Survival Rate Of Explants Research Articles

Клональное микроразмножение лесных ягодных растений рода Rubus

Cloudberry (Rubus chamaemorus L.) and arctic raspberry (Rubus arcticus L.) are highly nutritional and medicinal but lowyield berries, with some populations being on the verge of extinction. Micropropagation biotechnologies are cost-effective and may provide healthy and plentiful planting material for these valuable berries. Clonal micropropagation of cloudberry and arctic raspberry requires new methods adapted for Russian varieties. This research featured the effect of sterilizing agents on the survival rate of explants of R. chamaemorus and R. arcticus, as well as the effect of growth regulators in the nutrient medium on their organogenesis in vitro.
 Berries obtained from regenerant plants of R. chamaemorus (Arkhangelsk and Vologda varieties) and R. arcticus (Sofia and Galina varieties) underwent a biochemical analysis. Further research involved the effect of sterilizing agents and exposure time on the viability of explants, as well as the effect of the nutrient medium composition and the concentration of growth regulators on the development of microshoots and roots in vitro.
 In case of lateral buds, the highest survival rate of both types of explants (80–96%) belonged to the samples sterilized with 0.2% silver nitrate and 5% Lysoformin 3000 for 15 min. In case of etiolated shoots (79–100%), it was the samples treated with 0.2% silver nitrate, 0.01% Nika 2, and 5% Lysoformin 3000 for 10 min. The maximal total length of microshoots in R. chamaemorus (19.4–22.7 cm) was registered at 0.1 mg/L Cytodef in the Murashige and Skoog medium. The maximal total length of roots (46.0–56.6 cm) was obtained when the medium contained 0.5 mL/L indolylacetic acid. As for R. arcticus, the maximal total lengths of microshoots (22.4–22.8 cm) and roots (86.6–89.3 cm) occurred at the same concentrations of growth regulators on 1/2 Murashige and Skoog medium.
 In this research, Cytodef and indolylacetic acid applied in the process of clonal micropropagation of R. chamaemorus and R. arcticus made it possible to increase the yield of high-quality planting material for commercial plantations.

In vitro propagation of stone fruit rootstock cultivar Evrica 99 and its influence on some phytochemical traits of fresh apricot fruit

Background: The cultivation of stone fruits is of primary importance in Armenia. Their fruits contain antioxidants, fiber, potassium, vitamin A, C, E, minerals, etc., which have a beneficial effect on human health and prevent many diseases. The concentration of those components varies depending on ecological factors, cultivar, rootstock, cultural practices, etc. Clonal rootstocks are important for increasing orchard density, tree uniformity, and high yields, and they can also affect fruit quality. In vitro culture is a valuable method for rapid propagation of high-quality, virus free plant material. Objective: The purpose of this study was to develop in vitro production technology for the stone-fruit rootstock cultivar Evrica 99, and to determine if rootstocks affect some fresh fruit traits of ´Yerevani´ and ´Sateni´ apricot cultivars. Methods: The shoot apical meristem and lateral bud served as explants for shoot regeneration. Different sterilizing agents at various periods of exposure were used for the explant surface sterilization. Various concentrations of phytohormones, both individually and in combinations, were employed for in vitro regeneration and rooting of plants. The titratable acidity (TA), dry matter (DM), vitamin C, mineral content, total carotenoids (TC), and sugar contents were evaluated in fresh fruit. Results: The most optimal option for explant surface sterilization was the gradual application of calcium hypochlorite [Ca (ClO)2] (2.0% solution, exposure time 10 min) and ethanol (70% solution, exposure time 20 s), as a result of which we had 75.5% survival rate of explants. The efficient medium for in vitro shoot regeneration was MS supplemented with 6-Benzylaminopurine (BAP) 0.8 mg/l, Kinetin (Kin) 0.2 mg/l, and Gibberellic acid (GA3) 1.0 mg/l. The half-strength Murashige and Skoog (MS) medium containing 0.8 mg/l indole-3-butyric acid (IBA) was optimal for in vitro rooting. Rooted plants were successfully adapted with a survival rate of 85.0%. The defined method can be successfully used for 'Evrica 99' cultivar micropropagation. The results obtained showed that fruit quality strongly depended on both the varieties and the rootstock tested. Conclusion: In the current study, an alternative in vitro propagation technology for rootstock cultivar ´ Evrica 99 was developed by direct organogenesis, enabling mass-scale production of virus-free plants that is suitable for commercial purposes as well. The apricot cultivars ´Yerevani´ and ´Sateni´ grafted on the virus-free rootstock cultivar Evrica 99 showed higher fruit quality traits, which are essential for human health and diet. Keywords: ´Evrica 99, fucnctional foods, in vitro regeneration, micropropagation, plant growth regulators, stone fruit rootstock, tissue culture.

