Protocol For Callus Induction Research Articles

Optimization of callus induction protocol from leaf explants of P<i>ortulaca oleracea</i> and assessment of fatty acid profiles

A protocol for optimization of callus culture establishment from leaf explants of Portulaca oleracea L. was developed. The effect of ethyl methanesulphonate (EMS) on callus induction and synthesis of fatty acids was evaluated. Callus culture was initiated from leaf explants on Murashige and Skoog’s medium supplemented with 2,4‑dichlorophenoxy acetic acid (2,4‑D), a combination of 2,4‑D + 6‑benzyl adenine (BA) or 2,4‑D + kinetin. The maximum callus biomass was obtained at 2.5 µM BA + 2.5 µM 2,4‑D (20.22 g leaf explant–1 fresh weight). The leaves treated with EMS (0.1 to 0.4% h–1) differentiated callus on this optimized medium combination. The gas chromatography-mass spectrometry analysis of fatty acids indicated that the leaves had a high linolenic acid content (17.72%). The callus cultures synthesized heptadecanoic, oleic, and tricosanoic acids, which were otherwise absent in the leaves. Eicosanoic and docosanoic acids in callus cultures were 2.78 and 3.18 times higher than their content in the leaves, respectively. The diversity of fatty acids in treated callus cultures decreased with increased EMS concentration, but the content of a few individual fatty acids was enhanced. Callus at the dose of 0.1% EMS synthesized linoleic acid, which was two times higher than in the untreated callus, while at 0.2% EMS stearic acid was synthesized, which was absent in the untreated callus. It was concluded that the callus of P. oleracea accumulated different fatty acids, and EMS treatment enhanced the content of a few fatty acids in the cultures.

Effects of different growth hormones on Solanum tuberosum L. callus induction and regeneration

This study was carried out to develop a protocol for callus induction, regeneration, shoot, and root induction in two potato cultivars (Riviera and Almonda) that can be further used to develop genetically modified plants. Callus was induced from leaf and internodal explants on Murashige and Skoog's medium (MS) supplemented with different combinations of hormones. The highest percentage of callus induction was obtained from the internodal explants of Riviera cultivar was 100% in Callus Induction Media I (CIM I) supplemented with 2 mg/L Benzyl Adenine (BA), 0.2 mg/L ?-Naphthalene Acetic Acid (NAA), 2 mg/L 2,4-Dichlorophenoxy acetic acid (2,4-D), 0.2 mg/L Kinetin (Kin) and 0.2 mg/L gibberellic acid (GA3). After one-month calli were shifted to Shoot Induction Media I and II (SIM I and SIM II), the highest percentage for shoot induction was 33.3% with the leaf explants of Riviera cultivar on SIM II supplemented with 2 mg/L BA, 0.2 mg/L NAA, 0.5 mg/L Kin and 0.2 mg/L GA3. The root induction percentage was 100% for both cultivars. Regenerated potato plants had normal leaf shapes with uniform morphology. These findings provide valuable insights for developing tissue culture protocols for potato regeneration, which are essential for genetic engineering and potato breeding programs.

Efficient isolated microspore culture protocol for callus induction and plantlet regeneration in japonica rice (Oryza sativa L.)

BackgroundIsolated microspore culture is a useful biotechnological technique applied in modern plant breeding programs as it can produce doubled haploid (DH) plants and accelerate the development of new varieties. Furthermore, as a single-cell culture technique, the isolated microspore culture provides an excellent platform for studying microspore embryogenesis. However, the reports on isolated microspore culture are rather limited in rice due to the low callus induction rate, poor regeneration capability, and high genotypic dependency. The present study developed an effective isolated microspore culture protocol for high-frequency androgenesis in four japonica rice genotypes. Several factors affecting the isolated microspore culture were studied to evaluate their effects on callus induction and plantlet regeneration.ResultsLow-temperature pre-treatment at 4 ℃ for 10–15 days could effectively promote microspore embryogenesis in japonica rice. A simple and efficient method was proposed for identifying the microspore developmental stage. The anthers in yellow-green florets located on the second type of primary branch on the rice panicle were found to be the optimal stage for isolated microspore culture. The most effective induction media for callus induction were IM2 and IM3, depending on the genotype. The optimal concentration of 2, 4-D in the medium for callus induction was 1 mg/L. Callus induction was negatively affected by a high concentration of KT over 1.5 mg/L. The differentiation medium suitable for japonica rice microspore callus comprised 1/2 MS, 2 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 6 g/L agar. The regeneration frequency of the four genotypes ranged from 61–211 green plantlets per 100 mg calli, with Chongxiangjing showing the highest regeneration frequency.ConclusionsThis study presented an efficient protocol for improved callus induction and green plantlet regeneration in japonica rice via isolated microspore culture, which could provide valuable support for rice breeding and genetic research.

