Somaclonal Variation Research Articles

Current status of the cryopreservation of embryogenic material of woody species.

Cryopreservation, or the storage at liquid nitrogen temperatures (-196°C), of embryogenic cells or somatic embryos allows their long-term conservation without loss of their embryogenic capacity. During the last decade, protocols for cryopreservation of embryogenic material of woody species have been increasing in number and importance. However, despite the large experimental evidence proved in thousands of embryogenic lines, the application for the large-scale conservation of embryogenic material in cryobanks is still limited. Cryopreservation facilitates the management of embryogenic lines, reducing costs and time spent on their maintenance, thus limiting the risk of the appearance of somaclonal variation or contamination. Somatic embryogenesis in combination with cryopreservation is especially useful to preserve the juvenility of lines while the corresponding clones are being field-tested. Hence, when tree performance has been evaluated, selected varieties can be propagated from the cryostock. The traditional method of slow cooling or techniques based on vitrification are mostly applied procedures. For example, slow cooling methods are widely applied to conserve embryogenic lines of conifers. Desiccation based procedures, although simpler, have been applied in a smaller number of species. Genetic stability of the cryopreserved material is supported by multiloci PCR-derived markers in most of the assayed species, whereas DNA methylation status assays showed that cryopreservation might induce some changes that were also observed after prolonged subculture of the embryogenic lines. This article reviews the cryopreservation of embryogenic cultures in conifers, fruit species, deciduous forest species and palms, including a description of the different cryopreservation procedures and the analysis of their genetic stability after storage in liquid nitrogen.

An insight into tissue culture-induced variation origin shared between anther culture-derived triticale regenerants

BackgroundThe development of the plant in vitro techniques has brought about the variation identified in regenerants known as somaclonal or tissue culture-induced variation (TCIV). S-adenosyl-L-methionine (SAM), glutathione (GSH), low methylated pectins (LMP), and Cu(II) ions may be implicated in green plant regeneration efficiency (GPRE) and TCIV, according to studies in barley (Hordeum vulgare L.) and partially in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927). Using structural equation models (SEM), these metabolites have been connected to the metabolic pathways (Krebs and Yang cycles, glycolysis, transsulfuration), but not for triticale. Using metabolomic and (epi)genetic data, the study sought to develop a triticale regeneration efficiency statistical model. The culture’s induction medium was supplemented with various quantities of Cu(II) and Ag(I) ions for regeneration. The period of plant regeneration has also changed. The donor plant, anther-derived regenerants, and metAFLP were utilized to analyze TCIV concerning DNA in symmetric (CG, CHG) and asymmetric (CHH) sequence contexts. Attenuated Total Reflectance–Fourier Transfer Infrared (ATR-FTIR) spectroscopy was used to gather the metabolomic information on LMP, SAM, and GSH. To frame the data, a structural equation model was employed.ResultsAccording to metAFLP analysis, the average sequence change in the CHH context was 8.65%, and 0.58% was de novo methylation. Absorbances of FTIR spectra in regions specific for LMP, SAM, and GSH were used as variables values introduced to the SEM model. The average number of green regenerants per 100 plated anthers was 2.55.ConclusionsThe amounts of pectin demethylation, SAM, de novo methylation, and GSH are connected in the model to explain GPRE. By altering the concentration of Cu(II) ions in the medium, which influences the amount of pectin, triticale’s GPRE can be increased.

