Stages Of Micropropagation Research Articles

Clonal micropropagation of Thymus vulgaris L.

Thymus vulgaris L. is one of the widely known spicy aromatic and medicinal plants. Thyme plant material is widely used in medicine, cooking and perfumery. To increase the efficiency of breeding and seed production, it is necessary to develop biotechnological techniques, in particular, clonal micropropagation. The aim of the research is to optimize the composition of culture media for the main stages of propagation in vitro and to select adaptation ex vitro conditions for the development of Thymus vulgaris. clonal micropropagation. The article presents the results of studies of explant morphometric parameters cultivated on 20 variants of culture media at firstsecond stages of micropropagation. It was found that the optimal culture medium at the introduction stage is MS medium with 1.0 mg/l Kin and 1.0 mg/l GA3, on which, on average, 2.2 microshoots per explant with a length of 1.9 cm were obtained. Both high vitrification rate of microshoots and formation of small shoots (0.6-0.9 cm) were observed on media supplemented with BAP or TDZ. The most effective culture medium at the proper propagation stage is MS with 1.0 mg/l Kin, on which 4.6 shoots per explant and the multiplication index 12.8 were obtained. It is advisable to root microshoots at the 3rd stage of micropropagation on MS culture medium supplemented with 1.0 mg/l IBA or 1.0 mg/l IAA. It has been shown that it is possible to obtain high plant survival rate (89.5%) during adaptation ex vitro, using a substrate consisting of peat and perlite (1:1).

Application of bioactive compounds for increasing production of grape planting materials and higher germination of hybrid seeds

The paper presents the results of studying biological methods of increasing the yield of planting material for grapes and increasing the germination of hybrid seeds to accelerate the selection process. The impact of bioactive compounds (6-benzylaminopurine and gibberellic acid) in culture medium on production of grape clones during micropropagation stage was studied. Modified culture medium of the A.N. Rebrov’s patent was determined as having the most potential on the first stage of grape explant cultivation. The establishment of grape regenerant plants was up to 80%, depending on the variety. On the second and third passages Rebrov’s culture medium was modified by the addition of 6-benzylaminopurine in the concentration 0.35 mg/l and gibberellic acid in the concentration 0.1 mg/l. The method of prestratification soaking of seedlings seeds in the solutions of gibberellic acid of various concentrations was also studied to accelerate the breeding process. The production of seedlings increased to 91.2% at the concentration of gibberellic acid 0.01% that was higher than control production on 31.2%.

Application efficiency of Superstim modifications in low doses on Lonicera caerulea adaptation stage and it’s after-effect during subsequent growing

Relevance. In recent years, interest in the edible honeysuckle culture has increased in Russia, the wide distribution of which is hampered by the lack of quality planting material. The technology of clonal micropropagation allows for a short time to obtain a large amount of honeysuckle planting material, more than a thousand regenerated plants per year from one meristematic apex introduced into an in vitro culture. It is hundreds of times more than in traditional methods of vegetative propagation. Adaptation to non-sterile conditions is the final and most crucial stage of clonal micropropagation, the loss of which can be from 50 to 90%. It should be noted that there is practically no research on how the further development of adapted honeysuckle plants takes place during subsequent growing.Methods. Researching of growth regulators of the new generation Superstim 1 and Superstim 2 effect in low and ultra-low doses on the survival rates and development of honeysuckle plants at the stages of adaptation subsequent growing.Results. Superstim 1 is more effective at physiological concentrations – 1 x 10-7 and in the field of ultra-low doses – 1 x 10-14, 1 x 10-15%. At the stage of subsequent growing, a positive after-effect of physiological concentrations – 1x10-3 and 1x10-7 was observed, and an ultra-low dose – 1x10-17%. The growth regulator Superstim 2 at the stages of adaptation and subsequent growing is effectively used only in one concentration – 1x10-16%. The additional foliar treatments at the stage of subsequent growing are not necessary.

