Showing papers on "Zeatin published in 2011"

Evolution of cytokinin biosynthesis and degradation

Abstract: Cytokinin hormones are important regulators of development and environmental responses of plants that execute their action via the molecular machinery of signal perception and transduction. The limiting step of the whole process is the availability of the hormone in suitable concentrations in the right place and at the right time to interact with the specific receptor. Hence, the hormone concentrations in individual tissues, cells, and organelles must be properly maintained by biosynthetic and metabolic enzymes. Although there are merely two active cytokinins, isopentenyladenine and its hydroxylated derivative zeatin, a variety of conjugates they may form and the number of enzymes/isozymes with varying substrate specificity involved in their biosynthesis and conversion gives the plant a variety of tools for fine tuning of the hormone level. Recent genome-wide studies revealed the existence of the respective coding genes and gene families in plants and in some bacteria. This review summarizes present knowledge on the enzymes that synthesize cytokinins, form cytokinin conjugates, and carry out irreversible elimination of the hormones, including their phylogenetic analysis and possible variations in different organisms.

Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants

Abstract: Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis↔trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.

Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphylla

Abstract: Shoot regeneration and occurrence of hyperhydricity in Aloe polyphylla were greatly affected by the type of gelling agent The use of gelrite resulted in a significantly lower multiplication and almost four times higher hyperhydricity (65%) compared to agar-solidified medium Gelrite was further selected to evaluate if hyperhydricity can be overcome by altering the physical properties of the gel, as represented by increasing gelrite concentrations Four concentrations of gelrite (0, 24, 6 and 16 g l−1) were tested in combination with zeatin, N6-benzyladenine (BA) or thidiazuron (TDZ) Almost all explants grown in liquid media in the presence of cytokinins became hyperhydric and lost their ability to regenerate The greatest shoot formation was obtained on media with 24 g l−1 gelrite and 5 μM zeatin or BA, however hyperhydricity was very high Satisfactory reduction in hyperhydricity was achieved only at 16 g l−1 gelrite, under which conditions the multiplication also decreased The use of TDZ resulted in very low shoot regeneration and high hyperhydricity irrespective of the gelrite concentration

Release of Hormones from Conjugates: Chloroplast Expression of β-Glucosidase Results in Elevated Phytohormone Levels Associated with Significant Increase in Biomass and Protection from Aphids or Whiteflies Conferred by Sucrose Esters

Abstract: Transplastomic tobacco (Nicotiana tabacum) plants expressing β-glucosidase (Bgl-1) show modified development. They flower 1 month earlier with an increase in biomass (1.9-fold), height (1.5-fold), and leaf area (1.6-fold) than untransformed plants. Trichome density on the upper and lower leaf surfaces of BGL-1 plants increase by 10- and 7-fold, respectively, harboring 5-fold more glandular trichomes (as determined by rhodamine B staining), suggesting that BGL-1 lines produce more sugar esters than control plants. Gibberellin (GA) levels were investigated because it is a known regulator of flowering time, plant height, and trichome development. Both GA1 and GA4 levels are 2-fold higher in BGL-1 leaves than in untransformed plants but do not increase in other organs. In addition, elevated levels of other plant hormones, including zeatin and indole-3-acetic acid, are observed in BGL-1 lines. Protoplasts from BGL-1 lines divide and form calli without exogenous hormones. Cell division in protoplasts is enhanced 7-fold in the presence of exogenously applied zeatin-O-glucoside conjugate, indicating the release of active hormones from their conjugates. Whitefly (Bemisia tabaci) and aphid (Myzus persicae) populations in control plants are 18 and 15 times higher than in transplastomic lines, respectively. Lethal dose to kill 50% of the test population values of 26.3 and 39.2 μg per whitefly and 23.1 and 35.2 μg per aphid for BGL-1 and untransformed control exudates, respectively, confirm the enhanced toxicity of transplastomic exudates. These data indicate that increase in sugar ester levels in BGL-1 lines might function as an effective biopesticide. This study provides a novel strategy for designing plants for enhanced biomass production and insect control by releasing plant hormones or sugar esters from their conjugates stored within their chloroplasts.

