Maximum Growth Index Research Articles

Biology of <i>Pieris brassicae</i> (L.) on Cauliflower

Biology of Pieris brassicae (L.) was studied on cauliflower under screen house conditions on main and late season crops during 2021-22 in PAU, Ludhiana, Punjab. The total lifecycle was maximum for both male (50.11± 1.30) and female (52.24± 1.27) in the main season crop and minimum in the late season crop. Survival was maximum (27.67± 0.04%) in main season crop than (25.4± 0.06) in late season crop due to colder climatic conditions. The maximum growth index was observed in late season crop (1.54± 0.14) as compared to main season crop (1.13± 0.09) which indicated that P. brassicae developed better on late season crops.

Effect of seaweed elicitation and culture conditions on biomass and metabolite production in in vitro shoots of Withania somnifera

Withania somnifera is an important medicinal plant used in various herbal preparations due the presence of its key metabolites-withanolides. The present study focuses on the effect of seaweed extract and different in vitro culture conditions on the biomass and key metabolite content in in vitro shoot cultures of Withania somnifera . The commercially available seaweed extract powder of Kappaphycus alverizii with the trade name ’Magic vita - P’ was examined for its effect on solid, suspension and bioreactor cultures of Withania somnifera. The standardisation of elicitation conditions showed the exposure of shoot cultures to 0.25% seaweed extract for 7 days resulted in maximum biomass and withaferin A content which were 1.91 fold and 3.05 fold higher than that of untreated shoots. Further among the three culture conditions studied, the bioreactor cultures exhibited maximum growth index compared to the solid and suspension cultures thus producing maximum biomass which in turn resulted in higher accumulation of withanolides per batch of culture. Thus the seaweed treated shoots in bioreactor yielded 18.56, 8.96, and 7.29 fold increase in withaferin A, withanolide A and withanone content compared to control shoots in solid media. Further the effect of this elicitation and culture conditions on the accumulation pattern of the primary metabolites were studied using GC-MS. The Principal Component Analysis (PCA) of the GC-MS data exhibited a clear distinction between the control and seaweed treated samples. Thus our findings indicate a clear protocol for scaling up of withanolide production from in vitro shoot cultures of Withania somnifera . Withania somnifera being a traditional medicinal plant with wide array of medicinal properties poses a great demand in the international market. Hence there arises a need for alternate culture techniques to meet its rising demands which is still increasing past the COVID-19 pandemic. Thus this paper focuses on in vitro technique for mass production of Withania somnifera shoots. This main highlights of the paper includes: • Effect of a seaweed (organic fertilizer) on biomass and withanolides production in in vitro shoots of Withania somnifera. • The influence of various culture techniques on the biomass and metabolite production in in vitro shoots of Withania somnifera. • Correlation among the primary metabolites as obtained from the GC-MS analysis with that of withanolide content obtained using HPTLC technique.

Improved accumulation of betulin and betulinic acid in cell suspension culture of Betula pendula roth by abiotic and biotic elicitors

Betulin (B) and betulinic acid (BA) are two triterpenes with diverse pharmacological and physiological actions. Elicitation of Betula pendula Roth cell cultures by elicitors is an excellent strategy to increase B and BA levels. Six abiotic and biotic elicitors were studied to improve accumulation of B and BA in the cell culture of B. pendula. The B and BA production in treated cells was verified by HPLC. The results showed the maximum growth index (7) on day 3 in cells treated with 0.5 mg L−1 chlorocholine chloride (CCC). The increased accumulation of BA in the cells treated with 200 mg L−1 of chitosan was found to be 5.9 × (6.5 mg g−1 DW) higher over control cells. Treating the cells with 2 mg L−1 of CCC, after 7 days, led to 149.3× enhancement of B content (19.4 mg g−1 DW) over the controls. Production of this triterpenoid at a much shorter time with a much higher growth rate can be economic and lead to producing large amounts of B and BA for anti-cancer and HIV drugs preparation.

Effect of elicitation and precursor feeding on accumulation of 20-hydroxyecdysone in Achyranthes aspera Linn. cell suspension cultures.