In vitro micropropagation and ex vitro rooting of some potato varieties

The article presents the results on selection of optimal concentrations of nutrient media components and nutrient solution at the stages of clonal micropropagation of potato varieties Lyubava, Kemerovochanin, and Tuleevskiy (actual reproduction, rooting in vitro, adaptation to ex vitro conditions). The influence of some components of the nutrient medium (sucrose, agar-agar, growth regulators, namely α-naphthyl acetic acid, β-indolyl acetic acid, and β-indolyl propionic acid) was studied at the stages of reproduction and rooting in order to obtain regenerants of the studied potato varieties. The best development of plants on nutrient medium with addition of 4 g L-1 of agar-agar was revealed. The addition of sucrose in the concentration of 3-5% contributed to the formation of more internodes. The influence of naphthyl acetic acid, β-indolyl oil, and β-indolyl propionic acids in different concentrations on the rhizogenesis of regenerating plants of three potato varieties at the stages of reproduction and establishment of different types and concentrations of auxins was studied. The researches have shown that a one-stage method of adaptation of Solanum tuberosum L. varieties of Lyubava, Kemerovochanin, and Tuleevskiy to the conditions of ex vitro cultivation with the use of hydroponic installation is characterized by efficiency and allows to receive plants with well-developed root system. The efficiency of using the hydroponic plant for adaptation of potato regenerants at the final stage of micropropagation is shown.

Micropropagation of tree peony (Paeonia sect. Moutan): A review

Tree peony is a well-known ornamental plant that is also valued for its medical uses and edible oil production. A long breeding period and low propagation rate are the main hurdles hindering the development of the tree peony industry, for which micropropagation can offer a solution. This article reviews the advances in tree peony micropropagation during the past three decades, providing a detailed analysis of the conditions required for the four stages of micropropagation (initiation, multiplication, in vitro rooting, and acclimatization). Additionally, potential of tree peony micropropagation for commercial application was evaluated. This review provides valuable information for the successful micropropagation of tree peony, permitting more targeted and in-depth research into tree peony micropropagation. The review summarized in detail the results achieved in tree peony micropropagation over the last three decades. Furthermore, the potential of the existing micropropagation protocol was evaluated and the problems and the future perspectives were summarized.

Effects of silver nanoparticles and zirconium trisulphide nanoplates on the adaptation of woody species microclones to ex vitro conditions

The transfer of microclones to nonsterile conditions is one of the critical stages of micropropagation. Nanoparticles and nanomaterials can significantly improve effectiveness of this technique if used as substances increasing adaptive capabilities of the propagated plants. We have studied the impact of silver nanoparticles and zirconium trisulphide nanoplates on the adaptation of microclones of white poplar × aspen hybrid, hairy birch, crack willow, red oak and scots pine upon transfer to ex vitro conditions. The performed study has revealed that foliage application of colloidal aqueous solutions of zirconium trisulphide and silver nanoparticles at 3 μg/l concentration to in vitro microclones was more effective than the reference growth regulator. The best results were observed in the experiments with red oak microclones, where the number of surviving and adapted plants increased by 50-60 %. Increase in biomorphological parameters was accompanied by improvement of photosynthetic activity. The results indicate high potential of zirconium trisulphide nanoplates and silver nanoparticles for protecting woody species microclones during their transfer to non-sterile conditions of glasshouse.

Effect of cytokinins and sucrose concentration on the efficiency of micropropagation of ‘Zes006’ Actinidia chinensis var. chinensis, a red-fleshed kiwifruit cultivar

