Friable Callus Research Articles

Rosmarinic acid production in cell suspension cultures of Ehretia asperula Zollinger & Moritz

Plant cell cultures provide an alternative means for producing secondary compounds in food, cosmetic and pharmaceutical industries. Ehretia asperula Zollinger & Moritzi is used as a traditional medicine for the treatment of liver detoxification, ulcers, tumors, inflammation and enhancing the body's resistance in Vietnam. The study was carried out to select suitable callus line for cell suspension cultures of E. asperula Zollinger & Moritzi and investigate the effects of inoculum size, rotation speed and naphthalene acetic acid (NAA) on the proliferation of cell suspension cultures. In addition, the influence of light intensity on the growth and rosmarinic acid (RA) biosynthesis of cell suspension was also surveyed. After 4 weeks of culture, the white to pale yellow friable callus expanded significantly with a fresh weight (FW) of 0.788 g and a high RA content of 2.062 mg/g FW. An appropriate medium for cell proliferation was the liquid B5 medium, which contained 30 g/l glucose, 0.1 mg/l benzyl adenine (BA) and 0.4 mg/l NAA. The results also demonstrated that a 1:20 ratio (w/v) inoculum size, darkness and rotation speed of 90 rpm were the optimal conditions for the proliferation and RA accumulation to 188.217 mg/l in 4 weeks of culture. These findings showed that E. asperula Zollinger & Moritzi cell suspension cultures could be a potential alternative approach for RA production in vitro.

Influence of plant growth regulators and salicylic acid on the production of some secondary metabolites in callus and cell suspension culture of Satureja sahendica Bornm.

The impact of combinations of plant growth regulators (PGRs) on callus culture of Satureja sahendica Bornm. was investigated. In nodal explants, the response of secondary metabolite production to different concentrations of PGRs was analyzed regarding the presence and absence of polyvinylpyrrolidone (PVP). The explants were cultured on MS media in presence of auxins (2,4-dichlorophenoxyacetic acid and naphthylacetic acid) and cytokinins (thidiazuron and kinetin); which were used in equal concentrations of 0.5, 1, and 2 mg l-1. The treatment of 2 mg l-1 2,4-D + 2 mg l-1 Kin (MD3) led to the highest production of total phenolics (4.303 ± 0.449 mg GAE g-1) and flavonoids (24.903 ± 7.016 mg QE g-1). Moreover, the effect of salicylic acid (SA) on the production of secondary metabolites in cell suspension culture of Satureja sahendica was evaluated. The cell suspension culture was established by culturing the nodal-derived friable callus in the liquid medium containing different concentrations of SA (0, 100, 150, 200 µM). An inverse relationship exists between the fresh mass and secondary metabolites contents. In addition, there was a significant difference among concentrations of SA in the production of total phenolics and flavonoid compounds. SA enhances secondary metabolites production and decreases cell fresh mass.

Callus induction, clonal propagation and in vitro germplasm conservation of ‘hualtaco’ Loxopterygium huasango Spruce ex Engl. (Anacardiaceae)

Loxopterygium huasango Spruce ex Engl., of the Anacardiaceae family, classified critically endangered, is a forest tree with considerable value in the Seasonally Dry Tropical Forest (SDTF) in the northwest of Peru and south of Ecuador. The aim of the study was callus induction, propagation and germplasm conservation using in vitro tissue culture techniques. In vitro seed germination (90%) was obtained on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L-1 GA3. The cotyledonal nodes of seedlings were used as explants to initiate the growth of the apical shoots, in culture medium with the IAA-GA3 interactions, without observing growth of the axillary buds. The highest rate of friable callus induction (100%) was recorded on MS medium containing 0.1 to 5.0 mg/L-1 2,4-D in cotyledons and hypocotyls explants after 30 to 45 days of culture. The profuse roots formation was the only morphogenic response (10.8 to 13.8%) on MS medium with 0.5 mg/L-1 IAA, 0.5 mg/L-1 IAA 2.0 mg/L-1 BAP, KIN or 2iP or 2.0 mg/L-1 BAP, KIN or 2iP. The germplasm conservation of apical buds and nodal segments was achieved with 2.0 mg/L-1 AgNO3. This method could be used to conserve elite genotypes, rapid production of roots and establishment of cellular suspension for secondary compounds production.

