Sterilization Of Explants Research Articles

Enhancing Clonal Micropropagation of Zarya Alatau Apple

The purpose of this study was to improve the protocol of clonal micropropagation for effective mass production of the Zarya Alatau apple cultivar through the use of axillary buds. In Kazakhstan’s challenging climate, the Zarya Alatau apple thrives because of its unique traits, including fruit preservation until May, cold hardiness, and resistance to scab and powdery mildew. Micropropagation is essential for healthy mother tree establishment, and this research focused on key factors for successful in vitro propagation. The sterilization of explants was optimized: 1.6% solution of sodium hypochlorite effectively sterilized plant materials for 10 minutes. Nutrient media composition was evaluated for efficient shoot regeneration. The study examined axillary bud regeneration on Murashige and Skoog medium with different concentrations of hormones. A combination of 6-benzylaminopurine (0.5 mg/L) and gibberellic acid (0.5 mg/L) yielded optimal results, with shoots reaching 3.5 cm. Root induction was analyzed with varying indole-3-acetic acid (IAA) concentrations, and the best results were achieved with 1.5 mg/L IAA, resulting in an 85% rooting frequency. Adapting in vitro plants to ex vitro conditions is crucial given their sensitivity to environmental changes. Well-developed leaves and a robust root system are essential for successful acclimatization during transplantation into a soil substrate. This research provides valuable insights into the critical parameters for a successful transition of in vitro propagated plants to soil conditions, optimizing micropropagation practices.

THE PERSPECTIVITIES OF MICRICLONAL PROPAGATION OF ACER PLATANOIDES L. CRIMSON KING (ACEARCEAE JUSS.)

Clonal micropropagation – massive asexual propagation of plants from cell and tissue culture; the resulting plants are genetically identical to the original copy. The conservation of plants by tissue culture methods is of great interest and will probably become even more common in the future. The advantages of this method are numerous, but we find it most important that the initiation of cultivation requires only a single plant, a seed or a single explant (apex, meristem, bud, piece of leaf, stem etc.), thus, the available plant specimens (which can be only a few) will not be affected by harvesting them from their habitat. The state program “Developing of the scientific basis for the sustainable reproduction of valuable specimens of the botanical garden collection in in vitro culture” is being conducted in the laboratory of biotechnology of the Tashkent Botanical Garden of the Institute of Botany, Academy of Sciences of the Republic of Uzbekistan, directed at microclonal propagation of many valuable species presented in a single copy, as well as species in demand in urban greening programs. Acer platanoides L. Crimson King variety (Acearceae Juss.) is a deciduous, slow-growing tree, reaching 15 m with age. The crown shape is wide-round and even. The leaves are large, 5-7 lobed, deep purple, and do not change color throughout the growing season. The trunk is smooth, dark in color, with clearly defined longitudinal grooves. Because of its graceful shape, the Crimson King variety is extremely popular. It is actively planted in city yards, parks, and roadsides. Suitable for landscaping. Within this state program the protocol of microclonal propagation of A. platanoides Crimson King variety has been developed to propagate this species for Botanical Garden collection as well as for greening programs. Solutions of sodium hypochloride (4-6%), hydrogen peroxide (2-15%), silver nitrate (0.01%), Tween20, ethanol (70%), and some other sterilizing agents were tested for developing a protocol for sterilization of maple explants. “Belizna” solution, “Domestos” sterilizing soap, fungicides difenoconazole, mancoseb and metalaxyl, fludioxonil, propiconazole, antibiotics streptomycin, amoxicillin, gentamicin, and etc. were also implemented for explants sterilization. As a result of the studies, it was determined that the most optimal source of explants are branches with apical (10% regeneration) and lateral (100% regeneration) buds of the annual shoot of A. platanoides Crimson King variety. Plant growth environment is important in the success of in vitro propagation. Thus, explants obtained from trees growing in the arid areas of massive Kuylyuk in Tashkent, were characterized by low viability and a high degree of infection with fungal diseases in comparison with explants obtained from trees growing in Hastimom Square in Tashkent, where trees are watered daily. We found two ways of in vitro propagation of A. platanoides L. variety Crimson King: 1. With the use of microcuttings. 2. Indirect organogenesis with induction of callus. The best way for A. platanoides L. variety Crimson King became indirect organogenesis trough induction of the callus in the WPM - Wood Propagation Media, hormone free or with 6- benzilaminopurine in the media. The duration of the developing of a plant usually takes 6-9 months.