The effect of N6(3-hydroxybenzyl)adenine (meta-Topolin—mT) was compared with that of N6-benzylaminopurine (BAP) and zeatin at the proliferation stage of micropropagation of red-fleshed Actinidia chinensis var. chinensis ‘Zes006’ in two separate experiments. Shoot number, shoot weight, leaf number and leaf area were significantly higher in mT-supplemented media compared with BAP or zeatin. When transferred to rooting media, plantlets that were propagated in mT-supplemented media readily produced roots, enabling easy acclimation to the greenhouse, whereas none of the plantlets propagated in BAP- or zeatin-supplemented media produced roots. Using 12 pairs of Simple Sequence Repeat primers designed for A. chinensis var. chinensis, a very low rate of somaclonal variation was detected at some loci in plantlets produced in zeatin- (1.04%), BAP- (0.4%) as well as in mT- (0.2%) supplemented media. Overall, mT in equimolar concentrations was the better cytokinin for tissue culture of ‘Zes006’ kiwifruit and may well be applicable to many other kiwifruit genotypes. Supplementation of media with meta-Topolin for in vitro propagation of the red-fleshed kiwifruit cultivar ‘Zes006’ enhanced better shoot proliferation, giving healthy plantlets that were easier to acclimatize to the greenhouse environment compared with media supplemented with 6-benzylaminopurine or zeatin. It also induced a lower rate of somaclonal variation, as detected by SSR markers.

Алманың әр түрлі генотиптерін in vitro клондауды оңтайландыру

At each stage of plant clonal micropropagation appear difficulties, such as a failed sterilization, poor reproduction, abnormal development of microplants and phenolic oxidation plants in a nutrient medium. The consequences of such failures can lead to plant necrosis, and sometimes death. Successful micropropagation of plants depends on several internal and external factors, including ex vitro and in vitro conditions. It is important to create optimal conditions at each stage of micropropagation to achieve a high multiplication coefficient of in vitro plants. The article presents the results of improving the technology of clonal micropropagation of apple plants at different stages. Experiments conducted have shown that apple shoots are effectively sterilized with 0,2% HgCl2 at an exposure of 4 min. In this case, the number of explants capable of regenerating is 76% for variety Golden Delicious, 85% for variety Voskhod, 75% for variety Maksat. The best medium for isolating explants is MS medium containing double amount of iron chelate; vitamin C -1.5 mg/l; polyvinylpyrralidone - 250 mg/l; glycine - 2 mg/l; BAP - 0.5 mg/l; The IMC is 0.1 mg/l, 79% of regenerating apple shoots were obtained with such a composition of the nutrient medium. The optimal medium for micropropagation of apple is the mineral basis of MS with a hormonal composition: 1.0 mg/l BAP, 0.1 mg/l IMC, 0.1 mg/l HA. The multiplication coefficient of the studied cultivars ranged from 4 to 13 depending on the genotype. 36 cultivars and 9 wild forms of apple are introduced into in vitro culture and propagated. Key words: clonal micropropagation, in vitro, sterilization, nutrient medium, apple.

Роль углеводов при клональном микроразмножении садовых растений

Carbohydrates are the necessary component of nutrient medium in course of in vitro propagation. Along with sucrose, sugar and glucose can also serve as a source of carbohydrates in stages of micropropagation itself and rhizogenesis. Increasing the concentration of sucrose up to 40.0 g/l allows to prolong the storage time of pear rootstock PG 12 explants to 46 months and apple rootstock 62-396 storage up to 24 months.

In vitro propagation, clonal fidelity and phytochemical analysis of Rhodiola imbricata Edgew: a rare trans-Himalayan medicinal plant

The present study focuses on development of a micropropagation protocol for true to type plants of Rhodiola imbricata, an endangered medicinal plant found in trans-Himalayan Leh-Ladakh region of India. It also aims at analyzing the pharmaceutically important secondary metabolites and antioxidant potential of in vitro and in vivo plants. Various cytokinins and auxins were tested for shoot proliferation and in vitro rooting of the microshoots, respectively. Random primers were used for checking genetic uniformity at different stages of micropropagation. Pharmaceutically important secondary metabolites of R. imbricata such as Rosavin, total polyphenols and free radical scavenging activity were analyzed by HPLC. Among different cytokinins used, BAP (5 µM) and TDZ (1 µM) were found to perform better in terms of shoot proliferation, shoot length and number of leaves as compared to other concentrations. For rooting of microshoots, a lower concentration of NAA (0.5 µM) yielded more efficient rooting of micro shoots (17.33 roots per micro shoot). In vitro rooted microshoots were hardened and showed 60% survival rate. The content of gallic acid, chlorogenic acid and 4-hydroxybenzoic acid was higher in the in vivo plant. The amount of ferulic acid was higher in the in vitro raised plant when compared to field grown plant. Furthermore, caffeic acid and p-coumaric acid were higher in the in vitro raised plants as compared to field grown plants. This work will facilitate in conservation of this endangered herb and provide necessary plant materials for various biotechnological and pharmaceutical applications.

