Showing papers on "Zeatin published in 2016"

Cytokinin-induced promotion of root meristem size in the fern Azolla supports a shoot-like origin of euphyllophyte roots.

Abstract: The phytohormones cytokinin and auxin orchestrate the root meristem development in angiosperms by determining embryonic bipolarity. Ferns, having the most basal euphyllophyte root, form neither bipolar embryos nor permanent embryonic primary roots but rather an adventitious root system. This raises the questions of how auxin and cytokinin govern fern root system architecture and whether this can tell us something about the origin of that root. Using Azolla filiculoides, we characterized the influence of IAA and zeatin on adventitious fern root meristems and vasculature by Nomarski microscopy. Simultaneously, RNAseq analyses, yielding 36 091 contigs, were used to uncover how the phytohormones affect root tip gene expression. We show that auxin restricts Azolla root meristem development, while cytokinin promotes it; it is the opposite effect of what is observed in Arabidopsis. Global gene expression profiling uncovered 145 genes significantly regulated by cytokinin or auxin, including cell wall modulators, cell division regulators and lateral root formation coordinators. Our data illuminate both evolution and development of fern roots. Promotion of meristem size through cytokinin supports the idea that root meristems of euphyllophytes evolved from shoot meristems. The foundation of these roots was laid in a postembryonically branching shoot system.

Grain Filling Characteristics and Their Relations with Endogenous Hormones in Large- and Small-Grain Mutants of Rice.

Abstract: This study determined if the variation in grain filling parameters between two different spikelet types of rice (Oryza sativa L.) is regulated by the hormonal levels in the grains. Two rice mutants, namely, a large-grain mutant (AZU-M) and a small-grain mutant (ZF802-M), and their respective wild types (AZU-WT and ZF802-WT) were grown in the field. The endosperm cell division rate, filling rate, and hormonal levels: zeatin + zeatin riboside (Z+ZR), indo-3-acetic acid (IAA), polyamines (PAs), and abscisic acid (ABA) were determined. The results showed that there was no significant difference between the filling and endosperm cell division rates. These rates were synchronous between the superior and inferior spikelets for both mutants. However, the abovementioned parameters were significantly different between the two spikelet types for the two wild types. The superior spikelets filled faster and their filling rate was higher compared to the inferior ones. Changes in the concentrations of plant hormones were consistent with the observed endosperm cell division rate and the filling rate for both types of spikelets of mutant and wild type plants. Regression analysis showed a significant positive correlation between cell division and filling rates with the concentrations of the investigated hormones. Exogenous chemical application verified the role of ABA, IAA, and PAs in grain filling. The results indicate that poor filling of inferior spikelets in rice occurs primarily due to the reduced hormone concentrations therein, leading to lower division rate of endosperm cells, fewer endosperm cells, slower filling rate, and smaller grain weight.

Kinetic and structural investigation of the cytokinin oxidase/dehydrogenase active site.

Abstract: Cytokinins are hormones that regulate plant development and their environmental responses. Their levels are mainly controlled by the cytokinin oxidase/dehydrogenase (CKO), which oxidatively cleaves cytokinins using redox-active electron acceptors. CKO belongs to the group of flavoproteins with an 8α-N1-histidyl FAD covalent linkage. Here, we investigated the role of seven active site residues, H105, D169, E288, V378, E381, P427 and L492, in substrate binding and catalysis of the CKO1 from maize (Zea mays, ZmCKO1) combining site-directed mutagenesis with kinetics and X-ray crystallography. We identify E381 as a key residue for enzyme specificity that restricts substrate binding as well as quinone electron acceptor binding. We show that D169 is important for catalysis and that H105 covalently linked to FAD maintains the enzyme's structural integrity, stability and high rates with electron acceptors. The L492A mutation significantly modulates the cleavage of aromatic cytokinins and zeatin isomers. The high resolution X-ray structures of ZmCKO1 and the E381S variant in complex with N6-(2-isopentenyl)adenosine reveal the binding mode of cytokinin ribosides. Those of ZmCKO2 and ZmCKO4a contain a mobile domain, which might contribute to binding of the N9 substituted cytokinins.