Establishment of plant regeneration protocol through callus induction of sugarcane (Saccharum officinarum l.) var. China using leaf-sheath

Establishment of a plant regeneration system through direct organogenesis or via callus is also a prerequisite to further in vitro genetic manipulation of the cultivar through somaclonal variation, in vitro mutagenesis and genetic transformation. Production of sufficient numbers of plants of a unique genotype is possible using in vitro culture system. In this study, the effect of various concentrations and combinations of plant growth regulators for in vitro regeneration of sugarcane via callus was described using leaf-sheath as explants. Thus, a reproducible protocol for induction of callus and regeneration into plantlets were developed in sugarcane var. china using leaf-sheath as explants. Potential callus induction was observed on MS medium supplemented with 4.5 mg/l 2,4-D, in which 80% explants induced callus after culture initiation of 90 days. Shoot formation from the callus was optimum on MS + 1.0 mg/l GA3 + 0.5 mg/l Kin., in which 70% of the cultured calli formed shoots. The average number of shoot formed per culture callus was 27.0 ± 2.50 and the average shoot length of 6.5 ± 0.50 cm were achieved in this medium after culture of 60 days. Regenerated shoots rooted well when they were transferred into half strength of MS + 2.50 mg/l NAA, in which 90% shoots rooted within 30 days of culture. The average number of root produced per shoot was 18.0 ± 2.25 and the average root length of 7.50 ± 1.20 cm were observed in this medium. Ninety percent of the in vitro raised plantlets were survived in the natural environment.

Induction, Proliferation and Differentiation of Callus in Paris polyphylla Sm. through Leaf Culture

Paris polyphylla Sm. is a vulnerable medicinal plant employed in the treatment of various ailments. This study seeks to establish a protocol for callus induction, proliferation, and differentiation of P. polyphylla. Immature leaf explants were cultured on MS medium with varying concentrations of plant growth regulators (PGRs), including 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kn), 6-benzylaminopurine (BAP), Thidiazuron (TDZ), α-Naphthalene acetic acid (NAA), and Gibberellic acid (GA3), along with 10% coconut water. After 12 weeks of primary culture, the optimal callus induction was observed in MS medium supplemented with 0.25 mg/l 2,4-D + 0.5 mg/l Kn. In the secondary culture at 8 weeks, the best callus proliferation, as determined by callus weight or growth index, occurred in MS medium supplemented with 2.0 mg/l BAP alone, 2.0 mg/l Kn alone, 1.0 mg/l TDZ alone, combinations of 2.0 mg/l Kn + 1.0 mg/l BAP + 2.0 mg/l GA3, and combinations of 0.5 mg/l NAA + 2.0 mg/l BAP + 2.0 mg/l GA3, as well as 10% coconut water. Furthermore, callus differentiation into mini rhizomes with root primordia was successfully achieved in MS media containing 2.5 mg/l Kn and 10% coconut water. This study reports, for the first time, the formation and differentiation of callus from leaf explants in P. polyphylla. Large-scale callus generation from leaf explants has the potential to enhance the production of bioactive secondary metabolites for therapeutic purposes and facilitate the development of plantlets through organogenesis.