In vitro propagation of triploid Toxicodendron vernicifluum (Stokes) F. A. Barkl

AbstractTriploid lacquer trees have a higher yield of raw lacquer and greater stress resistance; however, it is challenging to develop efficient and rapid seedling culture techniques because of the influence of metabolites with raw lacquer. In this study, the stem segments of a triploid lacquer tree were used to investigate the effects of explant sterilization treatment, different combinations of exogenous hormone concentrations on the induction of axillary buds, seedling development, and rooting, as well as transplanting substrate ratios. The results showed that the optimal combination of disinfection treatment for triploid Toxicodendron vernicifluum (Stokes) F. A. Barkl explants involves 75% alcohol immersion for 10 s, followed by 2.0% benzalkonium chloride treatment for 1 min and 0.1% HgCl2 treatment for 4 min. The suitable medium for axillary bud induction was Murashige and Skoog (MS) containing 0.1 mg•L−1 zeatin (ZT) and 0.1 mg•L−1 6-benzylaminopurine (6-BA), resulting in a high induction rate of 95.0%. Use of 1/2 MS supplemented with 0.5 mg•L−1 6-BA and 0.3 mg•L−1 2,4-dichlorophenoxyacetic acid (2,4-D) was the most effective for axillary bud proliferation. The combination of 1/2 MS containing 0.1 mg•L−1 6-BA, 0.5 mg•L−1 2,4-D, 0.5 mg•L−1 indole-3-butyric acid (IBA), and 0.2% activated carbon (AC) was an optimum for hardening-off. With woody plant medium (WPM) supplemented with 0.5 mg•L−1 naphthaleneacetic acid (NAA) and 0.5 mg•L−1 2,4-D and 0.5 g•L−1 AC as a rooting medium, the rooting rate was 67.8%. Also, the appropriate transplanting substrate ratio for triploid Toxicodendron vernicifluum (Stokes) F. A. Barkl was grass charcoal:laterite:humus = 1:1:1. Collectively, the results provided the technical assurance for seedling scale multiplication and propagation, facilitating its application in production.

Development of a Suitable in vitro Regeneration Protocol for Rahangala Pear (Pyrus pyrifolia L.) in Sri Lanka

The limitation of planting material is the major drawback to expanding pear cultivation in Sri Lanka. Hence, an in vitro protocol was developed to produce planting material for the pear variety of Rahangala. Pear shoots were used as explants and surface sterilization of explants was done using 0.5% and 1% (w/v) AgNO3 for 15 min, 5% and 10% (v/v) NaOCl for 5, 10 and 15 min. In vitro shoot multiplication and root induction were found to be suitable in full and half strengths of MS medium respectively supplemented with various concentrations of BAP (1, 1.5, 2, 2.5, 3 mg/l), IBA (0, 0.5, 1, 2, 3, 4 mg/l) and NAA (0.1 mg/l). Acclimatization was conducted in pots containing different proportions of topsoil, compost, coir dust and sand. Completely Randomized Design (CRD) was used as an experimental design for this study. During sterilization of explants, the survival rate of 86.6% was recorded after 3 weeks where the treatment was done with 1% AgNO3, and 10% NaOCl for 10 min. BAP (3.0 mg/l) produced the best shoot multiplication (1.46 ± 0.2). Significantly (p<0.05), the highest mean number of roots (1.93 ± 0.98), average length (60.6 mm), and rooting percentage (68%) were observed in ½ MS medium with 4.0 mg/l IBA. After five weeks of transplantation in pots, 100% of plantlets survived in topsoil, compost, coir dust, and sand in a ratio of 1:1:1:1 Plant Tissue Cult. & Biotech. 34(1): 25-33, 2024 (June)

Callus induction of red ginger (Zingiber officinale var. rubrum) with 2,4-D and Kinetin combination to enhance total phenolic and flavonoid content

