Callus Initiation Research Articles

Transcriptional regulation of cell proliferation competence-associated Arabidopsis genes, CDKA;1, RID1 and SRD2, by phytohormones in tissue culture.

During organ regeneration, differentiated cells acquire cell proliferation competence before the re-start of cell division. In Arabidopsis thaliana (Arabidopsis), CDKA;1, a cyclin-dependent kinase, RID1, a DEAH-box RNA helicase, and SRD2, a small nuclear RNA transcription factor, are implicated in the regulation of cell proliferation competence. Here, we report phytohormonal transcriptional regulation of these cell proliferation competence-associated genes during callus initiation. We can induce the callus initiation from Arabidopsis hypocotyl explants by the culture on the auxin-containing medium. By RT-quantitative PCR analysis, we observed higher mRNA accumulation of CDKA;1, RID1, and SRD2 in culture on the auxin-containing medium than in culture on the auxin-free medium. Promoter-reporter analysis showed that the CDKA;1, RID1, and SRD2 expression was induced in the stele regions containing pericycle cells, where cell division would be resumed to make callus, by the culture in the medium containing auxin and/or cytokinin. However, the expression levels of these genes in cortical and epidermal cells, which would not originate callus cells, were variable by genes and phytohormonal conditions. We also found that the rid1-1 mutation greatly decreased the expression levels of CDKA;1 and SRD2 during callus initiation specifically at 28°C (restrictive temperature), while the srd2-1 mutation did not obviously decrease the expression levels of CDKA;1 and RID1 regardless of temperature conditions but rather even increased them at 22°C (permissive temperature). Together, our results implicated the phytohormonal and differential regulation of cell proliferation competence-associated genes in the multistep regulation of cell proliferation competence.

Efficient shoot regeneration of medicinal plant Haplophyllum tuberculatum by direct and indirect organogenesis and genetic fidelity assessment using Inter Simple Sequence Repeats markers.

In vitro plant cell and tissue cultures are potent tools to propagating germplasm resources in conserving and managing plant genetic resources. A reliable micropropagation protocol was developed for efficient callus proliferation and direct and indirect shoot regeneration of Meseika (Haplophyllum tuberculatum). With the applied sterilization procedure, immature, unopened H. tuberculatum seed pods can be identified as a potent explant with high viability and low contamination percentage. Multiple shoots were regenerated from leaf and stem explants through direct organogenesis on Murashige and Skoog’s (MS) + 3% sucrose medium amended with BAP. Indirect regeneration of several shoots was achieved on 1/2 MS + 1% sucrose media amended with 2 and 4 mg/l BAP. An efficient callus proliferation from both explants can be achieved by supplementing the MS media with NAA and BAP. All the cultures were incubated in a controlled growth chamber under 5/19 h light/dark photoperiod, temperature (25 ± 2°C), and 60% relative humidity (RH).10 ISSR (Inter Simple Sequence Repeat) markers were screened to test the genetic fidelity of regenerated H. tuberculatum shoots. Callus development was observed after 15 days and shoot regeneration was occurred after 30 days after callus initiation. 10 ISSR primers produced a total of 39 clear, distinct amplicons. 75, 60, 40, and 16% polymorphism percentages were recorded by the ISSR primer 11, 7, 5, and 4, respectively. The developed micropropagation protocol is appropriate for rapid in-vitro multiplication of H. tuberculatum shoots and callus.