Micropropagation of banana genetic resources adapted to the Argentine environment and generation of new variability for agronomic traits through somaclonal variation

Micropropagation of banana genetic resources adapted to the Argentine environment and generation of new variability for agronomic traits through somaclonal variation

Assessment of genetic stability of micropropagated Curculigo latifolia from Indonesia by RAPD and ISSR

Micropropagation of Curculigo latifolia is very important because this plant has high economic value including containing Curculin and Neoculin as sweetener and taste-modifying proteins. Micropropagated plants may experience genetic changes like somaclonal variation so it is necessary to assess their genetic stability. The purpose of this study was to obtain a micropropagation method of C. latifolia that is more efficient than the previous research and to assess the genetic stability of micropropagated C. latifolia. The plant material used was sterile leaves obtained from sterile seedlings raised from C. latifolia seed germination. Leaves were grown on a callus induction medium containing 5 mg l−1 indole-3-butyric acid and 3 mg l−1 BA in a dark room. Callus formed were subcultured to growth selection medium and shoots formed were subcultured to Murashige and Skoog-free growth regulator medium with the addition of 6 g l−1 agar and 3% sucrose. The cultures were incubated at 23ºC ± 2ºC under 1,500 lux white LED (PhilipTM TL) on a 16 hour/8 hour light/dark photoperiod. The cultures were incubated for 14 weeks. Genetic stability of in vitro propagated C. latifolia from Indonesia was assessed by molecular markers, RAPD and ISSR. The micropropagation method produces an efficient protocol because each growth stage occurs in a free growth regulator medium except for callus induction. Based on the results of the assessment using molecular markers, no polymorphism in DNA amplification using RAPD and a high percentage of genetic stability (66.67%) from ISSR analysis were obtained. In sum, the in vitro micropropagation protocol created can be used commercially to produce C. latifolia on a large scale that is genetically stable.

Comparative restriction enzyme analysis of methylation (CREAM) reveals methylome variability within a clonal in vitro cannabis population.

The primary focus of medicinal cannabis research is to ensure the stability of cannabis lines for consistent administration of chemically uniform products to patients. In recent years, tissue culture has emerged as a valuable technique for genetic preservation and rapid multiplication of cannabis clones. However, there is concern that the physical and chemical conditions of the growing media can induce somaclonal variation, potentially impacting the viability and uniformity of clones. To address this concern, we developed Comparative Restriction Enzyme Analysis of Methylation (CREAM), a novel method to assess DNA methylation patterns and used it to study a population of 78 cannabis clones maintained in tissue culture. Through bioinformatics analysis of the methylome, we successfully detected 2,272 polymorphic methylated regions among the clones. Remarkably, our results demonstrated that DNA methylation patterns were preserved across subcultures within the clonal population, allowing us to distinguish between two subsets of clonal lines used in this study. These findings significantly contribute to our understanding of the epigenetic variability within clonal lines in medicinal cannabis produced through tissue culture techniques. This knowledge is crucial for understanding the effects of tissue culture on DNA methylation and ensuring the consistency and reliability of medicinal cannabis products with therapeutic properties. Additionally, the CREAM method is a fast and affordable technology to get a first glimpse at methylation in a biological system. It offers a valuable tool for studying epigenetic variation in other plant species, thereby facilitating broader applications in plant biotechnology and crop improvement.

Callusogenesis and Organogenesis of Thymus Cultivars In Vitro

The article is devoted to the in vitro cultivation of three Russian cultivars of two Thymus species (Th. vulgaris and Th. serpyllum) in order to study the possibility of induction of callusogenesis and organogenesis. The developed technology opens up prospects for the induction of somaclonal variability, obtaining the source material for breeding, as well as regeneration of thyme plants after genetic transformation. For the study, we used nutrient media based on the mineral composition of the Murashige and Skoog medium with the addition of auxin (indole-3-butyric acid, indole-3-acetic acid) and cytokinin (6-benzyladenine, kinetin) substances. For the induction of callusogenesis, the use of stem explants on nutrient media supplemented with 2,4-dichlorphenoxyacetic acid (1 or 2 mg/L) can be recommended. The efficiency of shoot organogenesis also depends on the type of explant, and its rather low values in most experimental variants are apparently due to the accumulation of endogenous auxins and phenolic compounds. Thus, a cultivar-specific morphogenetic response to the use of various phytohormones and growth regulators was established, as well as the influence of the explant type on the efficiency of morphogenic processes.