Peranan Zat Pengatur Tumbuh dalam Perbanyakan Tanaman melalui Kultur Jaringan

Abstract: The Role of Growth Regulator in Tissue Culture Plant Propagation. Endang G. Lestari. In plant tissue culture, growth regulator has significant roles such as to control root and shoot development in the plant formation and callus induction. Cytokinin and auxin are two prominent growth regulator. Cytokinin consists of BA (benzil adenin), kinetin (furfuril amino purin), 2-Ip (dimethyl allyl amino purin), and zeatin. While auksin covers IAA (indone acetic acid), NAA (napthalene acetic acid), IBA (indole butiric acid) 2.4-D (2.4- dicholophenoxy acetic acid), dicamba (3,6 dicloro-O-anisic acid), and picloram (4-amino 3,5,6-tricloropicolinic acid). The emphasis of plant growth purposes decide the use of growth regulator. Cytokinin is applied mainly for the purpose of shoot, while auxin is mainly used for the purpose of root and callus. The application of growth regulator application is varied, depending on the genotype and physiological condition of the plant. The existence of a certain growth regulating substances can enhance growth regulator activity of other substances. The type and concentration of the appropriate growth regulators for each plant is not the same because it depends on the genotype and physiological condition of plant tissue. However so often both are frequently required depend on the ratio/ratio of auxin cytokines or vice versa. The existence of a certain growth regulating substances can enhance growth regulator activity of other substances. The type and concentration of the appropriate growth regulators for each plant is not the same because it depends on the genotype and physiological condition of plant tissue. For the propagation, multiple and adventive shoots along with embriosomatic formation could be applied. The seedling is obtained from one somatic cell. Here, strong auxin, such as dicamba and picloram 2.4-D, is utilized for callus production. For this reason, seedling per unit could be produced more than that of organogenesis.

Auxins or Sugars: What Makes the Difference in the Adventitious Rooting of Stored Carnation Cuttings?

Abstract: Cold storage of cuttings is frequently applied in the vegetative propagation of ornamental plants. Dianthus caryophyllus was used to study the limiting influences of auxin and sugars on adventitious root formation (ARF) in cuttings stored at 5°C. Carbohydrate levels during storage were modulated by exposing cuttings to low light or darkness. The resulting cuttings were treated (or not) with auxin and planted, and then ARF was evaluated. Carbohydrate levels in the cuttings were monitored and the influence of light treatment on indole-3-acetic acid (IAA) and zeatin (Z) in the basal stem was investigated. Dark storage for up to 4 weeks increased the percentage of early rooted cuttings and the final number and length of adventitious roots, despite decreased sugar levels in the stem base. Light during cold storage greatly enhanced sugar levels, particularly in the stem base where the Z/IAA ratio was higher and ARF was lower than observed in the corresponding dark-stored cuttings. Sugar levels in nonstored and dark-stored cuttings increased during the rooting period, and auxin application enhanced the accumulation of sugars in the stem base of nonstored cuttings. Auxin stimulated ARF most strongly in nonstored, less so in light-stored, and only marginally in dark-stored cuttings. A model of auxin-sugar interactions in ARF in carnation is proposed: cold storage brings forward root induction and sink establishment, both of which are promoted by the accumulation of auxin but not of sugars, whereas high levels of sugars and probably also of cytokinins act as inhibitors. Subsequent root differentiation and growth depend on current photosynthesis.

Effects of Nitrogen and 6-Benzylaminopurine on Rice Tiller Bud Growth and Changes in Endogenous Hormones and Nitrogen

Abstract: In the present study, 40 mg L -1 N (NH treatment) and 20 mg L -1 6-benzylaminopurine (6-BA; BA treatment) were used to stimulate tiller bud growth in rice (Oryza sativa L.). The hormone changes in the tiller nodes and tiller buds and the N levels in the leaf blades, leaf sheaths, and tiller nodes were measured. The results indicate that N and 6-BA promoted tiller bud germination and increased the indole-3-acetic acid (IAA) and zeatin (Z) plus zeatin riboside (ZR) levels in tiller buds and tiller nodes but decreased the abscisic acid (ABA) levels in tiller buds. Before tiller bud germination, the IAA and Z plus ZR levels increased but the ABA did not change, suggesting that IAA and Z plus ZR but not ABA may be the key regulators of tiller bud growth. From 4 d posttreatment, the tiller bud growth of the BA-treated plants markedly slowed but the buds of the NH-treated plants continued to grow quickly. We also found that the N levels in the leaf blades, leaf sheaths, and tiller nodes of the NH-treated plants increased significantly as compared to those of the control plants (the plants treated with 10 mg L -1 N). However, no significant differences in N levels were observed between the plants of BA and control treatments. This study suggests that N may regulate tiller bud growth in two ways: by regulating N metabolism and regulating endogenous hormones. External 6-BA promoted tiller bud germination by regulating endogenous hormones, but tiller bud growth was not maintained because it could not increase the N levels in the plants.