IN VITRO SELECTION BASAL MEDIUM USED FOR SEEDS GERMINATION, CALLOGENESIS INDUCTION OF LOCAL VARIETIES OF VICIA FABA L

One of the most significant winter crops consumed by humans in the Middle East is the faba bean (Vicia faba L.). As many other vetches and field crops. The ability of this plant to withstand such a biotic stress may be improved with the help of tissue culture. This investigation was carried out to create and selection basal medium used for seeds germination, callogenesis induction in three Faba bean varieties, Baraqat, Iraqi and Halabi, extensively grown for personal consumption in Iran, Iraq, and Syria respectively. First seeds were sterilized and planted on a growth-regulatory-free MS basal medium. Germination rates ranked between 24.6 to 63% because of the lethal effect of phenolic compounds. Five types of explants excised on the in vitro obtained seedlings were used for callus initiation: stems, shoot tips, leaves, epicotyls, and parts of the roots. They were put on MS basal medium that had been increased by BAP at a dosage of 0.5, 1.0, or 1.5 mg/l.. Depending on the genotypes, explants, and medium, callogenesis rates ranged from 33.33 to 100%. In terms of callogenesis rate and callus weight, shoot tip segments grown on media with BAP at 1 and 1.5 mg/l generated the highest outcomes of all three kinds. Even though it wasn't the goal of this study, the creation of somatic embryos was conceivably achievable (33%) throughout this study and happened just 2 months after calluses started, suggesting the embryogenic ability of such calluses for the 3 kinds examined.

Phytochemicals and Antioxidant Activities of Conventionally Propagated Nodal Segment and In Vitro-Induced Callus of Bougainvillea glabra Choisy Using Different Solvents

Bougainvillea, popularly known as ‘Bunga kertas’ in Malaysia, is thoroughly explored for nutritional and medicinal purposes. Bougainvillea has been shown to possess alkaloids and flavonoids which are widely used in folk medicine to treat different illnesses such as inflammatory, diarrheal, ulcer, and diabetic. Despite its major conventional therapeutic importance, only limited attempts have been made to investigate this species’ chemical and pharmacological properties in relation to its medicinal uses. Therefore, this study was conducted to determine the effect of in vitro-induced callus under different light conditions and plant growth regulators on phytochemical and antioxidant activities using different extraction solvents. Based on the results, the maximum days (17.67) to callus initiation were recorded when nodal was cultured on woody plant medium (WPM) supplemented with 7.5 µM 2,4-D + 0.5 µM BAP under light condition. On the contrary, the minimum days (7) to callus initiation were obtained when nodal was treated with 2.5 and 5 µM 2,4-D + 1 and 1.5 µM BAP under dark conditions. However, higher fresh and dry weight of callus was obtained when nodal was cultured on woody plant medium fortified with 7.5 µM 2,4-D + 1.5 µM BAP under dark and light conditions. In the analysis of the phenolics content and antioxidant activities, aqueous extract of conventionally propagated nodal part exhibited the highest phenolic content and antioxidant activities. However, the highest iron (II) chelating activity was produced from the aqueous extract of the calli induced under a dark condition. Hence, it can be concluded that the callus culture of Bougainvillea produced plant secondary metabolites and antioxidant activities comparable to the mother plants.

Simulated microgravity contributed to modification of callogenesis performance and secondary metabolite production in CannabisIndica

In vitro plant culture paves the way for meeting the industrial demand of pharmaceutically valuable secondary metabolites. This study intends to monitor how callus cells of Cannabis indica respond to the simulated microgravity (clinorotation; a Man-made technology). Callus initiation resulted from the culture of the leaf explant in a medium supplemented with kinetin (0.5 mgL−1) and 2, 4-D (2 mgL−1). Calli were treated with microgravity at three exposure times (0, 3, and 5 days). The microgravity treatments increased callus biomass about 2.5-fold. The clinorotation treatments transcriptionally induced the olivetolic acid cyclase (OAC) and olivetol synthase (OLS) genes about 6.2-fold. The tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes displayed a similar upward trend in response to microgravity. The applied treatments also stimulated the expression of the ethylene-responsive element-binding proteins (ERF1B) and WRKY1 transcription factors by an average of 7.6-fold. Moreover, the simulated microgravity triggered epigenetic modification in the DNA methylation profile. The HPLC-based assessment validated the high efficacy of the clinorotation treatments to increase the concentration of cannabinoids, including Cannabigerol (CBG) and Cannabidiol (CBD). However, the clinorotated calli contained a lower concentration of Tetrahydrocannabinol (THC) than the control group. The microgravity treatments increased concentrations of proline (79%), soluble sugars (61.3%), and proteins (21.4%) in calli. The biochemical assessment revealed that the clinorotation treatments slightly increased H2O2 concentration. The upregulation in the activities of peroxidase, catalase, and phenylalanine ammonia-lyase enzymes resulted from the microgravity treatments. Both HPLC and molecular assessments validated the significant efficacy of microgravity to enhance the production of cannabinoids.

