Hormone-free MS Medium Research Articles

Indirect in vitro Regeneration in Four Varieties of Potato (Solanum tuberosum L.) from Internodal Segments and Leaf Explants

Internodal segments and leaf explants from four varieties of potato (Solanum tuberosum L.) cultivated in Bangladesh, namely, Asterix, Diamant, Granola and Lady Rosseta were utilized for indirect in vitro regeneration of shoots through callus culture. The best responses regarding the reaeration of shoots were obtained when the explants were cultured on MS medium supplemented with 4.0 mg/l BAP and 1.0 mg/l IAA and 0.5 mg/l GA3. The highest frequency of multiple shoot regeneration was recorded from internodal segments rather than from the leaf explants. Hormone free MS medium was found to be the most effective for healthy root induction from the in vitro raised excised shoots. Following adequate root induction, the in vitro regenerated plantlets were successfully transplanted and established to soil for further growth and development of tubers. Plant Tissue Cult. & Biotech. 34(1): 83-92, 2024 (June)

Optimizing Green Globular Body Induction for Micropropagation of Microsorum pteropus ‘Windeløv’

Microsorum pteropus ‘Windeløv’ is a water fern from Asia commonly used in aquarium landscapes. This study aimed to develop a mass production method through the induction of green globular bodies (GGBs) from leaf explants of M. pteropus. Surface sterilization was performed on adventitious buds on the fronds of M. pteropus ‘Windeløv’ as explants. The results showed that the optimal explant disinfection condition was 1% sodium hypochlorite solution for 20 min, achieving a successful rate of 87.5 ± 9.6%. The most effective GGB induction was achieved with MS medium supplemented with 5.0 mg/L of NAA (1-Naphthaleneaceticacid) and 0.5 mg/L of 6-BA (6-benzylaminopurine), producing the highest number and size of GGBs, with a mean value of 4.0 ± 0.8 GGBs per explant and a diameter of 2.45 ± 0.61 mm after 8 weeks of culture. The regeneration of multiple sporophytes from the GGBs was only observed under hormone-free MS medium, with a mean value of 34.2 ± 7.3 sporophytes per GGB after 6 weeks of culture. Subsequently, rootless and separated sporophytes could be directly transplanted into the aquarium with a 100% survival rate.

Features of clonal micropropagation of plum PK SK 1 clonal rootstock

Methodological features of the main clonal micropropagation stages of plum PK SK 1 clonal rootstock (of NCFSCHVW selection) are described. The rootstock is characterized by a high adaptability to soil and climatic conditions of the region. In particular, it exhibits good winter hardiness, resistance to overwatered soils, tolerance to drought, and the capacity to grow on heavy soils. Plum seedlings on this rootstock develop a powerful root system. These advantages render the development and improvement of methods for clonal micropropagation of plum PK SK 1 clonal rootstock highly relevant. The research was carried out in 2019-2022. The period of active shoot growth, i.e., the first and second decade of may, was established to be the most optimal period for introduction of explants into in vitro culture. In this period, regeneration reaches 78.1–93.8 %. Eff ective shoot multiplication proceeds on MS medium with Fe-EDTA or MS medium with Fe-EDDHA and 6-BAP in the amount of 1 mg/L, with the highest number of shoots formed from one explant following the fourth passage (on average, 11.7 and 12.3 shoots per explant). A signifi cant eff ect of the 6-BAP concentration on the number of shoots formed was observed. The combined eff ect of the form of chelated iron and 6-BAP concentration on the number of shoots formed was established only after 3-5 passages on MS medium with Fe-EDDHA. The rooting stage of PK SK 1 rootstock can also be carried out on a hormone-free MS medium with Fe-EDDHA. The rooting effi ciency reaches 57.1–61.8 %. On average, 2.4 roots are formed per shoot with a length from 1.2 to 5.1 cm, with the root quality being high (not cartilaginous and not brittle). For the adaptation stage, rooted shoots with a height of at least 2 cm having two or more roots with a length of at least 2-3 cm are selected. A mixture of the Agrobalt plant nutrient soil, vermiculite, and perlite in a ratio of 3:1:1 can be used as a substrate. Following four weeks of adaptation, the number of adapted plants averaged 87.5 %.