PENGARUH 2,4- D TERHADAP PENINGKATAN KUALITAS DAN KUANTITAS KALUS YANG DIINDUKSI DENGAN KOMBINASI AUKSIN DAN SITOKININ PADA KULTUR DAUN RUMPUT BENGGALA ( Panicum maximum) VARIETAS TRICHOGLUME

To determine the effect various concentration 2, 4, 6 and 8 mg/l of 2,4- D (2,4- dicloropenoxy acetic acid) on callus induction of Benggala grass variety Trichoglume leaf culture on Murashige and Skoog (MS) standard medium and its organogenesis stimulated using deffent concentration of growth regulator, namely [N6- (2- isopenteny)- adenine] or 2iP (0, 0.2 and 1.0 mg/l ) and (l- naphthalene acetic ent, wacid) or NAA (0, 0.03, 0.16 and 3.0 mg/l) were performed. Percentage of callus were measured and organogenesis from callus were subjected to description analysis . The results showed that callus induction was optimum when 2,4-D was treated at 4 mg/l, friable callus were produced. Percentage production of callus week 4 was 46.2 percent, while using 8 mg/l 2,4 –D the callus production was about 59.7 percent yellowish coloured and more compact callus were produced. Combination of 0 mg/l 2iP ( auxin) + 3mg/l (cytokinin) at 3 weeks showed reseilted 100 % of calluses produced roots, the highest amaunt roots (9,0) was observed in combination 0 mg/l 2iP + 3 mg/l NAA, and the longest root (17,0 mm) was recorded in combination 0 mg/l 2iP + 3 mg/l NAA. Calluses yielded varied from white, yellowish to brown colour.

Genomic Methylated Cytosine Level during the Dedifferentiation and Cellular Competence in Coffea arabica Lines: Insights about the Different In Vitro Responses

Coffea arabica genotypes present distinct responses in vitro, and somaclonal variation occurrence has been reported. Global cytosine methylation is one of the epigenetic mechanisms that influences the Coffea in vitro responses. We aimed to establish the indirect somatic embryogenesis in C. arabica ‘Catuaí Vermelho’, ‘Caturra’ and ‘Oeiras’, associate the distinct responses to the methylated cytosine genomic level, and check the ploidy stability. Leaf explants were cultured in callus induction and proliferation medium. The resulted calli were transferred to the regeneration medium, and the mature cotyledonary somatic embryos were transferred to the seedling medium. ‘Oeiras’ exhibited the highest number of responsive leaf explants, followed by ‘Caturra’ and ‘Catuaí Vermelho’. Global methylated cytosine level increased over time in the ‘Catuaí Vermelho’ and ‘Caturra’ friable calli, remaining constant in ‘Oeiras’. ‘Oeiras’ did not regenerate somatic embryos, while ‘Catuaí Vermelho’ exhibited the highest number. Somatic embryo regeneration was associated with the increase of the methylated cytosine level. However, the ‘Catuaí Vermelho’ embryogenic calli showed a lower methylated cytosine level than ‘Caturra’. Recovered plantlets exhibited the same 2C value and chromosome number to the explant donors. Therefore, cytosine hypermethylation occurred during C. arabica indirect somatic embryogenesis, influencing cell competence and somatic embryos regeneration.

Callus induction and chemical characterization of cell suspension cultures of jojoba (Simmondsia chinensis L.)