Effects of a New Plant Growth Regulator on Callus Induction from Immature Embryo Explants of Korean Pine (Pinus koraiensis)

Somatic embryogenesis is currently the most promising technique for the large-scale production and breeding of conifer species. Nonetheless, the low explant induction rate in this process hampers the development of improved materials. In this study, three immature Korean pine (Pinus koraiensis) embryos capable of induction were used as experimental materials. Various concentrations of brassinolide (BL), melatonin (Mel), salicylic acid (SA), glutathione (GSH), and ascorbic acid (AsA) were added to induce embryogenic callus tissue. The results show that BL had the most significant positive effect on promoting induction and increasing explant survival. Mel was also found to slightly increase the induction and survival rates of explants. When 1.00 mg·L−1 BL was added to the explants derived from stock tree I, which had strong self-induction ability, for 30 days, the callus induction rate rose to 400% compared to the control group. Following the addition of 25 mg·L−1 Mel to stock tree I for 30 days, the callus induction rate further increased to 450% of the control group. The effect of GSH on callus induction was insignificant. The addition of 0.10 g·L−1 to stock tree I for 30 days resulted in only a 150% higher induction rate compared to the control group. When 1.00 mg·L−1 BL was applied to explants with weak self-induction ability (stock tree III) for 15 days, the callus induction rate increased to 600% of that of the control group. The callus induction rate further increased to 800% of the control group after 50 mg·L−1 BL were added to stock tree III for 15 days. This study presents a method to improve the induction of embryogenic callus tissue in Korean pine.

Survival and State of Garlic Explants of Two Lithuanian Cultivars after Cryopreservation

Cryopreservation features of Lithuanian garlic (Allium sativum L.) cultivars ‘Žiemiai’ and ‘Dangiai’ were investigated. The survival rate and state of explants, depending on the type of explant, and dehydration conditions were evaluated in the experiment. Explants isolated from cloves and bulbils were dehydrated with a plant vitrification solution 3 (PVS 3) containing 50% w/v glycerol and 50% w/v sucrose in liquid MS medium. Three different exposure durations of 1.5, 2, and 3 h in PVS3 solution were applied at 0 °C. Unloaded stem domes were planted in Petri dishes with MS medium supplemented with 1 mg L−1 naphthaleneacetic acid (NAA) and 6–dimethylallylamino purine (2-iP) on 0.8% plant agar. The obtained results showed that the cryopreservation in liquid nitrogen reduced the survival rate of explants by 20–40%. The average number of surviving explants after freezing reached 64.3% for both cultivars. Cultivar ‘Žiemiai’ showed a 24% higher capability for survival than ‘Dangiai’. The total number of surviving explants of ‘Žiemiai’ reached 76.1%. Explants from bulbils were 23% more effective for cryopreservation compared to cloves. Evaluation of the effect of dehydration duration showed that the survival rate of the explants from bulbils of both cultivars was highest after treatment with PVS3 for 3 h and reached 91%. Treatment in PVS3 solution for 2 h was sufficient for survival of explants from cloves. Obtained results confirm that the efficiency of garlic cryopreservation depends on complex factors.

