Showing papers on "Zeatin published in 2015"

The effect of cytokinins on shoot proliferation, secondary metabolite production and antioxidant potential in shoot cultures of Scutellaria alpina

Abstract: The present study evaluates the effects of various cytokinins on Scutellaria alpina shoot proliferation and production of polyphenolic metabolites (baicalin, wogonoside, luteolin, luteolin 7-O-glucoside, verbascoside). The shoots were induced from shoot tips on MS medium supplemented with IAA (indole-3-acetic acid, 0.57 µM) and various concentrations of 6-benzylaminopurine (BAP), kinetin, zeatin (1, 2, 4, 8 µM) or tidiazuron (TDZ) (0.2, 0.5, 1 µM). Among the cytokinins tested, BAP was the most effective for shoot induction, and the highest number of shoots (25 per explant) was achieved with 2 and 4 µM BAP. Maximum biomass production was also achieved on these media. Significantly higher baicalin, wogonoside and verbascoside contents were recorded in treatments containing cytokinins combined with 0.57 µM IAA, when compared to cytokinin-free medium. TDZ at a concentration of 0.5 µM was the most effective for polyphenol production. However, supplementation with cytokinins often results in the reduction of luteolin and its 7-O-glucoside production in the shoot culture of S. alpina. ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] and ferric reducing antioxidant power assays were used to identify the antioxidant potential of methanolic extracts from shoots cultured in the presence of different types and concentrations of cytokinins. In both tests, the shoots from medium supplemented with 0.5 µM TDZ demonstrated the strongest antioxidant activity. The results indicate that higher polyphenolic content correlated with greater reducing power and antiradical efficiency.

Hormonal requirements for effective induction of microspore embryogenesis in triticale (× Triticosecale Wittm.) anther cultures

Abstract: Effective microspore embryogenesis in triticale is determined by a specific hormonal homeostasis: low value of IAA/cytokinins, IAA/ABA and cytokinins/ABA ratios as well as proper endogenous/exogenous auxin balance, which favours androgenic structure formation and green plant regeneration ability. The concentration of plant growth regulators (PGRs): auxins (Auxs), cytokinins (CKs) and abscisic acid (ABA) was measured in anthers of eight DH lines of triticale (× Triticosecale Wittm.), and associated with microspore embryogenesis (ME) responsiveness. The analysis was conducted on anthers excised from control tillers at the phase optimal for ME induction and then after ME-initiating tillers treatment (21 days at 4 °C). In control, IAA predominated among Auxs (11–39 nmol g−1 DW), with IBA constituting only 1 % of total Auxs content. The prevailing isoforms of CKs were cis isomers of zeatin (121–424 pmol g−1 DW) and zeatin ryboside (cZR, 146–432 pmol g−1 DW). Surprisingly, a relatively high level (10–64 pmol g−1 DW) of kinetin (KIN) was detected. Cold treatment significantly changed the levels of all analysed PGRs. The anthers of ‘responsive’ DH lines contained higher concentrations of IBA, cis and trans zeatin, cZR and ABA, and lower amount of IAA and KIN in comparison with ‘recalcitrant’ genotypes. However, the effects of exogenous ABA, p-chlorophenoxyisobutyric acid (PCIB) and 2,3,5-triiodobenzoic acid treatments suggest that none of the studied PGRs acts alone in the acquisition of embryogenic competency, which seems to be an effect of concerted PGRs crosstalk. The initiation of ME required a certain threshold level of ABA. A crucial prerequisite for high ME effectiveness was a specific PGRs homeostasis: lower Auxs level in comparison with CKs and ABA, and lower CKs/ABA ratio. A proper balance between endogenous Auxs in anthers and exogenous Auxs supplied by culture media was also essential.

Effect of seaweed liquid extracts and plant growth regulators on in vitro mass propagation of brinjal (Solanum melongena L.) through hypocotyl and leaf disc explants

Abstract: The effect of seaweed liquid extracts (SLEs) made from Gracilaria salicornia, Padina gymnospora, Padina boergesenii, Gelidiella acerosa and plant growth regulators (PGRs) were examined on in vitro mass propagation using hypocotyls and leaf disc explants of brinjal (Solanum melongena L.) cultivar Pusa purple long. For the germination bioassay, seeds germinated with 20–40 % SLEs exhibited enhanced germination. Initially, hypocotyls and leaf discs were cultured on Murashige and Skoog (MS) medium containing 6-benzylaminopurine, zeatin and thidiazuron for shoot induction. The best responding cytokinin, 6-benzylaminopurine, was employed with different auxins (indole-3-acetic acid, indole-3-butyric acid and 1-naphthaleneacetic acid) for shoot proliferation. In a second experiment, all the four SLEs (10–60 %) combined with MS medium were studied for shoot propagation. Augmented shoots transferred to half-strength MS medium and supplemented with auxins and SLEs (10–70 %) individually to induce rooting. In these experiments high rate of shoot induction (96.2 %), proliferation (6 cm) and rooting (95.3 %) was found with 20–40 % of SLEs. Well-matured plantlets were transferred to soil cups, maintained in a growth chamber for a week to control humidity and then shifted to a greenhouse. This study demonstrated that SLEs could serve as an alternative to phytohormones as they were easy to extract and gave quick and high-frequency mass propagation.