Sucrose Concentration, Light Intensity, and CO<sub>2</sub> Concentration Affect Growth and Development of Micropropagated Carnation

Carnation (Dianthus caryophyllus L.) ‘Purple Beauty’ at micropropagation stage 3 was cultured under two levels each of medium sucrose concentration (0 and 30 g·L-1), photosynthetic photon flux density (50 and 200 μmol·m-2·s-1 PPFD), and CO2 concentration (350 and 1,000 μmol·mol-1), in a factorial experimental design, consisting of all eight possible treatment combinations. Shoot tip explants, obtained from in vitro-grown plantlets, were cultured on 50 mL agar-solidified Murashige and Skoog medium per container. Each culture container was sealed with a rigid lid vented by a high-efficiency particulate air filter attached to a 10 mm diameter hole made in the lid center, with an estimated number of 2.8 air exchanges per hour. All cultures were maintained for four weeks at 24°C/22°C (day/night) temperatures, 70%–80% relative humidity, and a 16 h/8 h (day/night) photoperiod was provided by white light-emitting diodes. The treatment combining high light intensity (200 μmol·m-2·s-1), high CO2 concentration (1,000 μmol·mol-1), and without supplementation of sucrose to the medium (i.e., photo-autotrophic conditions) resulted in better plantlet growth and development, with higher values being observed in terms of total fresh weight, tissue water content, leaf length, leaf width, and total chlorophyll content than in plantlets grown under the other treatments.

Effect of prolonged cultivation on the micropropagation in vitro of mint cultivars and breeding samples

The influence of the genotype and the number of subcultures on the development of meristem cultures of five mint cultivars and breeding samples – Krasnodarskaya 2, Diana, K37, No. 2.8.14, A2 (Mentha spp.) was studied. At the second stage of micropropagation, the analysis of morphometric parameters of explants during six subcultures of four genotypes showed an increase in the multiplication index at 4-5th passages, while in the sample A2 - at 3-4th passages.

Влияние питательной среды и спектрального состава света на размножение земляники in vitro

The research was conducted in 2016-2017 on the basis of a meristem laboratory. Object of the research is the micro-shoots of garden strawberry the Festival variety and remontant strawberry the Brighton variety at the stages of micropropagation and rooting. The following nutrient media were studied: Murashige and Skoog (control), Murashige and Skoog modified (microelements were replaced with a corresponding dose of microfertilizer “Siliplant”) and Boxyu; LED irradiators - with a ratio in the spectrum of red, blue and white light R2: B1:W1 and R1: B1: W1, respectively, and a programmable one with a changing spectrum. A luminescent lamp with white light served as the control. The highest rate of reproduction in the cultivation of garden strawberry in vitro was obtained using the Murashige and Skoog culture medium under a LED irradiator with a R1: B1: W1 combination of red, blue and white light and the irradiator with a changing spectral composition: 6.0 and 6.2 pieces/microcutting, respectively. The highest reproduction factor of remontant strawberry was provided by cultivation on the Murashige and Skoog nutrient medium using the LED light irradiator in ratio of red, blue and white as R1:B1:W1 and the irradiator with a changing spectral composition: 3.5 and 3.2 pieces / microcutting, respectively. A rooting of micro-shoots of garden strawberry on the modified Murashige and Skoog nutrient medium and Boxyu provided a significant increase in rooting ability to 100% and 97.5%, respectively. Cultivation on the Boxyu nutrient medium has increased the rooting ability of micro-shoots of remontant strawberry to 95.0%. All the LED irradiators provided a significant increase in the rooting ability of micro-shoots: for garden strawberries - up to 96.6-100%, for remontant - up to 83.3-90.0%.