Modification of plant regeneration medium decreases the time for recovery of Solanum lycopersicum cultivar M82 stable transgenic lines

Abstract: Tomato (Solanum lycopersicum) has rapidly become a valuable model species for a variety of studies including functional genomics. A high-throughput method to obtain transgenic lines sooner than standard methods would greatly advance gene function studies. The goal of this study was to optimize our current transformation method by investigating medium components that would result in a decreased time for recovery of transgenics. For this study, 6-day-old cotyledon explants from Solanum lycopersicum cultivar M82 in vitro-grown seedlings were infected with the Agrobacterium tumefaciens strain LBA4404 containing the binary vector pBI121. This vector contains the β-glucuronidase reporter gene and the neomycin phosphotransferase II selectable marker gene that confers resistance to kanamycin. Modification of our standard plant regeneration medium with indole-3-acetic acid (IAA) at concentrations of either 0.05 or 0.1 mg/l decreased the recovery time for transgenic lines by 6 weeks as compared to our standard medium that contains zeatin as the only plant growth regulator. We observed 50 and 54 % transformation efficiency on plant regeneration medium containing 0.05 and 0.1 mg/l IAA, respectively. Moreover, addition of 1 mg/l IAA to the root induction medium resulted in earlier root development than medium that did not contain IAA. Addition of IAA to the plant regeneration and rooting media did not have any negative effects on plant development. Recovery of transgenic lines in a shorter time results in higher throughput for the introduction of gene constructs and has the potential to decrease the time and resources needed to complete investigations of gene function.

Putative zeatin O-glucosyltransferase OscZOG1 regulates root and shoot development and formation of agronomic traits in rice

Abstract: As a ubiquitous reaction, glucosylation controls the bioactivity of cytokinins in plant growth and development. Here we show that genetic manipulation of zeatin-O-glucosylation regulates the formation of important agronomic traits in rice by manipulating the expression of OscZOG1 gene, encoding a putative zeatin O-glucosyltransferase. We found that OscZOG1 was preferentially expressed in shoot and root meristematic tissues and nascent organs. The growth of lateral roots was stimulated in the overexpression lines, but inhibited in RNA interference lines. In shoots, knockdown of OscZOG1 expression by RNA interference significantly improved tillering, panicle branching, grain number per panicle and seed size, which are important agronomic traits for grain yield. In contrast, constitutive expression of OscZOG1 leads to negative effects on the formation of the grain-yielding traits with a marked increase in the accumulation levels of cis-zeatin O-glucoside (cZOG) in the transgenic rice plants. In this study, our findings demonstrate the feasibility of improving the critical yield-determinant agronomic traits, including tiller number, panicle branches, total grain number per panicle and grain weight by downregulating the expression level of OscZOG1. Our results suggest that modulating the levels of cytokinin glucosylation can function as a fine-tuning switch in regulating the formation of agronomic traits in rice.

Regeneration of soapnut tree through somatic embryogenesis and assessment of genetic fidelity through ISSR and RAPD markers.

Abstract: Somatic embryogenic system was developed in Sapindus mukorossi Gaertn. using rachis as explants from a mature tree. Explants showed callus initiation on Murashige and Skoog medium supplemented with TDZ (1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea), zeatin or 6-benzylaminopurine. Induction of somatic embryogenesis was achieved on both MS basal medium and MS medium supplemented with 8.88 µM 6-benzylaminopurine. Hundred percent embryogenesis was observed on MS medium supplemented with 8.88 µM 6-benzylaminopurine with maximum intensity of embryogenesis (51.92 ± 0.40 a). Maximum maturation of somatic embryos (92.86 ± 0.34 a) was observed on induction medium supplemented with 0.0378 µM abscisic and treated for 21 days. Germination of somatic embryos was maximum (77.33 ± 0.58 a) on MS medium supplemented with 8.88 µM 6-benzylaminopurine. In vitro raised plantlets were hardened, acclimatized and transferred to the field. Survival frequency of plantlets was 80 % in field conditions. The genetic fidelity of in vitro regenerated plants was also evaluated and compared with mother plant using random amplified polymorphic DNA and inter simple sequence repeat. Both markers showed similarity in molecular profile of mother plant and in vitro regenerated plants.

Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin

Abstract: Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77 × 105 protoplasts (Pp) per gram fresh weight with 94 % viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5 % cellulase Onozuka R10, and 1 % pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 μM α-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3–5 × 105 Pp ml−1. Protoplast-derived microcalli became visible after 3–4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 μM BA plus 4.6 μM zeatin after 2–3 weeks, with an overall shoot organogenesis efficiency of 78–93 %. Rooting of microshoots on half-strength MS medium containing 4.9 µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62 %. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.

In vitro regeneration by callus culture of Anoectochilus elatus Lindley, an endangered terrestrial jewel orchid

Abstract: A process of organogenesis via callus with successful plantlet formation was developed for Anoectochilus elatus. Indirect organogenesis was achieved from in vitro-derived node, internode, and leaf explants. The explants were cultured on Mitra medium fortified with different concentrations and combinations of plant growth regulators such as cytokinins (N6-benzyl adenine [BA], thidiazuron [TDZ], kinetin [KN], N6-(2-isopentyl) adenine [2ip] and zeatin [ZEA]), auxins (2,4-dichlorophenoxyacetic acid [2,4-D], α-naphthalene acetic acid [NAA], indole-3-acetic acid [IAA], indole-3-butyric acid [IBA], and 4-amino-3,4,6-trichloro picolinic acid [Pic]), and additives (citric acid, trisodium citrate, peptone, coconut water, potato extract, and banana pulp). Organogenic callus proliferation was highest from internode (77.8 %), followed by node (69.7 %) and leaf explants (64.2 %), on Mitra medium supplemented with TDZ (1.0 mg L−1) and NAA (0.5 mg L−1). Organogenic callus derived from internodal explants produced an average of 41.8 shoots per explant, with average length of 2.5 cm, on Mitra medium supplemented with BA (1.0 mg L−1), NAA (0.5 mg L−1) and coconut water (10 %). In rooting experiments, a maximum of 3.2 roots per shoot was observed with an average length of 2.1 cm with 97.8% response on Mitra medium amended with AgNO3 (1.0 mg L−1). The rooted plantlets were acclimatized in a mixture of garden soil, sand, vermicompost, and used tea waste (8:4:2:1 [w/w/w/w]) in a greenhouse environment, with a 72.3% survival rate. Finally, the well-developed plants were transferred to the National Orchidarium, Yercaud, Tamil Nadu, a unit of the Botanical Survey of India, Southern Regional Centre, for further maintenance and establishment under natural conditions for conservation.

Somatic embryogenesis and plant regeneration from immature embryos of Triticum timopheevii Zhuk. and Triticum kiharae Dorof. et Migusch, wheat species with G genome

Abstract: The efficiency of immature embryo-derived in vitro culture of G genome wheats is significantly influenced by various auxins and sugars which are used for induction of embryogenic response, and by regeneration media composition for promotion of plant development from subcultured embryogenic calli. The embryogenic calli of Triticum timopheevii has demonstrated the highest regeneration ability when the initial explants were cultured on the media supplemented with 4 mg l−1 of Picloram (29.0 %), 4 mg l−1 of Dicamba (28.7 %) or 3 mg l−1 of 2,4-D (29.1 %). The media supplemented with 5–6 mg l−1 of Picloram were considered to be the most effective for promotion of embryogenic/regenerable callus production in Triticum kiharae cultures (73.7–75.0 %). Both T. timopheevii and T. kiharae embryogenic structures were characterized by the formation of green and albino plantlets. Generally the medium that was initially supplemented with Picloram promoted the formation of lower albino plants fraction rather than 2,4-D and Dicamba. As it was measured by the total green plant production per initial explant, the overall efficiency has been reduced when sucrose was substituted by glucose or maltose. The regeneration medium supplemented with 0.25 mg l−1 TDZ significantly enhanced the regeneration capacity of embryogenic callus in T. kiharae. In culture of T. timopheevii the difference between the medium lack of growth regulators and the medium supplemented with TDZ was not prominent, though both of the media have demonstrated the greater efficacy as compared to those supplemented with BA and Zeatin.

