Frequency Of Callus Formation Research Articles

OBTAINING ASEPTIC CULTURE IN VITRO OF RHODIOLA SEMENOWII L. FROM VARIOUS TYPES OF EXPLANTS

In vitro cultivation of callus cells allows for the stable production of secondary metabolites, being an alternative to the use of medicinal plant biomass from natural populations. Standardization of cultivation protocols and technological processes for the synthesis of secondary metabolites in vitro conditions determines significant interest in the use of tissue culture methods (PTC – Plant Tissue Culture). The aim of the study was to obtain an aseptic culture of Rhodiola semenowii in vitro from various types of explants, as well as the effect of the hormonal composition of the nutrient medium on the induction of callusogenesis in the culture of isolated organs of R. semenowii. As a result of the research, the optimal mode of obtaining R. semenovii aseptic cultures from various explants was determined. It has been shown that the use of 70% EtOH in combination with 0.01% HgCl2 or 20% NaClO makes it possible to obtain aseptic cultures from any type of R. semenovii explants. It has been established that the type of explant is R. semenovii influenced both the frequency of callus formation and the beginning of its formation. The least optimal types of R. semenovii explants are stem and rhizome segments, the frequency of callusogenesis varied from 5.8% to 75.6%. The induction of callusogenesis also depended on the hormonal composition of the nutrient medium: the highest percentage of callus formation in all types of explants was on media containing 0.5mg L−1 thidiazuron (TDZ) + 1.0 mg L−1 1-naphthaleneacetic acid (NAA) and 0.5mg L−12,4D + 2.0 mg L−1 6-Benzylaminopurine (BAP).

Plant regeneration from embryogenic callus-derived from immature leaves of Momordica charantia L

Bitter melon (Momordica charantia L.), a widely cultivated food and medicinal plant native to the world's subtropics and tropics, is a Cucurbitaceae rich in carotenoids. However, the low seed germination frequency and progeny variability associated with the production of this plant have a substantial impact on its growth and yield. These constraints affect the availability and exploitation of this crop, especially the fruits, which are rich in secondary metabolites such as β-carotene and α-carotene. In vitro regeneration would help overcome the obstacle linked to the germination of this plant and increase its yield and utilization. A reproducible in vitro organogenesis protocol was established using bitter melon embryogenic callus derived from immature leaf explants of in vivo grown seedlings and in vitro plantlets. Regeneration via callus was conducted on MSB5 media augmented with different plant growth regulator concentrations. The maximum frequency of callus formation (95.09 %) was produced in MSB5 media incorporated with 1.2 mg L−1 NAA augmented with 0.5 mg L−1 TDZ. MSB5 medium with no growth regulators was observed to be the most suitable for the shoot and root formation from the callus, producing a significantly high shoot percentage of 90.91 % and 21.53 shoots per explants, and the highest rooting frequency and root number of 88.92 % and 6.23 roots per explant, respectively, from leaf-derived callus of in vitro plantlets. The elongated plantlets had grown to a significantly higher average height of 12.20 cm on media added with 0.75 mg L−1 GA3. This reproducible method for regenerating bitter melon plantlets could facilitate mass multiplication, conservation, and commercial field production.

Assessment of the genetic fidelity of true-to-type regenerants of medicinal plant Rheum emodi using RAPD and ISSR molecular markers