The effect of plant growth regulator bacteria on micro propagation of grapevine rootstock with three different rooting abilities

In the study, 3 American rootstocks (110 R, 1103 P and 5 BB) with low, medium, and high rooting ability were used, 2 different bacterial isolates Bacillus cereus (ZE-7) and Pseudomonas putida (ZE-12) and their binary combination and the bottom of the cuttings. It was aimed to determine the effect of bacteria on rooting by treating the rooting part. During the rooting stage, hormone-free MS medium was treated with PGPRs, and in addition, hormone-free MS medium containing 1 mgl-1 IBA was used to determine the effects of PGPRs strains. At the end of the study, the root ratio of the explants was maintained; root length, number of roots, root fresh weight, root dry weight, shoot length, shoot fresh weight, and shoot dry weight data were examined. It was determined that PGPR applications generally gave higher values than control and IBA applications. Among the rootstocks, it was determined that they gave the best results in terms of root development. The highest values were obtained in the 5BB rootstock, and the lowest values were generally obtained in the 110R rootstock. Shoot development values varied according to the rootstocks and applications. In terms of the effects of the applications on root development, the highest rooting rate was 72.03%, the highest root number ratio was 1.95 on average, and the highest root fresh weight was 39.75 mg from the Bacillus Cereus application; Additionally, the highest root dry weight was obtained from B.cereus+P.putida application with 13.06 mg. The research highlighted this feature of 5 BB rootstock, which is considered among the easily rooting rootstocks, with its PGPR effect. 110 R, which is known as a difficult rooting rootstock and has high resistance to drought and active lime, which are the biggest threats today, showed a lower rooting rate, but when the values were examined, it was above the control and IBA applications.

Adaptability of Buckwheat in vitro

The research goal was to study the morphogenesis of common buckwheat plantlets obtained using different stressors in vitro. The research object was common buckwheat variety Izumrud – single-node cuttings (1.0-1.5 cm in length; two-three lowest internodes) of the plantlets that were obtained by subculturing on a hormone-free MS medium and of the lines that showed tolerance to CuSO4 Å~ 5H2O (161 and 184 mg/l) at the Laboratory of Agricultural Biotechnology. Selective media were prepared based on the Murashige and Skoog medium supplemented with a zinc salt (ZnSO4 Å~ 7H2O) at concentrations of 808, 909, 1010, 1111, 1212, and 1313 mg/l. To evaluate the morphological characteristics of the studied genotypes, their parameter values were calculated relative to the control and expressed as percentage. The genotypes were compared in the following groups depending on the exposure to a certain stressor: group 1 – the genotypes obtained without exposure to copper, group 2 – the genotypes obtained on the media containing the copper salt at a concentration of 161 mg/l, and group 3 – the genotypes obtained on the media containing the copper salt at a concentration of 184 mg/l. Plants from different groups demonstrated dissimilar growth rates under the toxic conditions caused by the studied concentrations of zinc. The morphological parameter values of the copper-tolerant plants (groups 2 and 3) were significantly higher than those of the plantlets obtained without exposure to copper. The plantlets tolerant to CuSO4 Å~ 5H2O (161 and 184 mg/l) showed a heightened resistance to the selective factor (Zn) as evidenced by their high regeneration and survival rates (95.7-100%).

