Viability Of Explants Research Articles

Ex vivo effects of corticosteroids on equine deep digital flexor and navicular fibrocartilage explant cell viability

To investigate the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on the viability of resident cells within the fibrocartilage on the dorsal surface of the deep digital flexor tendon (FC-DDFT) and fibrocartilage on the flexor surface of the navicular bone (FC-NB) of horses. 12 to 14 explants of FC-DDFT and of FC-NB from grossly normal forelimbs of 5 cadavers of horses aged 9 to 15 years without evidence of musculoskeletal disease. Explants were incubated with culture medium (control) or TA-supplemented (0.6 or 6 mg/mL) or MPA-supplemented (0.5 or 5 mg/mL) medium for 6 or 24 hours. Explant metabolic activity and percentage of dead cells were assessed with a resazurin-based assay and live-dead cell staining, respectively, at each time point. Drug effects were assessed relative to findings for the respective control group. Application of TA (at both concentrations) did not significantly change the cell viability of FC-DDFT explants. For FC-NB explants, TA at 6 mg/mL significantly reduced the metabolic activity and increased the percentage of dead cells at both time points. With either MPA concentration, FC-DDFT and FC-NB explants had reduced metabolic activity and an increased percentage of dead cells at 24 hours, whereas only MPA at 5 mg/mL was cytotoxic at the 6-hour time point. In ex vivo explants, TA was less cytotoxic to equine FC-DDFT and FC-NB cells, compared with MPA. Further work is warranted to characterize the drugs' transcriptional and translational effects as well as investigate their cytotoxicity at lower concentrations.

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.

Micropropagation on multiplication of native greenberries in South of Brazil

Rubus brasiliensis and Rubus erythroclados are commonly known as greenberries. This work aimed to test in vitro micropropagation protocols to greenberries. The multiplication experiment was conducted using BAP at concentrations 1 mg L-1, 2 mg L-1 and 3 mg L-1. The rooting experiment was carried out in ½ MS medium without the addition of hormones, using 0.1 mg L-1 NAA or 1.0 mg L-1 IBA. The highest rate of viability of explants was obtained with R. brasiliensis propagation using 1.0 mg L-1 of BAP. Rooting explants showed survival rates higher than 85%. Rooting rates in R. erythroclados was 87% while in R. brasiliensis it was 38%, The highest rates were obtained using 1.0 mg L-1 IBA in R. erythroclados.The acclimatization reached survival rates of 75% when rooted using NAA and ½ MS and 86% when rooted with IBA. In vitro culture shows to be a viable method to propagate greenberries.

Surface sterilization method for reducing contamination of Clinacanthus nutans nodal explants intended for in-vitro culture

Surface sterilization is a vital step in preparation of healthy and viable explants in tissue culture. Most surface contaminants can be eliminated by surface sterilization with a suitable sterilizing agent. The study aimed to present an effective disinfection method for Clinacanthus nutans shoot regeneration using nodal segments. A total of four different sterilization approaches were conducted by treating nodal explants with various concentrations of sterilizing agent. Sterilizing agents used were Rhizophora apiculata Pyroligneous acid (PA), sodium hypochlorite (Clorox) thiophanate-methyl (fungicide), and Mercuric chloride (HgCl2). Nodal explant then was cultured on plant growth regulator-free Murashige and Skoog (MS) basal medium. This study sterilizing agents revealed that PA showed strong bactericidal activity. However, it led to a high number of fungal contaminations. The pyroligneous acid did not exhibit a strong potential as a disinfectant for C. nutans nodal explant. Overall, HgCl2 exhibits the best reduction in fungal contamination and gives a significant result with thiophanate-methyl fungicide. Surface sterilization with mercuric chloride (0.2%) for 1 hour was the optimum concentration and duration, which resulted in the highest percentage of nodal explant survival and viability. All viable nodal segments developed into shoots. It had been concluded that the best surface sterilization agent was HgCl2.