Establishment of Stem Cell-like Cells of Sida hermaphrodita (L.) Rusby from Explants Containing Cambial Meristems.

The in vitro cultures of plant stem cells and stem cell-like cells can be established from tissues containing meristematic cells. Chemical compounds—as well as their production potential—is among the emerging topics of plant biotechnology. We induced the callus cell biomass growth and characterized the parameters indicating the presence of stem cells or stem cell-like cells. Four types of explants (stem, petiole, leaf, root) from Sida hermaphrodita (L.) Rusby and various combinations of auxins and cytokinins were tested for initiation of callus, growth of sub-cultivated callus biomass, and establishment of stem cells or stem cell-like cells. Induction of callus and its growth parameters were significantly affected both by the explant type and the combination of used plant growth hormones and regulators. The responsibility for callus initiation and growth was the highest in stem-derived explants containing cambial meristematic cells. Growth parameters of callus biomass and specific characteristics of vacuoles confirmed the presence of stem cells or stem cell-like cells in sub-cultivated callus cell biomass. Establishment of in vitro stem cell or stem cell-like cell cultures in S. hermaphrodita can lead to the development of various applications of in vitro cultivation systems as well as alternative applications of this crop.

Plant Regeneration of Malaysian Aromatic Rice MRQ88 Via Somatic Embryogenesis

Aromatic rice has become one of the most sought-after sources by today's consumers. As a result, by utilising mature seeds, an efficient somatic embryogenesis system for the MRQ88 variety has been constructed. Due to the highest percentage of callus initiation and no browning, MS-B5 medium supplemented with 1 mg/L 2,4-D and 10 mg/L NAA was the best medium for MRQ88 callus induction. Incubation of embryogenic callus on identical medium containing maltose 10 g/L and ABA 10 mg/L for 8 weeks resulted in the highest proportion of somatic embryo initiation (80%). The number of regenerated green plantlets on medium containing NAA and 2,4-D which was previously pre-treated with 5 to 10 mg/L ABA, 10 g/L maltose, showed good results, with 8-9 green plantlets developed.

Development of an efficient in vitro regeneration system in Costus speciosus - an important herbal insulin plant

Costus speciosus is being rapidly eliminated in its natural habitats in India and driven to a nearly threatened degree of extinction because of non-selective collection and over-exploitation. The commercial cultivation of C. speciosus is impeded owing to its poor seed viability, reduced germination rate, and weak rooting potential of vegetative cuttings. As costus could not be multiplied rapidly by conventional propagation methods, in-vitro regeneration technology was experimented with to standardize explants in addition to the combinations of plant growth regulators (PGRs) to fortify the growing media that helps to produce the enormous number of plants in a shorter duration. Several kinds of explants viz., leaves and nodes were cultured into the Murashige and Skoog (MS) medium reinforced with different combinations and doses of PGRs. A higher frequency (51.67 ± 1.7%) of the regenerated adventitious shoot was obtained from nodal segments cultured onto MS medium fortified with 0.5 mg/l Thidiazuron (TDZ) and 2.0 mg/l of 6-benzyl amino purine (BAP). TDZ in combination with BAP and Indole-3-acetic acid (IAA) exhibited a remarkable effect on shoot multiplication and elongation. Further, MS medium with 0.5 mg/l TDZ, 1.0 mg/l 2, 4-dichlorophenoxyacetic acid, and 0.5 mg/l IAA yielded a great range of callus initiation (86.66 ± 1.6%) from leaf explants. Leaf-derived callus results in the greatest shoot regeneration (56.66 ± 1.7%) response on TDZ (0.5 mg/l) and BAP (1.0 mg/l) reinforced MS medium. Elongated shoots from node and callus cultures when inoculated with the MS medium reinforced along with 1.0 mg/l of IAA and 0.5 mg/l of Indole-3-butyric acid (IBA) produced optimum rooting (88.33 ± 1.66%). The rooted young plantlets were effectively acclimatized to the environmental climate and their greenhouse survival rate was 85%.

MAX2-dependent competence for callus formation and shoot regeneration from Arabidopsis thaliana root explants.