Effect of Na, Mg, Ca chloride salts on mineral element, proline and total protein contents in rice (Oryza sativa L.) grown in vitro

In this study, the effects of different types and concentrations of salts on local Siverek rice plant (Oryza sativa L.) grown in vitro were investigated in terms of mineral elements (K, Ca, P, Mg, Na, Fe, Cu, Zn, Mn, Mo, Co), proline, and total protein content. Sterilized seeds were planted in hormone-free and salt-free MS medium. After one week, the seedlings were subjected to different concentrations of NaCl, CaCl2, and MgCl2 salts (0, 30 mM, 90 mM) in order to evaluate the effect of salinity on plant growth and development. In response to salt stress, a decrease in nutrient elements was observed for all three types of salt compared to the control group, which can be attributed to disruptions in ion balance. Changes in element levels generally showed varying levels of increase or decrease depending on both the type and concentration of the salt and these changes were statistically significant. The increase in proline level was found to be directly proportional to the changes in the amounts of Ca, Mg, K, and Na elements. Both total protein and proline content showed the lowest values for all salt concentrations with CaCl2, while the highest values were obtained with NaCl. In conclusion, the changes in the level of mineral elements, total protein, and proline content levels, which decrease or increase in different ratios, depending on the type and concentration rising of the salt, are associated with the varying tolerance of the plant to different types of salts.

Establishing a Long-Term Cultivated Embryogenic Culture of Papaver rupifragum Boiss. & Reut. and Its Cytological and Biochemical Study

An embryogenic culture of poppy Papaver rupifragum Boiss. Reut., which was initiated on the roots of seedlings grown from seeds on MS medium with the addition of 1 mg/L IBA, was obtained for the first time. Subsequent maintenance of the embryogenic culture was carried out on a hormone-free MS medium. Long-term cultivation and preservation of the embryogenic capacity of the culture (more than 5 years) was supported by cycles of adventitious embryoidogenesis, including the formation of callus on preexisting embryoids and the induction of new embryoids from their subsurface cells. In this regard, the resulting P. rupifragum culture can be considered as a differentiated culture in which the callus stage is an intermediate stage of development. It has been established that the surface of newly formed embryoids is covered with a surface network of extracellular matrix formed by polysaccharides, lipids, and terpenes. Histological analysis showed that embryogenic P. rupifragum culture is characterized by the formation of complexes of fused embryoids (CFE), which presumably arise either as a result of cleavage polyembryony or during the synchronous development of nearby embryoids. A study of the histology and ultrastructure of CFE revealed that the fusion of embryoids is caused by disturbances in the formation of the epidermis and cuticle. Histochemical studies have established that embryogenic P. rupifragum culture synthesizes and accumulates triacylglycerides, polysaccharides, phenolic compounds (PCs), terpenes, and alkaloids. It has been shown that the quantitative and qualitative composition of the PCs and alkaloids of the P. rupifragum culture depended on the age of the culture and its differentiation, adjustable by growing conditions (light, dark). Differentiated embryogenic P. rupifragum culture retains the ability to form embryoids on a hormone-free MS medium for a long time of cultivation and can be the basis for the further development of biotechnological methods for producing medicinal compounds for cosmetology and pharmacology.

Regenerative ability and micropropagation of <I>Petunia hybrid</I> in vitro

Scientific relevance. The garden petunia, Petunia hybrida, is a popular and wide spread ornamental crop from the family Solanaceae. It is a promising model plant for molecular and genetic research. In vitro micropropagation plays an important role in the distribution of the garden petunia because the survivability and quality of seed material decreases significantly in every subsequent generation. Besides, micropropagation reduces the cost of production substantially. Considering that very few researchers addressed this question in the Russian Federation, this direction of research is still worthy of attention.Materials and methods. The experiments were conducted by the Laboratory of Breeding and Genetic Research on Field Crops at FSBSI “Federal Scientific Center of Agricultural Biotechnology of the Far East named after A.K. Chaiki”. Seeds of Petunia hybrida (double-flowered) were used as primary explants. Liquid bleacher ACE diluted with distilled water in the proportion 1:9 was used as a sterilizing agent (the working solution contained 0.50% NaOCl). The total time of exposure was 15 minutes. The primary explants were subcultured onto a hormone-free Murashige and Skoog basal medium containing 20 g/L sucrose and 6 g/L agar. Isolated in vitro objects were cultured in test tubes with cotton-gauze plugs at an illuminance of 4000 lx, a temperature of 22–25 °C, and a 16h photoperiod in a culture room. The duration of one passage was 60 days. Micropropagation was carried out using 7- 10 mm cuttings with one or two nodes. The pot culture of the regenerants was established under controlled conditions in a light room (photoperiod was 16 hours, temperature was 23°С).Results. The optimal method for introducing Petunia hybrida into cell culture is the use of seeds treated with the solution of bleacher ACE that was diluted with distilled water in the proportion 1:9. The optimal time of exposure is 15 minutes. Petunia hybrida demonstrated a high regeneration rate on the hormone-free MS medium – it had a fast growth and development rate, and good rhizogenesis; the reproductive rate was 8.77. For the micropropagation of the garden petunia, it is advisable to use cuttings of test tube plants, which should be placed onto a hormone-free MS medium. The test tube plants of Petunia hybrida acclimatized successfully on a soil substrate. This shows the high plasticity of the culture.