The Development of Gametophyte Sterilization Method for Liverworts Acrolejeunea fertilis (Reinw., Blume and Nees) Schiffn. In vitro Culture

In vitro culture gametophytes of leafy liverworts often have problems in their sterilization process. These problems due to the high level of contamination and the fragile structure of the gametophyte leafy liverworts. The structures can be easily to damage after exposure to disinfectant. This study aimed to observe the concentration and the exposure time of “Bayclin” commercial bleach to suppress contamination with the viability of Acrolejeunea fertilisgametophytic explants. This research was conducted using control and 6 combination treatments with “Bayclin” concentration (1.00%, 1.25%, and 1.50%) and exposure time 60 and 120 seconds, then accompanied by the addition of Tetracycline 2.5 mg/ml. The qualitative parameters observed were the explant color, the type and location of contamination, and the growth of explants. The quantitative parameters were the percentage of contamination, the percentage of growth, and the number of new branches. The results showed that “Bayclin” 1.25% and 1.50% with 60 seconds exposure time has the lowest percentage of contamination which is 70% until the 7th days after planting. The most common type of internal contamination from the explant is bacteria and fungi. However, the growth of the new branch still occurs in some explants even though it has been contaminated and browned.

Features of sterilization of various explants of rhododendrons (Rhododendron L.) and their introduction in vitro

Purpose. To establish the influence of sterilization conditions and the type of explants for obtaining aseptic culture of representatives of the genus Rhododendron (L.).
 Methods. Biotechnological, laboratory, analytical, statistical.
 Results. It was found that for 70 % of the concentration of the soap solution and exposure for 10–15 minutes with three washes, it is possible to obtain additional sterile seeds up to 33 %, and seedlings 29 %. The most effective treatment was with a concentration of primary sterilizer of 50 %, which provided additional sterile seeds of 37 % and seedlings – 31 %. The worst was a solution of primary sterilizer 20% for which sterile seeds amounted for 15 % and seedlings for 8 %. At a concentration of 35 %, and exposition for up to 10 minutes it is possible to reach seed sterility of 78 % and seedlings of 65 %, of which viable seeds 67 % and viable seedlings 57 %, which was one of the best results of the studied treatments. The lowest rates of sterile explants were observed in the treatments with hydrogen peroxide (H2O2). Seeds were only 23 % sterile and seedlings 12 %. Seed viability was up to 14 %, and seedlings up to 34 %. The results of the studies indicate that with the combined use of 35 % Bilyzna and 50 % Bradofen, it is possible to obtain sterile seeds from 83 to 95 % and seedlings from 83 to 91 %. In addition, viable explants in this combination were founding in seeds from 76 to 85 %, and in seedlings from 67 to 82 %. The most effective combination is 35 % Bilyzna and 50 % Bradofen for 10 minutes of exposure. Sterility was 94 % in seeds and 90 % in seedlings, viability: 83 and 80 %, respectively. It is worth noting the lack of necrotic tissue in seedlings.
 Conclusions. Regardless of the concentration, the most optimal exposure was 10–15 minutes. Depending on the type of explant and the use of the primary sterilizer, a higher percentage of sterile seeds was obtained compared to seedlings. The mercury dichloride concentration of 0.4 % and 0.2 % provided a low percentage of viable explants for both seeds and seedlings. In the latter, its use led to partial burns of the leaf surface. Therefore, the use of mercury dichloride 0.4 % and 0.2 % was not appropriate for sterilization of plant material of the genus Rhododendron (L.). The most effective way to sterilize the seeds of the genus Rhododendron (L.) is to use a 35% solution of Bilyzna for 10 minutes. Seedlings should be sterilized for 8 minutes, and the increase leads to a higher percentage of necrotic material.

INFLUENCE OF THE STERILIZATION METHOD AND THE TIME OF INTRODUCTION INTO IN VITRO CULTURE ON THE VIABILITY OF EXPLANTS OF THE CLONAL APPLE STOCK 54-118

The stage of introducing explants into a sterile culture is difficult in the technology of clonal micropropagation of plants. The article shows the possible ways of sterilization and the introduction terms of explants of the clonal apple stocks 54-118 into in vitro culture in order to reduce planting infection and increase the yield of sterile viable explants. The best time for introducing clonal apple stocks 54-118 into sterile in vitro culture is the period of active shoot growth. Sterilization of explants with ethyl alcohol (70,0 %, 1 min) in combination with hydrogen peroxide (33,0 %) for 7 minutes and ethyl alcohol (70,0 %, 1 min) in combination with diacide (0,1 %) within 6 minutes contributed to the production of 63,0 % and 60,0 % of viable sterile explants.