Although the division of the pericycle cells initiates both lateral root development and root-derived callus formation, these developmental processes are affected differently in the strigolactone and karrikin/KARRIKIN INSENSITIVE 2 (KAI2) ligand signalling mutant more axillary growth 2 (max2). Whereas max2 produces more lateral roots than the wild type, it is defective in the regeneration of shoots from root explants. We suggest that the decreased shoot regeneration of max2 originates from delayed formation of callus primordium, yielding less callus material to regenerate shoots. Indeed, when incubated on callus-inducing medium, the pericycle cell division was reduced in max2 and the early gene expression varied when compared with the wild type, as determined by a transcriptomics analysis. Furthermore, the expression of the LATERAL ORGAN BOUNDARIES DOMAIN genes and of callus-induction genes was modified in correlation with the max2 phenotype, suggesting a role for MAX2 in the regulation of the interplay between cytokinin, auxin, and light signalling in callus initiation. Additionally, we found that the in vitro shoot regeneration phenotype of max2 might be caused by a defect in KAI2, rather than in DWARF14, signalling. Nevertheless, the shoot regeneration assays revealed that the strigolactone biosynthesis mutants max3 and max4 also play a minor role.

GhWOX11 and GhWOX12 promote cell fate specification during embryogenesis

The acquisition of transgenic materials relies on an efficient genetic transformation system, in which WUSCHEL-related homeobox genes play imperative roles in enhancing somatic embryogenesis potential. In our study, we isolated and characterized the cotton (Gossypium hirsutum L) homologues of Arabidopsis WOX11 and WOX12, namely GhWOX11_At, GhWOX11_Dt, GhWOX12_Dt and GhWOX12_At. Domain analysis indicated that the putative GhWOX11 and GhWOX12 proteins contain a highly conserved DNA-binding HOX domain or ACR domain. We have also demonstrated that callus initiation and adventitious root formation are simultaneously regulated by GhWOX11/12 genes. The asymmetric distribution of auxin leads to the differential expression of GhWOX11 and GhWOX12, which controls the formation of callus and presents a polar distribution at the ends of hypocotyl. Furthermore, overexpression of GhWOX11/12 significantly promotes the formation of callus and the fate transition from callus cell to pluripotent cell in G. hirsutum. On the other hand, expression of the chimeric GhWOX11/12 repressor in cotton callus inhibited embryogenic callus formation. Overexpression of GhWOX11 also promoted the formations of green callus, adventitious roots and adventitious shoots in Arabidopsis, corroborating its conserved functional role in inducing plant somatic embryogenesis and root development. Our results show that GhWOX11 and GhWOX12 genes play an important role in the process of cell fate transition in the early stage of somatic embryogenesis, providing a new perspective for the optimization of plant transformation systems.

Effectiveness of magnetic field in stimulation of biochemical and enzymes activities in seedling and callus of Nigella sativa

This current study investigated the influence of Magnetic Field from Winding coil magnet about ( 400 mT ) for different exposure periods ( 0, 30 , 60 , 90 , 120 , 150 min ) on seed germination, callus initiation, and callus fresh weight, protein, carbohydrate and proline content and vitality , in addition to the efficacy of some antioxidant enzymes . The results indicated that the highest seed germination percentage was when exposed for an (90 Minutes). Also found that the best exposure period for acceleration of callus initiation was (30 minutes). Fresh weight, protein, proline, carbohydrates content and callus vitality got a better result under (90 minutes) of magnetic field exposure . On the other hand, the antioxidant enzymes activity significantly increased with 150 minutes M.F treatment.

Uniconazole Augments Abscisic Acid in Promoting Somatic Embryogenesis in Cotton (Gossypium hirsutum L.).

During somatic embryogenesis (SE), somatic cells initiate embryogenic development under appropriate conditions. Uniconazole, a plant growth regulator, was found to inhibit the proliferation of callus but promoted the conversion of callus into an embryogenic callus (EC) in cotton. The supplementation of uniconazole in the culture medium significantly suppressed the endogenous auxin [indole acetic acid (IAA)] level in callus tissues in both the callus initiation and proliferation stage but enhanced the abscisic acid (ABA) level only in the callus proliferation stage. Exogenous ABA and uniconazole showed cooperative effects on promoting the differentiation rate of callus into EC. These findings were verified by RNA-seq analysis, which elucidated that the genes involved in the IAA biosynthesis, metabolism, and signaling, and ABA metabolism pathways were regulated by uniconazole during the callus development and SE. Overall, the results suggest that uniconazole could modulate callus proliferation and callus differentiation rate by regulating the endogenous levels of IAA and ABA.