Rheum emodi commonly known as rhubarb is mainly found in Northern Himalayas. It is a valuable medicinal plant having major pharmacological activities such as antimicrobial, anticancer, antioxidant, anti-inflammatory and is used extensively as purgative, stomachic and astringent tonic and improves gastro related problems. This herb is used by the local communities for medicinal as well as common eating purpose. This leads to its immense decline in its natural habitat and now this herb falls under threatened species and demands conservation. In this prospective, an efficient in vitro propagation method from callus culture has been achieved using leaf explants excised from the juvenile plant of R. emodi. Murashige and Skoog (MS) basal medium was used for regeneration procedure with different concentration of phytohormones. Maximum frequency of callus formation (84.44±0.27%) was observed in MS+36.19μM (2, 4-D) in combination with 11.10μM (BAP). The highest percentage of adventitious shoot regeneration was observed as 75.56±0.27% and the maximum number of shoots per explant that is 3.67±0.27 was achieved on MS basal medium containing BAP (35.5 μM) and Kn (11.61 μM). The maximum frequency of rooting was observed in MS full strength media + IAA (28.55 μM) + BAP (8.88 μM). The highest frequency of roots per shoot was observed as 5.0±0.47 with an average root length of 11±1.25mm. For ascertaining the clonal fidelity, 20 ISSR markers and 15 RAPD markers were assayed and employed to validate the true-to-type regenerants of Rheum emodi. Out of 15 RAPD and 20 ISSR markers, 7 markers and 15 markers produced distinct, clear and scorable bands with an average of 4.5 bands and 4.4 bands per marker respectively among the tissue cultured progenies. For each primer, the banding pattern was uniform and comparable to mother plant and showed about 99% homology. All the markers produce the monomorphic bands and no variation was detected among the micropropagated plants. Thus, the analysis of ISSR and RAPD patterns revealed that the bands were shared by both in vitro raised plants and parent clump confirming the genetic stability. DNA based molecular markers have proved to be versatile tools in diverse fields of biology. These markers proved to be model tools for routine analysis of clonal fidelity of micropropagated plants prior to commercialization.

In vitro propagation of Asplenium aethiopicum through tissue culture

Poor viability of spores and germination and slow growth in conventional vegetative propagation are major constraints in the in vitro culture of Asplenium aethiopicum, an important medicinal fern. In the present study, a procedure for in vitro propagation of this endangered fern through croziers has been established. Maximum sporophytes efficiency was obtained on KC media supplemented with growth regulators such as 0.5mg/l NAA & 0.5mg/l 2,4-D with 2 sucrose under photoperiod condition. Maximum frequency (33.2%) of callus formation and germination of sporophyte was observed on KC medium with growth regulators.

Metal Akümülatör Bitki olan Brassica nigra L.’nin in vitro Kallus İndüksiyonu ve Sürgün Gelişimi

This study established an efficient in vitro callus formation and plant regeneration protocol for a prevıously reported Cu accumulator, Brassica nigra, black mustard collected from Diyarbakir (Station site). Node explants from 10-day old mature plants were used for callus formation and shoot regeneration. The highest callus formation frequency (100%) was observed on Murasige Skoog (MS) medium containing 0.1 mg/L Benzylaminopurine (BAP) + 0.5 mg/L Naphthylacetic acid (NAA) (MS 2), 0.6 mg/L BAP + 0.2 mg/L NAA (MS 7), the highest shoot regeneration frequency (100%) was achieved on MS medium containing 0.6 mg/L BAP + 0.05 mg/L Indole butyric acid (IBA) (MS 8), 0.2 mg/L IBA + 0.2 mg/L NAA (MS 10) and the highest number of shoots per explant (3,25) was obtained on MS medium supplemented 0.6 mg/L BAP + 0.05 mg/L IBA (MS 8). After root, stem, and leaf formation from explants in MS medium, these plants were transferred to soil and grown in the plant growth room for one month. A dependable and effective shoot regeneration procedure was developed, laying the groundwork for genetic transformation in Brassica nigra.

In vitro selection of birch for tolerance to salinity stress

In vitro modelling of stress is one of the promising avenues for plant breeding for tolerance to negative environmental factors. In this study we examined the effect of NaCl (0.5%) on callusogenesis and morphogenesis of stem explants of different birch genotypes: Betula pendula Roth, B. pendula Roth var. carelica (Mercklin) Hämet-Ahti, B. pendula f. ‘dalecarlica’ (L.f.) Schneid., B. pubescens Ehrh. In our experiments we used pre-selected microclones from our in vitro collection on NaCl (0.2-1.0%) selective media. The clones were contrasted by the degree of their sensitivity to salinity (so-called ‘stable’ and ‘sensitive’ microclones). With the use of stem callus cultures we identified informative, simple and reproducible indicators for the selection of salt-tolerant genotypes. Among these indicators were the frequency of callus formation and the viability of callus cultures, which were significantly higher in ‘stable’ group of microclones. Polyploid birch clones (2n=4x=56, 2n=3x=42) were more resistant to salination compared to diploid clones (2n=28). Our study has shown that the selection of salt-tolerant birch lines can be based on the plants’ genetic diversity presented in the collection (various species, varieties, hybrids, polyploids) and manifested in the process of in vitro cultivation, as well as in the cellular heterogeneity of callus cultures.