Low expression of PIN gene family members is involved in triggering the dwarfing effect in M9 interstem but not in M9 rootstock apple trees

Abstract: In many perennial woody crops, true-to-type scion is propagated via grafting techniques. The vigour of apple trees can be reduced by grafting on to dwarfing rootstock like M9. However, the mechanisms as to how rootstocks induce dwarfing on their scion remain unclear. Here, members of the gene families encoding auxin polar transporters, PIN and LAX, were analysed in M9, Fuji, and Baleng Crab. Of the 13 members, MdPIN8, MdPIN10 and MdPIN13 were preferentially and differently expressed in the scion, interstem or rootstock in grafting complexes. The dynamic changes in phytohormone contents, relative expressions of MdPINs, and shoot growth after 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA) or N-1-naphthylphthalamic acid (NPA) treatments were evaluated in 3-year-old Fuji/M9, Fuji/M9/Baleng Crab and Fuji/Baleng Crab. In Fuji/M9, foliar or root application of NAA increased root auxin (NAA + IAA) levels but did not significantly increase root zeatin content or shoot growth. Leaf cytokinin (zeatin + BA) levels and shoot growth increased significantly after foliar or root application of BA. In Fuji/M9/Baleng Crab, root application of NAA induced a significant increase in root and leaf zeatin contents and shoot growth, but NAA foliar sprays did not, because the expression of MdPIN8 in the bark of interstem M9 did not increase. When stem auxin transport was inhibited by NPA in Fuji/Baleng Crab, root zeatin level and shoot growth declined dramatically. It was proposed that the dwarfing effect was initiated by inherently lower expression of MdPIN8 in M9 interstem, or by poor root zeatin synthesis in M9.

Assessing temporal flux of plant hormones in stored processing potatoes using high definition accurate mass spectrometry

Abstract: Plant hormones are important molecules which at low concentration can regulate various physiological processes. Mass spectrometry has become a powerful technique for the quantification of multiple classes of plant hormones because of its high sensitivity and selectivity. We developed a new ultrahigh pressure liquid chromatography-full-scan high-definition accurate mass spectrometry method, for simultaneous determination of abscisic acid and four metabolites phaseic acid, dihydrophaseic acid, 7'-hydroxy-abscisic acid and abscisic acid glucose ester, cytokinins zeatin, zeatin riboside, gibberellins (GA1, GA3, GA4 and GA7) and indole-3-acetyl-L-aspartic acid. We measured the amount of plant hormones in the flesh and skin of two processing potato cvs. Sylvana and Russet Burbank stored for up to 30 weeks at 6 °C under ambient air conditions. Herein, we report for the first time that abscisic acid glucose ester seems to accumulate in the skin of potato tubers throughout storage time. The method achieved a lowest limit of detection of 0.22 ng g(-1) of dry weight and a limit of quantification of 0.74 ng g(-1) dry weight (zeatin riboside), and was able to recover, detect and quantify a total of 12 plant hormones spiked on flesh and skin of potato tubers. In addition, the mass accuracy for all compounds (<5 ppm) was evaluated.

Adaptation to altitude affects the senescence response to chilling in the perennial plant Arabis alpina

Abstract: In annual plants with determinate growth, sugar accumulation signals high carbon availability once growth has ceased, resulting in senescence-dependent nutrient recycling to the seeds. However, this senescence-inducing effect of sugars is abolished at cold temperature, where sugar accumulation is important for protection. Here, natural variation was exploited to analyse the effect of chilling on interactions between leaf senescence, sugars, and phytohormones in Arabis alpina, a perennial plant with indeterminate growth. Eight accessions of A. alpina originating from between 2090 and 3090 m above sea level in the French Alps were used to identify heritable adaptations in senescence, stress response, sugars, and phytohormones to altitude. Accessions from high altitudes showed an enhanced capacity for sucrose accumulation and a diminished loss of chlorophyll in response to chilling. At warm temperature, sucrose content was negatively correlated with chlorophyll content, and sucrose treatment induced leaf senescence. Chilling resulted in lower indole-3-acetic acid, but higher zeatin and jasmonic acid contents. Interactions between sugar and phytohormones included a positive correlation between sucrose and jasmonic acid contents that may be involved in promoting the stress-dependent decline in chlorophyll. These findings reveal regulatory interactions that underlie adaptation in the senescence and stress response to chilling.

