot multiplication was observed in half-strength B5 medium containing 3% (w/v) sucrose and 5.77 M gibberellic acid (GA₃). Each rhizome gave rise to an average of 12 shoots. Shoot elongation and root induction from multiple shoots occurred on growth regulator-free half-strength B5 solid medium. Healthy plantlets were transferred to a peat moss:vermiculite mixture for acclimatization, which was successful. The concentrations of tropane alkaloids, hyoscyamine and scopolamine were determined in different tissues of native growing plants, in vitro-propagated plants and acclimatized plants by high-performance liquid chromatography. The analysis revealed that the levels of hyoscyamine and scopolamine were higher in in vitro-propagated plants than in the native growing plants. When the rhizome was cut into segments and transferred to optimal culture conditions for multiple shoot propagation, only 12 weeks were required to produce a mature plant. We conclude that in vitro propagation techniques through rhizome cultures provide an efficient and rapid method for shoot propagation of S. parviflora.Abbreviations BA Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - HPLC High-performance liquid chromatography - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid     

Accumulation of essential oils by Agrobacterium tumefaciens-transformed shoot cultures of Pimpinella anisum

Axenic transformed shoot cultures of Pimpinella anisum (anise) were established following inoculation of plant stems with the nopaline strain T37 of Agrobacterium tumefaciens. The stable incorporation of T-DNA in the transformed tissues was demonstrated by polymerase chain reaction. Total essential oil accumulated by transformed shoot cultures grown under continuous light was found to be 18% lower (per unit fresh weight of tissue) than that produced by untransformed shoot cultures incubated under similar conditions, but more than 89% lower than the yield of oil from the intact plant. The relative amounts of the principal components of the essential oil of the transformed shoot cultures, namely geraniol, -bisabolene, trans-pseudoisoeugenol-2-methylbutyrate and transanethole, were similar to those present in the parent plant, but significantly different from those of the untransformed shoot cultures.Abbreviations T-DNA transfer-DNA - MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - TY tryptone and yeast extract medium - t_D doubling time - GC-MS gas chromatography coupled with mass spectrometry - FID flame ionisation detector - PCR polymerase chain reaction - TE Tris-HCl, EDTA buffer - TBE Tris, borate, EDTA buffer     

Micropropagation of four cultivars of Saskatoon berry (Amelanchier alnifolia NUTT.)

In vitro culture establishment, shoot proliferation, ex vitro rooting and dormancy breaking of the newly rooted plantlets were examined on Saskatoon berry (Amelanchier alnifolia NUTT.) cultivars Northline, Pembina, Smoky and Thiessen. Shoot-tip explants taken from actively growing plants were better for culture initiation than dormant buds. MS gave the most satisfactory results of the media formulations. Optimal shoot proliferation occurred at 8.8 and 13.3 M BA. Higher BA concentrations caused culture deterioration during long-term maintenance. Auxin treatments significantly stimulated ex vitro rooting of shoots in all cultivars. The best rooting was achieved with IAA/NAA (2.8/1.1 M) mixture. Satisfactory results were also obtained with commercial powder formulation, Rootone F, containing IBA/NAA mixture. Foliar application of BA and GA₄₊₇ was successful in breaking dormancy of newly rooted plantlets. Combinations of these two growth regulators caused formation of axillary shoots and vigorous plant growth. There were significant differences in the cultivar responses to culture conditions and treatments with growth regulators. The best culture establishment and the highest rate of shoot proliferation was observed in cv. Thiessen; the best rooting and the most vigorous post-dormancy growth was recorded in cv. Smoky. Cultivar Northland gave the most erratic responses.Abbreviations BA benzyladenine - cv(s) cultivar(s) - GA gibberellin - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - MS Murashige & Skoog's medium     

The effect of leaf source and developmental stage on shoot organogenic potential of sweetgum (Liquidambar styraciflua L.) leaf explants