Callus Induction, Regeneration of Rice Variety BRRI Dhan29 and Determination of Lethal Dose Of Ethyl Methanesulfonate

Rice is an essential meal for a large section of the world's population including Bangladesh. BRRI Dhan29 is known as an excellent rice variety in Bangladesh but it is now susceptible to several diseases and pests, which might reduce rice yields. Therefore, it becomes necessary to explore and optimize different approach with the aim of enhancing yield, diseases resistance and overall genetic variability of this important rice variety. Ethyl methanesulphonate (EMS) produces a large number of non-lethal point mutations by alkylates guanine bases and leads to mispairing-alkylated guanine pairs with thiamine instead of cytosine. The LD50 of EMS is frequently used as a general indicator of a substance's acute toxicity and a lower LD50 is indicative of increased toxicity. This study aimed to develop a comprehensive protocol for callus induction, regeneration for the BRRI dhan29 rice and to determine the LD50% of EMS on callus survival for further genetic improvement. Mature embryos were used to induce callus by utilizing different concentrations (0.00, 0.5, 1.00, 1.50, 2.00, 2.50, 3.00 and 3.50 mg/L) of 2,4-Dichlorophenoxyactic acid (2, 4-D), while callus regeneration was studied using varying combine concentrations of 1-naphthaleneacetic acid (NAA: 0, 5, 10 and 15 μg/L) and kinetin (0.00, 1.00, 2.00 and 3.00 ml/L). The optimal concentrations for callus induction was 2.0 mg/L 2, 4-D and a combination of 10 μg/L NAA and 2.0 mg/L kinetin were identified for efficient callus regeneration. The LD50 of EMS on BRRI dhan29 calli was 0.31%, indicating its potential mutagen for creating genetic variations. Regenerated plants were successfully acclimatized at field conditions. These findings might provide valuable insights for tissue culture-based studies and genetic improvement efforts for the BRRI dhan29. Bangladesh J. Nuclear Agric, 37(1): 1-10, 2023

Callus induction studies in various explants in different genotypes of sesame (Sesamum indicum L.)

The goal of this study was to develop a suitable protocol for callus induction in sesame, Sesamum indicumwhich can be further used for regeneration studies. The experiments were carried out using three explants viz.,cotyledonary leaf, hypocotyls and primary leaf at different hormonal combinations and concentrations to studycallus induction. There was significant difference in the response of explants to different hormone treatmentsalthough there is no genotypic difference with regard to explants. Results showed that callus induction wassignificantly influenced by explants type. Hypocotyls responded well for callus induction in most of the genotypes.The callus induction frequency was significantly reduced (36.5%) when 0.2 mg BA was used along with 0.2 mg/lNAA. Replacing this hormonal combination with 2.0mg/l BA and 0.2 mg/l NAA greatly improved the callusinduction frequency (51.4%).

Effect of Plant Growth Regulators on Callus Induction of Black Rice (Oryza sativa L.)

Aims: Black rice of Manipur is known for its aroma and nutritional quality. As per the reports on tissue culture of rice, callus induction response of traditional cultivars was much lower than the modern high yielding varieties. Considering the importance of the black rice, and effort was taken to standardized callus induction protocol of black rice of Manipur.
 Study Design: Complete Randomized Design.
 Place and Duration of Study: Department of Seed Science and Technology, Faculty of Agriculture, Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar 736165, West Bengal, India, between June 2020 and July 2023.
 Methodology: Mature seeds of two black rice varieties used in this work. The seeds were dehusked manually, washed with running tap water. Then sterilized with 1% bavistin and again washed with running tap water. Seeds were surface sterilized with 0.2% HgCl2 for 15 minutes. Sterilised seeds were rinsed with sterile distilled water inside laminar air flow hood. The seeds were blotting dry using autoclaved tissue paper and sterile seeds were cultured with the scutellum pointing up wards on callus induction medium fortified with different concentrations and combinations of plant growth regulators.
 Results: MS medium fortified with 2,4-D was found to be better than picloram and TDZ to induce callus and the 2,4-D @ 1.00 mg/L and 2,4-D 2.00 mg/L was found to be the best concentrations. Callus health was also good when MS medium invigorated with 2,4-D.
 Conclusion: An efficient embryogenic callus induction and plantlet regeneration protocol for black rice was established by using mature seeds. In callus induction studies, with different concentrations of PGR, 2,4-D, higher callus induction frequency was observed with 1.00 mg/L 2,4-D and 2,4-D @ 2 mg/L for both rice varieties.