Pengaruh waktu induksi dan ZPT terhadap pertumbuhan kalus kotiledon kacang panjang (Vigna unguiculata subsp. sesquipedalis)

Yard-Long bean plants are an agricultural commodity that has great potential to be developed because they are easy to cultivate, their market share is also quite high but there are still problems in meeting the need for sufficient seeds and they are still susceptible to pests and disease. Therefore, efforts need to be made to increase production and induce variations. One method that can be used is tissue culture for the induction of somaclonal variations and/or the formation of transgenic plants. This study aims to determine the effect of variations in callus initiation time and the effect of Plant Growth Regulators (PGR) administration on the growth of long bean cotyledon explant callus. Seed sterilization was carried out using detergent, 10% sodium hypochlorite, 70% alcohol and sterile distilled water. The results of the research showed that variations in callus initiation time on MS media with the addition of 2,4-D PGR and the use of several variations of PGR in subcultures were able to initiate callus and influence callus size, callus color, and the percentage of browning of Yard-long bean cotyledon explants. Callus initiation time of 4 weeks with a combination of 2 ppm BAP concentration with 0.1 ppm IAA produced the largest callus size and brownish white callus color. The addition of PGR during subculture had a smaller browning percentage than the treatment without PGR.

Molecular characterization of Red banana and its somaclonal variant: a comprehensive study.

The online version contains supplementary material available at 10.1007/s13205-023-03868-6.

An efficient direct organogenesis protocol for in vitro clonal propagation of Rubia cordifolia L.

This comprehensive study delves into the intricate process of in vitro clonal propagation of R. cordifolia, with a particular focus on optimizing a method of direct organogenesis. The choice of direct organogenesis over indirect organogenesis is driven by the propensity for somaclonal variations associated with the latter. The presented research explores the critical factors influencing direct organogenesis, including the selection of appropriate explants and the optimum use of plant growth regulators. Notably, nodal explants have demonstrated great potential for regeneration, and the choice of cytokinins such as kinetin (KIN) and 2-isopentenyladenine (2-iP) for shoot induction has proven highly effective, with 1.0 mg/L KIN supplementation to MS media emerging as the most favourable choice resulting in 12.14 ± 1.58 shoots and 29.78 ± 1.93 axillary buds. Subsequent adventitious rooting using indole-3 butyric acid (IBA) and indole-3-acetic acid (IAA) resulted in a 100% rooting response. Among the concentrations tested, 1.0 mg/L IBA was found most effective, yielding the best rooting response (22.9 ± 1.06 roots/ micro-shoot). Acclimatization of in vitro cultures was achieved by gradually exposing them to natural conditions, maintaining high humidity and minimizing photorespiration. The morphological transformations observed, including robust leaf development contributed to the high acclimatization success (100% survival). Additionally, the genetic stability was confirmed using DNA-based ISSR markers, with complete monomorphism indicating no variations among the regenerants. The efficient direct organogenesis protocol developed in this study may prove a valuable contribution towards the mass clonal propagation and conservation of R. cordifolia, an industrially and medicinally important plant.

Somaclonal Variation in Callus Cultures of Rose Periwinkle, <i>Catharanthus Roseus L</i>. Under Induced Salt and Osmotic Stresses