Leaf explants harvested from shoot proliferating cultures and intact plants of Liquidambar styraciflua Variegata were placed on solidified Woody Plant medium supplemented with 0.1 mgl^-1 (0.5 M) naphthaleneacetic acid and 2.5 mgl^-1 (11.1 M) benzyladenine to initiate shoot meristems directly. Leaves from intact plants produced over 4 times more adventitious shoots than leaves from in vitro shoots and had a greater tendency to form shoots on the lamina. The relative developmental age of leaf tissue used dramatically influenced the shoot organogenic response observed for leaf explants from intact plants of L. styraciflua Variegata and Moraine.-Leaves that were either 20% or 50% of full size and still actively expanding were superior to other developmental stages for shoot organogenesis. As developmental leaf age increased throughout the period of leaf expansion, the number of shoots forming on the petiole stub remained constant, whereas shoot formation on the lamina increased 8 fold. Shoots derived from Variegata leaves rooted well and grew normally as plants. Differences in rooting ability and plant size could be detected between groups that had been separated according to explant source (in vitro vs. intact plant) and the location of shoot formation (petiole vs. lamina).     

Effect of exogenous plant growth regulators on in vitro seed growth,embryo development,and haploid production in a cross between H. vulgare (L.) and H. bulbosum (L.)

Exogenous plant growth regulators are known to increase the efficiency of interspecific and intergeneric crosses. In vitro floret culture provides a defined system for assessing the importance of various plant growth regulators on the determinants of haploid production efficiency (seed set, embryos per seeds, and plants per embryos) in Hordeum vulgare × Hordeum bulbosum crosses. The individual and combined effects of three plant growth regulators (2,4-D, GA₃ and kinetin) on in vitro seed growth, embryo development and haploid production efficiency were tested in floret culture of the cross H. vulgare, cultivar Klages × H. bulbosum. All treatments, except kinetin alone, produced larger seeds and more embryos/100 seeds than the control (no plant growth regulator). 2,4-D alone was superior to GA₃ alone in haploid production efficiency (70.6 vs. 51.5) as measured by the number of plants regenerated/100 florets pollinated. Although kinetin +2,4-D+GA₃ produced the largest seeds and embryos, no advantage over 2,4-D alone was observed in haploid production efficiency. 2,4-D alone or kinetin +2,4-D are recommended for the purpose of barley haploid production in floret culture using the bulbosum method.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Botanical and chemical evaluation of Atractylodes lancea plants propagated in vitro and by division of the rhizome

Summary A single plant of Atractylodes lancea, a perennial herb of Asteraceae, was propagated in vitro by shoot culture from floral buds and shoot tips. The reproduced plants were then grown under field conditions for up to four years and their botanical and chemical characteristics were compared to those of control plants that were propagated by dividing the rhizome of the parent plant. The first-year-plants of the micropropagated plants were scanty, but in the second year they were comparable to the control in most botanical features. On the other hand, the contents of two sesquiterpenes (hinesol and -eudesmol) and a polyacetylenic compound (atractylodin) in the rhizome of the micropropagated plants remained low for the first two or three years of cultivation and at the third or fourth year they reached to the same level as that of the control. Accordingly micropropagation yields clonal plants of A. lancea comparable to those obtained by conventional propagation methods within three or four years of cultivation.     

The effects of in vitro and ex vitro root initiation on subsequent microcutting root quality in three woody plants

In vitro- and ex vitro-rooted microcuttings of Acer rubrum L. Red Sunset, Betula nigra L., and Malux x- domestica Borkh McIntosh were distinguished by several important anatomical and morphological properties which continued to regulate both root system and whole plant quality in later stages of production. In vitro microcuttings formed adventitious roots in greater number and more quickly than ex vitro microcuttings. Roots produced in vitro were characterized by extremely enlarged cortical cells and, consequently, had a much greater diameter than ex vitro roots. However, the vascular system of in vitro roots was underdeveloped (primary vascular tissues only) as compared to ex vitro roots, which produced vascular cambium and secondary growth during the same early stage of production. At least 50% of the post-transplant in vitro adventitious roots either died immediately, or temporarily persisted during acclimatization without producing any further growth. For the surviving in vitro-produced roots, the cortex partially collapsed after transplant, and new root extensions with ex vitro-like structure were produced. Only then did the in vitro portion of the root begin to form secondary vascular tissues. Shoots from in vitro treatments continued to grow vigorously during adventitious root initiation and during acclimatization, so that the plants were significantly taller and had a greater shoot area than those receiving comparable ex vitro rooting treatment. In vitro rooting led to a horizontal root morphology which continued to distinguish these treatments from ex vitro rooted plants during later stages of production, when anatomical differences in the roots could no longer be detected.Abbreviations BA benzyladenine - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron - WPM woody plant medium     