Jojoba (Simmondsia chinensis L.) oil is also known as liquid wax or fixed oil. It is an important metabolite of jojoba having commercial importance in cosmetics as well as a potential biofuel source. We presented an efficient system for in vitro establishment of cell suspension cultures (CSC) from proliferating friable calluses. For this purpose, cotyledon, internode, and leaf explants were cultured on MS medium + 1, 2, 4, 6, 8 or 10 µM 2, 4-Dichlorophenoxyacetic acid (2, 4-D), α-Naphthalene acetic acid (NAA) alone or in combination with 1 or 2 µM N6-benzylaminopurine (BAP) or Kinetin. Results demonstrated that 100% healthy, friable and variegated calluses were obtained on 8 µM, 10 µM 2, 4-D or 2, 4-D 10 µM + 2 µM BAP and represented as callus lines (CL) CL-1, CL-2 or CL-3, respectively, after 38 days. One-gram callus tissue per CL was then immersed in the respective liquid medium and agitated on an orbital shaker at 60-70 rpm under the growth room conditions (25 ± 2 °C, 16 h light period) for the preparation of CSC. After 15 days, CSC was sieved and large clumps were removed. Growth measurement of CSC was determined by cell counting, packed cell volume (PCV) and cell viability. The highest number of viable cells was obtained at 2.57 OD with CL-3, where PCV was highest (0.35 ml) on CL-1 of 38 days old calluses. 2,3,5-Triphenyltetrazolium chloride was a reliable approach for the determination of cell viability of CSC.

Growth and Oleanolic Acid Accumulation of Polyscias fruticosa Cell Suspension Cultures.

Oleanolic acid is an oleanane triterpene found in many plant species all over the world. This compound is also a major saponin in leaves of Polyscias fruticosa and possesses several promising pharmacological activities, such as hepatoprotective effects, and antiinflammatory, antioxidant, or anticancer activities. The objective of the present work is to establish cell suspension culture of P. fruticosa, investigate the influence of several factors such as plant growth regulators and carbon source on cell growth, and determine their oleanolic acid content. Cell culture was established by using 2 g fresh weight of 30 day old friable callus derived from in vitro stem segment in 50 mL of liquid medium with a shaking speed of 220 rpm. The culture was then incubated at 25±2ºC with a shaking speed of 120 rpm in the period of 12 h daylight at a light intensity of about 6.75 μmol/m2/s. Cell growth was measured by fresh and dry biomass at 16 h day. Oleanolic acid content was determined using HPLC analysis. The study results showed that MS medium containing 2% sucrose as a carbon source, supplemented with 1 mg/L 6-benzylaminopurine and 0.5 mg/L 2,4-dichlorophenoxyacetic acid was the most appropriate growth medium. Cell biomass and oleanolic acid content reached the highest values of 0.43 g dry weight/flask and 25.4 mg/g dry weight, respectively. These results indicated the potential production of oleanolic acid, a compound with high pharmacological value, from P. fruticosa cell culture.

EFFECTS OF LIGHT ILLUMINATION AND SUBCULTURE FREQUENCY ON BIOMASS PRODUCTION IN CELL SUSPENSION CULTURES OF Clinacanthus nutans

Clinacanthus nutans (Burm. f.) Lindau is a medicinal plant rich in polyphenols and possesses multiple pharmacological properties. It is widely used as a side treatment for cancer and to prevent oxidative stress-related diseases. The present study aims to investigate the effects of light illumination and subculture frequency on the growth and proliferation of cells in the suspension culture of C. nutans. Friable callus induced from leaf explants C. nutans were cultured in liquid Murashige & Skoog medium supplemented with 0.25 mg/L 2, 4-dichlorophenoxyacetic acid, and 0.25 mg/L 6-benzylaminopurine to initiate the cell suspension culture. The suspension cultures were incubated under different conditions (16 hr photoperiod and continuous darkness). Subcultures were conducted every two weeks for ten subculture cycles. Results revealed that higher cell biomass was obtained under the exposure of 16 hr photoperiod with the highest biomass production at the fifth subculture cycle (growth index of 11.8) exhibiting stable growth from the eighth to the tenth subculture cycle. The suspension cells showed stable growth rates up to the 10th subculture cycle producing small aggregates and single cells. The present study indicated that light illumination significantly impacts the growth of suspension cultures for C. nutans in the production of valuable secondary metabolites.

Sterile Tissue Preparation and Callus Induction of Curcuma longa Linn.