GhLBDs Promote Callus Initiation and Act as Selectable Markers to Increase Transformation Efficiency.

Detached organs or differentiated tissues could form a mass of pluripotent cells termed as callus on an auxin-rich medium, the underlying molecular mechanism of which remains elusive in cotton. LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor is a key regulator of plant cell totipotency/pluripotency, and a number of cotton GhLBDs with high-level differential expression during the callus induction process have been identified. Their overexpression in cotton calli fostered promotions in and callus induction without exogenous auxin. Expression analysis and histological observation using paraffin sectioning suggested that the first 72 h on culture is a key time point for callus initiation, whereby the GhLBDs showed high transcript abundance and enlarged calli that were rapidly developed from procambium and cambium. GhLBDs’ expression level could be precisely modulated by the gradient concentrations of exogenous auxin, whereas auxin transport inhibitor 2,3,5-triiodobenzoic acid could severely inhibit its expression. The LBD-mediated callus formation was also dependent on the expression levels of GhLBDs. Further, a β-estradiol-inducible promoter pER8 was used to drive GhLBD18-1 expression, which led to rapid callus proliferation, suggesting that pER8/GhLBD18-1 could be used as a selectable marker system to replace the existing antibiotic/herbicide-resistance selectable markers in plant transformation. Our study provides new insights for callus initiation regulatory mechanism and strategies for improving transformation efficiency in cotton.

IN VITRO ASSESSMENT OF EMBRYO AGE AND NAPHTHALENE ACETIC ACID SUPPLEMENTATION OF MEDIA IN CALLUS GENERATION OF OIL PALM (Elaeis guineensis Jacq)

Callus generation from embryo explants of the oil palm (Elaeis guineensis Jacq) was observed in relation to age of plants at time of explants excision and types of media used. The two stages of explants were those at 15 and 18 weeks after anthesis. Eeuwens and Murashige & Skoog‘s (MS) media supplemented with Naphthalene acetic acid (NAA) were used. These gave two media variations viz: MS medium supplemented with NAA and Eeuwens‘ medium supplemented with NAA. The best combination for the generation of callus from the embryo explants within 56 days in culture was determined by observing type of response, time of initiation of callus and callusing explants. It was found that callus generation was best from embryo explants taken from 18 weeks after anthesis cultured in Eeuwens‘ medium, supplemented with NAA. The optimum concentration of NAA was found to be 50 mgl-1, which initiated callus within 39 days in culture. The percentage of explants producing callus was 80 %. The results revealed the importance of the supplementation of nutrient media with NAA and the usefulness of selecting embryo explants of about 18 weeks after anthesis for use in callus generation in the oil palm

Callus mediated in-vitro plant regeneration and clonal fidelity assessment of Lilium longiflorum cv. “Pavia” by ISSR markers

Aim: Developing a protocol for mass multiplication of Lilium (Lilium longiflorum) bulbs via indirect organogenesis and somatic embryogenesis for in-vitro plant regeneration and ISSR marker-based mapping to assess the fitness of true to type. Methodology: To induce organogenic callus, six combinations of 2,4-D and BAP were tested; while nine combinations of picloram and NAA were used for somatic embryogenesis. NAA and IBA were tested for root induction. The clonal fidelity of the in-vitro regenerated plantlets from both calluses were tested using polymerase chain reaction (PCR)-based ISSR markers analysis. All experiments were arranged in a complete randomized block design and replicated three times. Results: Two methods (direct and indirect organogenesis) were investigated to assess the best callus mediated plant regeneration for Lilium bulb multiplication. The maximum organogenic callus induction and the highest regeneration percent was recorded with BAP (0.5 mg l-1) + 2,4-D (3.0 mg l-1). However, MS medium containing 0.50 mg l-1 picloram and 0.20 mg l-1 NAA was found best for initiation of embryogenic callus and proliferation and was found superior over direct organogenesis. Clonal fidelity was assessed through ISSR markers comparing the mother plant and regenerated plantlets. Interpretation: Both organogenic callus andembryogenic callus are capable of developing true to type in-vitro plants and can be explored for mass multiplication of Lilium bulbs. Embryogenic callus can further be utilized in liquid suspension based bioreactor system. The present protocol has potential applications in micropropagation and genetic transformation studies in other Lilium spp.