Changes in distribution of zeatin and indole-3-acetic acid in cells during callus induction and organogenesis in vitro in immature embryo culture of wheat

Abstract: Plant organogenesis remains one of the most essential questions of plant developmental biology. Callus tissue in vitro is a valuable tool for the studies on hormonal aspects of plant organogenesis, especially its early events, and immunohistochemical analysis is one of the few approaches offering information on the localization and role of hormones during organ development. The localization of endogenous zeatin and indole-3-acetic acid was investigated during simultaneous bud and root formation in calluses derived from immature embryos of wheat (Triticum aestivum L.). Calluses were induced on Murashige and Skoog (MS) medium supplemented with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid. To stimulate simultaneous bud and root formation, calluses were transferred onto MS medium supplemented with 0.2 mg L−1 kinetin and 0.2 mg L−1 indoleacetic acid. Strong immunostaining for both hormones was detected in proliferating callus tissue, in developing meristematic centers and meristematic zones (whose cells were shown to be involved in organ formation), and at the sites of shoot and root apex initiation. During further development, shoot apexes with leaf primordia were heavily immunostained for zeatin, while immunostaining for indole-3-acetic acid was more intense at the sites of leaf primordia initiation and incipient primordia themselves. In the developing roots, immunostaining for both hormones reached a maximum in the root apex and gradually declined with increasing distance from the apex. Cells of developing procambial strands were also strongly stained for both zeatin and indole-3-acetic acid. These data suggest considerable similarity between patterns of hormone distribution in organs in vitro and in vivo. Thus, callus culture is a convenient and useful model for the study of fundamental biological questions such as how hormones regulate development.

Effects of cytokinins on physiological and biochemical responses of the agar-producing red alga Gracilaria caudata (Gracilariales, Rhodophyta)

Abstract: The red alga Gracilaria caudata J. Agardh (Gracilariales) is commercially exploited in Brazil as a source of raw material for agar production, and its cultivation is needed for sustainable biomass production to avoid overexploitation of natural populations. The objective of the present study was to evaluate the effects of aromatic and isoprenoid-derived cytokinins under two photon flux densities (PFDs) on growth rates, formation of lateral branches, pigment content (chlorophyll a and phycobiliproteins), and total soluble proteins in G. caudata. Aromatic cytokinins (benzylaminopurine, BA, and kinetin, K) and isoprenoid cytokinins (2-isopentenyladenine, 2iP, cis-zeatin, and trans-zeatin) were tested at 50 and 100 μmol photons m−2 s−1, temperature of 23 ± 1 °C, and light:dark cycle of 14:10 h. Cytokinins were added to the ASP 12-NTA synthetic medium (salinity 30 PSU, pH 8.0) in concentrations ranging from 0.0 to 50.0 μM. Growth rates of G. caudata were not affected by addition of isoprenoid and aromatic cytokinins. Zeatins (cis and trans forms) and BA at the concentrations of 0.5 and/or 5.0 μM stimulated the branch formation at 100 μmol photons m−2 s−1. In general, concentrations of isoprenoid cytokinins showed negative correlation with total soluble proteins and pigment contents at both PFDs. On the other hand, aromatic cytokinins did not affect protein contents, and K inhibited the pigment contents in both PFD levels. In conclusion, our results indicate that the isoprenoid and aromatic cytokinins and PFD levels did not affect the growth rates of G. caudata, but influence the branching and contents of pigments and total soluble proteins. Moreover, aromatic cytokinins and zeatins stimulated branch formation, which is a morphogenetic process useful for micropropagation of G. caudata, and could improve its cultivation in the Brazilian coast.

Roles of ethylene and cytokinins in development of defense responses in Triticum aestivum plants infected with Septoria nodorum