Preliminary study on in vitro shoot culture of Hibiscus coddii subsp. barnardii, an indigenous South African flowering plant

Abstract In vivo and in vitro grown plants of Hibiscus coddii subsp. barnardii were used as explant source for establishment of in vitro cultures. Nodal shoot explants derived from in vivo grown plants, both naturally and under controlled environmental conditions, showed high sensitivity to the surface disinfection treatment and poor survival in in vitro culture. In vitro grown seedlings proved successful as aseptic source of apical and basal shoot explants to establish contamination-free in vitro cultures. Sprouting of axillary buds was observed on 90% of apical shoot explants after four weeks of culture on full strength, plant growth regulator (PGR)-free Murashige and Skoog (MS) medium. However, further proliferation of short shoots, limited to the bud sprout at the explant base, occurred on only 50% of these explants. In contrast, all basal shoot explants attained 3-5 single primary axillary shoots (30-40 mm in length) while a clump of short (5-10 mm) shoots also formed at the base in 60% of these explants. In both explant types, addition of 0.25-1 mg L-1 6-Benzylaminopurine (BAP) to the MS medium resulted in a low frequency (10%-60%) of explants with short shoots (5-10 mm) that showed no further elongation. Moreover, explants cultured in the presence of BAP showed a high frequency of callus formation (up to 90%) and low survival (20%-60%). A lower frequency of callus formation (30%-40%) and higher survival (90%-100%) of both explant types occurred on BAP-free medium. Further subculturing of primary and secondary axillary shoots onto fresh MS medium (with and without BAP) did not improve shoot multiplication. Regenerated plantlets from PGR-free MS medium were successfully acclimatized and hardened-off.

Callus formation, phenolics content and related antioxidant activities in tissue culture of a medicinal plant colocynth (Citrullus colocynthis)

Callus cultures from stems, leaves and roots of colocynth were initiated on MS media supplemented with various combinations of 2,4 dichlorophenoxyacetic acid (2,4-D) with kinetin (KIN) and benzyladenine (BA) with α-naphthaleneacetic acid (NAA). The highest percentage of callus formation frequency (98.9%) was obtained from stem explants grown on MS media supplemented with (1.0 mg/L) 2,4-D + (1.0 mg/L) KIN. The total phenolics and flavonoid content of the colocynth callus cultures were measured. The results showed that the MS medium supplemented with 6.0 mg/L 2,4-D + 2.0 mg/L KIN (MD3) gave the highest content of total phenolics (19.2 mg/100g d.w.) in leaf-derived calli. The highest content of flavonoids (47.3 mg/100g d.w.) was obtained in stem derived calli grown on the same medium (MD3). Antioxidant activities of extracts were determined using different assays, including DPPH radical scavenging activity, hydrogen peroxide (H2O2) scavenging activity and ferric reducing power. Leaf-derived calli cultured on MS medium + 2.0 mg/L 2,4-D + 1.0 mg/L KIN (MD1) showed the highest DPPH radical scavenging activity (85.3%). The highest percentage of H2O2 scavenging activity (61.4%) was detected in leaf explant-derived calli growing on MD1. The leaf-derived calli growing on (MD3) gave the highest ferric reducing power (22.3 μg/g d.w.), compared to the activities of stems, leaves and roots of in vitro grown seedlings (3.28, 12.9 and 2.85 μg/g d.w.), which were used as controls. On the basis of the current findings, we conclude that MS media supplemented with different combinations of 2,4-D and KIN yields higher phenolics, flavonoids contents and antioxidant activities than MS media supplemented with BA and NAA.

Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly. The total number of somatic embryos development was highest with the 3% (w/v) sucrose treatment, which was found to be the optimal concentration for somatic embryo formation. Activated charcoal (AC) and 6-benzyladenine (BA) significantly increased the frequency of plantlet conversion from somatic embryos, but gibberellic acid (GA3) had a negative effect on plantlet conversion and subsequent development from somatic embryos. Even though the cell suspension cultures were maintained for more than 1 year, cell aggregates from embryogenic cell suspension cultures were successfully converted into normal somatic embryos with two cotyledons. To our knowledge, this is the first successful report of a plant regeneration system of E. alatus via somatic embryogenesis. Thus, the embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and production of pharmaceutical metabolite in E. alatus.

In vitro plant regeneration of Christmas cactus (Schlumbergera truncata (Haw.) Moran) by indirect morphogenesis

Abstract Plants of Schlumbergera truncata (Haw.) Moran were obtained by indirect morphogenesis from the segment section of shoots in vitro, they were multiplied and rooted. Also were determined the effect of the lighting regime, the composition of the nutrient medium on the consistency and frequency of callus formation. The studies were conducted during 2016–2018. The mode of effective sterilization (more than 90%) of S. truncata plant explants using 0.1% HgCl2 for 7–8 min was established. Optimal conditions for the induction of callus formation in stem node segments of S. truncata plants (rate more than 90% and significant growth) were created on MS (Murashige and Skoog 1962) nutrient medium supplemented with 1.0 mg/l BAP (6-benzylaminopurine) and 0.3 mg/l NAA (1-naphthylacetic acid) under conditions of placement on the nutrient medium and doing a significant number of cuts on the explants. The light intensity of 2.0–3.0 klx, obtained by a callus of dense consistency of dark green pigmentation, when using the thermostat condition without illumination, the callus had loose consistency, dark yellow pigmentation. It is established that the influence of the lighting regime and the composition of the nutrient medium on the frequency of callus formation is statistically significant. The largest number of shoots was obtained on the MS medium with the addition of 2.0 mg/l of BA. At the same times, shoot proliferation and root induction in such numbers were observed on MS culture medium with the addition of 0.5 mg/l BA and 0.5 mg/l kinetin (multiplication factor – 8.8±0.6 per 60-day cultivation cycle).

Effect of explant and phytohormone on in vitro regeneration of Solanum indicum, an important medicinal weed

The aim of this study was to observe the effect of different explants viz. apical shoot, nodal stem and leaf of Solanum indicum and the concentration of phytohormones on tissue culture responses during the in vitro regeneration and development of micropropagation protocol. For primary establishment of explants, 0.1% HgCl2 solution for 5 minutes was found to be most effective for surface sterilization and Murashige and Skoog (MS) medium having 3 mg/l kinetin and 3 mg/l 6-benzylaminopurine was found to be the best for all the explants. Among all three explants, leaf explants showed the best primary establishment. The explants showed callus formation after 10 to 12 days of culture. The frequency of callus formation was higher on MS medium supplemented with 3 mg/l 3-indoleacetic acid and 5 mg/l 6-benzylaminopurine by the leaf explant. The highest frequency of shooting was recorded on MS medium with 3 mg/l kinetin and 2mg/l 6-benzylaminopurine. Apical shoot showed higher potential of shooting than nodal stem and leaf explants. The in vitro regenerated elongated shoots were then excised from shoot clumps and transferred to rooting medium containing Indole butyric acid at 1.5 mg/l. The in vitro regenerated plantlets were shifted to the polycups having mixture of sterilized soil and farm yard manure in 1:1 ratio. Initially, these plants were kept under high humidity and progressively acclimatized to reduced humidity for their hardening and acclimatization. Plantlets, thus developed, were successfully established and finally transferred to a greenhouse.