Comparative determination of antimicrobial activity of the Balkan endemic species Stachys thracica Davidov during the process of ex situ conservation

Stachys thracica Davidov – Thracian woundwort is a Balkan endemic plant included in The Red Data Book of Bulgaria with conservational status “rare”. The plants from genus Stachys have a long history of use to treat various diseases, inflammatory conditions, coughs, ulcers, genital tumors, and infected wounds. Due to its limited distribution the information on the biological activity and chemical composition of S. thracica is rather scarce. The aim of the present research is the comparative determination of the antimicrobial activity of methanolic extracts obtained from in situ wild, in vitro cultivated and ex vitro adapted S. thracica plants. The in vitro shoot culture of the Thracian woundwort was maintained in hormone-free MS medium under controlled environmental conditions. The methanolic extracts from in situ, in vitro cultivated and ex vitro adapted S. thracica plants were active mainly against Gram-negative bacteria. All three extracts showed equal activity against Acinetobacter calcoaceticus. The establishment of in vitro shoot culture and its subsequent adaptation in ex vitro conditions was an appropriate alternative approach for the ex situ conservation of S. thracica as well as for the study of its biological activity.

Evaluation of Hippeastrum papilio (Ravenna) Van Scheepen potencial as a new industrial source of galanthamine

Galanthamine is a natural compound used for the treatment of Alhzeimer’s disease. Currently, it is extracted at industrial scale from Narcissus pseudonarcissus cv. Carlton, Lycoris radiata and Leucojum aestivum. Hippeastrum papilio is an ornamental plant which shows advantages in comparison to the currently used plants for galanthamine extraction. It is a plant without a period of dormancy with bulbs up to ca. 8 cm in diameter and leaves up to ca. 80 cm long. In the present study, H. papilio was successfully propagated in vitro on hormone-free MS medium supplemented with charcoal. Galanthamine content in the leaves of plants, cultivated for one growing season after ex vitro acclimation, was 0.54 ± 0.21% of the dry weight. The alkaloid fractions from leaves and bulb of a selected genotype were found to comprise 97.91% and 46.07% of galanthamine, respectively. At greenhouse conditions, the leaves, bulbs and roots comprise 39%, 38% and 23%, respectively, indicating that leaves and bulbs may equivalently contribute to the biomass of the plant. An effective propagation of selected genotypes together with adequate cultivation and biomass extraction techniques may introduce H. papilio as a new and competitive source for industrial production of galanthamine.

Development of an Efficient In vitro Regeneration Protocol for Chrysanthemum (Chrysanthemum morifolium Ramat)