Abstract Plant tissue culture perform as in vitro technique which use any parts of plants under environmental and nutritional control. Tissue culture provides a competitive systems for effective secondary metabolites production, including in red ginger which has many functional properties with high demand in health and pharmaceutical sectors. This study aims to develop an appropriate procedure to cultivate red ginger with in vitro technique to enhance its phenolic and flavonoid content. The rhizome buds were use as explants for callus induction by MS medium with some combination of 2,4-D and kinetin (1:0; 1:1; 1:5; and 5:1). The 70% ethanol, 5,25% sodium hypochlorite, and 10% povidone iodine were used for surface sterilization of bud explants. The result reveals that in 1:0 combination of 2,4-D and Kinetin, the compact callus were initiated on day-16. The other three combinations showed shorter callus forming time in day-14 and they were friable. The 1:1 combination showed the highest percentage (70%) of callus formation compared to other three combinations. In this combination also indicated no contamination occurs. From this in vitro tissue culture, the methanolic extract of callus obtained highest total phenolic content (204,793 µg/mL) than rhizome (130.77 µg/mL). Though, the total flavonoid content of callus extract (0,289 µg/mL) was lower than rhizome extract (6.74 µg/mL). This study showed that the in vitro tissue culture was quite appropriate to enhance total phenolic content in red ginger, although not at the flavonoid content.

The use of plant explants to obtain embryogenic callus clams as an early stage in the selection of Napier Grass cv Taiwan

Abstract This study aimed to obtain 50-100 mussels from a population of embryogenic callus derived from cultivars of Taiwan elephant grass. The research was conducted in three phases: 1) sterilization, which was accomplished by immersing the material in 70% alcohol solution, 20% chlorox, and rinsing it with sterile distilled water so that it could be cultured in the treatment media; 2) callus induction with explants of young leaves in culture medium supplemented with growth regulator (PGR) 2,4-D at a concentration of 2 mg/l and enriched with amino acids and casein hydrolysate; 3) callus proliferation and regeneration utilizing culture-based. The results indicated that the success rate of explant sterilization ranged from 10 to 78%, and the ability of the tissue to form callus ranged from 60 to 80%, with the highest callus-forming ability obtained from culture explants with chemical and physical combination sterilization by burning, while at the proliferation stage, it was obtained on average 5 times per series. After two weeks, the average callus weight was 1.72 to 1.76 g per bottle, increasing to 1.92 to 2.07 g per bottle for a total of 150 bottles, with 78.1 to 85.3% of callus cells capable of regenerating into shoots via organogenesis or embryogenesis. It was determined that the initial selection procedure for fodder plants from explants to obtain callus has the potential to be further developed.

Establishment of Explant Sterilization Protocol and the Effects of BAP and AgNO3 on In Vitro Multiplication of Kaempferia parviflora Wall. ex Baker

Establishment of Explant Sterilization Protocol and the Effects of BAP and AgNO3 on In Vitro Multiplication of Kaempferia parviflora Wall. ex Baker

Micropropagation of berry crops for creation of germplasm cryobanks.

One of the main stages of cryopreservation of meristematic tissues in vegetative plants is a clonal micropropagation, which includes isolating the explants of the raw material in vitro and optimizing the culture medium for micropropagation. As the result of our studies, the optimal periods for in vitro micropropagation are: first - isolation of explants from initiated shoots of dormant buds (blackcurrants and raspberries) in January-March; the second - from actively growing shoots (blackcurrants and raspberries) in May-June, from the formed mustache (strawberry) in July-August. The optimal drugs for sterilization of raspberry explants are: a) 0.1% HgCl2 (6 min), then 3% H2O2 (15 min); b) chlorine-containing bleach «Domestos» in the dilution of H2O 1:9 (10 min). For blackcurrant: a) 0.1% HgCl2 (5 min) in combination with 0.1% fungicide "Topaz" (30 min); b) 0.1% HgCl2 (5 min) in combination with the treatment with KMnO4 (30 min); c) "Domestos" in the dilution of H2O 1:5 (20 min). For strawberry: a) 0.1% HgCl2 (6 min) followed by treatment with 3% H2O2 10 (min); b) 1% deochlor (7 min), 3% H2O2 (10 min); c) "Domestos" in the dilution of H2O 1:5 (8 min) with subsequent treatment 0,1% HgCl2 -7 min, then 0,20 mg/l КМnO4 - 30 min. Optimal compositions of culture media for micropropagation of blackcurrant - Murashige and Skoog (MS) medium with 0.5 mg L-1 BAP, 0.5 mg L-1 GA3, 0.1 mg L-1 IBA and 20 g L-1 glucose. For raspberry -MS medium with 0.5 mg L-1 BAP, 0.1 mg L-1 IBA, 10 mg L-1 iron chelate and 30 g L-1 sucrose. For strawberry - MS medium with 0.3 mg L-1 BAP, 0.01 mg L-1 IBA, 0.2 mg L-1 GA3, 10 mg L-1 iron chelate and 30 g L-1 sucrose. Based on these studies, the cryobank was created, which include the germplasm of in vitro meristematic tissues in 66 cultivars, hybrids and wild-growing forms of blackcurrant, raspberry and strawberry. Therefore, the aim of the research was to obtain aseptic plants, clonal micropropagation and the creation of a cryogenic collection of germplasm based on the developed technology.