Effect of plant growth regulators on the accumulation of indolizidine alkaloids in Securinega suffruticosa callus cultures

Abstract: In the presented work, plant growth regulators (PGRs) were tested for their influence on biomass growth and accumulation of therapeutically-relevant indolizidine alkaloids in Securinega suffruticosa (Phyllanthaceae) callus cultures. The study included 9 auxins [2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid, indole-3-acetic acid (IAA), 4-chlorophenoxyacetic acid, indole-3-butyric acid, 4-amino-3,5,6-trichloropicolinic acid, β-naphthoxyacetic acid, 1-naphthaleneacetic acid, indole-3-propionic acid, one polar auxin transport inhibitor (2,3,5-triiodobenzoic acid) and 7 cytokinins [2-isopentenyladenine, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea, N-(2-chloro-4-pyridinyl)-N′-phenylurea, diphenylurea, zeatin, kinetin (KIN), 6-benzylaminopurine]. The respective PGRs were applied at 0.5 and 5.0 mg l−1, together with 5.0 or 0.5 mg l−1 KIN or 2,4-D (for auxins and cytokinins, respectively). The calli subjected to different phytohormone combinations (39 modifications in total) were evaluated for growth and alkaloid content, and the results tested for statistical differences. The highest concentrations of securinine (1.73 mg g−1 DW) and allosecurinine (3.11 mg g−1 DW) were recorded in the callus grown in the presence of 0.5 mg L−1 IAA and 5.0 mg L−1 KIN. To the best of the authors’ knowledge, this is the first such comprehensive report concerning the influence of PGRs on alkaloid accumulation in plant in vitro cultures.

Kinetin Is the Most Effective Cytokinin on Shoot Multiplication from Cucumber

Abstract: In vitro shoot multiplication of cucumber (Cucumis sativus) was examined from the nodal explants of 10-day-old aseptic plantlets using Murashige and Skoog (MS) media supplemented with different concentration (0, 0.5, 1, 2, 3 mg L-1) of cytokinins (6-Benzylaminopurine-BAP, Kinetin-Kn, Thidiazuron-TDZ, and Zeatin). Nodal explants of cucumber showed shoot induction and multiplication in response to all cytokinins tried. MS medium containing Kn was the most effective for inducing shoots from nodal explants of cucumber. The maximum rate of regeneration (83%), the highest number of obtained shoots (7.93 shoots/explant) and the longest shoots (3.61 cm) were obtained on MS medium fortified with 1 mg L-1 Kn. The lowest culture responses were recorded for media supplemented with either BAP or Zeatin. In conclusion, using Kn is strongly recommended than using Zeatin, TDZ and BAP to obtain the highest percentage of regeneration, the highest number of shoot/explant, and the highest shoot length for nodal explants of cucumber.

Effects of Exogenous Putrescine on Leaf Anatomy and Carbohydrate Metabolism in Cucumber (Cucumis sativus L.) Under Salt Stress

Abstract: The objective of this study was to identify the effects of exogenous putrescine (Put) on leaf traits, hormones levels, and carbohydrate metabolism in cucumber seedlings under salt stress. The stress of 75 mM NaCl decreased plant growth and photosynthetic carboxylation efficiency. NaCl also increased the leaf thickness but destroyed the leaf internal structure. The contents of indoleacetic acid, zeatin, and zeatin riboside were decreased and the abscisic acid level was increased by NaCl. In addition, NaCl stress caused the accumulation of sucrose and starch in leaves and led to the feedback inhibition of photosynthesis, which can be inferred from the lower maximum rate of RuBisCo-mediated carboxylation (V cmax). The analysis of starch-metabolizing enzyme activities suggested that the largely improved amylopectin contents contributed to the starch accumulation. Put regulated the levels and ratios of endogenous hormones, lowered the leaf thickness, and protected the leaf structure from damage caused by NaCl. Furthermore, Put decreased the accumulation of sucrose and starch caused by NaCl in leaves through regulating the activities of enzymes involved in carbohydrate metabolism. In conclusion, Put regulates the hormones levels, leaf structure, and carbohydrate metabolism and therefore improves the photosynthesis and growth of NaCl-stressed cucumber plants.