Interactions of bacterial cytokinins and IAA in the rhizosphere may alter phytostimulatory efficiency of rhizobacteria

Abstract: Phytohormones from rhizobacterial origin have been linked to their phytostimulation potential. However, while studying the efficacy of plant growth promoting bacteria, focus has always been on a single hormone. The role of plant hormones often overlay and they mutually modulate their effect. In current study focus was on the role of two hormones (cytokinins and indole acetic acid) in phytostimulation by rhizobacteria. Endogenous rhizosphere bacteria were isolated and screened for the presence of phytohormones. Bacterial strains from three different genera (Pseudomonas, Bacillus and Azospirillum) were screened positive for cytokinins and IAA. Phytohormones were simultaneously determined in SPE purified bacterial extract by ultra performance liquid chromatography (UPLC) coupled to a tandem mass spectrometer through electrospray interface. Cytokinins and IAA were determined in positive and negative mode, respectively with MRM scan. Zeatin, zeatin riboside and dihydrozeatin riboside were detected and quantified in the selected strains. Significant positive correlation between cytokinins and IAA in bacterial culture and plant endogenous hormones (r = 0.933 and r = 0.983; P = 0.01, respectively) was observed. However, strains with high IAA to cytokinins ratio could hardly enhance in-planta cytokinins, indicating antagonistic relation between the two hormones. Significant correlation of cytokinin with shoot length (r = 0.797; P = 0.01), fresh weight (r = 0.685; P = 0.01) and dry weight (r = 0.704; P = 0.01) was reported under axenic conditions. Bacterial IAA was correlated negatively to root length (r = 0.853; P = 0.01) and positively correlated to the number of roots (r = 0.964; P = 0.01). In natural conditions maximum increase in spike length (33%), number of tillers (71%) and weight of seeds (39%) was documented at final harvest in bacterially inoculated plants.

Cytokinin and explant types influence in vitro plant regeneration of Leopard Orchid ( Ansellia africana Lindl.)

Abstract: The effects of explant and cytokinin types on in vitro plant regeneration of Ansellia africana were investigated. The exogenous addition of cytokinins is not required for the proliferation of new protocorms from Trimmed protocorm cluster (TPC) explants. To the contrary, nodal and shoot-tip explants produced a single shoot in response to the addition of cytokinins. Overall plant growth in terms of shoot length, leaf number, frequency of root organogenesis, root length, and fresh weight/plant were significantly higher in media containing meta-Topolin Riboside (mTR) in both nodal and shoot-tip explants. Thidiazuron (TDZ) and 6-benzyladenine (BA) induced stunted and hypertrophied shoots at their highest level (15 μM). In addition root differentiation and root growth were significantly lower on P668 media with TDZ and BA. Zeatin was capable of inducing a significantly higher root organogenesis frequency and root length in TPC explants as compared to other cytokinins. However, TPC explants produced a significantly greater number of longer shoots (>3 cm) on P668 media with mTR. Hyperhydric shoots were produced from TPC explants. The occurrence of hyperhydricity is discussed with respect to the culture vessel used in this study.

EFFECTS OF PACLOBUTRAZOL (PBZ) ON FLORAL INDUCTION AND ASSOCIATED HORMONAL AND METABOLIC CHANGES OF BIENNIALLY BEARING MANGO (Mangifera indica L.) CULTIVARS DURING OFF YEAR

Abstract: ABSTARCT The physiology of floral induction in mango is still controversial and thus further work is needed for a better understanding of reproductive physiology of this important fruit tree. The objectives of this study were to investigate the role of a gibberellins biosynthesis inhibitor, paclobutrazol on floral induction of biennially bearing mango during off year and to examine the possible correlated hormonal and non-structural carbohydrate changes. Three distinctly biennially bearing mango cultivars were tested for two years under North Sudan climates. Results indicated the advantage of paclobutrazol on inducing flowering of the biennially bearing mango cultivars, Miska, Mahmoudi, and Totocombo during off year. Similar trends of hormonal changes were observed during the floral induction period on the tested cultivars. More-specifically, the levels of cytokinins (zeatin (z) + zeatin riboside (zr) and isopentenyl Adenosine (i-Ado) + isopentenyl Adenine (i-Ade)), and to a less extent the levels of abscisic acid (ABA) generally showed trends of increase during the floral induction period, while those of gibberellins (GA1+3+20) and auxin (IAA) were decreased during the same period. Starch levels in most of the cases were increased by the paclobutrazol treatment. Moreover, sucrose levels were generally increased during the floral induction period. To close, possible roles for some of the tested hormones and nonstructural carbohydrates on mango flowering are probably implicated.