BIOCHEMICAL COMPOSITION OF VEGETATIVE EXPLANTS AND CALLUS PINUS SIBIRICA DU TOUR

The methods of sterilization of annual shoots Pinus sibirica Du Tour and the conditions for their introduction into in vitro culture were studied. Induction of callusogenesis of aseptically viable explants of P. sibirica proceeds more intensively on the modified Murasige-Skoog medium: hormonal supply of 0.4% kinetin and 0.25% 6-benzylaminopurine; reduced sucrose concentration of 1.5%. The frequency of callus formation was 83%. Close quantitative indicators of extractive substances were established (36 and 33% of absolutely dry weight for callus and explant, respectively); easily hydrolyzable polysaccharides (18 and 16%) and proteins (11 and 10%).Callus P. sibirica has a higher content of ascorbic acid, flavanoids, tocopherols and ash elements compared to explants and a low amount of hard-hydrolyzable polysaccharides, lipids, tannins, pigments, and essential oils.The electrophoretic spectrum of water-soluble callus proteins is represented by eleven fractions: 63% of the total water-soluble proteins are fractions with a molecular weight of 33 kD and above. Fractions with molecular weights of 50 and 62 kD (20 and 17%, respectively) are represented as much as possible.In the explants of P. sibirica, low molecular weight fractions of proteins with molecular masses of 5 kD and lower (59%) predominate. The amino acid composition of calli and explants of P. sibirica is identical and is represented by fifteen individual amino acids. Callus tissue has a higher content of glutamic acid and two hydrophobic amino acids (proline and isoleucine) compared to the vegetative part of the plant and low tyrosine content.

Development of clonal micropropagation technique for Gloxinia hybrida hort. “Strawberry Ice-Cream”

The reported paper presents outcomes of clonal micropropagation of ornamental variety Gloxinia hybrida hort. “Strawberry Ice-Cream”. In the study conditions supporting in vitro implanting leaf blade fragments are developed with an outcome of 97 % viable explants. An optimal composition of the Murashige and Skoog growth medium is suggested; it contains BAP (0.5 mg/l) and IAA (0.2 mg/l) and brings about efficient adventive sprout regeneration. Implantation takes place in a growth medium; no root stimulating hormones are applied. The survival of regenerative plants is reported to be high after transplanting ex vitro.

Determination of an effective method of Citrus aurantifolia lime and Citrus lemon varieties plant explants sterilization for in vitro introduction into the culture

Potted citrus plants are in constant demand, but the propagation of such plants requires the use of a winter greenhouse and several well-developed mother plants to obtain planting material. The use of microclonal propagation can accelerate the production of seed material though it requires detailed development of in vitro cultivation techniques. The aim of the research was to study the effectiveness of sterilizing substances and methods of sterilization on the yield of viable micro shoots of lime plants (Citrus aurantifolia) as well as Meyer and Jubilee lemon varieties and their growth in vitro. The research was conducted in the interdepartmental "Biotechnological Laboratory" of the Agrobiotechnological Faculty of Bila Tserkva NAU. The source material was microshoots from lime plants (Citrus aurantifolia), and lemon varieties (Citrus lemon) Meyer and Jubilee. The objects of the study were selected according to the diversity of genotypes, development type and varietal characteristics. The possibility of using different parts of plants for reproduction is a feature of in vitro culture. We used microshoots with a bud in our research. To neutralize the exogenous bacterial and fungal microflora, we used 70 % ethanol solution C2 H5 OH, sodium hypochlorite 5 %, 15 % solution of hydrogen peroxide H2 O2 , sulema (0.1) and washed the material in sterile water (for 5–10 min) under several schemes. The study revealed the influence of different sterilization regimes for obtaining sterile explants and the introduction into plant culture of explants of the species of lime Citrus aurantifolia as well as Meyer and Jubilee lemon varieties. It was found that the use of step sterilization using 2.5 % solutions of sodium hypochlorite gives a high percentage of sterile viable material – 27.7 %, which is significantly higher than in other variants of the experiment. Key words: microshoots, lime, sterile material, micropropagation, sterilization, hydrogen peroxide, viability of explants.