Genetic fidelity assessment of in vitro -regenerated plants of Albizia julibrissin using SCoT and IRAP fingerprinting

Abstract: A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2 ± 3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3 ± 6.3%). Maximum rooting of shoots (73.3 ± 5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.

Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis

Abstract: Doubled haploid (DH) technology allows for the production of pure lines, useful for plant breeding, through a one-generation procedure that reduces considerably the time and resources needed to produce them. Despite the advantages of microspore culture to obtain DHs, this technique is still insufficiently developed in eggplant, where DHs are produced from microspore-derived calli through organogenesis. At present, very little is known on the best in vitro conditions to promote this process. This is why in this work we addressed the optimization of the process of regeneration of eggplant DH plants from microspore-derived calli. We evaluated the effect of different media compositions in the induction of organogenesis, in the promotion of shoot growth and elongation, and in root growth. According to our results, we propose the repeated subculture of the calli in MS medium with 0.2 mg/l IAA and 4 mg/l zeatin to produce shoots, and then the repeated subculture of the excised shoots in basal MS medium to promote their conversion into entire plantlets. This procedure yielded 7.6 plants per 100 cultured calli, which represents a ~4× increase with respect to previous reports. We also evaluated by flow cytometry and SSR molecular markers the effect of these in vitro culture conditions in the rate of DH plant production, finding that ~70 % of the regenerated plants were true DHs. These results substantially improve the efficiencies of DH recovery published to date in eggplant, and may be useful to those working in the field of eggplant doubled haploidy and breeding.

Histological and endogenous plant growth regulators changes associated with adventitious shoot regeneration from in vitro leaf explants of strawberry (Fragaria × ananassa cv. ‘Honeoye’)

Abstract: Few studies have been conducted on the links between histological and hormonal variation during shoot regeneration. Therefore, we investigated this link in strawberry (Fragaria × ananassa cv. ‘Honeoye’). Main plant growth regulators were measured using reverse-phase liquid chromatography–tandem mass spectrometry. Histological observations were conducted to understand the pattern of adventitious shoot regeneration from the leaf segments. After 14 days of dark culture, a mean shoot regeneration frequency of 94.7 % was obtained on MS medium supplemented with 2.0 mg L−1 thidiazuron, 30 g L−1 sucrose, and 6 g L−1 agar (pH 5.9). During shoot regeneration, indole acetic acid (IAA) concentrations increased, abscisic acid (ABA) decreased, and gibberellic acid (GA3) and zeatin showed peaks. The results could be correlated with the cell division and differentiation that occurred during shoot regeneration. Histological observation showed that the adventitious shoots were derived from subepidermal cells and the epidermal cells of the midrib near the cut. The meristemoids, primordia and shoots were formed sequentially on day 6, day 12, and day 18 after culture. During the meristemoids formation on day 6 after culture, IAA rapidly increased from 1.49 to 1.72 ng g−1 fresh weight (Fw), ABA rapidly decreased from 52.61 to 13.47 ng g−1 Fw, and zeatin increased from 1.68 to 5.98 ng g−1 Fw. During primordia formation on day 12 after culture, IAA rapidly increased to 1.88 ng g−1 Fw, ABA rapidly decreased to 2.69 ng g−1 Fw, GA3 peaked at 73.91 ng g−1 Fw, and zeatin peaked at 7.69 ng g−1 Fw. Our results suggested that the histological variations were consistent with the plant hormonal changes during shoot regeneration, and that changes in hormone concentration could be used as a reference to characterize the mode of shoot regeneration.

Micropropagation of Vaccinium myrtillus L. (Bilberry) naturally growing in the Turkish flora

Abstract: This study was designed to micropropagate Vaccinium myrtillus L., a naturally growing bilberry, in the Turkish flora. In order to determine the most effective basal medium, lateral buds were initially selected as explants, which were then individually cultured in Murashige and Skoog medium, Anderson's rhododendron medium, and McCown's woody plant medium (WPM) supplemented with 1.0 mg L-1 zeatin in combination with either 0.1 mg L-1 indole-3-butyric acid (IBA) or 0.1 mg L-1 \alpha-naphthalene acetic acid. WPM, supplemented with zeatin and IBA, was found to be the most effective basal medium tested. As shoot regeneration ability highly depends on the medium and cytokinin concentration, WPM basal media containing various concentrations (0.5, 1.0, and 2.0 mg L-1) of 3 different cytokinins, namely zeatin, thidiazuron, and N6-[2-isopentenyl] adenine, were employed together with IBA (0.1 mg L-1) and were compared with basal media containing none of the growth regulators. In terms of shoot multiplication, 2.0 mg L-1 zeatin was found to be superior to the other tested growth regulators when combined with 0.1 mg L-1 IBA. WPM was also used as a basal medium for rooting and was supplemented with different concentrations of IBA (0.25-2.0 mg L-1) with or without activated charcoal (AC). WPM supplemented with 0.5/1.0 mg L-1 IBA/AC was the best culture medium for rooting with 60% root formation.