An efficient and rapid in vitro regeneration protocol was developed for chrysanthemum (Chrysanthemum morifolium Ramat) using two local varieties of Bangladesh namely, BARI Chrysanthemum-2 (BARI Chry-2) and local yellow (Y). MS medium supplemented with nine different concentrations and combinations of BAP and IAA was employed to optimize regeneration protocol using young in vitro derived leaf explants. Direct organogenesis was observed from the leaf explants on MS medium supplemented with 0.5 mg/l BAP and 2.0 mg/l IAA (T6) for both the varieties. This treatment (T6) induced shoot buds directly on the adaxial surface of the leaf providing the highest regeneration percentage (90% for BARI Chry-2 and 94.73% for Y), the highest number of shoot/explant (7.6 for BARI Chry-2 and 8.6 for Y) and maximum length of the shoot after six weeks (3 cm for BARI Chry-2 and 2.9 cm for Y) of culture. Explants with initially regenerated shoots were subculture on hormone free MS medium for shoot elongation after 4 weeks of their inoculation. During elongation of shoots, 90-95% of the regenerated shoots produced roots spontaneously in hormone free MS medium within 7-8 weeks of their inoculation. Rooted plantlets were transplanted to the field following hardening where 100% plantlets were survived and produced flower without any variation. Plant Tissue Cult. & Biotech. 31(2): 161-171, 2021 (December)

Effect of Plant Hormones for Micro Propagation on Improved and Landrace Yam Varieties (<i>Dioscorea species</i>) Via Nodal Culture

Yam (<i>Dioscorea species</i>) is a monocotyledonous tuber forming tropical vine which belongs to the order <i>Liliflorae</i>, family <i>Dioscoreaceae</i>, and genus <i>Dioscorea.</i> Conventional propagation of yam (Dioscorea spp.) is limited due to low propagation rates and highly infected by pests. However, <i>In vitro</i> clonal propagation is the best alternative to mass multiplication over conventional propagation. Moreover, shoot multiplication and rooting induction experiments were incubated on hormone free MS medium within 30 g/l sugar and 7 g/l agar. For shoot multiplication the initiated shoots of both landraces were cultured on MS media supplemented with 0, 1, 2, 3, 4 and 5 mg/l benzyl amino purine (BAP). For root induction, shoot lets were cultured on MS media supplemented with 0, 1, 2, 3, 4, 5 and 6 mg/l indol-3-butyric acid (IBA). Finally, for acclimatization, <i>in vitro</i> multiplied plantlets were transferred to greenhouse for hardening off. The results showed that the interaction effects of both landraces with BAP and IBA concentrations was significantly influenced <i>in vitro</i> yam shoot multiplication and rooting induction respectively. The maximum shoot number (7.23±0.21) with a maximum shoot length 7.68±0.24cm and 6.79±0.09 shoot number with 7.47±0.47 cm were obtained on MS media supplemented with 3 mg/l BAP for 11/02 and Koffea cultivars respectively. The maximum root number 10.03±0.49 and root length 10.76±0.16 cm and 10.58±0.26 root number with 10.42±0.32 cm were recorded on MS media with 3 mg/l IBA for Koffea and 11/02 cultivar respectively. <i>In vitro</i> raised plantlets were acclimatized and recorded 86% and 90% of survival rate in Koffea and 11/02, respectively, on soil medium with combination ratio of 2:1:1 top soil, sand soil and compost, respectively.

Dracocephalum charkeviczii Prob. in vitro

We established sterile clones of a medicinal plant dragonhead Dracocephalum charkeviczii Prob. (Lamiaceae). We used shoots of the plants from natural habitat, notably leaf nodes with axillary bud, as explants, sterilized the explants in 0.1 % Diocide for 4 minutes and cultivated them on agarized nutrient half-strength MS medium with vitamins, 2 % sucrose, 0.4 % agar, 1 mg/l indolylbutyric acid (IBA), or 0.5 or 1.0 mg/l 6-benzyladenine (BA). Medium pH was 4.6 or 5.4. We took hormonefree half-salt MS medium as a control. We observed the maximum propagation coefficient equal to 9.4 in microplants cultivated on the medium with 1 mg/l BA. Application of hormone-free MS medium can be recommended for D. charkeviczii propagation due to its simplicity. The use of the medium protocol with 1 mg/l BA on the first step and with IBA on the second step will allow rapid multiplication and establishment of full-fledged plants of D. charkeviczii for different purposes such as restoration of natural populations, source of valuable metabolites, landscaping, etc.