In vitro regeneration of shoot and roots of the wild folkloric medicinal plant Ammannia baccifera L. via indirect organogenesis from leaf explant cultures

Ammannia baccifera L. is a wetland medicinal herb that belongs to the family Lythraceae. This plant is very notable to have a wide scope in pharmacological, therapeutic and medicinal properties. The current examination imparts an effective, proficient and fast strategy of commercial proliferation to satisfy the folklore medicinal and pharmacological demands of the market. A large-scale micropropagation of Ammannia baccifera L was done through a meticulous and a well-organized procedure. The leaf segments were cultured on MS medium augmented with different concentrations (0.5–2.5 mg/l) of 6- benzylaminopurine (BAP), (0.5- 2.0 mg/l) 1- naphthaleneacetic acid (NAA) and (1.0- 2.0 mg/l) 2,4- Dichlorophenoxyacetic acid (2,4-D) and were used for callus initiation. BAP at 1.0 mg/l combined with 0.5 mg/l NAA was found to be the best concentration and combination for initiation of high frequency of reddish green nodular callus (95.00±1.87) and good response for leaf explants. For shoot regeneration, MS medium blended with BAP at 1.5 mg/l and IAA at 0.6 mg/l produces maximum percentage of shoots from the callus derived by leaf explant. The leaf segments produce maximum percentage shoots (90.00±1.52), total number of shoots per explant (26.33±1.45) and length of shoot (9.63±0.73 cm). However, healthy established shoots were cultured on MS medium fortified with 1.5 mg/l indole-3-butyric acid (IBA) along with 2.0 mg/l. Indole-3-acetic acid (IAA) was found to be best for the root initiation and production in the leaf segments (95±1.57), number of roots (19.33±0.88) and root length (8.63±0.61cm). The regenerated plantlets were relocated to paper cups filled with vermiculite:sand:soil in the proportion of 1:2:1 (w/w) and were then transferred into outdoor conditions.

Mutagenic efficiency of gamma rays on in-vitro regeneration in Indian mustard (Brassica juncea)

In the study Indian mustard [Brassica juncea (L.) Czern. & Coss.] was treated with different doses (0, 2.5, 10, 20 and 100 Gy) of gamma rays (radioactive cobalt, 60Co) and the effects were investigated on callus initiation, seed germination, seedling growth, total chlorophyll, protein and proline contents of seedling leaves, which were grown from hypocotyl explant of non-irradiated and irradiated seeds. Experiment was conducted at School of Biotechnology, Gautam Buddha University during 2017–18. The results showed that irradiated seeds had increased seed germination per cent, seedling length, fresh and dry weight, total chlorophyll, protein and proline content in low dose exposure (2.5Gy). Callus cultures were established from hypocotyl explants of 15 days old seedlings obtained from irradiated and non-irradiated seeds of B. juncea on MS1 [MS+BAP (2 mg/ml) + 2,4 D (1 mg/ml)] and MS2 [MS+BAP (2 mg/ml) + 2,4 D (0.5 mg/ml)] medium. Cultured hypocotyls formed callus at their cut ends within a week and simultaneously differentiation started into nodular structures from their base. MS1 callus induction medium gave good callus induction percentage (58%) as compared to MS2. It was observed that the callus was green, compact and showed 83% induction frequency in 100Gy gamma dose in MS1, whereas in different dose treatment (2.5–20Gy), callus was white and friable. It turned light brown in 100Gy treatment in MS2 medium. Thus, results of the present study showed pre-sowing seed irradiation is an effective method of improving seed germination at the early developmental stage of the plant.