Shoot regeneration from in vitro-derived leaf and root explants of Centaurea ultreiae

Abstract: In vitro culture is currently used to produce plant material for ex situ conservation of endangered species. In this study, an efficient protocol for shoot regeneration from leaves and roots was developed for Centaurea ultreiae, a critically endangered species. Organogenesis from leaf and root explants was promoted by incubating these explants on half-strength Murashige and Skoog (MS) medium in the presence of one of four different cytokinins [6-benzyladenine (BA), zeatin, kinetin or N6-(2-isopentenyl) adenine (2iP)], each provided at five different levels. Shoot organogenesis was induced in both explants. The best response, 90% of leaf explants producing a mean of 2.48 shoots per explants and 94.3% of root explants producing a mean of 5.60 viable shoots per explants, was observed when explants were incubated on a medium containing 0.55 μM BA. Histological studies revealed connectivity between vascular tissues of regenerated shoots and cambial cells of leaf explants. Moreover, adventitious shoots were derived from pericycle cells of root explants and parenchymatic cells of callus tissues.

A Simple and Efficient Protocol for Plant Regeneration and Genetic Transformation of Tomato cv. Micro-Tom from Leaf Explants

Abstract: A simplified protocol to obtain transgenic tomato plants was established. The effects of culture media composition and Agrobacterium concentration were evaluated. The highest shoot-forming capacity index (5.6) was observed when leaf explants were cultured for 6 weeks with 2 mgL -1 zeatin, 0.1 mgL -1 indoleacetic acid, and 300 mgL -1 timentin. Shoot elongation and root formation were performed in one step on growth regulator-free media. The highest percentage (82%) of fully developed plantlets was obtained when shoots were cultured for 4 weeks with 0.5· Murashige and Skoog (MS) media and 15 gL -1 sucrose. A 100% of plant survival rate was observed after 4 weeks of being transplanted to ex vitro conditions followed by fruit production (15 fruits/plant) after 2 more weeks. Transient expression of b-glucuronidase was visualized in 100% of the leaf explants infected with Agrobacterium at an OD600 = 0.5 and cocultured for 48 h with 2 mgL -1 benzylaminopurine, 0.1 mgL -1 naphthaleneacetic acid, and 100 mM acetosyringone. Stable transformation was confirmed by histochemical glucuronidase assay and polymerase chain reaction (PCR) analysis with a total efficiency of 19.1%. The complete protocol, from shoot induction to fruit production of soil-adapted transgenic plants can be accomplished in only 4 months, and it seems to be very useful for both micropropagation and genetic transformation purposes.

Somatic embryogenesis and shoot organogenesis in the medicinal plant Pulsatilla koreana Nakai

Abstract: We have developed a system for the in vitro regeneration of pasqueflowers (Pulsatilla koreana Nakai). The system was based on somatic embryogenesis and shoot organogenesis. Over a growth period of 6 weeks, multiple shoots were initiated from leaf, petiole, and pedicel explants on Murashige and Skoog (MS) medium containing 0.5 mg l−1 indole-3-acetic acid (IAA) and zeatin (Zn), kinetin (Kin), or 6-benzyladenine (BA). We achieved 100% of adventitious shoot induced when petiole and pedicel explants were cultured on MS, 0.5–2.0 mg l−1 Zn, and 0.5 mg l−1 IAA. Somatic embryos developed from the explants and generated shoots on MS medium containing 0.25 mg l−1 Zn and 0.5 mg l−1 IAA. Globular and heart-shaped stages of somatic embryos were observed. Histological studies have revealed the stages of development of somatic embryos. For propagation and growth, the regenerated shoots from organogenic or embryogenic calluses were transferred to MS medium containing either (1) 1.5 mg l−1 Zn and 0.05 mg l−1 IAA or (2) 1.0 mg l−1 BA and 0.05 mg l−1 IAA. After the length of the shoots reached 3 cm, the shoots initiated by organogenesis as well as those initiated by somatic embryogenesis were transferred to the root induction medium. After 2 months of culture in half-strength MS with 1.5 mg l−1 α-naphthalene acetic acid (NAA), the rooting ratio was 93%. Finally, the rooted plantlets were acclimatized in a mixture of mountain soil and perlite.