Abstract: Effects of ethephon (2-chloroethylphosphonic acid, ET), which is a producer of ethylene, and 1-methylcyclopropene (1-MCP), which inhibits ethylene binding with the corresponding receptors, on defense responses caused by the causal agent of leaf blotch (Septoria nodorum Berk.) in leaves of soft spring wheat (Triticum aestivum L.) of cultivars contrast in the resistance to the pathogen were studied. After treatment with 1-MCP, an induction of wheat resistance to the disease, more prominent in the susceptible cv. Kazakhstanskaya 10 than in the resistant cv. Omskaya 35, was found. The rise in the resistance was accompanied by rise in zeatin content in leaves, enhanced generation of hydrogen peroxide (most likely, due to the decreased catalase activity and increased peroxidase activity), and accumulation of transcripts of marker genes of the salicylate signaling pathway (PR-1 and PR-2). On the contrary, in ET-treated plants, all the studied defense responses were inhibited, and the pathogen developed more intensively. The effect of ethylene on zeatin distribution in infected wheat leaves of the susceptible cv. Kazakhstanskaya 10 was also found. In the 1-MCP-treated wheat leaves, cytokinins were localized in mesophyll cells and cell walls. In the ET-treated leaves, cell walls were free of zeatin, and the hormone concentrated in developing hyphae of the pathogen. The results allow for the hypothesis that wheat plant resistance is controlled by antagonistic interaction of signaling pathways of salicylic acid and ethylene with participation of cytokinins.

Hormonal changes during flowering in response to paclobutrazol application in mango cv. Alphonso under Konkan conditions

Abstract: Studies were conducted to investigate the effect of soil applied paclobutrazol (PBZ) on the hormonal composition of auxin (IAA), abscisic acid (ABA), cytokinins and gibberellins in 12 years old Alphonso mango trees during the year 2011. Paclobutrazol treatment decreased IAA contain in shoots by 4.3 and 28.2 % at 15 days before bud break and at bud break stage, respectively. Abscisic acid content in PBZ treated trees was 59.85 and 41.11 % higher in leaf and bud, respectively, as compared to untreated trees, during flowering period. Fifteen days before bud break, total cytokinin contents (zeatin, dihydrozeatin riboside, zeatin riboside, isopentenyl adenine, isopentenyl adenosine) in leaf and bud were 25.93 and 37.54 %, respectively less than untreated trees, but at bud break and 15 days after bud break it increased by 31.92 and 36.37 % in leaf and bud, respectively. Paclobutrazol treatment decreased gibberellin contents in shoots. Total gibberellin contents at bud break stage was 51.71 % less in treated trees as compared with untreated trees, while 55.58 % reduction was observed in treated trees from 15 days before bud break to bud break. While in untreated trees slight increment in total gibberellin contents was observed. These results indicated that, PBZ application though decreased gibberellin and IAA contents, but caused increases in ABA and cytokinins in mango shoots to elicit flowering responses.

Micropropagation of globe artichoke (cynara scolymus l.) from underground dormant

Abstract: Side shoots excised from underground dormant buds of Cynara scolymus L. were used as primary expiants to establish in vitro cultures. A 3 X 3 factorial experiment with all possible combinations of three concentrations (0.5, 1.0, 2.0 mg/ liter or 2.22, 4.44, 8.88 \iM) of 7V6-benzyladenine (BA) and three concentrations (0, 0.1, 0.2 mg/liter or 0, 0.54, 1.07 ujlf) of 1-naphthaleneacetic acid (NAA) was used to determine the optimum growth regulator combination for shoot multipli cation. The highest rate of axillary shoots was induced on Murashige and Skoog agar medium supplemented with 0 mg NAA/liter and 1.0 mg BA/liter (4.44 \iM). Other cytokinins tested (kinetin, zeatin, and 2-isopentenyl-adenine were less effective than BA in inducing axillary shoot growth. Up to 60% of elongated microshoots rooted after 5 weeks on 1/2 MS agar medium supplemented with 2 mg/liter (11.42 \xM) indole-3-acetic acid (IAA). Seventy percent of rooted plantlets were transferred successfully into soil. Plants are under evaluation for their genetic uniformity and clonal fidelity.

In vitro seed germination, plantlet growth, tuberization, and syntheticseed production of Serapias vomeracea (Burm.f.) Briq.