Quantification and FTIR Analysis of Precursor Mediated Alkaloid Production in Cell Suspension Culture of Gnidia glauca (Fresen.) Gilg

The current investigation focuses on developing a reproducible protocol for callus induction and for studying the precursor L-phenylalanine impact on product enhancement for alkaloid production and measurement of pH. Alkaloids are nitrogen-containing compounds; individual alkaloids were purified and synthesized commercially as excellent substances with recognized and experimentally verified pharmacological attributes because of their antimicrobial, anticancer properties and other therapeutic values. The current studies explore the impact of Precursor on alkaloid production by cell suspension culture in G. glauca, resulting in an increase in the cell growth observed and quantified by HPLC, TLC analysis, and the presence of functional groups identified by FTIR profiling. Additionally, this finding offers a protocol for biomass production of alkaloids from medicinally important endangered plants aiding in the conservation of the plants as well as producing commercially valuable secondary metabolites.

Callogenesis of Moringa oleifera seed cotyledon

Moringa oleifera has gained much importance in the past two decades due to its multiple uses and benefits to agriculture and industries. All the parts of the moringa plant are used for medicinal and other purposes. The roots, flowers, bark, stem, leaves and seeds of moringa possess antimicrobial properties, and hence it’s being regarded as a miracle plant. Different combinations of growth hormones have been used for plant tissue culture studies in M. oleifera but the difficulty in predicting a suitable medium for callus induction using different growth regulators in different localities is a major challenge. Studying the effect of different concentrations of growth hormones on callus induction of M. oleifera seed cotyledon is necessary to develop a reliable protocol for callus induction which is a prerequisite for the source of new metabolites which are not present in the wild. The seed cotyledon of indigenous M. oleifera seeds was used as an explant for callus induction. Seeds were surface sterilized with 90% ethanol for 1 min, washed 3 times with sterilized distilled water, surface sterilized using 3.5% aqueous solution of sodium hypochlorite containing few drops of Tween-20 for 10 min, followed by rinsing three times with sterile distilled water. The cotyledon explant was cultured on MS medium supplemented with different concentrations of 6- Benzyl amino purine (BAP), 2, 4-dichlorophenoxyacetic acid (2, 4-D) and α-naphthaleneacetic acid (NAA). Callus formation was monitored for a period of 5 weeks. The highest callus formation (90%) occurred on medium containing 1.0 mg/L BAP + 2.0 mg/L 2, 4-D + 0.5 mg/L NAA while the lowest callus formation (66%) was on medium containing 1.0 mg/L BAP + 2.0 mg/L 2, 4-D + 1.0 mg/L NAA. There was no callus formation on the medium lacking plant growth hormones. The T2 and T4 hormone combination would provide the basis for callus culture for the production of bioactive compounds and further in vitro genetic transformation in M. oleifera.

Induction of organogenesis and callogenesis in Limbarda crithmoïdes L. (Asteraceae) explants cultured on MS media supplemented with various concentrations of Na+ and K+

The present work describes an efficient micropropagation and callus induction protocol of Limbarda crithmoïdes L. (Asteraceae), a halophyte species with medicinal and horticultural interests. The objective was to identify the culture media that support the best organogenesis and callogenic expressions of four types of aerial explants; nodal and internodal segments and leaf portions, by varying the mineral composition of twenty culture media by adding increasing concentrations of Na+ (from 0-100 mM) and K+ (from 0-50 mM). After two months of culture, parameters relating to the various expressions of organogenesis and callogenesis were measured. Results showed that the K+ and Na+ interactions affect the explants development. The combination of high concentrations of Na+ (50, 100 mM) and K+ (50 mM) ions allowed the most important regeneration of the axillary shoots (70.6-100%), root neoformation (82.6-96.1%) and callus induction (76.3-100%). Rooted plantlets with well-developed axillary shoots have been successfully acclimatized with a 93% of survival rate. The selected media would allow a large-scale multiplication of this medicinal species, without adding exogenous phytohormones, and could be used for the micropropagation of other threatened halophyte species or for the production of callus; rich in secondary metabolites.