Abiyotik stres koşullarında yetiştirilen Gypsophila pilulifera (Caryophyllaceae) tohumlarının in vitro çimlenme ve vejetatif büyüme özellikleri

Gypsophila pilulifera Boiss. &amp; Heldr. is an economically important critically endangered (CR) endemic species of Turkey. This species is a Mediterranean element and type sample is located in square C3 (Antalya). Plants are much more sensitive to abiotic stress factors, especially during germination and seedling development stages. In this study, in vitro germination and seedling growth properties of G. pilulifera Boiss. &amp; Heldr. (Caryophyllaceae) were investigated under salt and drought stress. The seeds were cultured in hormone-free MS media containing increasing doses of NaCl (50, 100, 150, 200 mM) and PEG 6000 (5%, 10%, 15% and 20%). Germinated seeds were counted every three days for the first week and every day for the second week. The root and hypocotyl length, number of leaves, fresh weight and vigor index of the plants were determined at the end of 21 days. A negative correlation was determined between increased salt and drought stress and all vegetative growth parameters. Although germination was obtained on MS medium with the highest level of drought (20%), there was no germination on MS medium containing 200 mM NaCl. As a result, it was determined that the plant was more sensitive to salinity stress than drought and high salt concentrations inhibited the germination by 100%.

In Vitro Multiplication and NMR Fingerprinting of Rare Veronica caucasica M. Bieb.

Micropropagation of rare Veronica caucasica M. Bieb. was achieved by successful in vitro cultivation of mono-nodal segments on MS medium supplemented with 1.0 mg L–1 6-benzylaminopurine (BA) and then transferring the regenerated plants on hormone free basal MS medium for root development. In vitro multiplicated plants were successively acclimated in a growth chamber and a greenhouse with 92% survival. The number of plastid pigments and the total phenolics content in in vitro cultivated and ex vitro adapted plants were unchanged, and no accumulation of reactive oxygen species (ROS) was detected by staining with 3-3′-diaminobenzidine (DAB) and 2′,7′-dichlorofluorescein diacetate (DCF-DA). Nuclear Magnetic Resonance (NMR) fingerprinting allowed for the identification of the major alterations in metabolome of V. caucasica plants during the process of ex situ conservation. Iridoid glucosides such as verproside, aucubin and catalpol were characteristic for in vitro cultivated plants, while in ex vitro acclimated plants phenolic acid–protocatechuic acid and caffeic acid appeared dominant. The successful initiation of in vitro and ex vitro cultures is an alternative biotechnological approach for the preservation of V. caucasica and would allow for further studies of the biosynthetic potential of the species and the selection of lines with a high content of pharmaceutically valuable molecules and nutraceuticals.

Збереження представників роду Drosera L. з використанням біотехнологічних методів

The peculiarities of obtaining planting material of rare representatives Drosera spatulate L. and Drosera aliciae L. using microclonal propagation in order to preserve and cultivate them in ex vitro conditions were studied. The method of sterilization of D. spatulate and D. aliciae explants with 80-90% obtaining aseptic material has been developed. The influence of different sterilization options on the development of microshoots has been studied. The best mode of sterilization is 0,1% solution of AgNO3 and 12.5% solution of H2O2. The features of organogenesis and regeneration of the whole organism from cultivated tissues and organs of Drosera L. was investigated. The effect of exogenous growth regulators at different stages of plant morphogenesis in vitro is shown. Improved conditions of rhizogenesis in vitro. It was found experimentally that MS nutrient media with the addition of 2 g∙l-1 PVP is optimal at the stage of introduction into culture in vitro D. spatulate and D. aliciae. The regeneration of microshoots of D. spatulate and D. aliciae depending on the type of explant and the composition of nutrient media was studied. Morphogenesis was most effective on nutrient media with the addition of 0.25 mg∙l-1 kinetin and on the hormone free MS media. Such cultivation conditions provided 100% regeneration of plants with a reproduction rate of 1:8. Studying the effect of cytokinins on the microclonal reproduction of D. spatulate and D. aliciae, it was found that the development and induction of multiple shoot formation in vitro is best performed on hormone free MS media. To induce the formation of the root system, it is necessary to add into MS nutrient media 0.5 mg∙l-1 IBA. According to the results of the research, a method of microclonal propagation was developed by cutting stem culture, which made it possible to obtain genetically stable, disease-free regenerating plants of D. spatulate and D. aliciae with an optimally formed root system and vegetative mass. The obtained homogeneous planting material can be used in floriculture, creation of terrariums, for pharmacological purposes and for the purpose of introduction. Keywords: Drosera L., microclonal reproduction, morphogenes.