Создание сорта шалфея мускатного с использованием методов клеточной инженерии. 1. Получение растений-регенерантов в каллусной культуре in vitro

To increase the efficiency in agricultural plant breeding, including clary sage – one of the main essential oil crops grown in Russia, it is necessary to use biotechnological methods. One of these techniques is based on the induction of somaclonal variability in the callus tissue culture. To develop it, it is necessary to optimize the conditions for obtaining plant-regenerants in vitro and their analysis. The aim of this work was to study the features of morphogenesis and regeneration of plants from callus cultures to develop cell technologies for creating an initial breeding material based on somaclonal variability in Salvia sclarea L. In the course of the research, we found that the optimal explants for obtaining morphogenic callus, from which shoots were regenerated, were segments of buds and stems with a node (isolated from seedlings in vitro). Cytological analysis of callus cultures revealed two types of morphogenesis – organogenesis (gemmogenesis) and somatic embryogenesis. The features of the morphogenic callus formation of six sage cultivars and samples during the long-term cultivation were studied. The maximum frequency of morphogenesis was noted in the 2nd passage (from 32.4 to 85.2 %, depending on the genotype). Then, to the 8–10th passage, this indicator decreased to 0.0–3.9 %.‘S-785’ and ‘Taigan’ cultivars showed the highest morphogenesis frequency (81.5–85.2 %) and duration of callus regeneration potential (up to the 10th passage). The analysis of callus cultures of six donor plants of ‘S-785’ cultivar helped us to reveal their heterogeneity in morphogenesis induction ability. The maximum frequency of morphogenic callus formation (76.3–91.5 % in the 2nd passage) and the duration of the morphogenic potential preservation (up to the 12th passage) were observed in plants No. 3 and 9, whereas in No. 2, regeneration with a frequency of 3.6–9.7 % was observed only during three passages. Analysis of plants obtained from calli showed their variability in morphology – up to 12.5 % of the samples had deviations compared to the initial cultivar ‘S-785’ in leaf shape, inflorescence structure, flower color, etc. Somaclonal changes in morphological and economically useful traits revealed in regenerants indicate that they are promising for use in sage breeding.

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.

ИЗМЕНЧИВОСТЬ И НАСЛЕДУЕМОСТЬ РЯДА ПРИЗНАКОВ У ПРОСТЫХ ГИБРИДОВ F1 КУКУРУЗЫ ПРИ ПОНИЖЕННОЙ ТЕМПЕРАТУРЕ И В ОБЫЧНЫХ УСЛОВИЯХ

The values of heritability coefficients of parental genotypes were determined for such characters as "frequency of callus formation", "frequency of somatic embryogenesis","frequency of plant regeneration" under normal and stressful in vitro conditions in simple maize F1 hybrids. The highest value of the coefficient of heritability of maternal genotypes in traits studied in vitro was found for “frequency of somatic embryogenesis” at low temperatures. The variability and heritability of characters during the initial stages of plant development under the actionof low temperatures, as well as quantitative characteristics of the plant and male gametophyte, were evaluated. Hybrids in which the inbred lines A239 and 092 were used as maternal genotypes showed under stress higher indices of the studied traits both invitro conditions and at the seed level. For the characters “number of grains per row” and “plant height”, the highest heritability coefficients of the interaction of maternal and paternal genotypes were noted. Hybrids A239xP502 and Co125xMK159 were selected by the higher values of the studied quantitative traits. Hybrid combinations with significantly high meanings of the most of the studied characters can be used to create cold-resistant and productive corn hybrids