Shoot tip regeneration and optimization of Agrobacterium tumefaciens-mediated transformation of Broccoli (Brassica oleracea var. italica) cv. Green Marvel

Abstract: A protocol of plant regeneration from shoot tips and optimization of Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) cv. Green Marvel have been developed. Shoot tip response was assessed on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin. The highest regeneration with a maximum of 13 shoots per explant was obtained on MS medium containing 1.5 mg l−1 zeatin. Primary selection of putative transformed explants was performed on the optimized regeneration medium (MS medium containing 1.5 mg l−1 zeatin and 80 mg l−1 kanamycin) for 60 days. The effects of pre-culture, acetosyringone and growth of bacterial culture were studied. Explants precultured on callus induction medium for 4 days prior to inoculation with A. tumefaciens with 200 μM acetosyringone resulted in improved transformation frequency. The Agrobacterium culture dilution of 1:5 and inoculation time of 30 min increased the efficiency of transformation of shoot tip explants. The results also indicated that 150 mg l−1 ampicillin alone was adequate to eradicate Agrobacterium growth in the SRM incorporated with the respective minimum inhibitory concentration of 80 mg l−1 kanamycin. The polymerase chain reaction (PCR) and Southern blot assays confirmed the transgenic status of the broccoli cv. Green Marvel regenerants. A transformation efficiency of 5 % was achieved based on the positive PCR results using the optimized procedure. The expression of luciferase reporter gene in the transformed cells and the transcription of AtHSP101 using RT-PCR further confirmed the transgenic status of the regenerated plants.

An efficient multiple shoot induction and genetic fidelity assessment of Exacum bicolor Roxb., an endemic and endangered medicinal plant

Abstract: Exacum bicolor, an endemic and endangered medicinal plant, belongs to the family Gentianaceae. A rapid protocol has been developed for efficient multiple shoot induction by testing nodal explants on Murashige and Skoog (MS) medium supplemented with various cytokinins. The cytokinins 6-benzyladenine (BA), 6-furfurylaminopurine (Kn), 2-isopentenyladenine (2-iP) and zeatin (Zn) were used individually and in combination at different concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 μM). The maximum number of shoots (19.33 ± 1.09 per explant) as well as their fresh weight (5.1 ± 0.68 g) and dry weight (216.83 ± 2.84 mg) were obtained with 10.0 μM BA + 2.0 μM Kn. After 4 wk, the multiple shoots from agar culture were subcultured into liquid medium containing the same growth regulator combinations. After 8 wk of liquid culture, the best treatment had about tenfold increase in shoot number (199.5 ± 1.14 per explant). The fresh weight (13.76 ± 0.14 g) and dry weight (909.33 ± 1.92 mg) were highest with full strength MS medium supplemented with 3% sucrose and containing 10.0 μM BA + 2.0 μM Kn. Maximum root development was observed after 30 d with 0.5 μM indole-3-butyric acid (IBA) supplementation. Regenerated plants were successfully transferred to pots containing coco peat:perlite mixture and showed a 75% survival rate. Genetic fidelity of in vitro plantlets compared to mother plant were assessed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. All the regenerated plants were genetically identical to their mother plant, showing no detectable genetic variation in the regenerated plantlets. Thus, this protocol could be successfully used for mass multiplication and germplasm conservation of E. bicolor.

Embryogenic response from anther culture of cultivars of loquat ( Eriobotrya japonica (Thunb.) Lindl.) from different origins

Abstract: Homozygous lines are very interesting for genetic studies and in plant breeding. These can be produced in a single step via haploidy. Production of haploids in loquat (Eriobotrya japonica (Thunb.) Lindl.) would be of considerable value. In this study, androgenesis by anther culture was used to regenerate loquat plants. Different variables related to the induction of embryogenesis were tested, including the temperature pretreatment of flower buds, various levels of growth regulators in the culture medium, and genotypic effects. Eight cultivars of loquat from different geographic origins were used. The first step, the association between floral bud size and the corresponding microspore/pollen developmental stages was established for the different cultivars. Bud sizes of 6.5–7.0 mm, corresponded to the uninucleate microspore stage and provided the highest callogenesis levels. Pre-treatment of flower buds at 4 °C for had a negative effect on anther response and only the 4-day pre-treatment resulted in callus formation at all. The highest percentage of morphogenetic calli was obtained on medium supplemented with 4.56 μM zeatin (Z) and 5.36 μM 1_naphthalene acetic acid (NAA) in cvs. ‘Changhong-3’ (27 %), ‘Jiefanghong’ (30 %) and ‘Moggi Wase’ (36 %). Following a transfer of morphogenetic calli to the embryo induction medium, six embryos were obtained from cv. ‘Jiefanghong’; and one of them developed into a plantlet. Flow cytometry and chromosome counts revealed that the plantlet was triploid. The single triploid plant obtained was unexpected and its origin is obscure. One possible explanation is that it arose from fusion of sperm nucleus and two vegetative nuclei.