20-Hydroxyecdysone is one of the most common ecdysteroids in plants with potential therapeutic applications. In this study, cell suspension cultures of Achyranthes aspera were raised in shake flasks to investigate the production of 20-hydroxyecdysone. The quantification and characterization of 20-hydroxyecdysone in the cultures were done by High performance liquid chromatography (HPLC) and Liquid Chromatography-quadrupole time-of- flight mass spectrometry (LC-Q-TOF) analyses. For raising the suspension, calli initiated from in vitro grown leaf explants were cultured in liquid Murashige and Skoog (MS) medium augmented with combinations of 2, 4-dichlorophenoxyacetic acid (1mgL-1) and α-naphthaleneacetic acid (1mgL-1). Maximum growth index of the cell suspension was 9.9, which was achieved during 20th day of culture (final phase of exponential growth). At this stage, the biomass accumulated was 1.09±0.09g dry weight (DW) and the 20-hydroxyecdysone concentration was 0.24mgg-1 DW. Eliciting the cultures with 0.6mM Methyl jasmonate for 6days; enhanced the production of 20-hydroxyecdysone production to 0.35mgg-1 DW. By augmenting the cultures with the precursors namely cholesterol (10mgL-1) and 7-dehydrocholesterol (10mgL-1), production of 20-hydroxyecdysone was boosted to 0.31mgg-1 DW and 0.28mgg-1 DW respectively.

Picroside I and Picroside II from Tissue Cultures of Picrorhiza kurroa.

Background:Picrorhiza kurroa (PK) belongs to Scrophulariaceae family and is a representative endemic, medicinal herb, widely distributed throughout the higher altitudes of alpine Himalayas from west to east, between 3000 and 4500 m above mean sea level.Objective:The objective of the present study is to assess the production of picroside I and picroside II from tissue cultures of PK.Materials and Methods:Auxiliary shoot tips of PK were incubated in Murashige and Skoog medium supplemented with indole-3-butyric acid and kinetin phytohormones. The callus produced was collected at different time intervals and was processed for extraction of picroside I and picroside II followed by thin layer chromatography and high-performance liquid chromatography HPLC analysis.Results:The maximum growth index was found to be 5.109 ± 0.159 at 16-week-old callus culture. The estimation of picroside-I and picroside-II was carried out by (HPLC) analysis; quantity of secondary metabolite found to be 16.37 ± 0.0007 mg/g for PK-I and 6.34 ± 0.0012 mg/g for PK-II.Conclusion:This is the first attempt to produce the Picroside-I and II in large amount by the tissue culture technique. It can be observed that the method of callus culture can be used in production of secondary metabolites Picroside-I and II from PKSUMMARYPicrorhiza kurroa is a high value medicinal herb due to rich source of hepatoprotective metabolites, Picroside-I and Picroside-II. The medicinal importance of P. kurroa is due to its pharmacological properties like hepatoprotective, antioxidant (particularly in liver), antiallergic and antiasthamatic, anticancer activity particularly in liver and immunomodulatory. Shoot apices which were produced a good response was inoculated on selected medium i.e., on MS medium containing 2, 4 D (mg/l) + KN (1mg/l) for induction of callus. The initiation of callus was observed after 4weeks and it was light green and fragile Maximum growth was observed with 3% w/v of sucrose supplement. The callus culture was maintained and growth index was recorded after every subculture. The growth index was calculated from the obtained final dried weight divided by initial weight.Abbreviations Used: PK-Picrorhizakurroa, IBA-Indole-3-butyricacid, KN-Kinetin, 2,4D-2,4Dichlorophenoxy acetic acid.