OPTIMIZATION OF THE NUTRIENT MEDIUM FOR THE PRIMARY STAGES OF COMMON WALNUT MICROCLONAL PROPAGATION IN VITRO

SummaryAim. To optimize the processes of microclonal propagation of Juglans regia in vitro by selecting the composition and consistency of the nutrient medium. Methods. The methods of in vitro culture establishment of initial explants and microclonal propagation were used. Parts of the stem and buds of young sprouts, germinated by the mini-greenhouse method from the seeds of Juglans regia, were used as initial explants. Murashige&Skoog (MS) and Driver&Kuniyuki (DKW) nutrient media were used to establish Juglans regia explants in vitro. Agar was used as a gelling agent. The solid, semi-liquid, and liquid media were prepared with addition of 7 g/l, 3.5 g/l and 0 g/l of agar, respectively. All experimental variants of the media were modified by adding the phytohormone of the cytokinin group 6-BAP. The pH of the medium was controlled at the level of 7.1–7.2. The established explants in vitro were cultivated at a temperature of + 25°C, a light intensity of 2500 lux, a relative humidity of 56–70% and a photoperiod 16/8 h light/dark. On the 3rd, 7th and 14th day, the survival rate, growth and development parameters of the initial explants were estimated. Results. It was found that the optimal nutrient medium for in vitro establishment and cultivation of Juglans regia is the DKW medium, on which the high survival rate of explants was observed (80%), as well as accelerated swelling of buds and their proliferation. It is proposed to use semi-liquid nutrient media for in vitro establishment of initial Juglans regia explants. The use of semi-liquid nutrient medium DKW for the initial stages of common walnut micropropagation in vitro contributed to the acceleration of the processes of reproduction of this culture by 2 days and improved the survival rate by 20% compared to solid medium. Conclusion. The use of semi-liquid nutrient medium DKW for introduction into the culture and cultivation of walnut (Juglans regia) in vitro is recommended to increase the survival rate of explants by 20%, as well as the accelerated activation of buds (2 days) and their further proliferation.

Óleos essenciais de plantas condimentares e medicinais no controle de contaminantes da micropropagação de Myrciaria dubia

Abstract The main limitation of the micropropagation of camu-camu (Myrciaria dubia) is related to in vitro contamination. In order to overcome contamination, the effect of essential oils was studied as an alternative to conventional chemical treatments. This study aimed to analyze the action of essential oils from four condiment and medicinal plants (Oregano, Origanum vulgare L.; Garlic, Allium sativum L.; Citronella, Cymbopogon nardus L.;and Ginger, Zingiber officinale Rosc.) in reducing microbial contamination growth and on the survival rate of explants in the micropropagation of camu-camu. The antimicrobial activity of essential oils was analyzed on the in vitro contamination of tissue culture from camu-camu at concentrations of 0.5, 1, 2, 3 and 4 µL mL-1 in woody plant medium (WPM), emulsified with 1% Polysorbate 80 (Tween 80). The antibacterial activity of essential oils on strains isolated from camu-camu tissue culture was also analyzed using agar diffusion and broth microdilution methods. The use of essential oils allowed a reduction in the rate of in vitro contamination in tissue culture, it being observed that, from the concentration of 2 µL mL-1, there was no manifestation of fungal contaminants, with a significant reduction in the rate of bacterial contamination, with the exception of ginger essential oil that showed significant contamination in all analyzed concentrations. Inversely in relation to the reduction in microbial growth in vitro, there is an increase in explant oxidation as concentrations increase, with citronella and oregano oils showing phytotoxic potential from the lowest concentrations. Garlic essential oil showed better balance, with lower oxidation rates and greater control of microorganisms in tissue culture at concentrations of 0.5 and 1 µL mL-1. Oregano and citronella essential oils showed better antibacterial activity in the agar diffusion test.