Establishment of an In Vitro Micropropagation Protocol for Hibiscus moscheutos L. ‘Berry Awesome’

Hibiscus moscheutos L. ‘Berry Awesome’ is a complex hybrid of the new Proven Winners Summerific series of varieties with highly ornamental characteristics. Micropropagation of highly ornamental varieties is important for mass production of planting material for commercial purposes. The traditional methods for propagating Hibiscus varieties, such as cuttings or seed propagation, however, do not guarantee high rates of production of high-quality seedlings. To solve this problem, an attempt was made to develop protocols for micropropagation of Hibiscus moscheutos L. ‘Berry Awesome’ in vitro on agar and liquid medium using a bioreactor system, followed by ex vitro adaptation of the regenerants. The optimal method for sterilization of nodal explants as well as the optimal composition of the initiation medium for shoot proliferation and rooting were determined. For micropropagation on a liquid medium, a rocker-type bioreactor was used, and its advantages over micropropagation on an agar medium were demonstrated. The results showed that the best sterilization method for nodal segment explants was as follows: pretreatment by rinsing with running tap water, sterile water, and distilled water for 70 min and soaking for 5 min in a mixture of solutions of ethyl alcohol (96%), hydrogen peroxide (38%), and water in a ratio of 1:1:2. In this case, live and sterile explants accounted for 62.6%. The optimal initiation medium for axillary buds in nodal segments was the Murashige and Skoog (MS) medium supplemented with 0.1 mg L−1 N-(2-chloro-4-pyridinyl)-N’-phenylurea (CPPU), which resulted in 73.3% of axillary buds being induced. The optimal solid proliferation medium was MS medium supplemented with 0.1 mg L−1 CPPU with a proliferation coefficient of 5.8. In a liquid medium, the optimal concentration of CPPU was 0.05 mg L−1 with a proliferation coefficient of 9.2. The best medium for rooting/shoots with agar and in bioreactors was MS medium with the addition of 0.1 mg L−1 indole-3-butyric acid (IBA). The highest rooting rate was 99.0% in both types of media, and the survival rate of plantlets was 88.7% in solid media and 98.7% in the bioreactor.

Combination of Planting Media and Fungicide Immersion on the Growth of Moon Orchid (Phalaenopsis sp.) Acclimatization Stage

Acclimatization is a critical stage in the propagation of tissue culture plants, because at this stage the plant must be able to develop and form strong tissue so that it can adapt to the new environment. The aim of this research was to determine the interaction of a combination of planting media and fungicide soaking on the growth of moon orchids (Phalaenopsis sp.) at the acclimatization stage. The method used was a factorial Completely Randomized Design (CRD) and was repeated three times. The first factor is a combination of planting media consisting of 100% white moss, 50% white moss + 50% fern, 50% white moss + 50% wood charcoal, and 50% white moss + 25% fern + 25% wood charcoal. The second factor is the length of soaking in fungicide, namely without soaking, soaking 6 minutes and 10 minutes. The data obtained were analyzed using ANOVA and continued with the 5% DMRT test. The results of the research showed that the combination of planting media treatment of 50% white moss + 25% fern + 25% wood charcoal not significant on all observed parameters. These results show that the planting medium has a very important role in the growth of orchid plants, sterilization of explants at the acclimatization stage only needs to be done if the source of contaminants is in the planting material.

WORTHY MICROPROPAGATION PROTOCOLS OF SEVEN CULTIVARS OF POMEGRANATE (PUNICA GRANATUM L.) CULTIVARS IN THE PROVINCE OF DUHOK, KURD ISTAN RIGION OF IRAQ.