MICROPROPAGATION OF AN ENDANGERED AND ENDEMIC MEDICINAL PLANT CAYRATIA PEDATA VAR. GLABRA

Objective: The objective of the present study was to develop standardization protocol for the successful in vitro mass propagation of Cayratia pedata var. glabra through leaf and stem explants, since it is a rare, endangered, and endemic medicinal plant using biotechnological involvements and to conserve this endangered species.
 Methods: The application of biotechnological principles for the establishment of micropropagation under in vitro conditions has been studied by following the methods. The explants, namely, leaf and stem harvested from in vivo plants were thoroughly washed and properly sterilized with sterilients. The explants were transferred to Murashige and Skoog (MS) medium supplemented with growth regulators 6-benzyladenine (BAP) and naphthalene acetic acid (NAA) in the concentration range of 0.5–3.0 mg/l which were tested for callus induction and morphogenesis. The elongated shoots were transferred to MS medium supplemented with NAA at different concentrations for root induction.
 Results: The explants collected from the field (shola) were treated in different steriliants with various concentrations at different time for sterilization. Among the various combinations tried, the Teepol treatment for 10 min followed by bavistin 20 min, antibiotics, namely, ampicillin and rifampicin for 20 min, 70% alcohol for 30 s, and 0.12 % HgCl2 for 3 min was found to be effective. The explants were cultured in MS medium supplemented with various concentrations of BAP and NAA. The results noted that an increase in the concentration of BAP concomitantly reduced the frequency of callus formation. The maximum callusing frequency and more number of shoot formation was observed in the lower concentration of BAP (0.5 mg/l) in combination with NAA (0.2 mg/l). The callus obtained from all the above combinations was sub-cultured on MS medium with same combinations of BAP and NAA. The maximum frequency of root formation in leaf callus was 85% and 75% in stem callus and both were achieved on MS medium with NAA (1 mg/l) after 2 weeks.
 Conclusions: The current investigation provides a competent in vitro propagation method for C. pedata var. glabra which could be commercialized for developing identical plants with high-quality mass multiplication rate and for better conservation of the germplasm. Both the methods described here are well suited for the mass multiplication of this critically endangered and endemic climber species.

Vegetative propagation of elite Eucalyptus clones as food source for honeybees (Apis mellifera); adventitious roots versus callus formation

Summer and autumn in Israel are highly arid with not enough plants in bloom offering nectar and pollen to support the local apiary. This leads to decline in colony health and honey production. To increase food sources for honeybees, we initiated a project to clone elite Eucalyptus trees exhibiting constant and rich blooming from late summer to early winter. We induced adventitious roots from cuttings of two mature Eucalyptus trees of which nectar production and honeybees’ attraction was measured: Eucalyptus brachyphylla and Eucalyptus x trabutii. During the rooting process, a high frequency of cylindrical callus formation instead of roots was obtained. To shed light on the inner anatomy of the callus chunks, we compared their cell organization and cell-wall composition to those of roots. Whereas in the root, cells were organized in circumferential symmetry, no symmetry was found in the callus. Instead, a more chaotic accumulation of meristematic-like cells with sporadic clusters of tracheary elements laid in different directions were observed. The outer cell layer of the callus often included swollen cells with thin cell walls. Most callus cells stained more strongly for cellulose and lignin than cells in the root meristem. In addition, specific antibodies to methylesterified and de-methylesterified pectin showed differential staining of callus vs. root cells indicating cell wall differences. Strikingly, roots were seen to differentiate from the chaotic cell organization of the callus, albeit at low rates. Further investigation of the cellular and molecular mechanisms underlying callus formation, are required.

Callus formation of sugar sorghum (Sorghum saccharatum) as affected by type and size of explantand level of ploidy