In vitro shoot multiplication and conservation of Caralluma bhupenderiana Sarkaria - an endangered medicinal plant from South India

Abstract: An efficient protocol was described for the rapid in vitro multiplication of an endangered medicinal plant, Caralluma bhupenderiana Sarkaria, via enhanced axillary bud proliferation from nodal explants collected from young shoots of six-months-old plant. The physiological effects of growth regulators [6- Benzyladenine (BA), kinetin (Kn), 2-Isopentyl adenine (2iP), zeatin (Zn), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA)], different strengths of Murashige and Skoog (MS) medium and various pH levels on in vitro morphogenesis were investigated. The highest number (8.40 + 0.50) of shoots and the maximum average shoot length (3.2 ± 0.31 cm) were recorded on MS medium supplemented with BA (8.87 μM) at pH 5.8. Rooting was best achieved on half-strength MS medium augmented with NAA (2.69 μM). The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing peat mass and garden manure in 1:1 ratio and grown in a greenhouse with 80% survival rate. The regenerated plants did not show any immediate detectable phenotypic variation. Key words: Apocynaceae, asclepiadoideae, conservation, micro-propagation, endangered, Caralluma bhupenderiana.

Direct regeneration from in vitro leaf and petiole tissues of Populus tremula ‘Erecta’

Abstract: Dormant buds from a mature tree of Populus tremula ‘Erecta’ were incubated on a Murashige and Skoog (MS) medium supplemented with 10 μM thidiazuron (TDZ) Induced shoots were then proliferated on medium of MS or Woody Plant Medium (WPM), or Driver and Kuniyuki Walnut (DKW) supplemented with varying levels of benzyladenine (BA) Overall, shoots grown on MS medium supplemented with 125–25 μM BA exhibited the highest frequency of shoot proliferation (>95%) and more than 60% of responding explants produced more than five shoots per explant Shoot organogenesis was induced from both leaf and petiole explants incubated on WPM medium containing BA, or TDZ, or zeatin Among the different cytokinins tested, zeatin induced the highest frequency (average 721%) of shoot organogenesis None of explants survived on media containing no cytokinins within 6–8 weeks following culture Overall, a higher frequency of shoot regeneration was obtained from petioles than from leaf explants The highest frequency of regeneration was achieved when petioles were incubated on WPM containing 10–20 μM zeatin Addition of naphthaleneacetic acid (NAA) did not have a significant effect on shoot regeneration in all treatments Shoot organogenesis was directly induced from petiole explants without intervening callus Regenerated shoots were easily rooted on all tested media supplemented with 05 μM NAA Rooted plants were transferred to potting mix and grown in the greenhouse

Direct shoot regeneration from leaf explants of Digitalis lamarckii, an endemic medicinal species

Abstract: Th is study reports, for the fi rst time, an effi cient in vitro plant regeneration protocol for Digitalis lamarckii Ivan. (dwarf foxglove) via direct shoot organogenesis. Two sets of experiments were carried out; the fi rst compared diff erent concentrations of 6-benzylaminopurine (BAP), kinetin, thidiazuron (TDZ), and zeatin alone using leaf explants excised from in vitro germinated seedlings, while the second set tested the combinations of indole-3-butyric acid (IBA) with BAP, kinetin, TDZ, and zeatin for shoot multiplication from the leaf explants, which were already cultured and developed numerous shoots during the fi rst set of experiments. For shoot regeneration (the fi rst set of experiments), TDZ was the most eff ective at 1.0 mg L -1

Influencing factors and structural characterization of hyperhydricity of in vitro regeneration in Brassica oleracea var. italica

Abstract: Yu, U, Zhao, Y-Q, Zhao, B, Ren, S and Guo, Y-D 2011 Influencing factors and structural characterization of hyperhydricity of in vitro regeneration in Brassica oleracea var italica Can J Plant Sci 91: 159–165 This study examines factors that affect the occurrence of hyperhydric tissue in in vitro cultures of Brassica oleracea variety italica The anatomy of normal and hyperhydric leaves of plantlets regenerated from the hypocotyls was compared using scanning electron microscopy and transmission electron microscopy In hyperhydric leaves palisade tissue was absent and the spongy mesophyll displayed large, unorganized intercellular spaces Hyperhydric leaves had abnormal stomata with deformed guard cells Significant ultrastructural differences were observed between chloroplasts in normal and hyperhydric leaves The effects of zeatin, indoleacetic acid, silver nitrate and sucrose on the formation of hyperhydric shoots were studied Zeatin was the most important factor, followed by sucrose conce