An efficient attempt was conducted to increase the mass production of seven local cultivars of pomegranate (Punica granatum L.) Masafik, Melisse, Radisho, Armishte, Diala, Halapja, and Dwarf pomegranate cultured in the Kurdistan region of Iraq from October 2021 to February 2023. Micropropagation technique was applied using eight combinations of plant growth regulators (PGRs) added to two media (MS and WPM). The results indicated that immersing explants in 70% Ethyl alcohol (EtOH) for (2 minutes) followed by immersion in (2.5%) Sodium Hypochlorite (NaOCl) plus 3 drops of tween 20 for 13 mins. was the more suitable combination for explant sterilization. MS and WPM media salts supplemented together with eight combinations of PGRs were carried out to evaluate the more suitable combination for the initiation stage. The obtained data clarified that the ½ strength of the MS medium (1/2 MS) supplemented with 0.5 BAP plus 0.5 mg/l Kinetin increases the pomegranate explant initiation response percentage. WPM basal medium was the preferred media to be used in the multiplication stage. The impact of different levels and protocols of Cytokinins, Auxins, and Gibberellins were investigated by eight protocols including using BAP, GA3, and adenine sulfate. The outcomes indicated that adding both concentrations of BAP (0.5 and 1.0 mg/l) separately supported by GA3 and adenine sulfate in amounts of (0.3 and 30 mg/l) respectively again were the most suitable protocol that affected the multiplication of regenerated shoots when compared to using BAP alone. Valuable results were found in rooting response after 6 weeks by using ½ strength of MS media along with two levels of Auxins (NAA, and IBA). Moreover, the visual observation records that around 85-90% of rooted cultures were successfully acclimatized by transferring the cultures from in vitro to the ex-vitro environment. Finally, Dwarf cultivars exceed all the other cultivars in studded parameters.

Pengembangan Metode Teknik Sterilisasi Eksplan Guna Meningkatkan Keberhasilan Kultur Jaringan Tanaman Stevia (Stevia Rebaudiana Bertoni)

Stevia rebaudiana Bert is a shrub native to the borders of Paraguay, Brazil and Argentina. The leaves of this plant produce steviol glycosides, a secondary metabolite which has a sweetness level of 200-300 times sweeter than sugarcane sucrose. Conventional stevia propagation methods cannot meet the increasing need for stevia seeds or seedlings. Tissue culture is able to fulfill this deficiency because it has the potential to produce large amounts of uniform plantlets. Tissue Culture Technique is a vegetative propagation of plants. Tissue culture includes the cultivation of cells or cell aggregates, tissues and organs of plants in a medium containing sugars, vitamins, amino acids, organic salts, water, growth regulators and compacting agents. The composition of the growing medium is also very beneficial for the growth of fungi and bacteria. Therefore, explant sterilization is an important factor in determining the success of a tissue culture program. The problem is that until now there is no optimal explant sterilization technique for Stevia tissue culture. The aim of this research is to find out the best explant sterilization method from a combination of several disinfectants on Stevia (Stevia rebaudiana Bertoni) explants which can remove contaminants on the explants. The main parameters observed were Source of Contamination (descriptive: shape and color), Percentage of Explants Alive, Percentage of Contamination, Percentage of Browning, and Time when contamination first appeared (Early). The results showed that the sterilization method F used running water, immersion in detergent for 10 minutes, immersion in 1.5% fungicide solution and 1.5% bactericide for 10 minutes, 70% alcohol immersion for 1 minute, 0.05% HgCl2 immersion 3 minutes and immersion in 10% and 5% NaOCl respectively for 5 minutes had the lowest contamination percentage of 20% and the highest survival presentation of 80% with 80% fungal and 20% bacterial contaminants and the initial contamination occurred in the first week of observation.

Explant Sterilization of Sandalwood (Santalum Album) and Teak (Tectona Grandis) Micropropagation with Commercial Disinfectant

Sandalwood (Santalum album) and Teak (Tectona grandis) are essential wood sources for furniture. Sustainable micropropagation methods can benefit wood availability. This study evaluates NaOCl and Povidone-iodine effects, immersion duration, and concentrations on teak and sandalwood micropropagation. Teak is treated with 10% & 15% NaOCl and 10% & 15% Povidone-iodine. No significant difference was observed between teak treatments; however, povidone-iodine shows potential as an effective teak sterilizing agent. Sandalwood's best sterilization: NaOCl at 10% & 15%.