Influence of the component composition of nutrient media upon the introduction into the culture in vitro of explants of Prikubanskoe and Kubanskoe Bagryanoe apple varieties

The work reflects the aspects of introducing two varieties of apple trees into the culture in vitro: Prikubanskoe and Kubanskoe Bagryanoe. The aims of study included testing a new disinfection protocol that accurately regulates the content of active chlorine in the disinfecting solution, as well as studying the regenerative ability of explants of apple trees of the Prikubanskoe and Kubanskoe Bagryanoe varieties on different culture medium: Murashige and Skoog (MS) ¼ NH4 NO3 , and Kvorin-Lepuavra (QL). The disinfection protocol used made it possible to obtain an insignificant level of explant contamination with fungal infection in passage zero, which indicates the effectiveness of the tested extraction protocol. The contamination of explants in passage zero by fungal infection was insignificant, which indicates the high efficiency of the sterilizing agent. The level of bacterial infection is higher in the Kubanskoe Bagryanoe variety (21.3 % of the total amount of explants). The influence of the component composition of nutrient media on the primary growth of apple micro-shoots was revealed. The following results were obtained: the yield of viable explants of the Prikubanskoe variety grown on nutrient medium MS ¼ NH4 NO3 was 44,4 %, which is 16,4 % higher than explants grown on QL medium (28 %). Explants of the Kuba nskoe bagryanoe variety, in turn, have a yield of vital microplants grown on MS ¼ NH4 NO3 16.7 %, which is 30.4 % lower than explants grown on QL medium (47.1 %). The combined effect of the genotype and the composition of the nutrient medium on the development of explants was statistically significant for the studied parameters. The share of the cumulative effect of factors of the variety x medium is 74.93 %, the influence of the genotype is 20.72 %. The data obtained indicate the variety specificity of the nutrient medium during clonal micropropagation of these apple varieties.

Culturing Articular Cartilage Explants in the Presence of Autologous Adipose Tissue Modifies Their Inflammatory Response to Lipopolysaccharide.

The purpose of the current study was to explore the effect of autologous adipose tissue on cartilage responses to lipopolysaccharide (LPS). We hypothesized that LPS elicits an inflammatory response in cartilage, and that response is augmented in the presence of adipose tissue. Furthermore, we hypothesized that this augmented inflammatory response is due, at least in part, to increased exposure of cartilage to adipose tissue-derived c3a. Porcine cartilage explants from market-weight pigs were cultured in the presence or absence of autologous adipose tissue for 96 hours, the final 48 hours of which they were stimulated with LPS (0 or 10 μg/mL). Adipose tissue explants were also cultured alone, in the presence or absence of LPS. Media from all cartilage treatments was assayed for c3a/c3a des Arg, PGE2, GAG, and NO, and the viability of cartilage tissue was determined by differential fluorescent staining. Media from adipose tissue explants was assayed for c3a/c3a des Arg and PGE2. LPS produced a significant increase in PGE2, GAG, and NO production when cartilage was cultured in the absence of adipose tissue. Coculture of adipose tissue prevented a significant increase in PGE2 in cartilage explants. There was no effect of adipose tissue on LPS-induced GAG or NO, but the presence of adipose tissue significantly reduced cell viability in LPS-stimulated cartilage explants. Adipose tissue explants from lean animals reduced inflammatory responses of cartilage to LPS via a c3a/c3a des Arg-independent mechanism and were associated with a significant decline in cell viability. Thus, contrary to our hypothesis, adipose tissue from lean animals does not augment the inflammatory response of cartilage to stimulation by LPS. The mechanism of modulatory effects of adipose tissue on LPS-induced increase in PGE2 and decline in chondrocyte viability requires further research but appears to have occurred via a mechanism that is independent of adipocentric c3a/c3a des Arg.