Somatic embryogenesis in cell suspension cultures of olive Olea europaea (L.) ‘Chetoui’

Abstract: Somatic embryogenesis of olive Olea europaea (L.) ‘Chetoui’ was studied using cell suspension cultures initiated from mature leaf-derived calli. Calli were developed on half-strength MS medium supplemented with 10 μM NAA and 2.25 μM 2i-P in the dark. Different combinations of three plant growth regulators (2,4-D, NAA and zeatin) were tested to determine cell proliferation and somatic embryogenesis induction and differentiation. Embryogenic suspension cultures were established in olive-modified medium for embryogenesis (OMe) containing 2.5 μM 2,4-D and 2.5 μM zeatin. Pre-globular and globular embryos were induced from mature olive tissue in liquid medium. In addition, the nitrogen form as inorganic (reduced; (NH4)2SO4 or oxidized; KNO3) and organic (CH) was used separately or in combination to improve the cell growth and proliferation. The most effective growth rate and cell proliferation were obtained with the medium containing inorganic and organic nitrogen forms.

In vitro Production of Benzylisoquinoline from Stephania tetrandra Through Callus Culture under the Influence of Different Additives

Abstract: Plant secondary metabolites have enormous potential for research and new drug development. Many secondary metabolites have complex and unique structure and their production can be enhanced by introducing different types of additives into the basal media. An efficient in vitro callus induction system in Stephania tetrandra S. Moore (Fan fang ji, an important Chinese medicinal herb) was established on MS medium supplemented with 3% sucrose and different concentrations (0.5 to 2.0 mg/L) of plant growth regulators i.e. auxins (2,4D, IAA and NAA) and cytokinins (BA, kinetin, TDZ and zeatin) in the dark. The effects of various auxins and cytokinin on the growth and accumulation of benzylisoquinoline alkaloids i.e. Fangchinoline (Fan) and Tetrandrine (Tet) were investigated. MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L TDZ supported callus growth and its proliferation. A maximum amount of dry biomass (7.8 fold) was produced 45 days after culture. High Performance Liquid Chromatographic analysis of methanol extracts from callus revealed an accumulation of Fan and Tet alkaloids. The addition of casein hydrolysate (500 mg/L) and coconut milk (10%), respectively, enhanced Fan and Tet accumulation. The established in vitro callus induction system in S. tetrandra can be utilized for biomass production of pharmaceutically important alkaloids such as Fan and Tet.

Free endogenous growth regulators in Pistachio (Pistacia vera L.)

Abstract: This research was carried out in order to observe seasonal changes of internal plant growth regulators in pistachio. This research was conducted during three years at a commercial pistachio orchard located in FA±rat valley in Nizip, Gaziantep. ‘KA±rmA±zA±’ pistachio cultivar trees grafted on Pistacia vera L. were used as plant material. Changes in auxin (IAA), cytokinin (Zeatin), gibberellin (GA3) and abscisic acid (ABA) were observed in leaves in May, July and September of the first (heavy cropping year - 'on'-year) and second (subsequent light cropping year ' off '-year) years. Besides, yield and flower bud abscission rate were observed during three years to determine the relationships among the level of plant growth regulator, yield and flower bud abscission. It was observed that the IAA, zeatin and ABA levels in the leaf were high in the first year, and was low in the second year. The annual GA3 levels were lower in the ‘on’ year and higher in the alternate bearing, ‘off’ year. The correlation analysis between the hormones and fruit bud abscission rates showed that increase in the ABA levels caused also increase in fruit bud abscission at all months in the first year. Key words: Auxin, zeatin, gibberellic acid, abscisic acid, pistachio.

Mixture screening and mixture-amount designs to determine plant growth regulator effects on shoot regeneration from grapefruit ( Citrus paradisi macf.) epicotyls