Somatic embryogenesis mediated micropropagation of polyembryonic cultivars of mango Vellaikolumban and Olour using nucellus tissue

Abstract Somatic embryogenesis mediated micropropagation from nucellus tissue was attempted in polyembryonic mango at the ICAR-Indian Institute of Horticultural Research, Bengaluru during 2016–2019. Studies were conducted to optimize the different stages of somatic embryogenesis namely induction, proliferation, conversion, maturation, germination of somatic embryos and ex vitro establishment of plantlets using nucellus tissue. For culture initiation, various developmental stages of fruits ranging from less than 20 days post-pollination to more than 60 days-post pollination of cultivars Vellaikolumban and Olour were utilized. Fruits of 30–40 days post-pollination in Vellaikolumban and 40–50 days post-pollination in Olour gave better percentage callusing with fewer days for callus initiation and callus formation. Embryonic calli developed on induction medium containing Rugini olive (RO) constituents, 6% sucrose, and 5 ppm each of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and Giberellic acid (GA3). Upon shifting to proliferation medium (RO with 6% sucrose, 5 ppm each of 2, 4-D and GA3, 400 mgL−1of glutamine and 20% v/v coconut water) pro-globular and globular embryos were formed within 4–5 weeks. Further development of somatic embryos through early heart, late heart and cotyledonary stages occurred in conversion medium (half strength B5 salts, full RO organics, 5 ppm of Indole-3-Acetic Acid (IAA), 20% coconut water and 200 mgL−1 of casein hydrolysate). Maturation of embryos was obtained by using 0.01 mg L−1of Abscisic acid (ABA) and 100 mg L−1of Polyethylene glycol (PEG). Germination of embryos with shoot and root initiation was observed in the presence of 5 ppm of zeatin. Our results show that somatic embryogenesis can be used as an alternative method to conventional propagation methods for rapid multiplication of uniform planting material in polyembryonic mango cultivars.

Особенности индуцированного морфогенеза и регенерации растений из соматических тканей люцерны сортов отечественной селекции в культуре in vitro

The authors have studied Morphogenesis and regeneration features of domestic breeding cultivars of alfalfa and conditions for obtaining regenerant plants from the morphogenic callus tissues. As a result of the study, the efficient sterilization protocol of alfalfa seeds was established with the use of sterilizing agents as 70% ethanol and 10% sodium hypochlorite which resulted in the production of 100% aseptically viable seedlings for the further in vitro studies. Callus initiation from cotyledons and hypocotyls was implemented on a Gamborg B5 nutrient medium containing 2,4-dichlorophenoacetic acid (2,4-D), kinetin in a concentration of 5,0 mg/l, and naphthaleneacetic acid (NAA) of 0,1 mg/l. Induction of morphogenic callus was achieved on a Gamborg B5 medium with 0,2 mg/l 6-benzylaminopurine (BAP). Further shoot initiation occurred on a hormone-free Gamborg B5 medium with the addition of Gamborg B5 vitamins, and on a half-strength medium with the addition of 0,2mg/l kinetin. It was found that established method of obtaining regenerated plants from the callus cultures of Medicago Varia proved itself effective.

The Role of Growth Regulators in The Multiplication of Stevia Rebaudiana Bertoni Shoot and Callus Induction in Vitro

The research was conducted to investigate the effect of plant growth regulators on the vegetative multiplication of the Stevia plant shoot and the induction of callus from it. The results indicated that the interaction between 2.0 mg. L-1 BA with 0.5 mg. L -1 Kin gave the highest rate number of shoot with 6.32 branches and the highest average of leaves number Were 9.60 leaves compared to the lowest average for the number of shoot and leaves Were 1.40 shoot and 3.80 leaves respectively. As for the length of the shoot, the interaction between (1.0 mg. L-1 BA and 0.5 mg. L-1 Kin) gave the highest average shoot length 4.31 cm compared to the control which gave 2.20 cm. in connection with the callus induction, the concentration 3 mg. L-1 of NAA gave the highest percentage of callus induction from leaves 100%, and the lowest mean of days that for callus initiation was 9 days compared to the control which reached to 20 days. As for the wet and dry weights of callus tissue, the interaction between 2.0 mg. L-1 NAA and 0.5 mg. L -1BA gave the highest wet weight rate 3.68 g and the average dry weight was 0.31 g compared to the control which gave the lowest rate 0.95 g, 0.08 g respectively.