Pengaruh Metode Sterilisasi Permukaan terhadap Kultur Biji Jeruk Japansche Citroen (JC) Citrus limonia Osbeck

Effect of Surface Sterilization Methods on Seed Culture of JC Citrus limonia Osbeck Japansche Citroen (JC) Citrus limonia Osbeck is one type of citrus that is commonly used as rootstock. JC seed propagation through in vitro culture will be more effective. The initiation stage is the first step of in vitro culture to obtain microbial-free explants and initiate the initial growth. At this stage the problem of contamination becomes the main limiting factor. This study aims to obtain the appropriate method of sterilizing of JC orange seed explants in in vitro culture propagation. A total of four sterilization methods have been tested at the Tissue Culture Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Hasanuddin University. The sterilization material used is tween 80, fungicide, 20-30% NaOCl and 90% alcohol. The variables observed in this study were the time of appearance of contamination, the percentage of types of contaminants (bacteria and fungi), the percentage of sterile explants and the percentage of live explants after 28 days of culture. The results showed that the fourth method was the optimal sterilization method with the lowest contamination level of 30% of the other three methods with the highest explant survival rate of 60% compared to the other three methods.

Micropropagation of Citronella mucronata D. Don, a Vulnerable Chilean Endemic Tree Species.

Citronella mucronata (C. mucronata), a tree species endemic to Chile, has become threatened in its natural habitat and is currently listed as vulnerable. Tree population parameters have deteriorated due to indiscriminate logging and other anthropogenic activities, warranting research on mass propagation as a means of recovery. This study, unprecedented for this native species, has developed a successful method for its micropropagation. The objective was to establish a protocol for in vitro propagation of C. mucronata to produce large quantities of high-quality seedlings in an accelerated plant acquisition process. The best results were achieved by growing explants on Murashige and Skoog (MS) basal culture medium supplemented with 4.44 μM 6-benzylaminopurine (BAP) and 14.76 μM indole-3-butyric acid (IBA). Explant survival rate was 78 %, the average shoot length reached 3.2 cm, the number of lateral shoots was 3.9, and rooting rate was 60%. Furthermore, stimulation with red and blue light in a 1:2 ratio, supplemented with 14.76 μM IBA, improved the rooting rate to 93%. The survival rate of rooted explants reached 100% in the acclimatization stage when using peat and perlite substrate (1:1 v/v).

Tetraploid induction through somatic embryogenesis in Panax vietnamensis Ha et Grushv. by colchicine treatment

• Somatic embryos regenerated from AgNPs-sterilized leaf explants of Ngoc Linh ginseng. • Tetraploid induction by colchicine in Panax vietnamensis Ha et Grushv. from somatic embryo. • Tetraploid plantlets had vigorous growth compared to the diploid plantlets. In the present study, the protocol of polyploid induction via somatic embryogenesis of the Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) (2n = 2x = 24) was developed. Ex vitro leaf explants were disinfected with AgNPs at concentrations of 0.05, 0.1, 0.2 and 0.5 g/L for 5, 10, 15, 20 and 30 min and cultured on MS medium supplemented with 1.0 mg/L 2,4-D, 0.5 mg/L NAA and 0.2 mg/L Kin for somatic embryogenesis. Globular embryos induced from the leaf explants were treated with colchicine by immersed method at different concentrations of 0, 0.1, 0.2, 0.3, 0.5 and 0.7% for 24, 36, 48 and 72 h. Then, these embryos were cultured on medium supplemented with 1.0 mg/L 2,4-D, 0.5 mg/L NAA and 0.2 mg/L Kin for secondary embryos induction. The ploidy level was identified by counting chromosomes of the root tip of plantlets derived from these secondary embryos. The results showed that the survival rate and somatic embryo induction the highest (65.56% and 29.89%, respectively) at 0.2 g/L AgNPs for 20 min. For colchicine treatment, the increase in the concentration and exposure time of colchicine decreased the survival rate of explants and the number of secondary embryos but increased the abnormal secondary embryo formation. Identifying the ploidy level showed that the treatments with colchicine concentrations from 0.3% to 0.5% for 48 h lead to tetraploid induction rates from 22.22% to 25.92%. The results also revealed that the tetraploids (2n = 4x = 48) had larger stomatal size, lower stomatal density, denser stomatal chloroplast density, and better growth than did the diploids.