Purpose. Investigate the frequency of callus formation of different starting material of sugar sorghum depending on the type and size of explant and the level of ploidy.Methods. Biotechnological, laboratory, field, analytical, statistical.Results. In diploid forms of sugar sorghum in vitro, callus formation from boot was found at 11.7 %, a lower percentage of 9.8 and 9.6 % were respectively found in adapted in soil mixtures and field sorghum plants. The same pattern was observed in triploid forms: 13.6 % with respect to 12.5 and 12.3 %. Tetraploid forms of sugar sorghum had the highest percentage of callus formation compared to diploid and triploid, 16.6 % and 15.8 and 15.5 %, respectively. That is, the above mentioned pattern is observed in all sorghum plants regardless of the growing conditions. Regardless of the ploidy of sugar sorghum starting material and the given explant size (3–5.0 mm; 5.0–8.0; more than 8.0) of leaf blade and boot, a low level of callus genesis was observed, from 1.9 to 5.3 %. Within these sizes, diploid forms of callus formation amounted to only 4.0 % in cultivated plants, 3.3 % in adapted soil mixtures, and 3.1% in field-grown plants. In triploid forms of the above mentioned starting material, callus genesis rates were 3.0 and 3.3 and 3.9 %, respectively. A slight increase in callus formation in tetraploid starting material was from 5.3 to 4.2 %. The type of boot explant provided, regardless of the ploidy of the formation material, 4.5 pcs and leaf blades 17.9 pcs of regenerative explants. However, given the material ploidy, it was found that in diploid forms the number of regenerative explants from laminas in cultivated plants reached 11 pcs, adapted in soil mixtures 8 pcs, grown under field conditions 7 pcs, in triploid forms 21 pcs and 17 and 15 pcs., respectively. Tetraploid forms of sugar sorghum allowed to obtain 31 pcs and 27 and 24 pcs of regenerative explants depending on the type of starting material. The frequency of callus genesis, depending on the level of starting material ploidy, indicates that the highest percentage is established in tetraploid forms of sugar sorghum. Plants in vitro of diploid form had a frequency of callus genesis from leaf blades at the level of 34.1 ± 7.1 %, triploid 54.1 ± 7.2 %, tetraploid – 62.4 ± 5.7 %. Sugar sorghum material grown in vitro conditions and adapted in soil mixtures had a frequency of 28.1 ± 4.6%, triploid forms 35.8 ± 4.2 %, tetraploid – 51.3 ± 6.1 %. In plants grown under field conditions, the lowest frequency of callus formation was established, which in diploid forms was 24.3 ± 2.1 %, triploid 33.1 ± 2.4 %, tetraploid 48.7 ± 3.4 %. The frequency of callus formation in boots of sugar sorghum had the same pattern as that of laminas.Conclusions. The induction and frequency of callus genesis of sugar sorghum were influenced by the type and size of explants and the level of ploidy of the starting material. Genotypic features have been found to influence the formation of callus structures. In diploid forms, the lowest percentage of callus induction was observed and in tetraploid form, the highest. This pattern is observed in all sorghum plants, regardless of the growing conditions. The level of ploidy and starting material influence the regenerative capacity of sugar sorghum. The effect of different sizes of sugar sorghum explants on sorghum callus suggests that it is most appropriate to use 5.0–8.0 mm laminas for both sheath and leaf. Callus formation in sugar sorghum, depending on size, was most intense in polyploid forms compared to diploid ones. Studies indicate that the use of sorghum explants with a sugar size greater than 8.0 mm is not appropriate. The frequency of callus genesis, depending on the level of starting material ploidy, indicates that the highest percentage is established in tetraploid forms of sugar sorghum.

Embryogenic Callus Induction of Aquilaira Malaccensis Lam . a nd Aquilaria Subintegra Ding Hou

Aquilaria malaccensis Lam. and Aquilaria subintegra Ding Hou belong to the family of Thymelaeaceae which is commonly known as gaharu or agarwood. It is a commercially important tree and identified as a potential aromatic plant. The overwhelming responses in the lodging sector reduce gaharu species in the forest. Mass propagation through plant tissue culture technology will substitute this problem. The present study was conducted to investigate the embryogenic callus induction between these two species. The most optimum sterilization method for both species was sodium hypochlorite 5.0% which gave the highest percentage of aseptic culture (95%) with the absence of tissue browning. The leaves of both species were cultured on Murashige and Skoog, (1962) (MS) media supplemented with combination of various concentrations of 6-benzylaminopurine (BAP) (0.5, 1.0, 2.0 and 2.5 mg/L) and 2,4-dichlorophenoxyacetic acid (2, 4-D) (0.5, 1.0, 1.5 and 2.0 mg/L) and kept under dark condition. The explants produced embryogenic, white and compact callus at the end cut of the explants after two weeks of culture in all treatments. The highest frequency of embryogenic callus formation was observed in explants cultured on 2.0 mg/L BAP and 0.5 mg/L 2,4-D for both species. From the present study, the optimum sterilization technique and embryogenic callus induction for A. malaccensis Lam. and A. subintegra were established.

Callus formation and organogenesis in tissue culture Deschampsia antarctica E. Desv.