In vitro regeneration of Cyrtanthus species: ornamental plants with medicinal benefits

Abstract: Cyrtanthus (Amaryllidaceae) is a genus of perennial geophytes, endemic to the southern African region. Destructive and indiscriminate harvesting of bulbs for medicinal and ornamental purposes has led to intensive decimation of the populations of most of these species in their natural habitats. The aim of this study was to develop in vitro regeneration systems for Cyrtanthus contractus, Cyrtanthus guthrieae, and Cyrtanthus obliquus using twin-scale explants from mature bulbs. Twin scales from the three species were cultured on solid Murashige and Skoog (MS) medium with different concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA) under 16/8-h light/dark conditions at 25 ± 2°C. The best shoot induction responses were obtained on MS medium containing 4.4 μM BA + 1.1 μM NAA (3.1 shoots/explant) for C. contractus and C. guthrieae and 6.7 μM BA + 2.7 μM NAA for C. obliquus. When the best shoot induction medium for each species was investigated for the effect of cytokinins on shoot organogenesis, using different concentrations of BA, kinetin (Kin), meta-topolin (mT), zeatin (ZT), and thidiazuron (TDZ), the medium that produced the best shoot induction for C. guthrieae also produced the highest number of shoots per explant, whereas the highest numbers of shoots per explant were obtained with 10 μM TDZ for C. contractus and 10 μM BA for C. obliquus. Superior quality shoots in all three species were obtained from MS medium supplemented with Kin and mT. Regenerated shoots were rooted successfully on half- and full-strength MS medium without plant growth regulators, transferred to organic soil mix, and successfully acclimatized in the greenhouse. The micropropagation protocols described provide rapid and cost-effective methods for in vitro propagation for application to the conservation and domestication of Cyrtanthus species.

Effect of MgCl 2 and double concentration of Murashige and Skoog medium on in vitro plantlet and root cultures generation in halophytic grasswort Salicornia brachiata

Abstract: An improved micropropagation protocol has been developed for halophytic grasswort Salicornia brachiata by using double concentration of Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (DMS) supplemented with plant growth regulators. Best shoot bud induction (90 %) from nodal explants was observed on DMS supplemented with 3.0 mg L−1 6-benzyl aminopurine (BAP) and 0.5 mg L−1 Zeatin with >3 shoot buds. Shoot proliferation and elongation was achieved on DMS supplemented with 1.0 mg L−1 thidiazuron (TDZ) and 1.0 mg L−1 α-naphthalene acetic acid (NAA) with multiple shoot buds after 30 days of culture. Best callus response (78 %) was observed when stem explants were cultured on DMS supplemented with 2.0 mg L−1 2, 4-dichlorophenoxyacetic acid and 0.01 mg L−1 BAP. Regeneration from the callus was achieved when callus was cultured on DMS supplemented with 0.5 mg L−1 TDZ. These shoot buds were elongated on shoot proliferation and elongation medium. Elongated shoots (5 cm) could be rooted on DMS supplemented with 0.5–1.5 mg L−1 NAA, 0.5–1.5 mg L−1 indole-3-acetic acid and 0.5–1.5 mg L−1 indole-3-butyric acid. DMS medium supplemented with 0.5 mg L−1 NAA found to be best for rooting and the addition of 20 g L−1 magnesium chloride (MgCl2) to the medium resulted in highest percentage of rooting and not with the addition of sodium chloride (NaCl). Rooted plants could be established in soil with 55 % survival. In another set of study on root culture generation, many roots initiated from explants cultured on DMS supplemented with 2.0 mg L−1 NAA. Addition of 10 g L−1 MgCl2 was highly beneficial in stimulating root initiation and proliferation as compared to NaCl.