Identification of Best Exposure Timings of Sodium Hypochlorite in Surface Sterilization of Different Explants in invitro Propagation of Annona muricata

The most crucial and delicate stage in plant tissue culture is the surface sterilization of the explant. An inappropriate concentration of sterilant may have deadly effects on explants by altering cell division and other metabolic processes that limit the growth and development of explants. Considering the explants observations and sterilization treatments of Sodium hypochlorite (4%), T6 (7 min), T7 (8 min) and T8 (9 min) are selected for green nodal explants as highest response, least days for initiation of response, good health with less contamination and moderate survival of explants. Whereas, T5 (6 min) T6 (7 min) and T7 (8 min) are selected for leaf with lower contamination with good health and survivability. Internode was not selected due to no response with higher contamination and less survivability. Brown nodal explants responded very less and less survivability with higher contamination as compared to other explants

Optimizing Surface Sterilization for Micropropagation of Gerbera jamesonii: A Study on Explant Survival and Contamination Control

In the commercial production of gerberas, micropropagation enables the large-scale production of plants in a limited space, as well as the obtaining of disease and pest-free uniform plants. This technique also ensures precision in production schedules and product quality, regarding plant homogeneity and vigour. Nevertheless, the occurrence of contamination in an in vitro culture is frequent, and might result in high damage. Plant materials have different kind of microbes i.e., exogenous and endogenous in nature which is the main reason for contamination in tissue culture. Pre-treatments to explants with fungicides and bactericides can be effectively used to eliminate the surface contamination. Surface sterilizing agents, their levels and duration of exposure are known to impact the in vitro culture establishment. In the present experiment, pre-treatment of explants was carried out with Teepol BleachTM solution (0.1%) and BavistinTM (0.1%) for 15 and 20 minutes respectively for effective control of fungal contaminations in all the cultures. For the control of bacterial contamination antibiotics were added in culture medium. Surface sterilization of explants with 0.1% mercuric chloride (HgCl2) for five minutes gave the maximum survival of the growing cultures.

Establishment and Optimization of Micropropagation System for Southern Highbush Blueberry

The optimization of micropropagation for blueberries is crucial due to the growing blueberry industry and demand for plantlets. This study focused on four stages: explant sterilization, in vitro establishment, in vitro proliferation, and ex vitro rooting, aiming to establish an efficient in vitro propagation system for southern highbush blueberry cultivar ‘ZY09’. The most effective explant sterilization method was a 60 s treatment with 75% ethanol and a 5 min treatment with 4% NaClO. During the establishment of the in vitro culture, the modified woody plant medium was found to be suitable. The replacement of NH4NO3 in woody plant medium with (NH4)2SO4 facilitated the proliferation of blueberry microshoots. The optimal combination of plant growth regulators for the in vitro proliferation of blueberry microshoots was indole-3-butyric acid (0.1 mg·L−1), thidiazuron (0.0005 mg·L−1), and zeatin (1 mg·L−1). Perlite was the most suitable substrate for ex vitro rooting. The best ex vitro rooting performance was observed without immersion in growth regulators. Indole-3-butyric acid enhances root formation and suppresses root elongation in blueberries. The findings of this study can be applied to large-scale in vitro propagation of southern highbush blueberry and provide a reference for the genetic transformation of blueberries.

Effect of Explants Type and Plant Growth Regulators on the Initiation and Multiplication of Vegetative Shoots of Stevia rebaudiana Bertoni in vitro

This study was carried out in the Plant Tissue Culture Laboratory of the College of Agriculture - Karbala Unaiversity for the period from 2022 to 2023. The study included the use of explants and different combinations in the sterilization, initiation and multiplication of the vegetative shoots of the Stevia plantiv vitro, the study was carried out in three phases: the first included sterilization of explants (shoot tips and lateral buds) using sodium hypochlorite at concentrations of (0, 0.5, 1, 1.5, 2)% for periods of (5, 10, 15) minutes. The second included the initiation of shoots cultures by cultivating the shoot tips and lateral buds on the MS medium with different concentrations (0, 0.5, 1, 1.5) mg l-1 BA and (0, 0.1, 0.2, 0.3) mg l -1 NAA, the third stage was carried out by cultivating the growths resulting from the shoot tips cultivated in the previous stage on the MS medium prepared with different concentrations (0, 1, 2, 3, 4) mg l-1 BA and (0, 0.1, 0.2, 0.3, 0.4) mg l-1NAA. The results of the study showed the superiority of the developing apical in achieving the lowest contamination rate of 38% compared to the side buds that achieved a contamination rate of 40.67%, and the concentration of 2% of NaOCl achieved the lowest pollution rate of 8.33%, and the duration of 15 minutes achieved the lowest pollution rate of 24%. The results of the initiation experiment showed the superiority of the shoot apical as a explants that achieved the highest response rate to emergence of 70% compared to the lateral shoots that recorded a response rate of 54.69%, and the concentration of 1 mg l-1 BA was significantly superior and achieved a response rate of 82.5% compared to the comparison treatment that gave a rate of 82.5%. A response of 32.5%. As for the results of the vegetative multiplication experiment, the results showed the superiority of BA at a concentration of 3 mg l-1 in achieving the highest rate in the number and length of branches was 33.35 branches Plant-1, 8.20 cm respectively. compared to the comparison treatment that achieved the lowest rate, and the concentration of 0.3 mg l-1 NAA was superior in achieving the highest average of the number and length of the vegetative total reached 30.84 plant branch-1, 7.65 cm respectively, while the comparison treatment achieved the lowest mean for the studied traits.