Curcuma longa Linn. (family Zingiberaceae), commonly known as ‘turmeric’, is native to Southeast Asia. Turmeric has been used for color, flavor as a spice in cuisine and employed for treatment of various diseases. The major component in yellow-pigmented fraction of turmeric is curcuminoids. Curcuminoid production in callus of C. longa Linn. is our focus of study. Sterile techniques to obtain germ-free of C. longa Linn. explants were investigated and the results showed that immersing rhizome buds in 70% ethanol for 5 min, followed by 0.10% HgCl2 for 10 min offered approximately 66% survival rate. Multiple shoots were generated from the aseptic rhizome explants cultured on Murashige and Skoog (MS) agar medium fortified with 3.00 µM of 6-Benzylaminopurine (BA) and 0.50 µM of 1-Naphthaleneacetic acid (NAA) at 25 ± 2°C under a photoperiod of 16 h light and 8 h dark. The sterile leaf sheath and root were subsequently used for callus induction which produced various responses when cultured on MS agar medium fortified with different concentrations of 2,4-dichlorophenoxy acetic acid (2, 4-D), Thidiazuron (TDZ) and BA. The highest induction yields of friable callus were obtained from leaf sheath segments cultured on MS agar medium fortified with 0.50 mg/l 2, 4-D which are the conditions proposed for successful production of callus culture of C. longa Linn. Keywords: Callus induction, Curcuma longa Linn., Turmeric, Plant tissue culture

In-vitro Callogenesis and Screening of Antimicrobial Activity of Callus and Seed of Caesalpinia bonducella F.: A Threatened Medicinal Plant of Western Ghats

Aim: Caesalpinia bonducella Flem is a dioecious scrambling woody liana of Caesalpinoideae, a subfamily of Leguminosae. The plant is threatened and distributed in the deciduous forests of the Western Ghats of India. Being an important medicinal plant Caesalpinia bonducella F. attracted many scientists to exploit various activities associated with a number of phytoconstituents. The present study was undertaken to evaluate the most suitable media and suitable concentrations of plant growth regulators for in vitro Callogenesis and screening of antimicrobial activity of callus and seed of Caesalpinia bonducella.
 Materials and Methods: Callus was initiated from stem explants, on 1x and 0.5x MS medium plus supplements. The effects of plant growth regulators on callus cultures were studied and observations were made. The in vitro antibacterial activity was performed by using extracts of callus and seed of Caesalpinia bonducella in petroleum ether and methanol against multidrug resistance organisms. The organic extracts of seed and callus of the plant at concentrations of 0.02 mg/ml - 0.1 mg/ml were taken and their activities were measured. 
 Results: The combination of 2.5 mgL-1 2,4-D with 2 mgL-1 BAP, resulted in the highest frequency and the highest mean percentage of callus formation (2.35 ± 0.294) with yellow friable callus. The results revealed that all the extracts had a variable degree of antibacterial activity.
 Conclusions: It was observed that 2,4-D at 2.5 mgL-1 in combination with BAP,2.0 mgL-1 BAP resulted in early initiation, highest induction percentage, with frequency highest mean percentage of callus formation, Antimicrobial tests with methanol and diethyl ether extract of Caesalpinia bonducella seed powder against the clinical isolates showed the zone of inhibition for all the pathogens tested with concentration of methanolic extract of C. bonducella seed powder.

The Influence of Various Growth Regulator of Growth Media on Biomass and Callus Induction in Elephantopus scaber Linn