Toxicity of anticancer drugs in human placental tissue explants and trophoblast cell lines

The application of anticancer drugs during pregnancy is associated with placenta-related adverse pregnancy outcomes. Therefore, it is important to study placental toxicity of anticancer drugs. The aim of this study was to compare effects on viability and steroidogenesis in placental tissue explants and trophoblast cell lines. Third trimester placental tissue explants were exposed for 72 h (culture day 4–7) to a concentration range of doxorubicin, paclitaxel, cisplatin, carboplatin, crizotinib, gefitinib, imatinib, or sunitinib. JEG-3, undifferentiated BeWo, and syncytialised BeWo cells were exposed for 48 h to the same drugs and concentrations. After exposure, tissue and cell viability were assessed and progesterone and estrone levels were quantified in culture medium. Apart from paclitaxel, all compounds affected both cell and tissue viability at clinically relevant concentrations. Paclitaxel affected explant viability moderately, while it reduced cell viability by 50% or more in all cell lines, at 3–10 nM. Doxorubicin (1 µM) reduced viability in explants to 83 ± 7% of control values, whereas it fully inhibited viability in all cell types. Interference with steroid release in explants was difficult to study due to large variability in measurements, but syncytialised BeWo cells proved suitable for this purpose. We found that 1 µM sunitinib reduced progesterone release to 76 ± 6% of control values, without affecting cell viability. While we observed differences between the models for paclitaxel and doxorubicin, most anticancer drugs affected viability significantly in both placental explants and trophoblast cell lines. Taken together, the placenta should be recognized as a potential target organ for toxicity of anticancer drugs.

Effects of DDT, DDE, aldrin and dieldrin on prostaglandin, oxytocin and steroid hormone release from smooth chorion explants of cattle

Chlorooganic xenobiotics (XBs) such as DDT, DDE, aldrin and dieldrin interfere with release of hormones from chorionic villi that are necessary for sustaining the normal course pregnancy: prostaglandins (PGs), oxytocin (OT), progesterone (P4) and estradiol (E2). Approximately 20 %–40 % of these hormones originate from the smooth chorion. The aim of current studies was to investigate effects of these XBs on synthesis and release of PGE2, PGF2α, OT, P4 and E2 from explants of smooth chorion of cattle, obtained during the120–150 and 151–180 day gestational period. Explants were incubated with DDT, DDE, aldrin or dieldrin at concentrations of 1 and 10 ng/mL for 24 h, and concentrations of PGE2, PGF2α, OT, P4 and E2 in post incubation medium and the relative abundances of COX-2, PTGES, AKR1B1, NP-I/OT, PAM, HSD3B, and CYP19A1 mRNA transcripts in tissue explants were determined. The XBs did not have effects on cell viability in explants (P > 0.05), however, there were effects on prostaglandins, OT and P4 secretion and relative abundance of mRNA transcript for genes encoding the main enzymes involved in synthesis of these hormones (P < 0.05). The XBs that were evaluated did not have effects on E2 synthesis and secretion (P > 0.05). In summary, XBs evaluated in the present study had effects on the pattern of prostaglandin secretion, and can increase OT and P4 release from smooth chorion explants. Because XBs inhibit hormonal action throughout the chorion, there is an increase in risk of abortions or premature births in animals.

Molecular and Biochemical Differences in Leaf Explants and the Implication for Regeneration Ability in Rorippa aquatica (Brassicaceae).

Plants have a high regeneration capacity and some plant species can regenerate clone plants, called plantlets, from detached vegetative organs. We previously outlined the molecular mechanisms underlying plantlet regeneration from Rorippa aquatica (Brassicaceae) leaf explants. However, the fundamental difference between the plant species that can and cannot regenerate plantlets from vegetative organs remains unclear. Here, we hypothesized that the viability of leaf explants is a key factor affecting the regeneration capacity of R. aquatica. To test this hypothesis, the viability of R. aquatica and Arabidopsis thaliana leaf explants were compared, with respect to the maintenance of photosynthetic activity, senescence, and immune response. Time-course analyses of photosynthetic activity revealed that R. aquatica leaf explants can survive longer than those of A. thaliana. Endogenous abscisic acid (ABA) and jasmonic acid (JA) were found at low levels in leaf explant of R. aquatica. Time-course transcriptome analysis of R. aquatica and A. thaliana leaf explants suggested that senescence was suppressed at the transcriptional level in R. aquatica. Application of exogenous ABA reduced the efficiency of plantlet regeneration. Overall, our results propose that in nature, plant species that can regenerate in nature can survive for a long time.