Aim. The aim of the work was to determine the optimal conditions for induction and proliferation of tissue culture obtained from D. antarctica plants from various localities of the Maritime Antarctica. Methods. Tissue and organ culture techniques. Results. The media В5 supplemented with 2 mg/l 2,4-D + 0,1 mg/l BAP, В5 supplemented with 10 mg/l 2,4-D + 0,2 mg/l BAP and МС, supplemented with 5 mg/l 2,4-D + 0,1 mg/l Kin were optimal for callus induction from different types of explants. The media with a reduced concentrations of auxins and cytokinins were the most effective for maintenance of continuous tissue culture compared to the media for callus induction: B5 + 2 mg/l 2,4-D mg/l + 0,1 mg/l BAP and MC + 1 mg/l 2,4-D + 0.1 mg/l Kin. Tissues from shoot growth point and leaf explants of genotypes DAR12a and G/D12-2a on medium B5 with 2 mg/l 2,4-D + 0.1 mg/l BAP and B5 with 10 mg/l 2,4-D + 0,2 mg/l BAP demonstrated the ability to spontaneous organogenesis and formed separate shoots. Conclusions. Conditions have been determined for the induction and proliferation of tissue culture from leaf, root, and shoot growth point explants of D. antarctica. The frequency of callus formation depended on the mineral composition of medium, ratios and concentrations of growth regulators, type of explant, and genotype of a donor-plant. As a result of spontaneous organogenesis, regenerated plants were obtained, conditions for their rooting in vitro were elaborated. The proposed methods for induction and proliferation tissue culture of D. antarctica in vitro can be used to produce the plant material useful for a various investigations. Keywords: Deschampsia antarctica E. Desv., tissue culture, organogenesis in vitro, frequency of callogenesis.

Callus formation, organogenesis and microclonal reproduction in different species of the genus Linum L. in vitro

Purpose. To reveal the frequency and intensity of callus formation and organogenesis, the effectiveness of microclonal reproduction of various species of the genus Linum L. (Linaceae) in vitro. Methods. For in vitro induction of callus formation and organogenesis, hypocotyl segments of species Linum usitatissimum L. convar. elongatum and convar. usitatissimum, L. tenue Desf., L. bienne Mill., L. corymbulosum Pchb., L. nervosum Waldst. & Kit., L. flavum L., L. campanulatum L., L. perenne L., L. austriacum L., L. grandiflorum Desf., L. strictum L. were cultivated on Murashige and Skoog medium supplementedwith 0.05 mg/l 1-naphthylacetic acid and 1.0 mg/l 6-benzyl aminopurine at 22–24 °C, relative humidity of 60–80%,with 16 hours photoperiod (2500 flux). For microclonal reproduction Murashige and Skoog, White, Gamborg and Eveleigh media and their modifications were used. The measurement results were interpreted by the arithmetic mean, standard error for the sample mean, the leastsignificantdifference and ranked. Results. Different species of the genus Linum to a large extend are capable of forming callus and regenerating shoots under the specified cultivation conditions. The frequency of callus formation for the studied samples on the 35th day of cultivation varied within 81.25–100%, the mass of callus from one explant – 0.21–1.64 g, the frequency of organogenesis – 12.50–100%, the number of shoots – 1.8–7.6 pcs. and the height of the shoots was 0.82–2.12 cm. The following species: L. usitatissimum convar. elongatum, L. tenue, L. bienne and L. strictum were distinguished by a high intensity of callus formation. Intensive organogenesis was pecular to L. tenue, L. bienne, L. flavum, L. austriacum and L. grandiflorum. The efficiency of somaclone obtaining was quite low in L. nervosum and L. campanulatum. In total, for the microclonal reproduction of species of the genus Linum Murashige and Skoog, Gamborg and Eveleighmedia supplementedwith 12.5 g/l glucose were optimal. At the final stages of microclonal propagation, before transferring microclones in vivo, it is advisable to use White medium, which contributes to a high frequency of rhizogenesis. Varieties of L. usitatissimum convar. elongatum and convar. usitatissimum had diffe­rent responses to in vitro culture. Conclusions. The frequency and intensity of callus formation and organogenesis, the effectiveness of microclonal reproduction depended on the genotype of a particular species; therefore it is advisable to select the composition of the nutrient medium and growth regulators for each of them. Some species of the genus Linum have not yet been studied in vitro, so the obtained results allow expanding the scope of their use in practice, in particular in breeding as a new source material with somaclonal variation, interspecific crosses, and ornamental floriculture.