Elephantopus scaber has the potential to be developed and isolated of secondary metabolites by tissue culture techniques. Secondary metabolites of E. scaber include scabertopin, scabertropinol, luteolin, flavonoids, deoxyelephantopin, phenols, saponins, tannins, elephantopins which can be isolated from the callus, so that various growth media are used to get the largest E. scaber callus. The aim of this study is to describe the biomass and induction of E. scaber callus in various growth media in vitro (tissue culture techniques), and to determine the proper media to produce E. scaber callus biomass optimally. E. scaber culture experiments were conducted descriptively, using 5 growth media, namely 1) MS + BAP media, 2) MS + Kinetin media, 3) MS + 2.4 D media, with concentrations of BAP, Kinetin and 2.4 D 0.5; 1; 1.5; and 2 mg/L respectively, 4) MS + BAP + 2.4 D and 5) MS + Kinetin + 2.4 D, with the ratio of BAP + 2.4 D and Kin + 2.4 D are 0.5 + 0.5; 1 + 0.5; 1 + 1; 1.5 + 1.5 mg/L. Each medium contained 25 experimental bottles, each bottle are containing 5 E. scaber explants. Stages of research method are include explant sterilization, manufacture and sterilization of media, and planting explants into growth media. The research parameters are callus induction and callus biomass. The data were analyzed by quantitative descriptive. The results showed that the use of MS + 0.5 mg/L BAP growth media gave the fastest callus induction periode, 14 days after planting, while MS + 1.5 mg/L BAP + 1.5 mg/L 2.4 D growth media gave the largest callus biomass that is 0.0797 grams with a friable callus texture.

Growth kinetics, metabolite yield, and expression analysis of biosynthetic pathway genes in friable callus cell lines of Rhodiola imbricata (Edgew)

The plant cell culture provides an efficient technique for growth and production kinetics studies of bioactive compounds present in plants. Therefore, the present study investigated, for the first time, to identify the specific culture days required for higher metabolite yield in Rhodiola imbricata. A transcript-level gene expression study was conducted to understand the molecular cues associated with metabolite biosynthesis. The leaf callus cell line showed an optimum growth rate (FW: 20 g/100 mL, DW: 1.21 g/100 mL), growth index (19.00), salidroside (3.68 mg/g DW, rosavin (0.21 mg/g DW and rosarin (0.08 mg/g DW) on 24th day of the incubation period. However, salidroside, rosavin, and rosarin metabolite yield followed the growth-dependent manner in both callus cell lines, while tyrosol and p-coumaric acid showed growth independent accumulation pattern. Moreover, the antioxidant study displayed maximum phenolic content (8.07 mg/g DW) on day 18, flavonoid content (9.73 mg/g DW) on day 3, and DPPH activity (IC50; 0.79 mg/mL) on day 24 in leaf callus cell line. The RT-qPCR analysis showed upregulation of PAL, 4-HPAAS, 4-HPAAR, and UDPGT genes associated with metabolite accumulation. Expression analysis revealed a positive correlation with the metabolites yield. Furthermore, the optimized parameter could be exploited for a higher yield of specific metabolite and sequential scale-up studies. This is the first-ever study on the specific growth phase and accumulation pattern of metabolites in the friable callus cell lines of Rhodiola imbricata. Similarly, molecular signatures were also investigated to elucidate key genes associated with salidroside and rosavins biosynthesis. Furthermore, current optimized parameters lay the foundation for a cell suspension study, scale-up, and year-round metabolite production.

Evaluation of bioactive flavonols and ent-kaurenes in the 2,4-dichlorophenoxyacetic acid-induced calli of Senegalia nigrescens using FTIR and GC–MS

The increasing demand for plant-derived pharmacologically active compounds calls for sustainable alternative sources and conservation of medicinal plant species. To this end, the present study investigated the development of a callogenesis protocol for Senegalia nigrescens. The plant growth regulators (PGRs), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ) at concentrations of 0.5 and 1.0 mg L−1 were tested for callus induction in the leaf, stem, cotyledon and root explants of S. nigrescens. 2,4-D was the more effective PGR across the different explant sources with 85 to 100% stem, root and cotyledon explants forming callus (EFC). Hard and compact calli with nodular structures were obtained from the stem explants, the root-derived calli were soft and moist while the cotyledon explants produced glassy and friable calli. Methanol extracts of calli obtained from the stem and root explants were characterised using fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC–MS). The FTIR spectra and GC–MS profiles revealed the presence of the four compounds evaluated, quercetin, quercetin-3-O-methyl ether, ent-kaur-15-en-18-ol and ent-kaur-15-ene-18,20-diol in both 4 and 12-week-old, root-derived calli. This study, therefore, provides preliminary information for the propagation and phytochemical harvesting in S. nigrescens.

