Addition Of Zeatin Research Articles

Особенности органогене за труднокул ьтивируемых ни зкорослых сортов вишни обыкновенной (Prunus ceras us L.) в кул ьтуре in vitro

The article presents the results of research on clonal micropropagation of sour cherry (Prunus cerasus L.) which is difficult to reproduce at the proliferation stage by traditional methods. A number of modern winter-hardy domestic cultivars of P. cerasus are promising for plantation cultivation of this crop in the Nonchernozem zone of the European part of Russia. The method of clonal micropropagation is advisable to obtain a large amount of healthy and genetically homogeneous rootstocks of fruit crops. Regenerated plants of P. cerasus of low-growing, mid-season and winter-hardy cultivars Assol’ and Shokoladnitsa were used as research objects. The growth and developmental features of P. cerasus regenerated plants were studied during in vitro cultivation on a QL nutrient medium supplemented with growth regulators of the cytokinin group (6-BAP, thidiazuron, zeatin) in different concentrations. The highest indicators for the length of microshoots (average 13.4 mm) and the number of leaves (average 6.0 pcs.) of P. cerasus plants in in vitro culture are recorded for the Shokoladnitsa cultivar. The maximum length of microshoots of P. cerasus Assol’ cultivar is observed on the 60th day of cultivation on a QL nutrient medium with the addition of zeatin at a concentration of 0.3 mg/l (10.5 mm) and thidiazuron at a concentration of 0.3 mg/l (9.8 mm), while the maximum number of leaves (7.8 pieces) is observed with addition of 0.3 mg/l zeatin. The maximum length of microshoots of P. cerasus of the Shokoladnitsa cultivar is observed when grown for 60 days on a QL nutrient medium with the addition of 6-BAP at concentrations of 0.5 mg/l (17.7 mm) and 1.0 mg/l (14.2 mm), while the maximum number of leaves (7.2 pieces) is observed with addition of 0.3 mg/l zeatin.

КЛОНАЛЬНОЕ МИКРОРАЗМОЖЕНИЕ VACCINIUM CORYMBOSUM L. СОРТА БЛЮ БЕРРИ В КУЛЬТУРЕ ТКАНЕЙ

For efficient clonal reproduction of Vaccinium corymbosum L. of Blue Berry variety, the surface explant sterilization approaches have been developed - the apical part of the stem and stem segments using “Belizna” commercial bleach solution and nutrient media for explant cultivation have been selected. The maximum survival of the explants (100%) was observed during sterilization with a 15% of “Belizna” bleach solution with an exposure time of 15 minutes. The nutrient media Murasige-Skuga, WPM and Anderson were compared for effective breeding. The highest breeding rate of blueberry was on WPM medium with the addition of zeatin and indolyl-3-butyric acid. The maximum number of axillary shoots per explant was 3.80, with an average length of 3.26 cm, obtained on WPM medium containing 1.0 mg/l of zeatin and 0.1 mg/l of indolyl-3-butyric acid. It has been shown that with an increase in the number of passages, growth rates improve. The maximum increase in the number of healthy axillary shoots is observed at the fourth passage, while the phenomenon of vitrification begins to appear from the fifth passage.

INDUKSI PERTUMBUHAN TUNAS DARI EKSPLAN ANGGREK Dendrobium Heterocarpum Lindl. DENGAN PEMBERIAN HORMON ZEATIN DAN NAA

Dendrobium heterocarpum Lindl. is a native orchid in Indonesia. Its population continue to decline due to unsustainable exploitation. One solution to increase its population is via in vitro technique propagation. This research aimed to determine effect of hormone addition (Zeatin and NAA) to induce shoot multiplication of D. heterocarpum Lindl. grown in Murashige and Skoog (MS) basic medium. The research was conducted in Tissue Culture Laboratory, Department of Biology, Faculty of Mathematics and Science, Udayana University from November 2015 until March 2016. The experiment employed Completely Randomized Design (CRD) with 20 combinations of treatments, with three replicates each treatment. The treatment werer addition of Zeatin and NAA, with five concentrations of Zeatin (0, 0.1, 0,5, sweet corn and hybrids corn as the natural Zeatin); four NAA concentrations (0, 0.05, 0.1 and 0.5). Variable recorded include such as the number of shoots, roots and explant’s vigor. The quantitative data which include number of leaves and shoots after 12 weeks, were analyzed using Analysis of Variance (ANOVA); and if significantly different, tested further using Duncan Multiple Range Test (DMRT) at 5% level. Z5N3 medium revealed significantly different on amount of leaves and shoot height and also the best medium on amount of roots. Best hormone combination that can induce multiplication of new shoots are medium Z4N3 and Z5N1.