Clonal propagation of black currant (Ribes nigrum L.)

At the stage of transfer to soil, a scheme is defined that provides production of improved plantlets of thestudied varieties. 'Kuharsky's Memory' and 'Kanakhama' black сurrant varieties are characterized by high regenerativecapacity in vitro (the proportion of viable explants is 93.5 % and 96.4 %, respectively). At the multiplication stage, the influence of growth regulators (6-benzylaminopurine, gibberellic acid) on the morphogenesis of currant plantlets was studied. It shows their impact on such morphological indicators of development of plantlets as the height (cm), quantity, theaverage length of roots (cm). At the rooting stage, the concentration (0.5 mg/l) of auxin (β-indolylbutyric acid) was selected, the introduction of which into the rooting medium provided the maximum indicators of rhizogenesis: the numberand length of roots.

Methods of experimental polyploidization and their use in apple breeding

The article presents methods of experimental polyploidy of fruit, berry and ornamental plants. The purpose of this review is to highlight the problems and prospects of polyploidization of plants in the open ground and in vitro culture and the possibility of their application for apple trees. For the purpose of obtaining apple tetraploids as donors of diploid gametes, seed seedlings were treated with a solution of colchicine in concentrations of 0.1-0.4 % for 24 and 48 hours. Colchicine concentrations of 0.3 % and 0.4 % at 48 hours of treatment had a detrimental eff ect on their development. As a result, tetraploids and chimeras were obtained from seeds from free pollination of the varieties Orlik, Svezhest, Kandil Orlovsky, as well as from seeds obtained from crossing the varieties Svezhest×Bolotovskoe, Moskovskoe Оzherel’e×Imrus, Girlyanda×Venyaminovskoe. The optimal concentration of colchicine was 0.1 %. Methods of colchicine treatment have been studied: 1) adding to the nutrient medium, colchicine concentration: 0.01%, 0.02%, exposure time 24h-19 days; 2) applying amitotic solution to the growth point, colchicine concentration: 0.1 %, 0.2 %, exposure time 24h-7 days. To increase the penetration of colchicine through the cell walls, a 0.1 % dimexide solution was used. Studies have shown that high concentrations and prolonged exposure to colchicine reduce the viability of explants.

POTENTIAL OF AQUEOUS OZONE FOR THE IN VITRO STERILIZATION PROCEDURE OF SOLANACEAE

Objective: This research aimed to verify the use of aqueous ozone as an alternative sterilizer for in vitro sterilization procedure.&#x0D; Methods: Three species of Solanaceae were selected based on their trichomes. They are thorn apple (Datura metel L.), eggplant (Solanum melongena), and tomato (Lycopersicon pimpinellifolium). The treatment used four levels of concentrations; K0 (NaOCl – control), K1 (AO 0.1 ppm), and K2 (AO 0.5 ppm), K3 (1 ppm) and it employed three types of leaves, namely, D1 (thorn apple), D2 (eggplant), and D3 (tomato). The data were analyzed by analysis of variance univariate two factors and followed by Duncan’s multiple comparisons test.&#x0D; Results: This assay showed that the effect of ozone was almost immediately against microorganisms, and the optimum ozone concentration depends on the types of the leaf. Both levels of ozone concentration and types of leaves had a significantly different effect (p&lt;0.05), and there is no interaction between aqueous ozone concentration and types of leaves (p&gt;0.05). The level of sterility in K0 is 77, 78%, K1 is 22, 22%, K2 is 44, 44%, and K3 is 100%. Ozone 1 ppm had the highest result, but there is no viable explant because they got damage in their cells.&#x0D; Conclusions: Aqueous ozone showed good potential to inhibit the growth of microorganisms for the in vitro sterilization procedure of Solanaceae.