Analysis of Growth and Enzyme Contents of Papain Callus Papaya (Carica papaya L.) Through Tissue Culture Engineering with Saccharomyces cerevisiae Elicitors on MS and VW Media

Papaya plants are the main source of chemical compounds commonly used in the pharmaceutical industry and food additives. The use of papaya plants in both industries is due to the enzyme content of papain which contains bio-molecular protein compounds as a catalyst to accelerate inorganic chemical reactions either as a treatment or food treatment purpose. Papaya’s papain enzyme is a proteolytic enzyme that is secreted through the isolation of papaya’s sap. One of the efforts to increase papain content in papaya is through tissue culture using papaya leaf explants and elicitation techniques with the addition elicitor of Saccharomyces cerevisiae in MS and VW medium. This study to analyze: (1) papaya callus quantias with the added elicitor of S.cerevisiae on MS and VW medium; (2) compact or friable callus quality with the added elicitor of S.cerevisiae on MS and VW medium; (3) the results of the increase in the enzyme content of papain in papaya leaf callus in MS and VW media which had been added with S.cerevisiae elicitor. The research method used a completely randomized design (CRD) factorial of two factors with four samples and six replications consisting of four samples. Factorial treatment as follows: Factor I (Media) consisting of M1 (MS); M2 (VW). Factor II S.cerevisiae in MS and VW medium. The results showed that: (1) the treatment of MS medium and S.cerevisiae elicitor of 25 mg dry matter (DM)/L was able to produce the number of callus realization of other treatments; (2) All treatments of S.cerevisiae elicitor in medium produced compact callus quality; (3) Addition of S.cerevisiae elicitor of 25 mg DM/ L and VW medium as the highest producer of papain enzyme content in papaya leaf callus.

Establishment of a Cell Suspension Culture of Eysenhardtia platycarpa: Phytochemical Screening of Extracts and Evaluation of Antifungal Activity.

Eysenhardtia platycarpa (Fabaceae) is a medicinal plant used in Mexico. Biotechnological studies of its use are lacking. The objective of this work was to establish a cell suspension culture (CSC) of E. platycarpa, determine the phytochemical constituents by spectrophotometric and gas chromatography‒mass spectrometry (GC‒MS) methods, evaluate its antifungal activity, and compare them with the intact plant. Friable callus and CSC were established with 2 mg/L 1-naphthaleneacetic acid plus 0.1 mg/L kinetin. The highest total phenolics of CSC was 15.6 mg gallic acid equivalents (GAE)/g dry weight and the total flavonoids content ranged from 56.2 to 104.1 µg quercetin equivalents (QE)/g dry weight. The GC‒MS analysis showed that the dichloromethane extracts of CSC, sapwood, and heartwood have a high amount of hexadecanoic acid (22.3–35.3%) and steroids (13.5–14.7%). Heartwood and sapwood defatted hexane extracts have the highest amount of stigmasterol (~23.4%) and β-sitosterol (~43%), and leaf extracts presented β-amyrin (16.3%). Methanolic leaf extracts showed mostly sugars and some polyols, mainly D-pinitol (74.3%). Compared with the intact plant, dichloromethane and fatty hexane extracts of CSC exhibited percentages of inhibition higher for Sclerotium cepivorum: 71.5% and 62.0%, respectively. The maximum inhibition for Rhizoctonia solani was with fatty hexane extracts of the sapwood (51.4%). Our study suggests that CSC extracts could be used as a possible complementary alternative to synthetic fungicides.