The experiment was carried out at the laboratories of the National Commission for Biotechnology in Syria during the period 2020-2022 to detect the somaclonal genetic variation occurring in callus cultures at different ages and to compare them with the <em>In vitro</em> growing plants<em> </em>and<em> </em>introduced species of Rose Periwinkle that grew up in the Syrian environment, in addition, exploring the effect of abiotic stresses in causing genetic variations in callus, by the Inter Simple Sequence Repeats technique (ISSR) using 21 primers. Seeds were germinated on an MS nutrient medium devoid of growth regulators; explants were transferred to a propagation medium fortified with Naphthalene Acetic Acid (NAA) (1 mg L<sup>-1</sup>) and 6-Benzyladenine (BA) (2 mg L<sup>-1</sup>). Callus was developed from Rose periwinkle <em>Catharanthus roseus L</em>. leaves using 5C01 medium fortified with NAA and Kinetin (Kin). After that, callus was exposed to gradually increasing levels of PEG-6000-induced osmotic stress and salt stress (NaCl) with a higher level every 30 days. The molecular study results proved the effectiveness of all the used primers (21 primers) in presenting polymorphism between the studied samples at a rate of 92.16%. The cluster analysis showed separating the analyzed samples into two main clusters. The first cluster contained the osmotic stressed callus, while the second cluster separated into two sub-clusters that had the rest of the studied samples. It was found that there were genetic variations between <em>In vitro</em> plants and the callus. In addition, the stressed callus (salt and osmotic) was compared to the unstressed callus, and this confirmed that callus cultures were an essential source of genetic variations, and the ISSR technique was an effective tool for detecting these variations.

Callus stage and morphology affect the DNA yield on the DNA extraction from the sugarcane in vitro callus

The main concern with the micropropagation of sugarcane (Saccharum officinarum) with a complex polyploid genome is the frequent occurrence of somaclonal variations. Therefore, genetic stability analysis is often required to ensure the uniformity of the micropropagated sugarcane, as early as in the callus stage. DNA extraction is the initial and important step of the genetic stability analysis. In this experiment, we compared the yield of DNA extraction from the sugarcane embryogenic and non-embryogenic calli. The sugarcane non-embryogenic callus was initiated from immature rolled leaf explants of the 6 months-old plant cane in the callus induction media, that were incubated in the dark room. The embryogenic calli were obtained by routine subcultures of the non-embryogenic calli every six weeks. The results showed that the extraction yield from the non-embryogenic calli was far lower than that from the embryogenic calli, most likely due to the high-water content of the non-embryogenic calli. Furthermore, histological analysis using Safranin and Fast Green staining revealed the low lignin content and nuclei in the non-embryogenic calli cell mass, causing the transparent and watery appearance of the calli and might influence the low DNA extraction yield.

Study of the activity of antioxidant enzymes in somaclonal potato variants for use in selection for resistance to the PVS virus

The article presents the results of a study into the activity of the antioxidant system of potatoes in response to infection with the PVS virus. The PVS virus is widespread in the Omsk region. The work set out to study the activity of antioxidant enzymes in somaclonal potato variants when infected with PVS virus to obtain valuable varietal characteristics for use in breeding selection. Potato varieties Yermak, Alena and the Khozyayushka were selected from the Omsk Agrarian Scientific Centre breeding lines as objects of research. Differences in resistance to viral infection in varieties of different susceptibility and obtained somaclones were studied using the example of changes in the activity of the isoenzyme composition of antioxidant enzymes. The results showed that somaclone EC1, which was derived from the susceptible Yermak variety, had two additional isoenzymes upon infection and an increase in isoperoxidase activity compared to the control. Although the number of isoperoxidases did not increase in the samples obtained from the stable HC94 and the moderately susceptible AC91 variety, an increase in their activity was noted. Activation occurs in infected plants of the additional 3rd isoforms of catalase compared to the control – 1 isoform, while the 4th isoform of catalase was detected in the EC1 somaclonal sample. When studying the superoxide dismutase spectra, the control group showed the activity of Mp-SOD and Fe-SOD, while two forms of the enzyme were identified in the HC94, AC91 and EC1 somaclonal samples: Feand Cu/Zn-SOD. These forms play a significant role in the neutralisation of the superoxide radical.

Marker-assisted enhancement of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in a salt-tolerant rice variety for sustaining rice production of tropical islands.