Abstract: Orchids are considered recalcitrant plants in in vitro propagation. Due to the lack of appropriate micropropagation techniques for mass production and damage to their ecological distribution posed by local gatherers, these species are threatened with extinction, including Serapias vomeracea (Burm.f.) Briq. In this research, we put forward a complete micropropagation method covering in vitro micropropagation, synthetic seed formation, germination in soil, and acclimatization to ambient conditions. To the best of our knowledge this is the first report of successful synthetic seed formation and germination of S. vomeracea. Initially, seeds were germinated in different culture media and also media supplemented with different concentrations of plant growth regulators. Effects of plant growth regulators on tuber formation, glucomannan contents, and different growth parameters were evaluated throughout the study. The best germination rate (84.03%) was achieved on Orchimax including activated charcoal medium and supplemented with 2.0 mg/L 6-benzyladenine. The longest shoot elongation amongst plantlets was observed on the same medium supplemented with 0.25 mg/L thidiazuron, whereas 2.0 mg/L indole-3-butyric acid favored leaf formation. Higher indole-3-butyric acid concentrations were found to be more effective in the formation and elongation of roots. Orchimax medium supplemented with zeatin (2.0 mg/L) was superior to the others in terms of tuber formation and glucomannan content therein. Adaptation of seedlings to soil conditions and germination abilities of synthetic seeds were also studied and seedlings were successfully acclimatized and adapted to soil conditions.

Roles of plant hormones and anti-apoptosis genes during drought stress in rice (Oryza sativa L.).

Abstract: We previously identified the rice (Oryza sativa) senescence-associated gene OsSAP which encodes a highly conserved protein involved in anti-apoptotic activity. This novel Bax suppressor-related gene regulates tolerance to multiple stresses in yeast. Here, we show the effects of drought stress on leaf and root tissues of plants over-expressing OsSAP in relation to the levels of phytohormones, abscisic acid (ABA), jasmonic acid (JA), indole-3-carboxylic acid (ICA), gibberellic acid (GA3), and zeatin. Results showed that rice plants over-expressing SAP were tolerant to drought stress compared to wild type and the plants over-expressing AtBI-1, which is a homolog of the human Bax inhibitor-1 in Arabidopsis. ABA and JA levels in OsSAP and AtBI-1 transgenic plants consistently increased up to at least 3 days after drought treatment, whereas lower GA3 levels were recorded during early drought period. Comparison between control and transgenic plants overexpressing anti-apoptosis genes OsSAP and AtBI-1 resulted in different patterns of hormone levels, indicating that these genes are involved in the plant responses to drought stress and present an opportunity for further study on drought stress tolerance in rice and other plant species.

Genetic transformation of chilli (Capsicum annuum L.) with Dreb1A transcription factor known to impart drought tolerance

Abstract: Chilli, an important commercial crop, is recalcitrant to in vitro regeneration. In the present study, an attempt has been made to optimize the in vitro regeneration of two chilli local cultivars, viz., G4 and LCA334, and transform G4 with a transcription factor dreb1A under the control of a desiccation inducible promoter rd29A, known to impart desiccation tolerance, using binary vector pCAMBIA 2301. Different phytohormones and their concentrations in MS medium were evaluated for in vitro response; 0.25 mg L -1 zeatin and 2 mg L -1 phenyl acetic acid (PAA) supported the highest regeneration rate (37.86%). Among the explants, cotyledonary leaf exhibited a higher (51%) regeneration response compared to that of hypocotyls (24.73%), and genotype G4 had better (33.80%) regeneration rate compared to LCA334. Rooting of the shoots was the highest in MS medium with 2 mg L -1 indole butyric acid (IBA) compared to other hormones. The presence of transgene was confirmed by polymerase chain reaction (PCR) and Southern blotting. The acclimatized transformants were grown in pots and screened for drought tolerance. Some of the transformants showed improved tolerance to drought by lower wilting compared to the control plants. The transformation and regeneration protocol described in the present study may help in optimization of transformation of chilli for agronomic traits of interest.

Effects of Organic Additives and Plant Growth Regulators on Protocorm Development of Dendrobium lowii

Abstract: A simple and efficient growth protocol was develop forDendrobium lowii,an endangered and Borneo’s endemic epiphyte orchid, using four month old protocorms as explant sources produced by asymbiotic seeds germination. Protocorms of Dendrobium lowii were cultured on Knudson C (KC) media supplemented with organic additives (coconut water, tomato juice and banana pulp) or plant growth regulators (NAA, Zeatin and BAP) at different concentrations and observed for protocorm development. Among all organic additives tested, medium containing banana pulp at 25g/L induced the highest growth index value of 593.3 after 240 days of culture. This treatment also promoted 100% production of shoot and 93.3% of root formation compared to other treatments. Addition of 2g/L peptone or 15% (v/v) coconut water had significantly induced 16.7% protocorms proliferation. The supplementation of 6 μM NAA promotes similar responses for growth index of 563.3. The treatment induced up to 86.7% and 83.3% of protocorms forming shoots and roots, respectively. The study also revealed that the addition of 2 or 4 μM of NAA and 4 or 6 μM BAP is suitable for shoot induction, however with poor rooting formation. This finding is important for conservation and horticultural manipulation of the species.