Abstract: The objective of this study was to improve shoot regeneration from grapefruit. Because many commercially grown citrus types are apomictic, important in vitro applications such as Agrobacterium-mediated transformation commonly use epicotyl explants from in vitro seedlings; thus, adequate adventitious shoot production is an important prerequisite for efficient use of these applications. Eight plant growth regulators were studied—six cytokinins (6-benzylaminopurine, kinetin, zeatin trans-isomer, 6-[γ,γ-dimethylallylamino] purine, zeatin riboside trans-isomer and meta-topolin) and two auxins (α-naphthalene acetic acid and indole-3-acetic acid). An iterative design strategy was followed that included mixture and mixture-amount experimental designs suitable for resolving proportional and concentration effects; in vitro effects of cytokinins and auxins are affected by both proportion and concentration. One-centimeter-long explants were excised from the epicotyl of etiolated, in vitro-grown seedlings. Explants were placed onto experimental formulations and cultured in growth cabinets at 27°C over 6 wk, which included 2 wk in the dark followed by 4 wk in the light. The results indicated that (1) 6-benzylaminopurine or zeatin riboside were the most effective cytokinins for inducing shoot regeneration in citrus; (2) zeatin riboside singly or in combination with indole-3-acetic acid resulted in the highest quality, the greatest number of explants with buds/shoots, and the greatest shoot number; and (3) 6-benzylaminopurine and indole-3-acetic acid improved shoot regeneration vs. 6-benzylaminopurine at a considerably lesser cost than zeatin riboside and indole-3-acetic acid.

In vitro culturing and assessment of somaclonal variation of Solanum tuberosum var. desiree

Abstract: Aim: Genetic variability frequently occurs in micropropagated plants. In the present study, random amplification of polymorphic DNA technique is used to assess somaclonal variation in Solanum tuberosum var. desiree. Detection of somaclonal variants at an initial phase of growth can be valuable in establishing better tissue culture and transformation system in potato by quality control. Materials and Methods: Callus culturing conditions optimized at different hormonal concentrations. Three growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and zeatin were used in different concentrations and combinations. Maximum callus regeneration (90%) was observed at 2.5 mg/L 2,4-D while minimum growth was monitored at 1.5 mg/L Zeatin and 1.0 mg/L BAP combination. Potato plant was also regenerated from callus cells with best results on Murashige and Skoog basal media supplemented with both 1.0 mg/L BAP and 1.5 mg/L Indole acetic acid. Genetic variability in in vitro cultured callus at different hormonal concentrations was assessed by using random amplification of polymorphic DNA technique. Results: Ten different decamer oligonucleotide primers generated 111 reproducible amplified products. The similarity coefficient values calculated through Simqual subprogram of numerical taxonomy system of multivariate software ranged from 0.419-0.838. Cluster analysis divided five samples into two groups, an in group and an out group. Maximum polymorphic bands were revealed by using hormonal combination Zeatin 1.5 mg/L and BAP 1.0 mg/L in calli cultures. Conclusion: Results indicate that random amplification of polymorphic DNA is an effective technique for the assessment of genetic variability in in vitro cultured calli.

Effects of cytokinins on secondary somatic embryogenesis of selected clone Rayong 9 of Manihot esculenta Crantz for ethanol production

Abstract: In 2005, the Rayong-FCRC released a new high-yielding cassava cultivar designated Rayong 9 (R9) for ethanol production. However, the rate of distribution to farmers has been limited by the traditional vegetative propagation method of this crop which does not always satisfy the needs in planting material. The objective was to improve secondary somatic embryogenesis of the cassava clone Rayong 9 (R9) selected in Thailand by the Rayong Field Crops Research Center (Rayong-FCRC) for its suitability to produce ethanol. Fragments of cotyledon-stage somatic embryos were subcultured onto MS medium supplemented with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) at 4 mg/l in sight of inducing secondary somatic embryogenesis. Five different cytokinins, 6-benzylaminopurine (BAP), kinetin, zeatin, isopentenyladenine (2-iP) and adenine were added at 1 mg/l to the induction and maturation media to test their efficiency. Onto cytokinin-free media, 70 to 80% of explants produced embryoids, each explant giving 2 to 4 new embryoids within 7 weeks. The conversion rate of the embryoids into plantlets ranged from 11 to 26% depending on the type of cytokinin. With the exception of adenine, the other cytokinins inhibited the intensity of somatic embryogenesis, by 75% in the case of zeatin and 30% in the case of kinetin. Addition of adenine did not significantly improve the number of embryoids per explant. However, at 10, 20 and 40 mg/l adenine tended to improve the process relatively to embryoid sizes and plantlet survival rates in the greenhouse. Key words: Adenine, biofuel, cassava, cotyledonary-stage, embryoids, icrocuttings, micropropagation.