In vitro propagation and plant regeneration of Torenia crustacea (L.) Charm. & Schltdl; an important ethnic medicinal plant

Torenia crustacea (L.) Charm. & Schltdl is an important medicinal herb used in India, Indonesia and Malaysia. This herb is used by Bodo tribes in the Bodoland Territorial Region, Assam to treat various diseases like diabetes. The present experiment developed an efficient protocol for in vitro mass propagation technique for Torenia crustacea species using its nodal explants. For tissue culture, the rapidly growing nodal explants of Torenia crustacea were used. Effective explant surface sterilisation was found at 15 min. of treatment with 2% sodium hypochlorite resulting in a maximum explant survival rate and a lower rate of explant contamination after 21 days of explant culture. Media containing full strength MS (Murashige and Skoog's) + BAP (6- Benzyl amino purine) (1 mg/l) were found most effective for the establishment of the explant. The highest shoot proliferation and multiplication were observed in the media containing full strength MS + BAP (1 mg/l) + NAA (Naphthalene acetic acid) (0.2 mg/l); in this combination, an average of 16 shoots formed per nodal explant. The rooting of explants was observed highest in MS media along with 0.4 mg/l concentration of NAA and 1 mg/l BA. The in vitro multiplied shoots were grown in plastic pots containing vermicompost fertiliser and soil mixture and successfully grown in the open field condition.

In vitro amphidiploid induction of a distant hybrid Populus simonii × P. euphratica cv. ‘Xiaohuyang-2’ and its effect on plant morphology and anatomy

Abstract Highly gametic sterility of a distant hybrid Populus simonii × P. euphratica cv. ‘Xiaohuyang-2’ restricts its utilization in breeding programs of Populus. Amphidiploid induction by somatic chromosome doubling is expected to restore its gametic fertility. In this study, nodal-segment and leaf explants of ‘Xiaohuyang-2’ were used to induce chromosome doubling with colchicine in vitro. Although chromosome doubling of the nodal-segment explants only produced mixoploids, the treatments of leaf explants on adventitious bud regeneration medium successfully produced 4 amphidiploids, which might be attributed to the direct organogenesis of the adventitious buds on the leaf explants. This is the first report of amphidiploid induction in a distant hybrid between Populus section Tacamahaca and sect. Turanga. The highest amphidiploid induction frequency was 16.7 %. Both the explant survival rate and polyploidization frequency were significantly affected by colchicine concentration and exposure time. The amphidiploid plants significantly differed from the diploid and mixoploid plants in morphological and anatomical characteristics. They had larger, thicker, and greener leaves than the diploids and mixoploids. The increase in ploidy level also resulted in changes in stomatal features. The induced amphidiploid plants of the distant hybrid ‘Xiaohuyang-2’ are expected to play important roles in breeding programs of Populus in the future, which can be used as a bridge parent with the ability of unreduced gamete formation to cross with fast-growth germplasms to produce triploids pyramiding desirable traits of fast growth, easy cutting propagation, and salt and drought tolerances.

Prolongation of viability of isolated skin explants by exposing them to their own transformed physical fields: experimental study

Objective. To study the effect of materials with optical-electrical properties on the survival of full-layer skin explants when they are isolated from the body with completely stopped trophic activity.
 Material and methods. In rabbits of both sexes at the age of 5-6 months, full-layer skin flaps were excised from the back area, which were dissected into fragments-explants measuring 1.01.0 cm. All samples were thermostated for 2 days at 37 C near transducers represented by aluminum foil sheets and silicon single crystals. In the first series of experiments, the explants were directly in contact with the transducers; in the second and third series, screens made of light-proof black paper and thin glass were installed between the explants and the transducers, respectively. In each series, the experiments were repeated 5 times with each transducer. After thermostating, the explant viability index was evaluated by histological method in points.
 Results. In the first and second series of experiments, the highest survival rate of explants with a viability index from 3.6 to 3.8 points with a norm of 4.0 points) was revealed depending on the type of transducer. In series III, when screening silicon crystals with thin glass, the explant survival rate decreased slightly to 3.3 points. However, the screening of aluminum foil with glass led to a sharp decrease in the viability of explants to 0.2 points.
 Conclusions. A long-term viability of skin explants can be explained only by the effect of their own physical fields on them produced by the biostructures of explants and transformed by interaction with nearby materials possessing optical and electrical properties.