EFFECT OF GENOTYPE ON THE in vitro REGENERATION OF Stevia rebaudiana VIA SOMATIC EMBRYOGENESIS Efecto del genotipo sobre la regeneración in vitro de Stevia rebaudiana a través de embriogénesis somática

Abstract: Stevia rebaudiana (Asteraceae) is a plant of economic importance because of its medicinal properties and the presence of sweetener compounds on its leaves. These compounds can be a substitute for sucrose in a wide variety of products used by persons with diabetes and obesity problems. To standardize an efficient and effective propagation method for the different Stevia genotypes grown in Colombia, this study evaluated the effect of different combinations of the plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 6-(gamma, gamma-dimethylallylamino) purine (2iP) and Zeatin on the induction and development of somatic embryos. Adenine and coconut water were also evaluated as supplements in the basal culture medium Murashige and Skoog Basal Salt Mixture (MS) with glutamine. The combination of 2,4-D (18.09 µM) and 2iP (7.38 µM) produced the highest number of somatic embryos per explant, which had well-defined characteristics. The genotype showed a significant effect on the embryogenic response. In the "SRQ-93" genotype, the formation and development of somatic embryos was achieved, whereas the genotypes "Bertoni" and "Morita II" only yielded embryogenic and non-embryogenic calli, respectively. The conversion to seedlings was achieved on the regeneration medium containing gibberellic acid (GA 3 ) (0.29 µM) and activated charcoal.

Dependence of root biomass accumulation on the content and metabolism of cytokinins in ethylene-insensitive plants

Abstract: Diverse functions of ethylene in plants may depend on its ability to interact with other hormones. We studied the participation of ethylene in the regulation of accumulation and metabolism of cytokinins comparing ethylene-insensitive mutant plants of arabidopsis (Arabidopsis thaliana [L.] Heynh., etr1-1) with the plants of original ecotype Columbia (Col-0). Because cytokinins can regulate growth of both leaves and roots, we determined the weights of these organs and the ratio between them. The content of zeatin and its riboside in the roots of etr1-1 plants was two times greater than in Col-0 plants, which could be accounted for by inhibition of conversion of these forms of cytokinins into 9-N-glucosides. In the leaves of mutant plants, expression of IPT3 gene responsible for the synthesis of cytokinins was more intense than in Col-0 plants, which could also contribute to a rise in the content of cytokinins. In this case, the weight of roots in etr1-1 mutants was lower than in the plants of original ecotype. Because high concentrations of cytokinins can inhibit root growth, suppression of accumulation of their biomass in mutant plants may be related to a greater content of cytokinins therein. The obtained results suggest that ethylene can suppress accumulation of cytokinins and, thereby, maintain redistribution of biomass in favor of the roots, which is important for plant adaptation to a shortage of water and ions.

Expression analysis of D-type cyclin in potato ( Solanum tuberosum L.) under different culture conditions

Abstract: Taken together, this study suggests that potato D-type cyclin genes are expressed differentially between in vitro and planta caused by intrinsic differences in physiology and gene action at the cellular and whole organism levels. Cyclin performs a pivotal role in control of the cell cycle by forming a complex with cyclin-dependent kinases (CDKs). In this study, the potato cyclin D3 genes (StCycD3.1, StCycD3.2, and StCycD3.3) were isolated and analyzed. A sequence analysis showed that the potato cyclin D3 genes shared high levels of sequence homology with tomato cyclin D3 genes. The potato cyclin D3 genes revealed organ-specific expression; StCycD3.1 was strongly expressed in above-ground organs, whereas StCycD3.2 was strongly expressed in underground organs. The expression patterns of the potato cyclin D3 genes were analyzed under a variety of environmental conditions such as different carbon sources and hormones. Glucose (as a carbon source) and zeatin (as a hormone) were effective single factors for increasing potato cyclin D3 expression. Sucrose and zeatin were the most effective combination for high-level induction of the genes. Time-course-based gene expression patterns were evaluated in treatments of leaf explants with combinations of different light conditions and hormones. The potato cyclin D3 genes were expressed abundantly in the presence of hormones during the late stage of long-term dark conditions. The gene expression patterns were also monitored in entire potato plants under different light conditions. The gene expression levels were consistently low under the different light regimes but potato cyclin D3 genes were up-regulated during a shift in illumination from dark to light.