ANALISIS SESAR-SESAR MAJOR SEMENANJUNG MALAYSIA DENGAN TEKNIK GRAVITI DEKONVOLUSI EULER 3D

The application of biotechnology in upland rice improvement programs depends on the availability of efficient regeneration protocols. Although protocols for shoot regeneration of upland rice are available, none has been reported for pigmented cultivars. This study reports on a protocol for callus induction and regeneration of Tadong, a pigmented upland rice cultivar from Sabah. For callus induction, immature embryos were cultured on media containing 2,4-Dichlorophenoxyacetic (2,4-D) at various concentrations (0 – 2.5 mg/L) and on different types of media (MS; MSB5; N6B5; N6). To induce shoot regeneration, callus explants were cultured on MS medium supplemented with combinations of 6-Benzylaminopurine (BAP) at various concentrations (0 – 3.0 mg/L) and 1-Naphthaleneacetic acid (NAA) at 1.0 mg/L. To induce shoot development, callus explants were pre-treated with Thidiazuron (TDZ) at various concentrations (0-1.0 mg/l) and exposed to different desiccation periods (0 – 72 hours). 2,4-Dichlorophenoxyacetic at 2.5 mg/L and N6B5 medium resulted in the highest percentages of explant forming callus which were 60.3 ± 17.0 % and 58.7 ± 9.8 % respectively. The regeneration media failed to induce shoot on callus explants, instead, green spots were formed on the surface of the callus. The green spots were stimulated to develop into shoots when the callus explants were pre-treated with 0.5 mg/L TDZ or exposed to partial desiccation for 24 h, the percentages of explant forming shoot were 35.7 ± 4.8 % and 47.7 ± 6.8 % respectively. Shoots developed into complete plants on hormone-free MS medium and acclimatized.

CALLUS FORMATION AND PLANT REGENERATION FROM IMMATURE EMBRYOS OF PIGMENTED UPLAND RICE (Oryza sativa cv. Tadong)

The application of biotechnology in upland rice improvement programs depends on the availability of efficient regeneration protocols. Although protocols for shoot regeneration of upland rice are available, none has been reported for pigmented cultivars. This study reports on a protocol for callus induction and regeneration of Tadong, a pigmented upland rice cultivar from Sabah. For callus induction, immature embryos were cultured on media containing 2,4-Dichlorophenoxyacetic (2,4-D) at various concentrations (0 – 2.5 mg/L) and on different types of media (MS; MSB5; N6B5; N6). To induce shoot regeneration, callus explants were cultured on MS medium supplemented with combinations of 6-Benzylaminopurine (BAP) at various concentrations (0 – 3.0 mg/L) and 1-Naphthaleneacetic acid (NAA) at 1.0 mg/L. To induce shoot development, callus explants were pre-treated with Thidiazuron (TDZ) at various concentrations (0-1.0 mg/l) and exposed to different desiccation periods (0 – 72 hours). 2,4-Dichlorophenoxyacetic at 2.5 mg/L and N6B5 medium resulted in the highest percentages of explant forming callus which were 60.3 ± 17.0 % and 58.7 ± 9.8 % respectively. The regeneration media failed to induce shoot on callus explants, instead, green spots were formed on the surface of the callus. The green spots were stimulated to develop into shoots when the callus explants were pre-treated with 0.5 mg/L TDZ or exposed to partial desiccation for 24 h, the percentages of explant forming shoot were 35.7 ± 4.8 % and 47.7 ± 6.8 % respectively. Shoots developed into complete plants on hormone-free MS medium and acclimatized.