Influence of climatic conditions of the introduction period and varietal characteristics of black currant (Ribes nigrum L.) on the effectiveness of culture initiation in vitro

The article presents data from the effectiveness studies of the initial introduction stage of black currant (Ribes nigrum L.) into in vitro culture depending on the introduction period and the corresponding climatic conditions. The research objects were varieties of black currants selected by the Russian Research Institute of Fruit Crop Breeding: Azhurnaya, Orlovskaya serenada, Ocharovanie, Chudnoye mgnovenye. The introduction into in vitro culture was carried out in several periods characterized by different physiological states of the explants: the period of dormancy release (mid-March), the period of active growth (June), and the period of growth decay (mid-September). The source material in the spring and autumn periods were the buds of one-year stiffened shoots, in the summer introduction period - the buds of growing green shoots. The cultivation was carried out on Murashige-Skoog medium supplemented with 6-BAP (0.5 mg/l). It was noted that the survival rate of explants is determined by the physiological state of the source material due to the corresponding agro-climatic conditions during the introduction period, as well as the genotypic characteristics of the varieties. Thus, explants isolated during the active growing season are characterized by a higher and more stable survival rate. When explants were cultivated in spring and autumn, the physiological state of the explants and their survival rate were influenced by the genotypic response of varieties to the corresponding agroclimatic conditions.

Optimization of stages of clonal micropropagation of garden strawberry varieties Nelly and Kemiya breeding of NCFSCHVW

The production of strawberry planting material using the method of clonal micropropagation of plants is promising, modern and already widely used. This paper presents the results of assessing the breeding potential of garden strawberry varieties Nelly and Kemiya (selection of FSBSI NCFSCHVW) in in vitro culture. Apexes from growing rosettes of strawberries served as the starting material for the initiation. The best survival rate of explants was noted during the initiation in the second decade of August, which were in Nelly variety - 90%, Kemiya - 83.3%. At the stage of proliferation, 6-Benzylaminopurine (6-BAP) was injected into the culture medium at various concentrations (0; 0.5; 1.0 and 1.5 mg / L). To increase the efficiency of micropropagation of strawberries Nelly and Kemiya varieties, it is advisable to add 6-BAP into the culture medium with the amount of 1.0 mg / l. In this variant, 10.6 shoots were formed in the explants in Nelly variety, 5.9 in Kemiya variety. With an increase of the concentration of 6-BAP to 1.5 mg / l, the number of vitrified shoots were raised. According to the results of three subcultures, it was noted that the Nelly variety has a higher reproduction potential compared to the Kemiya variety. At the stage of rhizogenesis, the addition of auxins to the medium was not required, since the roots of regenerated plants formed independently.

Polyploidization of Miscanthus sinensis using dinitroanilines with low phytotoxicity

Aim. The aim of the work was to obtain polyploid lines of Miscanthus sinensis with enhanced biomass productivity using new dinitroaniline compounds. Methods. The work was carried out in in vitro conditions. Aseptic M. sinensis shoots were cultured on shoot multiplication medium containing various compounds of dinitroanilines class. Classic dinitroanilines (oryzalin and trifluralin) were used as a control. Results. When aseptic shoots were cultured on medium supplemented with oryzalin and trifluralin at concentration 10 mM, survival rate of explants was between 23.52 and 44.40%. However, when the medium was supplemented with tested dinitroanilines at the same concentration, survival rate of explants was in the range of 95.0–96.5%. Conclusions. Possibility of new compounds of dinitroanilines class utilization for M. sinensis polyploidization has been studied. In terms of explants survival rate, application of new compounds of dinitroanilines class appeared to be significantly effective than application of classic dinitroanilines.
 Keywords: dinitroanilines, in vitro, polyploidization, Miscanthus sinensis.