Начальные этапы клонального микроразмножения in vitro цитрусовых башкирской селекции.

Начальные этапы клонального микроразмножения in vitro цитрусовых башкирской селекции.

Rooting affects the photosystem II activity: in vitro and ex vitro studies on energy hybrid sorrel

Rumex tianschanicus × Rumex patientia is a high-biomass-yielding plant suitable for fuel and biogas production. The protocol of the hybrid sorrel micropropagation was used to study the changes in the photosystem II (PSII) activity as well as to analyse the ultrastructure of the chloroplasts. The lowest effective PSII quantum yield [Y(II)] and an apparent electron transport rate of PSII [ETR(II)] were observed for adventitious shoots that had been regenerated in vitro, before rooting. These fluorescence parameters were higher and similar for both the leaves of the same adventitious shoots that had been rooted under in vitro conditions and for the shoots that had been acclimated and grown in ex vitro conditions. The analysis indicated that the PSII activity strongly depends on the formation of properly functioning roots and that in vitro or ex vitro culture conditions are, at least to some degree, less important. TEM analysis revealed that chloroplasts from plants rooted in vitro were sufficiently mature and acclimatization processes have less impact on their development. This is the first report concerning the analysis of PSII activity and the ultrastructure of the chloroplasts at all of the stages of micropropagation, i.e. adventitious shoot formation in vitro, rooting in vitro and acclimation to ex vitro conditions. It strongly indicated that rooting under in vitro conditions, rather than the acclimation to ex vitro conditions, plays a key role in the development of a completely functional photosynthetic apparatus in hybrid sorrel.

Cryopreservation of red raspberry cultivars from the VIR in vitro collection using a modified droplet vitrification method

The goal of the present study was to analyze the post-cryogenic recovery of 12 red raspberry cultivars from the N.I. Vavilov All-Russian Institute of Plant Genetic Resources in vitro collection. The 1.1–1.8 mm shoot tips of microplants were subjected to cryopreservation using the modified droplet vitrification method. The current modifications to the droplet vitrification protocol included the elimination of the initial pretreatment stage of the microplants, and the use of modified media at the stages of initial micropropagation, explant isolation, and post-cryogenic regeneration. The optimized method reduced the duration of some cryopreservation stages compared to the initial protocol, and reduced the total procedure from 14 to 11 wk. This modified cryopreservation method also demonstrated a relatively high level of post-cryogenic regeneration. Depending on the genotype, the shoot recovery of explants after rewarming varied from 24.2–89.3% and averaged 58.8 ± 5.3%. There was a statistically significant influence of the genotype on the shoot recovery after rewarming. No differences in inter simple sequence repeats and in start codon targeted marker spectra were found between post-cryopreservation microplants and donor in vitro plants from two red raspberry cultivars.

Bioefficacy Test of Different Chemotherapeutic Substances against Aspergillus sp. and Chrysosporium sp. Contaminants of Tissue-Cultured Abaca (Musa textiles NEE.) during Initial Stage of Micropropagation

Bioefficacy Test of Different Chemotherapeutic Substances against Aspergillus sp. and Chrysosporium sp. Contaminants of Tissue-Cultured Abaca (Musa textiles NEE.) during Initial Stage of Micropropagation

Evaluation of genetic homogeneity of Jatropha curcas L. hybrid at an early stage of shoot bud formation from petioles using ISSR marker