Development of treated palm oil mill effluent (POME) culture medium for plant tissue culture of Hemianthus callitrichoides

Hemianthus callitrichoides is an ornamental aquatic plant that could be used for phytoremediation and aquarium decorations. In this study, POME culture medium was developed for the micropropagation of H. callitrichoides. The tissue culture of H. callitrichoides was carried out under temperature of 25°C with 24h of photoperiod for 15 days. Optimum strength of liquid MS medium for culturing the plant was determined. Liquid full strength MS medium had recorded maximum growth index of 7.34±0.37g/g in H. callitrichoides as compared to 1/2 MS, 1/4 MS and 1/8 MS. For different strength of POME culture medium, 1/2 strength POME had recorded highest growth index of 3.92±0.37g/g in H. callitrichoides as compared to full strength POME, 1/4 strength POME and 1/8 strength POME culture media. The MS components were introduced into POME culture medium to improve the propagation of H. callitrichoides. 1/2 strength POME medium with liquid full strength MS medium as supplements (Media E) was proven to be better than full strength MS medium (Media A) and other culture media due to its ability in suppressing the morphological changes that occurred in full strength MS medium and promote the maximum growth of H. callitrichoides. The reliance on treated POME as additional supplement towards the healthy growth of H. callitrichoides. Most importantly the availability of free treated POME do not contribute towards the additional cost of cultivation and prevent the reliance on high cost of commercial supplement.

Artificial neural network-based model for the prediction of optimal growth and culture conditions for maximum biomass accumulation in multiple shoot cultures of Centella asiatica.

An artificial neural network (ANN)-based modelling approach is used to determine the synergistic effect of five major components of growth medium (Mg, Cu, Zn, nitrate and sucrose) on improved in vitro biomass yield in multiple shoot cultures of Centella asiatica. The back propagation neural network (BPNN) was employed to predict optimal biomass accumulation in terms of growth index over a defined culture duration of 35days. The four variable concentrations of five media components, i.e. MgSO4 (0, 0.75, 1.5, 3.0mM), ZnSO4 (0, 15, 30, 60μM), CuSO4 (0, 0.05, 0.1, 0.2μM), NO3 (20, 30, 40, 60mM) and sucrose (1, 3, 5, 7%, w/v) were taken as inputs for the ANN model. The designed model was evaluated by performing three different sets of validation experiments that indicated a greater similarity between the target and predicted dataset. The results of the modelling experiment suggested that 1.5mM Mg, 30μM Zn, 0.1μM Cu, 40mM NO3 and 6% (w/v) sucrose were the respective optimal concentrations of the tested medium components for achieving maximum growth index of 1654.46 with high centelloside yield (62.37mg DW/culture) in the cultured multiple shoots. This study can facilitate the generation of higher biomass of uniform, clean, good quality C. asiatica herb that can efficiently be utilized by pharmaceutical industries.

Hairy root induction and polysaccharide production of medicinal plant Callerya speciosa Champ.

A reliable and efficient hairy root induction system for the important medicinal plant Callerya speciosa was established by optimizing several parameters that affected the efficiency of Agrobacterium rhizogenes-mediated transformations. Four strains, A4, LBA9402, R1601 and C58C1, and four types of explants, hypocotyls, cotyledons, leaves and excised stems, were examined and the highest rate of transformation (68.3 %) was obtained in cotyledons using strain LBA9402. Therefore, the optimum co-cultivation time and acetosyringone (AS) concentration were evaluated for LBA9402-infected cotyledons and determined to be 2 days and 100 µM AS, respectively, resulting in 64.4 and 66.7 % hairy root transformation efficiency rates, respectively. Transgenic hairy root lines were confirmed by PCR amplification and Southern blot. The growth trend of hairy roots was “slow-quick-slow” and peaked after 30 days of culturing, with increases of 11-fold and 11.4-fold of fresh and dry weights respectively, compared with initial inoculation. Hairy roots showed the ability to synthesize polysaccharides. The effects of different media Murashige and Skoog (MS), ½ MS, WPM and B5, and carbon sources, sucrose, glucose, fructose and maltose on the growth and polysaccharide production of hairy roots were also studied. The ½ MS medium was found to be superior, resulting in the highest accumulation of biomass (10.9 g L−1 fresh weight and 1.1 g L−1 dry weight), the maximum growth index (9.96) and polysaccharide content (22.56 mg g−1 dry weight). Sucrose at 30 g L−1 was optimal for both biomass accumulation and polysaccharide production.