In Vitro sterilization protocol for callus induction of Baccaurea courtallensis (Wight) Müll.Arg. - An endemic tree of Southern Western Ghats, India

Experiments were carried out to establish in vitro culture using terminal and lateral buds of the endemic tree Baccaurea courtallensis of Phyllanthaceae which presented formidable difficulties. Besides the profuse phenol exudation, cultures were infected with intense fungal contamination. Efforts directed to control the schedule by adjusting the concentration of disinfectants NaOCl and HgCl2 with additional rinsing of explants with 70% ethanol and pre-soaking of shoots in various concentrations of the fungicide Bavistin never fetched any success. The intricately connected fungus seems to have found a niche amidst the leaf primordia in shoot apices and the axillary crevices of the nodal regions completely establishing itself in cultures. Perplexed with no remedy in sight, an interesting attempt of quick surface washing with 70% acetone was introduced in the surface sterilization schedule. Fresh explants were quickly rinsed with 70% acetone, followed by 0.1% mercuric chloride, 1% bavistin along with 4% PVP in succession and later wash with sterile distilled water enabling effective establishment of culture. Callus formation was observed on MS and WPM supplemented with auxin and cytokinin. Callus induction from the nodal region was observed at all concentrations and combinations of PGRs supplemented. Maximum induction of callus was obtained from MS medium augmented with BAP 4μM alone and in combination with IBA 1 μM +TDZ 4 μM.

In vitro Induction and Proliferation of Callus in Piper longum L. through Leaf Culture

Piper longum L. (Family Piperaceae) is a well- known health promoter used to treat cough, chronic bronchitis, asthma, and diabetes mellitus. The study is aimed to develop a protocol for callus induction and proliferation in P. longum. The leaf explants from mature plants were cultured on an MS basal medium supplemented with various concentrations of plant growth hormones, viz. 2,4-Dichlorophenoxyacetic acid (2, 4-D), Kinetin (KN), 6-Benzylaminopurine (BAP), and α-Naphthalene acetic acid (NAA), as well as 10% coconut water. In primary culture, the callus was compact and light white. The best callus induction and growth were observed in the MS basal medium containing1.0 mg/L 2,4-D+ 2.0 mg/L KN at 12 weeks of primary culture. At eight weeks of secondary culture, the MS medium containing 2.0 mg/L BAP alone and 0.5 mg/L NAA + 2.0 mg/L BAP and 10% coconut water had the best callus proliferation. Compared to 2,4-D and KN alone, BAP alone supported rapid callus growth in the MS medium. In P. longum, large-scale callus formation from leaf explants could be exploited to produce, isolate, and increase bioactive secondary metabolites for therapeutic purposes.

Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation.

Paeonia lactiflora Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for P. lactiflora research. The purpose of the present study focuses on obtaining an efficient and stable genetic transformation method using callus as the receptor and exploring an efficient protocol for callus induction and proliferation associated with P. lactiflora. Callus induction and proliferation were performed using MS medium with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (6-BA) and thidiazuron (TDZ). The sensitivity of callus to kanamycin and cefotaxime was determined. Several parameters such as Agrobacterium cell density, infection time and co-culture duration were studied to optimize transformation efficiency. Agrobacterium strains EHA105 and pBI121 binary vector harboring the β-glucuronidase (GUS) gene were used for transformation. Expression of the GUS reporter gene was detected by GUS assay, polymerase chain reaction (PCR) and Quantitative Real-time PCR (RT-qPCR). The MS medium containing 1.0 mg·L-1 NAA, 0.5 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was optimal for callus induction and MS medium containing 0.5 mg·L-1 NAA, 1.0 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was the best for callus proliferation. The concentrations of kanamycin and cefotaxime used for screening positive callus were 125 mg·L-1 and 200 mg·L-1, respectively. Among various combinations analyzed, the best transformation result was obtained via the 25 min of infection of Agrobacterium at 0.6 OD600 and 3 d of co-culture. Overall, this study provided technical support and theoretical guidance for improving the callus induction and proliferation efficiency and the study of gene function in P. lactiflora.