Effects of Plant Growth Regulators and Sugars on Ehretia asperula Zoll. et Mor. Cell Cultures

Background: Belonging to the Boraginacae family, Ehretia asperula Zoll. et Mor., called “Xa den”, is a precious medicinal plant also known as the “cancer tree” by the Muong ethnic group in Hoa Binh Province, Vietnam. Xa den has been demonstrated to inhibit the development of malignant tumors, reduce oxidation and enhance the human immune system. This research focused on examining friable callus induction from young stems of Ehretia asperula Zoll. et Mor. Methods: Samples of Xa den were less than two years old. Young stems with 2 to 6 leaves served as explants for callus induction. Explants placed on autoclaved B5 nutrients incubated at 25oC, in the dark. The testing factors were concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D) and Benzyl adenine (BA), types and concentrations of sugars.Result: Friable callus was induced on B5 medium with 0.4 mg/L of 2,4-D, 0.1 mg/L of BA and 30 g/L of glucose at the highest rate (100%). Additionally, callus grew best after 5 weeks of culture weighing 0.194 g. Friable callus was used as material for cell suspension cultures. After two weeks, the Xa den cell suspension cultures contained single cells and small cell clumps. The liquid medium had changed from dark yellow to light brown.

Enhanced gymnemic acids production in cell suspension cultures of Gymnema sylvestre (Retz.) R.Br. ex Sm. through elicitation

Gymnemic Acids (GAs) belong to the class of triterpenoid saponins present majorly in the leaves of Gymnema sylvestre. These bioactive compounds are responsible for hypoglycemic activity. Therefore, it is expansively used in Ayurveda treatment for diabetes. This study is aimed to develop an effective method for suspension culture and to investigate the effect of sodium nitroprusside (SNP), salicylic acid (SA) and methyl jasmonate (MeJA) on the enhancement of deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII in G. sylvestre cell suspension culture. To induce callus, in vitro-derived leaf explants were cultivated on Murashige and Skoog's (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene-acetic acid (NAA) either alone or in combination with 6-benzylaminopurine (BAP) or kinetin (Kin) at 1.0 to 5.0 mg/L. The highest callus initiation frequency (95.30 ± 1.40%) and better friable callus observed on MS medium supplemented with 3.0 mg/L (2,4-D) + 1.0 mg/L (Kin). Optimized callus induction medium was used to develop cell suspension culture, growth kinetic study, elicitors treatment and quantify the production of major bioactive compounds. At 40 days, the highest fresh cell suspension culture biomass was 339.08 g/L fresh weight (FW). Biomass production, 366.68 g/L FW and 30.30 g/L DW were the highest in the treatment of 20 μM SNP among the tested elicitors. The highest accumulation of deacylgymnemic acid (2936.90 μg/g DW, 96 h treatment) and gymnemic acid XVII (2414.20 μg/g dry weight (DW), 48 h treatment) were obtained in 20 μM SNP treatments which were 6.1 and 5.2 fold higher than the control. Furthermore, the highest values of gymnemagenin (1179.77 μg/g DW at 50 μM treatment) and gymnemic acid IV (731.04 μg/g DW at 10 μM treatment) were obtained in SA treatment which was 4.12 and 5.11 fold greater than the control was studied through our newly developed HPLC method. The findings of the present study showed that the SNP could be a good strategy for large-scale industrial production of triterpenoid saponins in G. sylvestre cell suspension cultures.

Explants selection for in vitro propagation of Pachyrhizus erosus L.

Pachyrhizus erosus tuber is rich in protein asides its agronomical value as a legume, but the seeds by which it is propagated have very low viability. This study established sterilization protocol and effect of various concentrations of auxins and cytokinins on callus production and shoot regeneration from explants of P. erosus. Explants and seeds were sterilized using sodiumhypochlorite (NaClO) solution (5, 10 and 15% v/v) for 5 and 10 mins. Nodal, stem and leaf explants from in vitro germinated P. erosus and tuber from field grown plant were sterilized and cultured on Murashige and Skoog (MS) medium (control) and MS combined with different concentrations of auxins (NAA and 2, 4-D) and cytokinin (BA and Kinetin) and the cultured explants were monitored in terms of degree of callus formation, morphology and colour of callus and also for shoot induction. The results showed that seeds of P. erosus sterilized with 10% NaClO solution for 10 mins and germinated in vitro is the best way of getting sterile nodal, stem and leaf explants for the in vitro propagation of the plant, while tuber explants could be sterilized with 15% NaClO for 10 minutes. Nodal explants inoculated in MS medium supplemented with 1.0 mg/L BA gave the highest shoot regeneration response, while stem explants inoculated on MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA also gave the highest amount of friable callus. The study concluded that in vitro germinated seeds were the best way of getting explant for P. erosus.