IN VITRO RESPONSE BY Terminalia arjuna GENOTYPES DURING MICROPROPAGATION

Terminalia arjuna is an important tree of the medicinal and sericulture industry, commonly known as Arjun. It’s bark is rich in secondary metabolites makes this plant highly valuable in medicine industry to treat cardiovascular disease. Overexploitation due to high demand in medicine, low seed germination, limitations of the conventional method of propagation push this plant towards being endangered. To conserve germplasm of such tree species and meet the requirement in medicinal industry, some non-conventional propagation method like micropropagation has been developed. The present work highlighted the effect of three genotypes (G-1, G-2, and G-3) on tissue culture of T. arjuna situated at Jodhpur, Rajasthan, India. In vitro shoot proliferation was achieved on a modified MS medium enriched with BAP + additives. Among the tested genotypes, Genotype -1 showed maximum bud break response (100%) followed by G-3 (93.33 %) and G-2 (86.66%). Further multiplication of these shoots on modified MS medium containing BAP + NAA + additives gave 11.38±0.26 (G-1), 9.44±0.21 (G-2) and 10.22±0.32 (G-3) shoots. In vitro rooting was done by pulse treatment with IBA for 10 min prior to transfer on hormone free half strength MS medium containing 0.1% activated charcoal. Maximum in vitro rooting was obtained in G-1 (80%) followed by G-3 (71.11%) and G-2 (68.88%). In the present study, it was observed that optimum growth in all three genotypes required different doses of Plant Growth Regulator. Thus, by identifying and multiplying the best performing genotypes the gap between demand and supply of such medicinal plant can be fulfilled.

Rooting technique of double haploids obtained in culture of microspore &lt;i&gt;in vitro&lt;/i&gt; for European radish

Relevance. Doubled haploids (DH-plants) are excellent material for genetic research and breeding due to their complete homozygosity. The genus Raphanus from the Brassicaceae family is the toughest to produce doubled haploid plants through isolated microspore culture in vitro (IMC). The study of the causes of disturbed root formation and the development of elements of this stage of technology will significantly increase the effectiveness of the IMC technology for European radish.Methods. The study included three varieties from the collection of the Federal State Budgetary Scientific Institution Federal Scientific Vegetable Center (FSBSI FSVC): Teplichny Gribovsky, Rozovo-krasniy s belim konchikom and Rhodes. The experiments used a standard protocol for obtaining DH plants using IMC technology in a standard form and with a modification of the rooting stage. The solid MS medium (with agar 7g/L): MS without hormones, MS medium supplemented with IAA at concentrations of 0.5; 1 and 2 mg / L and liquid MSm medium supplemented with 0.1 mg / L kinetin were used for rooting of regenerated plants. All media were supplemented with 20 g/L sucrose. We used three types of techniques for transplanting plant explants onto a solid hormonefree MS medium: planting micro-shoots with their basal part immersed by 2-3 mm into the medium; planting in a well made in a nutrient medium using tweezers under sterile conditions; and landing on the surface of the medium without embedment.Results. In this work, we studied the features of the stage of rooting of regenerated European radish plants in vitro conditions. The transplant technique has been proven to be important for the successful establishment of radish micro-shoots. Plant explants must be planted strictly on the surface of a solid hormone-free nutrient medium MS, without embedment. The use of tubes with bridges made of filter paper and MSm liquid medium with the addition of 0.1 mg/L kinetin for the induction of root formation also showed high efficiency. For plants prone to the formation of root-like structures (RLS) with secondary tumors (ST), multiple dissection of abnormal formations with successive transplants is necessary. Modification at the rooting stage of micro-shoots growing has increased the percentage of successfully adapted DH plants in vivo conditions from 0-14% to 95-98%.