Improved Protocol for Agrobacterium-Mediated Transformation of Pea (Pisum sativum)

Pea is one of the most important legume crops whose production is constantly threatened by field and storage pests and diseases. Developing insect resistant transgenic pea plants through Agrobacterium-mediated transformation is a promising solution to maintain crop yield. However, the transformation efficiency is still low. Therefore, there was an attempt to enhance transformation efficiency by optimizing infection time, co-cultivation period and in vitro regeneration system. Transformation was performed using segments of embryonic axes from mature pea seeds (Pisum sativum L. cv. Sponsor). The segments were inoculated with the hyper virulent EHA105 strain of Agrobacterium tumefaciens. The Agrobacterium strain harboured a binary vector pGII35S containing the bar gene which confers resistance to phosphinotricin. The selection medium contained P2 medium with increasing concentrations of phosphinothricin. To improve the regeneration efficiency, 4.5 μM zeatin was added to the selection medium. The highest transformation efficiency (7.89%) was achieved with infection time of 90 min and co-cultivation period of 2 days. The shoots elongated well and the number of shoots/explants was increased (6 folds) after addition of zeatin. Resistant shoots were grafted onto rootstocks in soil and grafting success rate was 100%. The integration of cry1Ac gene in T0 transgenic plants was confirmed primarily by Polymerase Chain Reaction and further analysed by Southern blotting.

Induction of somatic embryogenesis and biochemical characterization of Cordyline australis (G. Forst.) Endl. ‘Red Star’ callus

The current study presents a method for the plant regeneration from stem segments of Cordyline australis (G. Forst.) Endl. ‘Red Star’ through somatic embryogenesis. We tested the effect of 3 kinds of auxin 4-amino-3,5,6-trichloropicolinic acid (picloram), 2,4-dichlorophenoxyacetic acid (2,4-D), and 2-methoxy-3,6-dichlorobenzoic acid (dicamba), with a combination of 2 kinds of cytokinin, 6-benzyladenine (BA) and thidiazuron (TDZ), on the induction of embryogenic callus and its capacity to induce somatic embryogenesis under dark culture condition. The highest percentage of explants forming callus (nodular and friable) was observed on medium containing 25μM picloram and 0.5μM BA. The embryogenic callus, with white-yellowish nodular structures, was proliferated in the initial media and finally produced somatic embryos on all media with picloram and 2,4-D. Somatic globular embryos were obtained with a higher frequency from explants cultured on MS medium supplemented with 25μM picloram and 0.5μM BA. On this medium, a mean number of 23.0 embryos developed per gram of callus. Globular embryos developed most efficiently into cotyledonary stage on MS medium supplemented with 5μM picloram and 1μM BA. The addition of zeatin (5 and 2.5μM) and NAA (0.5 and 0.25μM) to the medium was found to be essential for the highest conversion of somatic embryos into plants. Plantlets were successfully acclimatized in the greenhouse with 90% survival rate. Callus was tested for phenolic compounds, sugars, peroxidase activity and protein contents in order to find if these compounds might be the indicators of further embryos development. The embryogenic callus of C. australis with various stages of embryo development had higher level of protein than the non-regenerable one, whereas level of sugars increased only in callus with embryos in cotyledonary stage. Callus with globular embryos contained high concentration of phenolics and low activity of peroxidase, comparing to other types of callus.

An Improve Method for Somatic Embryogenesis of Schisandra chinensis (Turcz.) Baillon

A somatic embryogenesis protocol for plant regeneration of Schisandra chinensis (Turcz.) Baillon was established. Increased callus induction was obtained from mature zygotic embryos on Murashige and Skoog (MS) medium supplemented with 1.0 mg L(-1) N-phenyl-1,2,3-thidiazol-5-yl-urea (TDZ) or 2.0 mg L(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of Zeatin (Zt) promoted the formation of embryogenic calli. To induce somatic embryogenesis, 2,4-D, TDZ and Zt were incorporated in the medium alone or in combination. Development of the maximum number of somatic embryos (81 globular, 37 heart, 52 torpedo and 37 cotyledon-stage) and germination of the highest number of embryos (50%) was observed on 1/2 MS medium supplemented with 1.0 mg L(-1) TDZ and 0.2 mg L(-1) Zt. Further development of somatic embryos into plantlets was completed in 1/2 MS medium free of plant growth regulators.