Somatic embryogenesis and plantlet regeneration from the seaweed Kappaphycus striatus

The seaweed Kappaphycus striatus is a well-known source of carrageenan and other valuable bioactive compounds. To improve the efficiency of propagation of this important natural resource, we studied the aseptic efficiency of silver nanoparticles (AgNPs) in the explant sterilization process. We further evaluated the effects of culture media, plant growth regulators (PGRs), light intensity, and rotary shaking on callus induction and proliferation, somatic embryogenesis, and micropropagules regeneration of the red algae K. striatus. The treatment with AgNPs at a concentration of 500 ppm optimally resulted in the absence of contaminations. A high explant survival rate (80.0%) and callus induction rate (54.4%) were obtained. Healthy calli were transferred separately into MS (Murashige and Skoog), PES (Provasoli’s enriched seawater), and MPI media for callus proliferation. The results indicated that the PES medium was suitable for callus induction with higher yields of fresh and dry biomass (112.00 mg and 11.50 mg, respectively) compared with the MPI medium (79 mg fresh and 6.73 mg dry weight). Calli cultured using the MS medium failed to grow. The optimal condition for callus proliferation was the PES medium supplemented with 1.0 mg⋅L−1 1-naphthaleneacetic acid (NAA) and 1.0 mg⋅L−1 6-benzylaminopurine (BAP). Somatic embryo (SE) induction was best obtained when cultured calli were transferred onto PES semi-solid medium supplemented with 1.0 mg⋅L−1 NAA and 2.0 mg⋅L−1 BAP. To promote plantlet development, SE clumps were placed in PES liquid medium on a rotary shaker at 100 rpm for separating individual embryos. These embryos were collected and allowed to develop under cool white fluorescent tubes at a light intensity of 55 µmol⋅m−2⋅s−1. The regenerated plantlets had a 50% survival rate, light brown in colour and multibranched. The plantlets reached a length of 2 cm after 2 months of culture.

Doubled haploid production in onion (Allium cepa L.): from gynogenesis to chromosome doubling

Bulb onion (Allium cepa L.) is an allogamous diploid (2n = 16) important for its culinary uses, nutritional value, and medicinal benefits. Despite its economic importance, onion yields and bulb quality are declining, emphasizing the need for new and improved strategies for maintaining and enhancing overall crop quality. Development of inbred lines in onion through traditional breeding is often difficult due to its biennial life cycle, inbreeding depression, and comparatively high heterozygosities. Moreover, genetic research in onion has been hampered by large nuclear genome size. In this regard, gynogenic doubled haploids promise several advantages over inbred lines in support of onion breeding programs and genetic studies. These include complete homozygosity in doubled haploid lines, reduced DNA methylation, elimination of deleterious alleles, and amenability to genetic analysis. This review focuses on the application of in vitro gynogenesis for producing doubled haploids in onion. Factors influencing haploid induction, methods for inducing chromosome doubling and ploidy assessment, evaluation of haploid progenies and doubled haploid lines, and features of doubled haploids potentially useful in crop improvement and genetic studies, are discussed. We identify four major limitations to the success and efficiency of in vitro gynogenesis in onion and discuss strategies for mitigating the negative impacts they pose. This review may be useful to research programs producing doubled haploids in onion or other Allium species using in vitro gynogenesis. In onion, doubled haploids (DHs) were generated by gynogenesis using in vitro culturing of unfertilized ovules, ovaries or whole flowers. Genotype, geographical origin, photoperiodicity and genetic structure all affect gynogenic efficiency in onion. Despite higher doubling efficiencies, there are several disadvantages with induced chromosome doubling inlcuding low survival rates of explants, cytotoxicity, vitrification and polyploidization.