Genetic modification of potato development using Ri T-DNA

Summary Forty-two potato plants were regenerated from a hairy-root line obtained after infection of a shoot of Solanum tuberosum cv Desiree with Agrobacterium rhizogenes strain LBA 9402 (pRil855). Transformed plants were uniform and had a distinct phenotype and development compared with untransformed controls. Their growth was vigorous, especially early in their development, their roots were abundant and showed reduced geotropism, their leaves were slightly crinkled and glossy and they produced longer tubers with more frequent, prominent eyes. Cytological examination showed that ten of the forty-two transformed plants had either 47 or 49 chromosomes instead of the normal 48. In two of these aneuploids structural changes were observed.     

An efficient in vitro method for mass propagation of a woody ornamentalIxora coccinea L.

Various factors that affect culture establishment, shoot growth, proliferation and rooting ofIxora coccinea L., a woody shrub, were studied. Stem cuttings (decapitated shoot, three nodes) were the most suitable explants for multiple-shoot proliferation, and when cultured on a woody plant medium (WPM) containing 2.5 M BA produced axillary shoots which branched repeatedly, yielding an average of 27 shoots per explant after 6 weeks in culture. Kinetin, 2-iP, zeatin and thidiazuron all induced multiple-shoot formation, but were less effective than BA. While the presence of IAA in the multiplication medium was detrimental to shoot proliferation, shoot growth was not affected by IAA. The production of large amounts of basal callus and vitrification of shoots were the major problems to be avoided in proliferating shoot cultures. Addition of TIBA to the multiplication medium markedly reduced basal callusing, while sealing the culture vessels with a fluorocarbon polymer (tetrafluoroethyleneperfluoroalkyl vinyl ether) film (Neoflon PFA film) almost completely eliminated vitrification. A reduction in the number of vitrified shoots was also achieved with AVG treatment. Following this protocol of using BA-supplemented WPM and Neoflon film, it would be possible to produce more than 100,000 plants from a single stem cutting in 1 year.Abbreviations AVG Aminoethoxyvinylglycine - BA N⁶-benzyladenine - BM basal medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2-tiP N⁶-(2-isopentenyl)adenine - KIN kinetin - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - SRM shoot regeneration medium - TDZ thidiazuron - TIBA 2,3,5-triiodobenzoic acid - WPM woody plant medium - ZEA zeatin     

Indole alkaloids in clonal propagules of Rauwolfia serpentina benthe ex kurz

Plantlets were succesfully regenerated from shoot cultures of Rauwolfia serpentina initiated from auxillary meristems on medium containing BA (4.44 M) + NAA (0.54 M). Rooting was initiated in White's basal medium supplemented with NAA (0.54 M). Tissue culture derived piants of R. serpentina (RSTC) were similar to normal plants (RS) in their morphological characteristics and chemical consitution. The biomass of the RSTC plants was higher (47.11 gms) than the normal plant (18.23 gms) on a dry weight basis. Five RSTC plants were cloned and the cloned plants were similar in biomass and alkaloid content to the normal plant.Abbreviations BA benzyl adenine - NAA naphthalene acetic acid - MS Murashige and Skoog - TLC thin layer chromatography - HPLC high performance liquid chromatography - F.W. fresh weight - D.W. dry weight - RS normal field grown plant established from stem cuttings - RSTC tissue culture plants established from shoot cultures - R reserpine - Aj ajmalicine - A ajmaline - S serpentine     

Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation

A total of 35 independent transgenic tobacco plants were produced using the Agrobacterium tumefaciens-leaf segment co-cultivation method followed by selection with kanamycin for the nptII gene. The vector also carried the tobacco feedback-insensitive anthranilate synthase gene (ASA2). Many of the lines showed increased ASA2 mRNA levels but only three contained increased free tryptophan (Trp) and many lines contained lower Trp than the untransformed control. The line with the highest Trp level (threefold that of the untransformed control) contained increased anthranilate synthase activity (AS) both in leaves and a cell suspension culture derived from the plant while the feedback insensitivity was most evident in the suspension culture. Other kinetic data also indicated that the ASA2 encoded AS -subunit was more abundant in the tissue culture than in leaves. Progeny seedlings from this line were resistant to certain toxic Trp analogs, especially -methyltryptophan (MT) and less so to the most commonly used analog, 5-methyltryptophan. Shoots formed more readily from leaves of two of the transgenic lines than from leaves of the untransformed control on MT, indicating that it might be possible to use ASA2 as a selectable marker gene and MT as the selection agent.     