An efficient protocol for mass multiplication of Centella asiatica (L.) Urban and determination of its phenolic content

The present study was focused on standardizing a protocol for callus induction as well as regeneration in Centella asiatica from leaf and stem as explants. Stem and leaf explants have been inoculated in B5 media supplemented with BAP (0.1-2.5 mg/l), kn (01-04 mg/l) and NAA (0.1-0.5 mg/l), 2, 4-D (0.2mg/l) for callus induction. The combination of BAP and NAA leads to the formation of green, brown, compact and friable calli while Kn and 2, 4-D induced brown calli. Highest shooting was obtained from BAP (1.5 mg/l) and NAA(0.5 mg/l).When the shoots were inoculated in half strength of B5 media fortified with 0.1 mg/l BAP and 0.5 mg/l NAA showed cent percent rooting with the highest number of roots per shoot (11.05 cm) and maximum root length (1.86 cm). Stem showed the best explants for callus induction as compared to leaf explants. A low concentration of plant growth regulators was unable to induced callus response in leaf and stem explants. Phenolic content analysis showed that calluses contain more amounts of phenol (0.81 mg/gmdw) as compared to both leaf (0.63 mg/gmdw) and stem (0.59 mg/gmdw) explants.

Mass propagation through direct and indirect organogenesis in three species of genus Zephyranthes and ploidy assessment of regenerants through flow cytometry.

Genus Zephyranthes consists of economically important plant species due to their high ornamental value and presence of valuable bioactive compounds. However, this genus propagates by asexual division only which gives slow propagation rate. Plant tissue culture has the potential to provide efficient techniques for rapid multiplication and genetic improvement of the genus. In this work, a dual in vitro regeneration system through callus mediated shoot regeneration and direct shoot regeneration in species Zephyranthes candida, Zephyranthes grandiflora and Zephyranthes citrina was investigated. Bulb, leaf and root explants were cultured on Murashige and Skoog (MS) medium amended with different plant growth regulators (PGR's) viz. 2,4-dichlorophenoxyacetic acid (2,4-D), 1-Naphthalene acetic acid (NAA), 6-benzyl amino purine (BAP), N-phenyl-N'-1,2,3 -thiadiazol-5-ylurea (TDZ), 6-Furfuryl- aminopurine (KIN) alone or in combinations for callus induction and regeneration. Only bulb explants showed callus induction and regeneration response on different PGR combinations with a varied response in callus induction percentage, callus color and callus texture. Creamish compact callus (CC) was induced on 2 mg L[Formula: see text] 2,4-D, brown friable callus (BF) on 2 mg L[Formula: see text] NAA + 1 mg L[Formula: see text] BAP and green friable callus (GF) callus on 1 mg L[Formula: see text] KIN + 3 mg L[Formula: see text] NAA. The maximum shoot multiplication from different callus types (indirect organogenesis) was achieved on 2 mg L[Formula: see text] BAP alone without combinations. Bulb explants of Z. grandiflora induced maximum callus induction percentage (86.4%) and shoot regeneration percentage (83.5%) with the maximum 08 shoots per 150 mg callus mass. The induction and regeneration response was followed in the order of Z. grandiflora > Z. candida > Z. citrina. Similarly, maximum direct organogenesis from bulb explants was obtained in Z. grandiflora (93.3%) followed by Z. candida (91.5%) and Z. citrina (90.4%) on 3 mg L[Formula: see text] TDZ amended MS media. Adventitious root induction was achieved on 2 mg L[Formula: see text] IBA with a maximum of 8 roots per shoot. The in vitro raised plantlets were successfully acclimatized in the field with 85% survival efficiency. The genome size (2C DNA content) of the field-grown plants and in vitro regenerated plants, evaluated through flow cytometry technique, were similar and showed no ploidy changes. An efficient mass propagation protocol was established for obtaining plants with unaltered genome size in the three species of Zephyranthes.