Effect of growth hormones in the micropropagation of banana cv. matti

Abstract: Banana is an important fruit crop of the family musaceae. In Kanyakumari District, many varieties of banana are cultivated. Matti is one among them which fetches good price in the market and farmers called, it as money spinner. This study carry out the micro propagation of banana cv. Matti. The sword suckers of three months old were used as explants. The explants sterilised thoroughly and MS media supplemented with the cytokinins like BAP, kinetin, zeatin and adenine sulphate in different concentration (0.5 to 5.5 mg/l) were used for the multiple shoot generation and shoot growth. Among the tested cytokinins BAP in medium concentration performed well and higher concentration of all tested cytokinins showed declining effect. Generated shoot initials were transferred to the rooting media after three sub cultures. For rooting MS media was supplemented with different levels of IBA. Rooting was highly promoted in the medium concentration of IBA. After 115 th day of inoculation the plantlets were transferred to green house for hardening and subsequently planted in the field after 30 days of acclimatization.

Phytotoxical effect of Lepidium draba L. extracts on the germination and growth of monocot (Zea mays L.) and dicot (Amaranthus retroflexus L.) seeds.

Abstract: Laboratory experiments were performed to determine phytotoxic potentials of white top (Lepidium draba) methanol extracts (root, stem and leaf) on germination and early growth of corn (Zea mays) and redroot pigweed (Amaranthus retroflexus). Furthermore, the effects of different methanol extracts of L. draba on the phytohormone (indole-3-acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA) and zeatin) levels of corn and redroot pigweed were investigated. It was observed that all concentrations of methanol extracts of root, stem and leaf of L. draba inhibited germination, radicle and plumule elongation when compared with the respective controls. Besides this, the degree of inhibition was increased in concert with increasing concentrations of extracts used. On the other hand, phytohormone levels changed with the application of different extract concentrations. Comparing with the control, the GA levels significantly decreased while the ABA levels increased in all the application groups. Zeatin and IAA levels showed changes depending upon the applied extracts and concentrations.

Effects of plant growth regulators and carbohydrates on callus induction and proliferation from leaf explant of Lippia multiflora Moldenke (Verbenacea)

Abstract: Lippia multiflora Moldenke is a tropical plant used in traditional medicine for many diseases treatment (malaria, hyperthermia, blood pressure, etc.). The aqueous extract and essential oils obtained from the leaves have pharmacological, pesticide and cosmetic properties. This study aims to develop a protocol of callus induction and proliferation from leaf explants of L. multiflora. Murashige and Skoog (1962) basal medium was respectively supplemented with different concentrations (0,5; 2,5 ; 5 and 10 µM) of the plant growth regulators 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA) and Picloram (PIC). The best auxin was then combined respectively with three types of cytokinins (kinetin (KIN), benzylaminopurine (BAP) and zeatin (ZEA)). The best hormonal combination was used to test the effect of type and concentration of carbohydrate on callus formation. The results showed that the medium supplemented with 5µM PIC expressed a callus induction rate and a fresh and dry weight of callus significantly higher than the other tested auxins. Medium supplemented with 5 µM PIC + 25 µM KIN was more suitable for callus induction and proliferation. Among the carbohydrates, the maximum rate of callus formation was expressed by the leaf explants on medium containing 3% fructose. The protocol for callus induction developed in this study would allow the in vitro production of essential oil of L. multiflora from cell suspension cultures.

In vitro regeneration of the medicinal plant Cassia absus L.

Abstract: SummaryCassia absus L. is a medicinal plant with commercial value. In order to establish a protocol for its regeneration from nodal explants, C. absus was proliferated in vitro using 1.0_ Murashige and Skoog (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar, 0.05% (w/v) charcoal, and different concentrations of 6-benzylaminopurine (BAP), kinetin, zeatin, or isopentenyl adenine (2iP) under controlled temperature, humidity, and light conditions. Shoot induction was achieved at various rates on all media tested. MS medium containing 0.5 mg l-1 BAP gave the optimum results for shoot initiation and proliferation, with an average shoot length of 12.4 mm and an average of 2.7 leaves per shoot.The effect of 0.5 mg l-1 BAP combined with 0.1 mg l-1 gibberellic acid (GA3) was also tested under the same conditions to improve growth and increase shoot length. Shoots induced on this medium were significantly longer (13.4 mm) with more leaves (3.3 per shoot) than on 1.0_ MS medium containing 0.5 mg l-1 BAP alone....

Effect of Cytokinins on In vitro seed Germination and Changes in Chlorophyll and Soluble Protein Contents of Teak (Tectona grandis L.)