Influencing factors and structural characterization of hyperhydricity of in vitro regeneration in Brassica oleracea var. italica

Abstract: Yu, U., Zhao, Y.-Q., Zhao, B., Ren, S. and Guo, Y.-D. 2011. Influencing factors and structural characterization of hyperhydricity of in vitro regeneration in Brassica oleracea var. italica. Can. J. Plant Sci. 91: 159-165. This study examines factors that affect the occurrence of hyperhydric tissue in in vitro cultures of Brassica oleracea variety italica. The anatomy of normal and hyperhydric leaves of plantlets regenerated from the hypocotyls was compared using scanning electron microscopy and transmission electron microscopy. In hyperhydric leaves palisade tissue was absent and the spongy mesophyll displayed large, unorganized intercellular spaces. Hyperhydric leaves had abnormal stomata with deformed guard cells. Significant ultrastructural differences were observed between chloroplasts in normal and hyperhydric leaves. The effects of zeatin, indoleacetic acid, silver nitrate and sucrose on the formation of hyperhydric shoots were studied. Zeatin was the most important factor, followed by sucrose concentration, AgNO3 and indoleacetic acid. The process of hyperhydricity was found to be reversed by increasing the agar concentration and eliminating NH4NO3 from the macro-elements in the MS medium. This is the first report of hyperhydricity in Brassica oleracea, and our study gives a better understanding of the factors that influence hyperhydricity during in vitro regeneration in Brassica crops.

Rapid and efficient plant regeneration of eggplant ( Solanum melongena L.) from cotyledonary leaf explants

Abstract: The aim of this study was to develop an efficient protocol for establishment of plant regeneration through cotyledonary leaf explants of Eggplant ( Solanum melongena L.). Two varieties of brinjal [Pusa purple long (PPL) and Black beauty (BB)] were used. High frequency and rapid regeneration protocol was developed from cotyledonary leaf explants on MS medium supplemented with either 6-Benzyl amino purine (BAP), Kinetin (Kn), Thidiazuron (TDZ) or Zeatin (Ze). The highest number of shoots (23.3±0.10) was obtained on MS medium containg 2.0 mg/BAP+0.5 mg/L Kn. The in vitro regenerated small shoots were further elongated on MS medium supplemented with gibberellic acid (GA 3) at 1.5 mg/L. Elongated shoots were then excised from shoot clumps and transferred to rooting medium containg indole butyric acid (IBA) at 3.0 mg/L. The rooted plantlets were hardened on MS basal liquid medium and subsequently transferred to polycups containg vermiculate:soil:sand (1:2:2). Plantlets, thus developed were successfully established and finally transferred to a greenhouse. The plantlets showed high survival rate (80%) in the soil.

Effect of cytokinin type and concentration on in vitro shoot proliferation of hazelnut (Corylus avellana L.)

Abstract: Corylus avellana L. (hazelnut) is traditionally multiplied by layering because it is difficult to propagate vegetatively using shoot cuttings or grafting. Over the past two decades, in vitro micropropagation has become a successful technique for rapidly building large numbers of many new plant cultivars, but success with hazel remains mixed. The limiting factor for hazel has been low rates of in vitro shoot proliferation. Our study of hazel cultivar ‘Daviana’ examined the response of shoot multiplication to different cytokinin types and concentrations with the aim of maximizing new plant yields for commercial benefit. Four cytokinins (BAP, kinetin, zeatin, IPA) were evaluated at three concentrations. BAP at 5 mg/L (22.2 µM) was effective for initiation and elongation of new shoots when used with ‘Knoxfield2’ medium formulated for hazel. New shoot initiation was lowest when the medium contained kinetin.

Changes in cytokinin levels and metabolism in tobacco (Nicotiana tabacum L.) explants during in vitro shoot organogenesis induced by trans-zeatin and dihydrozeatin

Abstract: The uptake and metabolism of trans-zeatin and/or dihydrozeatin, in correlation with cytokinin oxidase/dehydrogenase (CKX) and β-glucosidase activity, were studied in leaf segments derived from wild-type (WT) and transgenic (T) tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) during in vitro induction of shoot organogenesis. T explants harbored the maize gene Zm-p60.1β-glucosidase. Higher levels of shoot regeneration were observed on T explants in the early stages of cultivation. In WT explants, the content of cytokinin (CK)-O- and N-glucosides increased. In T explants, a higher content of Z-9-riboside and Z-9-riboside-5′-monophosphate and higher CKX activity during the early stage of cultures were found. A positive correlation was obtained for bioactive CK content and the organogenic response in T explants. Our results indicate a connection between the organogenic capacity of tobacco explants, metabolism of endogenous CKs and uptake of exogenous CKs from the cultivation medium.