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae is a major disease of rice, specially in the tropical regions of the world. Developing rice varieties with host resistance against the disease is the most effective and economical solution for managing the disease. Pyramiding resistance genes (Xa4, xa5, xa13,and Xa21) in popular rice varieties using marker-assisted backcross breeding (MABB) has been demonstrated as a cost-effective and sustainable approach for establishing durable BB resistance. Here, we report our successful efforts in introgressing four resistance genes (Xa4, xa5, xa13, and Xa21) from IRBB60 to CARI Dhan 5, a popular salt-tolerant variety developed from a somaclonal variant of Pokkali rice, through functional MABB. Both BB and coastal salinity are among the major challenges for rice production in tropical island and coastal ecosystems. Plants with four, three, and two gene pyramids were generated, which displayed high levels of resistance to the BB pathogen at the BC3F2 stage. Under controlled salinity microplot environments, the line 131-2-175-1223 identified with the presence of three gene pyramid (Xa21+xa13+xa5) displayed notable resistance across locations and years as well as exhibited a salinity tolerance comparable to the recurrent parent, CARI Dhan 5. Among two BB gene combinations (Xa21+xa13), two lines, 17-1-69-334 and 46-3-95-659, demonstrated resistance across locations and years, as well as salt tolerance and grain production comparable to CARI Dhan 5. Besides salinity tolerance, five lines, 17-1-69-179, 46-3-95-655, 131-2-190-1197, 131-2-175-1209, and 131-2-175-1239, exhibited complete resistance to BB disease. Following multilocation testing, potential lines have been identified that can serve as a prospective candidate for producing varieties for the tropical Andaman and Nicobar Islands and other coastal locations, which are prone to BB and coastal salinity stresses.

Pitaya Nutrition, Biology, and Biotechnology: A Review.

Pitaya (Hylocereus spp.) is a member of the cactus family that is native to Central and South America but is now cultivated throughout the sub-tropical and tropical regions of the world. It is of great importance due to its nutritional, ornamental, coloring, medicinal, industrial, and high consumption values. In order to effectively utilize and develop the available genetic resources, it is necessary to appreciate and understand studies pertaining to the usage, origin, nutrition, diversity, evaluation, characterization, conservation, taxonomy, and systematics of the genus Hylocereus. Additionally, to gain a basic understanding of the biology of the plant, this review has also discussed how biotechnological tools, such as cell and tissue culture, micropropagation (i.e., somatic embryogenesis, organogenesis, somaclonal variation, mutagenesis, androgenesis, gynogenesis, and altered ploidy), virus-induced gene silencing, and molecular marker technology, have been used to enhance pitaya germplasm.

Micropropagation and assessment of genetic fidelity of Decalepis hamiltonii Wight & Arn using RAPD markers

Somaclonal variation in micropropagated plants improves crops. Genetic transformation research relies on micropropagated plant genetic integrity. Somaclonal variation frequently occurs in micropropagation, prompting an investigation of plant genetic stability. The high number of shoot buds was seen when Decalepis hamiltonii shooting tip explants were inoculated with reversed polarity in Murashige and Skoog (MS) medium supplemented with 0.5 mg L-1 benzylaminopurine (BAP). The shoot buds increased in length on an MS medium supplemented with 0.5 mg L-1 Benzylaminopurine (BAP) and 0.1 mg L-1 indole-3-acetic acid (IAA). The lengthened shoots were established in Murashige and Skoog (MS) medium supplemented with 1 mg L-1 Indole-3-butyric Acid (IBA). Well-rooted plants underwent greenhouse hardening. Micropropagated and seedpropagated plants have similar physical features. Regenerated plant genetic stability was assessed using ten primers in a Random Amplified Polymorphic DNA (RAPD) assay. Eighty-five bands were identified. Monomorphic features and original-type clones were found in field-grown plants and bands. The regeneration approach might help study D. hamiltonii micropropagation

Evaluation of somaclonal variation in triticale plants regenerated from mature embryos by in vitro culture and treatment with gamma-rays