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco

Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 mol PPFD m^-2 s^-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mol PPFD m^-2 s^-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.Abbreviations IPT isopentenyl, transferase - rbcS-3A gene encoding a small subunit protein (SSU) of Rubisco from Pisum sativum - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

The <Emphasis Type="Italic">rolB</Emphasis> gene promotes rooting <Emphasis Type="Italic">in vitro </Emphasis>and increases fresh root weight <Emphasis Type="Italic">in vivo</Emphasis> of transformed apple scion cultivar ‘Florina’

Transgenic apple (Malus × domestica Borkh.) Florina plants were obtained by Agrobacterium-mediated transformation. The efficiency of gene transfer was 7.9%, calculated as a number of explants producing at least one transgenic shoot, after co-cultivation of leaf explants from in vitro-grown shoots in a thin layer of the A. tumefaciens C58C1 strain with the binary vector pCMB-B:GUS. Polymerase chain reaction revealed that all the clones contained the nptII and rolB genes, while four of them did not contain the gus gene. Southern blot analysis confirmed the integration of the nptII and rolB genes, with one to three copies per genome being present. All independent rolB-transgenic lines were able to produce roots in vitro on the hormone free medium, while the plants, transformed with the vector pIB16.1, or untransformed control plants did not root, and only half of shoots of MM106 rootstock rooted on this medium. The average root number in the rolB-transgenic clones ranged from 4 to 7.7. Pretreatment with indole-3-butyric acid caused root formation in all transgenic and control plants and significantly increased root number in the rolB-transgenic lines, compared to untransformed plants. RolB-transgenic plants, grown in vivo in greenhouse for 2 years, did not differ phenotypically from the wild type line with the exception of root parts. All rolB-transformed plants produced altered root systems containing more fine roots leading to significantly increased fresh root weight in five plant lines.     

Plant regeneration from somatic tissue of Rhododendron laetum x aurigeranum

The influence of the source of plant material (greenhouse-grown plants or in vitro shoot cultures), the type of tissue explant (shoot-tip, single-node stem segment, whole leaf, leaf strip or half-leaf section) and growth regulator concentration on shoot regeneration from somatic tissue of Rhododendron laetum × aurigeranum was evaluated. No regeneration response was obtained on explants from greenhouse-grown plants. Adventitious shoots were obtained from callus produced at the basal end of shoot-tip and single-node stem segment explants derived from in vitro-grown shoots cultured on Anderson's medium supplemented with 22.8 M IAA and 73.8 M 2iP. The greatest percentage of adventitious shoot regeneration (77%) was induced on leaf sections cultured in the presence of 22.8 M IAA and 147.6 M 2iP. Plant regeneration was accomplished with minimal callus formation. This technique represents a further step toward gene manipulation of Rhododendron.Abbreviations IAA 1-H-Indole-3-acetic acid - 2iP N-(3-methyl-2-Butenyl)-1H-purin-6 amine     

In vitro shoot regeneration of Centranthus ruber DC from hairy roots induced by Agrobacterium rhizogenes,and determination of valepotriate content

ABSTRACT

Hairy roots were induced by infection with Agrobacterium rhizogenes strain LBA 9402 containing the plasmid 1855 from the valepotriate-producing medicinal plant Centranthus ruber. Plants were regenerated from callus derived from the hairy roots. The induction of shoot domes was obtained when hairy root calli, after a period of 3 months of dark incubation in MS (Murashige & Skoog, 1962) medium without growth regulators, were transferred for one week on the same medium under a 16-h light / 8-h dark photoperiod. Shoot regeneration increased with benzyladenine alone applied monthly at 2.5 µM concentration after the 3 months period of dark incubation. No bud formation was observed in untransformed tissues grown under the same light and hormonal conditions. Callus cultures of non-transformed plants was obtained by using leaves and roots as source material in the presence of 10.7 µM NAA and 1 µM kinetin as growth regulators, and 9 µM BA and 2.6 µM NAA for the next phase of shoot regeneration. BA and NAA together were not used for plant regeneration from transformed tissue. The morphological characteristics of the transgenic plants were analysed during two years of ex vitro growth. In the greenhouse, the transgenic plants showed pale pink flowers, heterostyly, leaves smaller than those of wild type plants, and a larger amount of roots. Roots of transgenic plants continued to produce valepotriates.     