Callus Production and Analysis of Isoflavonoids and Some Terpenes from Bituminaria bituminosa

Background: Bituminaria bituminosa is a long-day plant. The plant has a high pastoral value and palatability. It is grazed by cattle, goats and sheep. Leaves and stems contain four furanocoumarins’ psoralen, angelicin, xanthotoxin and bergapten. The seed is covered by the fruit. Germination was not observed in beaked fruits. A cut with a scalpel can increase the germination rate. According to the literature there are not enough studies about the culture media and convenient conditions. Methods: In our study, we tried six medium for callus production with different hormone concentrations including 2,4-D, BAP and kinetin. In this study, a protocol for callus induction and analysis of isoflavonoids and terpenes of Bituminaria bituminosa has been developed. Result: Maximum callus induction frequency was observed on Murashige and Skoog (MS) medium supplemented with 0.75 mg/L 2,4-D + 2.5 mg/L kinetin in young primary leaves. 0.75 mg/L 2,4-D+2.5 mg/L kinetin medium is more successful in the production ofses of flavonoids that can be produced in vitro and is more successful in producing phenols. In terpene production, it is seen that 1.25 mg/L 2,4-D+3.5 mg/L BAP, which is the best callus producing medium containing BAP, is the most successful medium among all tested mediums.

Salinity Stress Response of Rice (Oryza sativa L. cv. Luem Pua) Calli and Seedlings.

Soil salinity limits plant growth and production. This research investigated a suitable medium for callus induction and plantlet regeneration in the Luem Pua rice cultivar. The effect of salt stress on seedling growth was determined using in vitro culture and soil conditions. An efficient protocol for callus induction has been developed by culture sterilized seeds on the Murashige and Skoog (MS, 1962) medium containing 0.5 mg/l benzyladenine (BA) with 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) that resulted in a 100% callus induction. Plantlet regeneration percentage of 49% was recorded on the MS medium containing 4 mg/l BA with 0.5 mg/l 1-naphthaleneacetic acid (NAA) after 4 weeks. For salt stress investigation, the calli were treated on an induction medium containing various concentrations of NaCl (0, 50, 100, 150, and 200 mM), while two-week-old rice seedlings were planted in soil and treated with the same concentration of NaCl for 4 weeks. In vitro culture revealed that callus survival percentage decreased when NaCl concentration increased, similar to soil culture. Seedling growth under salinity treatment also decreased when NaCl concentration increased, while other physiological parameters such as total chlorophyll, chlorophyll a, chlorophyll b, green intensity, and chlorophyll fluorescence under light conditions increased under salinity stress. These changes define the growth and physiological salinity tolerance characteristics of Luem Pua rice calli and seedlings. They can be utilized as a baseline for demand-driven in vitro rice propagation, providing useful information that can be combined with other agronomic features in rice development or breeding programs to improve the flexibility of abiotic stress-tolerant cultivars.

Optimization of protocol for callus induction and whole plant regeneration for developing somaclonal variants in sugarcane cv. COC 671

The present study was carried out to optimize in vitro callus induction protocol and whole plant regeneration for developing red rot resistant somaclones in commercial sugarcane variety COC 671 using immature leaf sheaths as explants. In the whole experiment, the best medium for callus induction was MS medium supplemented with 2, 4-D (2.0 mg/l) recorded callus induction frequency of 100 per cent in eight days with a maximum callus fresh weight of 13.08 g/10 calli. The compact and friable embryogenic calli were regenerated in MS medium supplemented with KIN (1.0 mg/l) producing 21 shoots in 10 days with a shoot length of 8.69 cm. The well grown shoots were rooted in MS medium supplemented with NAA at 3.0 mg/L with 9 roots in 9 days which had an average root length of 3.63 cm. The regenerated plantlets were successfully acclimated under field conditions. This protocol would further be used for developing red rot resistant somaclonal variants in sugarcane. Keywords: Sugarcane, red rot, somaclonal variants, callus induction, regeneration

In vitro plant regeneration and metabolite profiling of an aromatic medicinal plant Ruta graveolens L. by using GC-MS