In vitro sterilization and callus induction from Cananga odorata forma genuina steenis explants

Cananga odorata (ylang-ylang) is a plant with numerous uses, including ornamental use, traditional medicinal ingredient, as fragrance, and cosmetics. The high market demand for ylang-ylang essential oil needs to be supported by information on the in vitro process of callus formation to supports the secondary metabolites production without having to cut or harvest the trees so that biodiversity is maintained. The aims of this study were to analyze the explants sterilization method using sodium hypochlorite (NaOCl) solution that was most suitable for the ylang-ylang initiation, and to analyze the effect of BAP and NAA treatment on the formation of ylang-ylang callus. The design used is a completely randomized design. The methods used included the preparation, sterilization, and callus induction using a combination of BAP and NAA hormones with twenty four treatment combinations. The results are: the sterilization of explants with sodium hypochlorite (NaOCl) solution at a concentration of 20% (v/v) 10 minutes followed by a concentration of 15% (v/v) 20 minutes was able to produce sterile explants in as much as 67.5% of cases. At the callus induction stage, the highest score of callus formation in leaf explants was in media-23 (BAP 4 ppm plus NAA 5 ppm).

Optimization of Key Technologies for Induction of Embryogenic Callus and Maturation of Somatic Embryos in Korean Pine (Pinus koraiensis)

Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low induction and proliferation rates, hindering the production of improved plant materials. We investigated the effects of the explant sterilization method, 4 °C cryopreservation, basal medium, ethylene removal, liquid medium supplementation, and a combination of PGRs on embryogenic callus (EC) induction of Korean pine, using immature embryos of Korean pine as explants. The effects of sucrose and maltose on EC proliferation and maturation were investigated. The differences in the maturation ability of EC somatic embryos before and after cryopreservation were evaluated using the induced embryonic cell lines. The results showed that zygotic embryos (ZEs) performed better than megagametophytes (MGs) as explants. The induction rate of EC was significantly increased after 28 days of cryopreservation at 4 °C. The induction rate of EC in the #5 family increased from 10.00% to 62.8%. The EC induction rate of the five families cultured with the DCR basal medium was higher than that with the mLV basal medium. Among them, the induction rate of the #5 family cultured with the mLV basal medium was 23.3%, while that with the DCR basal medium was 60.9%, an increase of 2.6 times. There was no significant difference in the maturation ability of EC somatic embryos before and after cryopreservation. In conclusion, this study provides a method to improve the EC induction rate and maturation ability of Korean pine.

Obtaining sterile Calligonum aphyllum material for in vitro culture preservation

The comparative evaluation’s results on the efficiency of surface treatment of Calligonum aphyllum (Pall.) Guerke explants, a perspective plant species in the restoration of degraded lands, are presented. Sterilizers «Lysoformin 3000», silver nitrate, hydrogen peroxide, and «Belizna-econom» are used at the introduction stage of the plants in vitro. As a result, 2 of 11 effective sterilization modes were found when C. aphyllum was introduced into the culture in vitro: No. 10 (hydrogen peroxide solution 10% v/v, 5 min) and No. 11 (hydrogen peroxide solution 6% v/v, 15 min). These modes had the highest rates of sterile and regenerative explants. The microflora was also isolated from the studied explant samples. Determined the preparation «Lysoformin 3000» effectively acts on all isolated microorganisms. «Belizna-econom» and hydrogen peroxide (concentrations of 10% v/v) do not act on 80% of isolated bacterial strains. Shown the silver nitrate solution was not sensitive in 100% of cases. The obtained information can be used to draw up a complete protocol for C. aphyllum explant sterilization at the introduction stage in vitro.