The protein of rolB gene enhances shoot formation in tobacco leaf explants and thin cell layers from plants in different physiological states

The objective was to determine whether the protein of rolB affects shoot formation and whether this potential relationship depends on the developmental stages of the plant and/or on the culture conditions. Thin cell layers (TCL) and leaf explants were excised from tobacco plants in the vegetative and flowering stages and cultured under various hormonal conditions. In TCLs of vegetative-stage plants, the expression of rolB enhanced the formation of the shoot buds under hormone-free conditions and with specific concentrations of auxin and/or cytokinin. Histological examination showed that the induction of the shoot meristemoids was particularly enhanced by rolB protein and that meristemoid growth was accelerated. In leaf explants from vegetative-stage plants, the expression of rolB increased the formation of shoot buds in the presence of 1 M IAA plus 1 or 10 M cytokinin. With BA alone, at a 0.1 M concentration, shoot formation occurred in the transgenic explants only, whereas with concentrations ranging from 0.5 to 10 M, it was higher in these explants than in controls.RolB protein enhanced the formation of shoot buds in TCLs from flowering plants under all hormonal conditions. In the presence of 1 M IAA and kinetin, the protein also increased the flowering response. In leaf explants from flowering plants, the expression of rolB increased the number of shoot buds in the presence of 1 M IAA with 10 M BA.In conclusion, rolB protein promotes shoot formation; it seems to have a positive interaction with cytokinin and an effect on the induction of the meristematic condition.     

Miconazole: An effective antifungal agent for plant tissue culture

Miconazole at concentrations between 5 and 20 mgl^-1 inhibited hyphal growth and sporulation in a wide range of fungi commonly associated with plants. These fungi included 4 Oomycetes, 11 Deuteromycetes, 4 Ascomycetes and 4 common airborne contaminants of plant tissue culture. The phytotoxicity of 20 mgl^-1 miconazole was also tested against a wide range of in vitro plant cultures. Shoot cultures from 15 species showed either no response or a slight growth reduction in the presence of miconazole. Although the growth of shoot cultures in 2 other species was significantly reduced by miconazole, a positive growth rate was maintained. Callus and hairy root cultures from 5 species were more sensitive to miconazole than shoot cultures, although they also retained a positive growth rate in the presence of the antifungal agent. Reconstruction experiments demonstrated the effectiveness miconazole had for the rescue of plant material from in vitro cultures contaminated from fungi.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - EBI ergosterol biosynthetic inhibitor - IAA indole-3-acetic acid - MZ miconazole - NAA -naphthaleneacetic acid     

An efficient adventitious shoot regeneration system for Zhanhua winter jujube (Zizyphus jujuba Mill.) using leaf explants

The direct induction of adventitious shoots from leaf explants obtained from adult plants of Zhanhua winter jujube, an elite variety of Zizyphus jujuba Mill., is reported. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when 10-day-old leaves were explanted onto Woody Plant Medium and maintained initially in the dark. The plant growth regulator thidiazuron (TDZ) was effective in stimulating shoot regeneration from leaf explants of Zhanhua winter jujube. The highest efficiency of shoot formation was observed with a 20-day culture in the dark on WPM containing 4.54 M TDZ and 2.85 M indoleacetic acid (IAA). The regenerated shoots were transferred to MS medium containing 0.89 M benzyladenine and 5.77 M gibberellic acid for growth. When the shoots were about 2 cm in height, they were transferred to Nitsch medium supplemented with 1.14 M IAA and 2.46 M indolebutyric acid to induce rooting. This system of adventitious shoot production from leaf explants of adult plants could be useful for the genetic engineering and polyploidization of winter jujube.