Determination of Abscisic Acid, Gibberellic Acid, Indole-3-Acetic Acid, and Zeatin Riboside in Masson Pine (Pinus massoniana L.) by Accelerated Solvent Extraction and High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Abstract: A highly sensitive and reproducible method without derivatization is reported for the determination of abscisic acid, gibberellin A3, gibberellin A4, indole-3-acetic acid, zeatin, and zeatin riboside in Masson pine (Pinus massoniana L.). The method combined accelerated solvent extraction, solid-phase extraction, and high-performance liquid chromatography with electrospray ionization–tandem mass spectrometry to determine these hormones. The optimal conditions for accelerated solvent extraction were 4:1 methanol/water acidified with 1.0% glacial acetic acid as the solvent with three 15-min cycles at 25°C and 1300 psi. The recoveries from the pine samples were from 86.8 to 105.4%. The limits of detection were from 0.005 ng L−1 for abscisic acid to 0.500 ng L−1 for gibberellin A4. The intraday precision values were 2.2–7.7%, with interday precision from 3.3 to 10.6% for all analytes. The developed method allows the identification and determination of endogenous hormones in tree samples.

In-Vitro Organogenesis in Tomato (Solanum Lycopersicum) Using Kinetin

Abstract: This research was undertaken for optimizing simple and reproducible protocol on indirect regeneration in tomato using kinetin. The effect of kinetin for efficient callus production was studied and its results were interpreted. Hypocotyls and cotyledons were used as explants and effect on callus derived regeneration was greatly influenced by addition of kinetin in media. The individually positioned explants on MS media supplemented with two cytokinins BAP (2.0mg/L) and kinetin (1.75mg/L) proved to produce good embryogenic calli and developed into whole plantlets. Lower and higher kinetin concentrations produce lesser calli and results derived using mean values in this experiment suggest closely equal concentrations of kinetin to other cytokinin can be a potential calli inducer in tomato regeneration. Multiple shoot induction data was recorded and its mean values interpreted. Shoots transferred in shooting elongation media MS+ IAA (0.1mg/L) + Zeatin (2.0mg/L) gave better shooting response (89.4%) and after two weeks these elongated plantlets were subjected to rooting. Therefore kinetin with combination of BAP majorly influences in-vitro organogenesis in tomato plant regeneration.

In vitro multiplication of lingonberry - Short Communication

Abstract: Paprstein F., Sedlak J. (2015): In vitro multiplication of lingonberry – Short Communication. Hort. Sci. (Prague), 42: 102–106. Although plants of Vaccinium genus have not been cultivated on a large scale in the Czech Republic, there is a potential for commercial lingonberry production in some mountain regions. The purpose of this study was to develop an efficient in vitro system for a quick multiplication of lingonberry cvs Koralle, Linnea, and Runo Bielawskie. McCown woody plant medium (WPM), Anderson’s rhododendron medium (AN) and half-strength Murashige and Skoog medium (half-MS) containing cytokinin zeatin in concentrations 0.5, 1 or 2 mg/l were tested for repeated subcultures. The number of newly formed shoots varied with the cultivar, medium tested and concentration of zeatin. Across all experiments, the highest multiplication rate (8.9 ± 0.6) was obtained for cv. Runo Bielawskie on WPM medium with the highest concentration 2 mg/l of zeatin. The lowest multiplication rates 1.1 ± 0.0 were noted on half-MS medium with the lowest concentration of zeatin (0.5 mg/l). In conclusion, micropropagation techniques described in this paper increased multiplication mainly in lingonberry cv. Runo Bielawskie on WPM medium. However, some cultivars of lingonberry would still require further research to optimize proliferation media.