Ruta graveolens L. is an endemic plant of the Mediterranean region. It has been used for centuries as a medical preparation and has a variety of roles because of its varied chemical composition. In vitro culture is a useful tool for both multiplication and study of important secondary metabolites. The present study was aimed to develop an effective and reproducible protocol for callus induction and indirect plant regeneration of Ruta graveolens (L.) by using leaf explants and to analyze chemical components present in different extracts of Ruta graveolens. The leaf explants were cultured on MS medium augmented with different combinations and concentrations of auxins and cytokinins for callus induction, shoot multiplication and rooting. The optimum plant growth regulator concentration for callus induction, shoot multiplication and root formation was recorded in MSM+2,4-D(1.5mg/L)+NAA(1.5 mg/L), MSM+ BAP (1.5mg/L) +IBA (1.0 mg/L) and half strength MSM+IBA(0.50 mg/L) respectively. The rooted Plantlets were successfully acclimatized and established in earthen pots. The leaves, stem, roots and callus of Ruta graveolens were extracted by using Acetone and Ethyl acetate solvents followed by volatile compound analysis using GC-MS. The phytochemical assay showed that extracts of Ruta graveolens contain various phytoconstituents having potential bioactivity. The major compounds found were 1, 3-Dioxolane-4-propanol, 2, 2-Dimetheyl-, kokusaginine, Bergaptene, 2 Undecanone, 3-Hexene-2-one, Alpha, - 1- Arabinopyranose, 1, 2:3,4-bis-o, and 1- (1,3-Benzodioxol-5-ylmethyl)-3-Nitro-1 which might be primarily contributing in the biological activity of the plant. The results of this study will make a way for the production of herbal medicines for various ailments by using callus cultures of Ruta graveolens L.

An efficient protocol optimization for callus induction, regeneration and acclimatization in sugarcane cv. CO 94012

Sugarcane's vast and complex genome, photosensitivity, low seed setting, prolonged crop duration, and complex environmental effect make it difficult to create diverse genetic variability by conventional breeding. However, the in vitro culture system can play a vital role in boosting yield, quality, and making the crop climate smart through in vitro approaches. In the present study in vitro culture using immature leaf sheath of Sugarcane (Saccharum officinarum L. cv- CO 94012) as an ex plant was used to standardize the protocol for callus induction and whole plant regeneration. MS medium supplemented with 2.0 mg/l, 2-4 D, recorded the highest percentage of callus induction. The highest number of multiple shoots was produced on MS medium fortified with KIN 1.0 mg/l. Rooting was more profuse when in vitro regenerated shoots were inoculated on MS basal medium supplemented with 3.0 mg/l NAA. Rooted shoots were hardened in the greenhouse before being transplanted to the field, where 90% survival rate.

In vitro propagation and genetic fidelity evaluation in LA Lilium

The present experiment was conducted at the Division of Floriculture and Landscaping, IARI, New Delhi, Indiaduring 2015–17 to develop a protocol for callus induction, PLB formation and plant regeneration from LA LiliumBrindisi using in vitro leaf segments, and to assess the genetic stability using SSR marker. Micropropagation of LALilium Brindisi led to compact calluses of dark brown to black colour. The in vitro regenerated leaves were inoculated at different concentration of 6-BAP and 2,4-D. Along with calluses, protocorm like bodies were also induced from the surface of cultured leaf segments, which further developed into shoots. MS medium fortified with 6-BAP (0.25 mg/l) and 2, 4-D (5 mg/l) recorded maximum callus formation. The mean number of shoot per callus clump ranged from 1.12 to 3.88, maximum number of shoots were recorded with 6-BAP (4 mg/l) and NAA (0.25 mg/l). Rooting ranged from 72–100% in IBA medium. Twenty regenerates were randomly selected for testing the fidelity. Out of 18 screened markers, only 10 produced clear and reproducible bands. A total of 244 bands were generated from 10 SSR primers in which seven primers were found polymorphic. Dendrogram generated by data analysis using Darwin 6 software package clearly indicated that the in vitro raised plants through leaf explant via callus phase were divided into three main clusters. The result of cluster analysis was supported by principal coordinate analysis (1/2 axis) where all the genotypes were distributed over different quadrangles. The total somaclonal variation was estimated to be 1.9% which indicated that even the plantlets raised through callus phase exhibited low frequency of somaclonal variation in case of LA hybrids of Lilium.