Abstract: Teak (Tectona grandis L.) is amongst the precious tropical trees produces luxurious wood items and metabolites of great medicinal value. However, teak growth is hampered due to high dormancy and poor viability of the seeds under the normal field conditions. Here we investigated the effect of various cytokinins on seed germination and growth under In vitro and ex vitro conditions of seedlings. Completely randomized design was used and data were analyzed to ascertain whether or not the tested cytokinins can improve seed germination and growth of the teak plants. Seeds were cultured on MS medium supplemented with various concentrations (0.08, 0.22, 0.35, 0.80, 2.20 or 3.50 μM) of adenine sulphate (ADS), N6-benzyleadenine (BA), kinetin (KIN), thidiazuron (TDZ), and zeatin (ZEA) under In vitro conditions for 40 days. Highest (100%) germination was obtained at 0.22 μM BA with 4.41 cm shoot and 4.67 cm root lengths as well as 75.32% highest seedling’s survival was observed in glasshouse. TDZ inhibited seedling’s growth and induced hyperhydricity (16.36–35.36%). Improved growth of seedlings in both conditions may be linked with enhanced production of chlorophyll (Chl) both In vitro (1.86 Chl a, 1.30 Chl b mg/g FW) and in acclimatized seedlings (1.91 Chl a, 1.70 Chl b mg/g FW) at 0.22 μM BA. The same concentration of BA also produced highest soluble proteins in In vitro (7.52 mg/g FW) and acclimatized seedlings (8.88 mg/g FW). Under specific tissue culture conditions, seed germination and subsequent growth of teak seedlings can be improved with BA.

An efficient adventitious shoot regeneration system for potato (Solanum tuberosum L.) using leaf discs

Abstract: Adenine-type cytokinins, such as zeatin (Z) and its riboside (ZR) are usually used for in vitro plant regeneration in potato (Solanum tuberosum L.), with the latter being more potent for both organogenesis and somatic embryogenesis. Here, we report an efficient ZR-free adventitious shoot regeneration (ASR) system for potato. In this two-step ASR system, callus was induced from leaf explants on Murashige and Skoog medium supplemented with 1.0 mg l−1 N6-benzyladenine (BA), 1.0 mg l−1 α-naphthaleneacetic acid (NAA), 1.0 mg l−1 calcium D-pantothenate, 100.0 mg l−1 polyvinylpyrrolidone (PVP) and 30.0 g l−1 sucrose followed by multiple ASR on the second medium containing 3.0 mg l−1 BA, 1.0 mg l−1 thidiazuron (TDZ) and 1.0 mg l−1 gibberellic acid (GA3), with the other additives remaining the same. The efficiency of this ZR-free ASR system was compared with that of the ZR-based one where the growth regulators in the second medium were replaced by 5.0 mg l−1 ZR. There was no significant difference in mean number of days to regeneration between the two ASR systems. Depending on the genotypes, mean number of days to regenerate were 12.6–23.6 days and 13.0–23.0 days for ZR-based and ZR-free ASR systems, respectively. However, in two out of four genotypes tested, significantly higher number of shoots (17.0–19.2) developed per callus in ZR-free ASR system. In vitro regenerants derived from the ZR-free ASR system did not show any morphological variation having comparable competence for tuberization in vitro and maintained genetic fidelity as revealed by inter-simple sequence repeat (ISSR) analysis. In this study, we have developed a leaf-based ZR-free ASR system which can reliably be used for potato transformation.

IAA and zeatin controls cell division and endoreduplication process in quiescent center cells of Allium cepa root

Abstract: Role of endogenous IAA and zeatin level in regulation of cell division and endoreduplication processes was studied in quiescent center cells of Allium cepa using colchicine by considering morphological and cytological parameters. This resulted in formation of c-tumor at root tips, which contained endoreduplicated cells. Different concentrations of NAA and BAP (1–250 μM) were exogenously applied to these endoreduplicated root tips to depolyploidize endoreduplicated quiescent center cells. Exogenous NAA treatment showed negative response on depolyploidization of quiescent center cells of root tips. However, elongation of cells, and decrease in nucleus size was observed after exogenous NAA treatment. There was no change in length of endoreduplicated roots, and growth of root at the tip of c-tumor. Exogenous BAP showed positive response in depolyploidization of quiescent center cells of root tips. There was remarkable increase in length of roots, growth of root at tip of c-tumor and decrease in cell size and nucleus size after BAP treatment. It induced cytokinesis process in endoreduplicated meristematic cells and started cell division and differentiation process in quiescent center cells. Endogenous IAA and zeatin measured from normal, endoreduplicated and depolyploidized quiescent center root tip cells suggested that when endogenous IAA/zeatin ratio is high in meristematic cells then cell cycle shifts towards endoreduplication process but when it decreases cell cycle shifts towards cell division process.