The Triticale regenerants (SC0) of 3 genotypes (Ingen 35, Ingen 93, 188TR5021) were obtained by calluso-genesis from irradiated mature embryos with gamma rays (150 Gy). The evaluation of the somaclones revealed changes in the quantitative traits: plant height, spike length, apical internode length, number of productive tillers and internodes per plant, number of grains, grains weight per plant. It was found that in vitro culture and gamma irradiation induced significant variations at the level of 95 – 99,9% of the characters studied in triticale SC1 and SC2 somaclones The mean value of 4 traits (plant height, spike length, number of grains and grains weight) of somaclones, SC2 (Ingen 93, in vitro and 188 TR5021, RAD) were higher than the control. The analysis of variance shows that the maximum contribution was manifested by the genotype (30,77%) and the interaction of genotype x radiation (17,28-41,62%) and genotype x in vitro culture (21,32-40,45%) depending on the character (99,9%). The obtained results confirm the contribution of researched factors to increase the genetic variability in triticale

Impact of Iron Nanoparticles Application on Date Palm In-vitro Regeneration Stability

The use of nanoparticles is critical in a number of industries, including manufacturing, healthcare, and agriculture. Iron nanoparticles (Fe NPs) are one of the main sources of plant nutrition because they are effective in releasing a variety of pH values. To study the effect of different concentrations of Fe NPs (0, 0.5, 1.0, 2.0, 4.0 ml/l) on the regeneration and rooting of Bartamoda cv. date palm, as well as to ascertain the content of carbohydrates and chlorophyll in shoots, an experiment was conducted at the Central Laboratory for Research and Development of Date Palm, Agricultural Research Center Giza, Egypt. Adding treatments of Fe NPs at 1.0 and 2.0 ml/l to MS medium containing BA at 2.0 mg/l and NAA at 0.5 mg/l significantly enhanced growth (number of shoots/cluster, number of leaves/cluster, and leaf length), according to Results. MS medium supplemented with NAA at 1.0 mg/l and Fe NPs at 1.0 ml/l showed the highest rooting percentage (89.0%). Treating the shoots with varying doses of iron nanoparticles led to a significantly higher iron content compared to the untreated control shoots. The carbohydrate content improved steadily and significantly with increasing Fe NP concentration. Compared with the control group, treatments with Fe NPs at 1.0 and 2.0 ml/l enhanced the levels of chlorophyll a, chlorophyll b, and carotenoids.
 Through the use of random amplified polymorphic DNA (RAPD) profiles, the somaclonal variation in shoots that can be caused by Fe NPs during the multiplication stage was examined. To amplify DNA from various shoots, seven random 10-mer primers were applied. The shoots' RAPD patterns matched those of the original plant mother, proving that Fe NPs did not cause somaclonal variation that could be seen using the RAPD method.

In Vitro Conversion of Coffea spp. Somatic Embryos in SETIS™ Bioreactor System.

Somatic embryogenesis (SE) is an excellent example of mass plant propagation. Due to its genetic variability and low somaclonal variation, coffee SE has become a model for in vitro propagation of woody species, as well as for large-scale production of vigorous plants that are advantageous to modern agriculture. The success of the large-scale propagation of an embryogenic system is dependent on the development, optimization, and transfer of complementary system technologies. In this study, two successful SE systems were combined with a SETIS™ bioreactor immersion system to develop an efficient and cost-effective approach for the in vitro development of somatic embryos of Coffea spp. This study used an efficient protocol for obtaining somatic embryos, utilizing direct and indirect SE for both C. canephora and C. arabica. Embryos in the cotyledonary stage were deposited in a bioreactor to complete their stage of development from embryo to plant with minimal manipulation. Following ten weeks of cultivation in the bioreactor, complete and vigorous plants were obtained. Different parameters such as fresh weight, length, number of leaves, and root length, as well as stomatal index and relative water content, were recorded. In addition, the survival rate and ex vitro development of plantlets during acclimatization was assessed. The best substrate combination was garden soil (GS), peat moss (PM), and agrolite (A) in a 1:1:0.5 ratio, in which the bioreactor-regenerated plants showed an acclimatization rate greater than 90%. This is the first report on the use of SETIS™ bioreactors for the in vitro development of somatic embryos in Coffea spp., providing a technology that could be utilized for the commercial in vitro propagation of coffee plants. A link between research and innovation is necessary to establish means of communication that facilitate technology transfer. This protocol can serve as a basis for the generation and scaling of different species of agroeconomic importance. However, other bottlenecks in the production chains and the field must be addressed.