Exogenous zeatin accumulation in wheat-root cells and its role in regulation of cytokinin transport

Here we have shown that 24 hours after addition of zeatin to the nutrient solution the cytokinin content in xylem sap of wheat plants appears to be about 10 times lower that in the nutrient solution. Cytokinins accumulated mostly in roots and not in shoots of treated plants. These data demonstrate the existence of some barrier on cytokinin pathway from the nutrient solution to the plant shoot. With the help of Sudan III an increase in lignin and suberin deposition in the endodermis could be detected, being stronger with the increase in the distance from the root tip. The increase in deposition of suberin and lignin coincided with the decrease in cytokinin immunolabeling in root cells revealed with the help of monoclonal cytokinin antibodies and the second gold-labelled antibodies. Simultaneously exogenous cytokinins accumulated in root stele cells showing that the Casparian band was not only barrier on cytokinin pathway to plant shoot. It is concluded that high cytokinin immunolabe ling in the stele parenchyma cells around the stele vessels demonstrated accumulation of cytokinins by these cells, which could be important in regulation of cytokinin loading to the xylem vessels during there transport to the shoot. The role of cytokinin transporters is discussed.

High-frequency regeneration via somatic embryogenesis of an elite recalcitrant cotton genotype (Gossypium hirsutum L.) and efficient Agrobacterium-mediated transformation

A highly efficient and reproducible regeneration system based on somatic embryogenesis in Gossypium hirsutum cv. Narasimha (NM), which has superior fiber qualities and is also used as a female parent in several hybrid cottons, has been developed. Embryogenic callus was obtained form both hypocotyls and cotyledonary leaves on Murashige and Skoog (MS) medium containing kinetin and 2,4-dichlorophenoxyacetic acid. Somatic embryogenesis was observed on hormone-free MS medium, but embryos did not grow well beyond globular stage. However, somatic embryos germinated well on MS medium containing B5 vitamins; addition of zeatin was found to be beneficial for their normal development. Most importantly, the media and culture conditions developed for NM were also found to be suitable for high-frequency somatic embryogenesis in Coker 310. In addition, the newly developed regeneration protocol has been successfully tested for genetic transformation through co-cultivation with Agrobacterium using embryogenic calli as explants. Molecular analysis confirmed the stable integration and expression of marker gene, green fluorescent protein (GFP). These results show that it is now possible to introduce foreign gene(s) directly into elite cultivar Narasimha with similar efficiency to in traditionally used Coker lines in a relatively short period of time. Development of efficient regeneration and transformation systems as demonstrated here should augment the introduction of new traits directly into cultivated varieties/hybrids, reducing the time required for back-crossing and the costs for seed production, besides aiding genomic research in cotton.

In vitro development and germination of immature olive embryos

SummaryIn vitro culture methods were used to germinate olive embryos prior to maturation. Fruit, seed and embryo development were established with consecutive sampling from 20 to 100 days after bloom. For that same period, embryo development and germination success were determined by in vitro culture trials using one-third strength MS medium with or without the addition of zeatin. For early developmental stages, when isolation of the embryo was difficult, a cut portion of the seed containing the embryo was used for culture. The embryos cultured within the cut seed portions germinated and formed normal plantlets. Histological observations indicated a close similarity between the natural and in vitro immature embryo differentiation pattern, progressing through preglobular, globular, heart-shaped and torpedo-shaped stages. In some cases, however, the in vitro immature embryos developed or germinated abnormally. The presence of zeatin (0.25 mg l–1) in the culture medium and the use of a cut seed-portion containing the immature embryo allowed in vitro germination sooner after bloom than previously obtained. On the contrary, zeatin was a handicap for mature olive embryo in vitro germination, which reached 100% seedling formation when no plant growth regulators were used.