ABSTRACTGenetic homogeneity is known to be the most important prerequisite in the micropropagation of Jatropha curcas L. to produce true-to-type plants. The detection of genetic homogeneity in clonal micropropagation for elite plants at an early stage is required, to avoid any increase in variation in the next stage of micropropagation. The genetic homogeneity was assessed during shoot bud formation from petiole explants of J.curcas (P1 × P3) hybrid with different concentrations of thidiazuron (TDZ) in a range of 0.5–4.0 mg/L using inter simple sequence repeat (ISSR) markers. Out of 23 ISSR primers, 16 primers produced clear, distinct and reproducible bands. A total of 96 bands, ranging in size from 100 to 1013 bp were generated. Based on the band data, a total of 94 bands were monomorphic (98%) and two bands were polymorphic (2%). All banding patterns from the shoot buds induced by 0.5, 1.0 and 2.0 mg/L TDZ were monomorphic, but 4.0 mg/L gave 2% polymorphism. These findings indicated that concentrations of 0.5, 1.0 and 2.0 mg/L TDZ did not trigger any somaclonal variation and could, therefore, be considered suitable for application in clonal micropropagation of J. curcas hybrid using petioles as explant material.

Carbon nanotubes impact on date palm in vitro cultures

Among the recent line of technological innovations, nanotechnology takes a promising position in agriculture and food production. Nanotechnology permits definite advances in agricultural research, such as reproductive science and technology. This investigation is interested in studying the in vitro effect of carbon nanotubes (CNTs) on callus, embryogenesis, embryo germination and elongation as well as rooting stage of date palm. Carbon nanotubes concentrations were investigated as 0.0, 0.05 and 0.1 mg/l. Results showed that CNTs affect all stages of micropropagation of date palm. Callus fresh weight showed the optimum value at 0.05 mg/l. In embryogenesis stage, CNTs decreased the number of embryos compared with the control while, increased number of germinated embryos and root number. Carbon nanotubes gave a significant enhancement for shoot length and leaf number in elongation stage. Similarly, it enhanced root number, root length, plantlet length and hairy roots. Chemical analysis as chlorophyll a, b, carotenoids, flavonoids, antioxidant enzymes and nutrients concentration and uptake were determined.

Increasing In Vitro Shoot Elongation and Proliferation of ‘G.30’ and ‘G.41’ Apple by Chilling Explants and Plant Growth Regulators

These studies were conducted to determine the most effective methods for increasing shoot elongation during the initial proliferation stage of micropropagation in two dwarfing apple, Malus ×domestica (Borkh.), rootstock cultivars. Several experiments were conducted to compare explant collection date, exposure to chilling (5 ± 1 °C) temperatures, and varying concentrations of plant growth regulators in Murashige and Skoog (MS) media. Microshoot growth of ‘Geneva 41’ (‘G.41’) was very low and unaffected by chilling duration from 0 to 8 weeks or by gibberellic acid (GA3) concentration from 0 to 1.0 mg·L−1, but was improved by an additional subculture which increased shoot length from 1 to 15 mm. In ‘Geneva 30’ (‘G.30’), shoot elongation was most affected by date, chilling explants, and by optimizing cytokinin concentration and type. Explant collection date in April increased shoot growth compared with August or November. Microshoot growth of ‘G.30’ was increased by chilling nodal explants for 4 and 6 weeks when explants were collected in August and November, but not in April. Eight weeks chilling was detrimental for explants collected in April, and generally had little or no effect with August and November. The cytokinin 6-benzylaminopurine (BA) increased shoot number to a greater extent than thidiazuron (TDZ) or zeatin (ZT), and was also more effective for increasing shoot elongation with concentrations of 0 to 2.0 mg·L−1. In ‘G.30’, GA3 increased shoot growth at the optimum concentration of BA, but not with lower concentrations. ‘G.30’ microshoots were fewer and shorter with 24-epi-brassinolide (EBR) at concentrations of 0.1 and 1.0 mg·L−1. Chemical names: N-phenyl-N’-(1,2,3-thiadiazol-5-yl)urea (TDZ), 6-(4-hydroxy-3-methylbut-2-enylamino)purine (ZT).