In Vitro Culture Technology and Advanced Biotechnology Tools Toward Improvement in Gladiolus (Gladiolus species): Present Scenario and Future Prospects.

In the world's flower trade, gladiolus (Gladiolus spp.) is ranked first among bulbous flowers and eighth among cut flowers, with more than 30,000 different cultivars being grown. Mass multiplication and commercialization are restricted by the traditional propagation methods. However, the large-scale proliferation and improvement of the gladiolus have been accomplished with the aid of plant tissue culture and other biotechnological techniques. The current review includes a thorough examination of the growth and development parameters required for successful in vitro gladiolus development as well as cormel formation. Moreover, focus is being given to various techniques and methods such as in vitro cytogenetic stability and modification of chromosome number, in vitro mutagenesis and selection of pest resistance, in vitro identification and selection to develop virus-free germplasm, cryopreservation, synthetic seed technology, identifying virus diseases by RT-PCR, somaclonal variation, and protoplast and somatic hybridization. Molecular markers and their applications for genetic diversity analysis, relationships between different genotypes, and clonal stability analysis in Gladiolus species have been conducted by several research groups worldwide and are also being discussed.The article also covers efforts to enhance the functionality of plant phenotypes through genetic transformation. Future prospects for further improvement of ornamental gladiolus are also explored.Overall, the current review provides insight into the applications of basic and advanced biotechnological tools for gladiolus improvement.

In vitro somatic embryogenis and plant regeneration from cell suspension culture of endangered plant ‘Virali’ Dodonaea viscosa

An effective protocol was developed for in vitro propagation of ‘Virali’ via somatic embryogenesis in cell suspension culture. Embryogenic callus was obtained on Murashige and Skoog (MS) medium by a relatively moderate concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (4 mg/L), high concentrations of proline and glutamine (both 300 mg/L) and coconut water to develop induction of calli from explant. A suspension culture of the callus-derived embryogenic cells in Murashige and Skoog (MS) basal medium (pH 5.3) with 2 mg/L 2,4-D, 1 mg/L indole-3-acetic acid (IAA), 1 mg/L NAA, 45 g/L sucrose, and 20 g/L maltose resulted in synchronously proliferating cells with the capacity to be induced into somatic embryos on an 8g/L agarose, 20 g/L maltose, 10 g/L dextrose, 40 g/L sucrose, 1 mg/L naphthaleneacetic acid (NAA), and 0.2 mg/L. On an 8 g/L agarose-solidified MS basal medium (pH 5.7) with 1 mg/L NAA, 100 ml/L coconut water, 30 g/L sucrose, 30 g/L maltose, and 100 mg/L glutamine, the young somatic embryos differentiated into regenerable mature somatic embryos. In a 2.5 g/L gelrite solidified MS-based medium with 2.5 mg/L 6-benzylaminopurine (BAP), 1 mg/L gibberellic acid (GA3), 100 mg/L L-glutamine, 30 g/L sucrose, and 2.5 mg thidiazuron, the mature somatic embryos regenerated into plantlets (TDZ). For the formation of plantlets with a height of 10 to 12 cm that could endure the hardening process, it took a total of 6 months from the explant stage. The plantlets’ regular morphology suggests that there was no somaclonal variation in their development. The current regeneration approach for “Virali” has a significant deal of potential for micro propagating endangered germplasm and improving it through transgenic technology.