РАЗРАБОТКА ПРОТОКОЛА ВВЕДЕНИЯ РАСТЕНИЙ ЗЕМЛЯНИКИ В КУЛЬТУРУ IN VITRO

The research was conducted in order to develop an effective protocol for introducing strawberry plants into in vitro culture. The objects of the research were the most popular varieties of strawberries of domestic and foreign selection: Tsaritsa, Bereginya, Florence, Frida, Kimberly, etc. Mercurial preparations mertiolate at a concentration of 0.01% and sulema at a concentration of 0.1% were used as sterilizing agents. The isolation of explants was performed in several periods: the beginning of the growth was in February, active growth was in June, the decline of growth was in August. The studies have shown that the maximum aseptic cultures were obtained when processing strawberry plant material with mercurycontaining sulema preparation in the concentration of 0.1%. At the first stage of micropropagation, explants had a high viability during all periods of the isolation, the average survival rate for varieties was 74.8-80.7%. A significant influence of the genotype (varietal characteristics) on the survival rates of explants was noted. The number of explants suitable for cloning did not depend on the overall level of regeneration. Stabilization of the crop during winter introduction was much faster than in other periods. Using the winter term of the isolation of strawberry explants allowed to increase the yield of explants capable of further cloning, accelerate the stabilization of the culture in vitro and reduce the time for obtaining micro-plants suitable for planting in non-sterile conditions. On average, 75.2% of explants capable of further cloning for the varieties were obtained. As a result of the research, the conditions and methods for obtaining the largest number of viable sterile strawberry explants were worked out, which will be included into the process of reproduction in vitro and further research.

Micropropagation of rootstocks of stone fruit cultures in vitro

The usage of the method of clonal micropropagation of plants is currently the most promising for obtaining virus-free plant material, including clonal rootstocks of stone fruit cultures. The introduction of clonal rootstocks of stone fruit cultures PK SK1, PK SK 2, AI 1 into invitro culture is best doneduring the active shoot growth period in the second decadeof May. During this period, the highest level of explants regeneration was noted: in the rootstock PK SK 1 – 94,6 %, in the rootstock AI 1 78.2%, in PK SK 2 80.4%, in Gisela 5 (control) 85,7%. From the third decade of May, the survival rate of explants begins to decline. At the stage of multiplication on the Murashige-Skoog medium, with the addition of 6-BAP 1 mg/L, the average multiplication factor of clonal rootstocks to the fourth passage was 1: 12 for PK SK1, for Gisela 5 and for AI 11: 8.

Establishment of an Open, Sugar-Free Tissue Culture System for Sugarcane Micropropagation

Tissue culture has been used for many years to produce a large number of plant species by micropropagation. Unfortunately, microbial contamination and production costs are major problems associated with this technology. We have developed a low-cost, open tissue culture system for rapid sugarcane micropropagation that uses a combination of broad-spectrum antimicrobial compounds, a sugar-free medium, and CO2 gas fertilizer as carbon source. A novel antimicrobial compound called “QX1” that contains nicotine, garlic extract, carbendazim, and “Yipeilong” was developed in this study. Non-autoclaved induction medium containing 0.5% (v/v) QX1 had a significantly lower microbial contamination and a significantly higher explant survival rate in comparision to that of autoclaved induction medium. Sugar-free medium containing 0.5% (v/v) QX1 and 1.0 g/L CO2 gas fertilizer was used for multiplication and rooting under the open tissue culture environment. Propagation efficiencies were similar in the newly developed and conventional tissue culture systems, but contamination was significantly less in the new system during both multiplication and rooting. Also, plantlet survival rate, fresh weight, and net photosynthetic capacity were increased significantly, and production costs decreased dramatically using the new tissue culture system compared with the conventional method. Thus, the low-cost, open, sugar-free tissue culture system can be adapted to produce sugarcane plantlets on a commercial scale.