In vitro development of young maize ears

The effects of sucrose, cytokinin and auxin on in vitro growth and development of immature maize ears (female inflorescences) isolated from the first and second nodes of a prolific and a nonprolific hybrid were evaluated. The explants were cultured on an agar-solidified Murashige and Skoog medium and tested at four sucrose concentrations, five cytokinin (6-benzylamino purine and zeatin) concentrations, and six zeatin/indole-3-acetic acid (IAA) combinations. Sucrose at 60 g l−1 resulted in the best ear growth and development in both hybrids and both node positions. Ear lengths from the first and second nodes of the prolific hybrid were further increased by 100 and 62% respectively, by the addition of zeatin (1 mg l−1) + IAA (1.5 mg l−1), whereas the ears of the nonprolific hybrid increased by 45 and 33%, respectively. Ear development was also increased by these treatments (114, 166, 57 and 57%, respectively), compared with the control without hormones. When growing in vivo, the second ears of the prolific hybrid were able to reach anthesis (stigma exposition) while in the nonprolific they could not develop beyond the style elongation stage.

Medium optimization for efficient somatic embryogenesis and plant regeneration from immature inflorescences and immature scutella of elite cultivars of wheat, barley and tritordeum

Media have been developed for somatic embryogenesis and plant regeneration from immature inflorescences and immature scutella of elite cultivars of wheat, barley and tritordeum. For wheat and tritordeum inflorescences, regeneration from embryogenic calluses induced on medium with picloram was almost twice as efficient as regeneration from cultures induced on 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of zeatin at 5 or 10 mg l−1 to regeneration media had a positive effect on regeneration. For scutella, the highest frequencies of embryogenesis (85%) and regeneration (50%) was obtained using an induction medium containing 2 mg l−1 of 2,4-D and half concentration of aminoacids. The morphogenetic capacities of 19 different cultivars of wheat, barley and tritordeum were compared, and clear differences were found both between explants and genotypes. In wheat, embryogenic capacity from inflorescences (average of 92%) was higher than from immature scutella (average of 62%). However, shoot regeneration from scutella was clearly higher than from inflorescences (averages of 63%, and 18% respectively). Frequencies of regeneration in wheat and barley varied widely among the cultivars tested and in both species no difference was found between spring and winter varieties.

Improved Callus Quality and Prolonged Regenerability in Model and Recalcitrant Barley (Hordeum vulgare L.) Cultivars.

In vitro performance of immature embryo-derived callus of two barley cultivars, the amenable cultivar, Golden Promise (GP) and the recalcitrant commercial cultivar, Galena (GL), was tested on fourteen media containing combinations of two auxins, dicamba and 2, 4-dichlorophenoxyacetic acid (2, 4-D) and two cytokinins, 6-benzylaminopurine (BAP) and zeatin. Generalizations for both cultivars include the fact that callus-induction frequencies on dicamba and 2, 4-D alone and dicamba plus zeatin were nearly 100%. Qualitative appearance for both cultivars was nearly identical on comparable media but markedly better on BAP-containing media. The addition of zeatin with either auxin supported vigorous growth due to the proliferation of nonregenerable callus; the presence of BAP produced higher quality callus although it inhibited the relative growth rate. GP calli grown on 2, 4-D plus 0.01mg/L BAP regenerated the largest number of green shoots at most time points relative to other media, while GL tissue on 2, 4-D plus 0.1mg/L BAP yielded the most green shoots at all but one time point. Over time regenerability was lost more rapidly in GL than GP except on 2, 4-D plus BAP media; GL had a higher propensity toward albinism than GP. In both cultivars, the use of 2, 4-D and BAP yielded tissue that proliferated for prolonged periods in a regenerable state.

Precocious Germination of Tomato Embryos in vitro

Precocious germination of tomato ( Lycopersicon esculentum Mill.) embryos from self or intraspecific pollination was studied in an attempt to speed up breeding programs. Growth of embryos excised from fruits 2 weeks post-anthesis was slow. Three week old embryos developed into transplantable plantlets 3 weeks earlier than the time needed for ripe seed produced plantlets. Optimal development of the embryos was achieved on medium without hormones. Exogenous indoleacetic acid (0.1 or 1.0 mg 1 -1 ) neither supported nor interrupted growth, whereas addition of zeatin (Z, 0.02 or 2.0 mg 1 -1 ) reduced growth in positive correlation with Z concentration. In two-week-old embryos Z caused abnormal growth and adventitious bud development on their hypocotyl. Adventitious buds rarely appeared on plantlets from 3week-old embryos and never on plantlets from older embryos grown on Z containing media. The results show that rapid development of embryos into transplantable plantlets depended on the embryos age and did not need exogenous hormones.