REGENERATION OF APPLE VARIETIES IN VITRO CULTURE

The process of clonal micropropagation is influenced by the genotypic characteristics of plants, the composition of nutrient media and the hormonal background at various stages of cultivation. The purpose of the research was to study the regenerative capacity of apple explants of promising varieties. The objects were colon-shaped, immune and scab-resistant apple varieties. The study was carried out in the laboratory of biotechnology of VNIISPK using generally accepted methods. The largest number of viable explants was obtained during the active growth phase, averaging from 72.1 to 88.5%. Maximum contamination was observed in explants introduced in April, regardless of the sterilizing agents. The highest infection rate was in the varieties Orlovskoe рolesie (up to 56.7%), Bolotovskoe (up to 51.4%), Poeziya (up to 43.3%). In the explants introduced in June, the infection was insignificant. Thus, the Imrus and Poeziya varieties showed a small degree of infection after using 0.1% mertiolate solution (1.2% and 2.4%), and the garland and Priokskoe varieties had a 100% yield of pure apexes. Among immune varieties, the highest necrosis was evident in the variety of Imrus (42,1%) and the smallest in the variety Kandil оrlovsky (5,1%) have the largest column-the variety Priokskoe (65.6 per cent) and Vostorg (65,6%). In most of the studied varieties, the greatest necrosis was observed after using 0.1% mertiolate solution. The study was carried out on the background medium QL and Fardzinova recommended for pears. Concentration of BAP 1.0 and 2.0 mg/l. Among the immune varieties, the content of BAP 2.0 mg / l in the QL medium caused the greatest proliferative activity in the varieties Kandil оrlovsky (3.4), Bolotovskoe (4.5) and Orlovskoe рolesie (2.9). Varieties of Imrus and a Veteran of the highest rate of multiplication were obtained in Fardzinova on medium containing BAP in both concentrations. In all varieties, the concentration of BAP 1.0 mg/l contributed to the formation of shoots more than 1 cm, i.e. suitable for rooting.

Responses to cryopreservation of recalcitrant seeds of Ekebergia capensis from different provenances

Recalcitrant seeds are shed at relatively high water contents and are metabolically active, initiating germination without exogenous provision of water. There is an urgency for these seeds to be conserved in light of the rapidly diminishing numbers of indigenous species and associated genetic diversity. This may be achieved by long term storage of whole seeds or seed germplasm. This study centred on the development of a cryopreservation protocol for embryonic axes (explants). Indicators of stress i.e. superoxide (·O2−), hydrogen peroxide (H2O2) and total aqueous antioxidants (TAA) levels were measured, as well as explant viability and shoot and root ultrastructure in response to exogenously applied cathodic water after each cryopreparative step.Explant viability gradually decreased after each cryopreparative step indicating the cumulative effect of the stresses imposed by cryopreservation. In conjunction with reduced viability, the levels of ·O2− and H2O2 increased gradually after each of these steps. Total aqueous antioxidant levels remained relatively unchanged throughout the cryopreservation process. The ROS levels in cathodic water treated explants were significantly lower compared with those that were not treated with cathodic water. A small number of explants from seeds obtained in Port Elizabeth (30%) survived after cryopreservation, while none of the explants from seeds obtained in St Lucia did.Root ultrastructure across explants from seeds of both provenances was well preserved after each cryopreparative step. Organelles were well developed and the ultrastructure was typical of metabolically active tissue. However, gradual ultrastructural deterioration was observed in the shoot meristem after each cryopreparative step. Typical of a stress response, increased vacuolation and a lack of visible organelles were noted. Root and shoot production (or lack thereof) was supported by ultrastructural observations.This study reports that the explants of PE origin were able to survive cryopreservation while those of St Lucia did not. The key findings of this study support the role of cathodic water in regulating ROS and indirectly preserving explant ultrastructure and survival, and its exogenous application during cryopreservation of recalcitrant seeded germplasm is recommended.