In-vitro Callus Induction and Rosmarinic Acid Quantification in Callus Culture of Satureja khuzistanica Jamzad (Lamiaceae).

In the present study, an efficient protocol has been developed for callus induction and production of RA in callus culture of Satureja khuzistanica for the first time. In-vitro callus induction was achieved from young shoot tip explants cultured on MS and B5 media supplemented with different concentrations of IBA (0.1, 1.0, 2.0 and 5.0 mgL(-1)) solely or in combination with cytokinins BAP and KIN (1.0, 2.0 and 5.0 mgL(-1)). B5 medium supplemented with 1.0 mgL(-1) IBA plus 5.0 mgL(-1) BAP and MS medium fortified with 2.0 mgL(-1) IBA and 2.0 mgL(-1) BAP were the most favorable media for callus formation with the highest induction rate (96%). Maximum growth index (2.89 and 2.63) and maximum callus biomass (2.34 and 2.33 g fresh weight) were obtained from the callus cultured on B5 medium supplemented with 1.0 mgL(-1) IBA plus 5.0 mgL(-1) BAP and MS medium fortified with 1.0 mgL(-1) IBA plus 1.0 mgL(-1) KIN, respectively. Determination and quantification of RA in cultured calli were performed by HPLC UV/MS analysis. Calli induced from the plant and maintained on supplements of IBA and BAP in the absence of light produced RA 7.5% based on dry weight (DW). No differentiation was observed in any callus during the course of this study.

In Vitro Shoot Multiplication of Stevia and Assessment of Stevioside Content and Genetic Fidelity of the Regenerants

A reliable protocol for micropropagation was developed for an anti-diabetic medicinal plant species, Stevia rebaudiana. In the present investigation the highest percentage of response was shown by nodal segment (98.00 ± 1.22) and both MS full and half strength performed well and were almost equally effective. Callus along with multiple shoots was obtained from nodal segments of S. rebaudiana on half strength Murashige and Skoog (MS) media supplemented with various concentrations of benzyl amino purine (BAP) and indole-3-butyric acid (IBA) (0.2, 0.5, 1.0, 1.5 mg l−1 each). The in vitro shoots produced along with callus were re-cultured on one-fourth strength MS media supplemented with kinetin (Kn) (0.5 mg l−1), IBA (1.0 mg l−1), activated charcoal (50 mg l−1), polyvinylpolypyrrolidone (100 mg l−1) and gibberellic acid (1 mg l−1) to obtain optimum shoot regeneration. Shoot multiplication and simultaneous rooting of S. rebaudiana was observed. The results obtained showed multiple shooting (18–20) with shoot length (6.5–7.2 cm), number of leaves (42–50), number of roots (12–14) and root length (5.8–6.2 cm), after 4 weeks of re-culturing of callus. Among different auxins and cytokinins used for secondary callus formation in half strength MS media. 2, 4-D (0.5 mg l−1) showed maximum growth index (266.384), after 8 weeks of callus culture. In vitro raised plantlets were transferred for hardening in plastic pots containing garden soil and perlite (1:1). A total of 80 % survival rate was observed. Quantitative analysis of stevioside content of in vitro and field grown plants was carried out by HPLC which showed increase of about 2 % stevioside content in the regenerants as compared to field grown plants. The genetic fidelity of micropropagated plantlets was confirmed by ISSR analysis confirming that in vitro plants obtained from callus showed genetic variation during the period of culturing, whereas those from nodal segments did not. Overall, this system results in mass multiplication of S. rebaudiana with simultaneous production of callus within short period. The high stevioside content in micropropagated plants may be of immense use to the pharmaceutical industry.

Isolation, quantification and anti‑microbial screening of flavonoids from callus of Vitex negundo Linn

Context: Natural habitats for medicinal plant are disappearing fast and together with environmental instabilities, it is increasingly difficult to acquire plant‑derived compounds. This has prompted scientists to consider the possibilities of using cell cultures as an alternative supply for the production of plant natural products. Aim: The present investigation deals with the establishment of callus cultures from leaf explants of Vitex negundo, to extract flavonoids from the raised callus and to assess their anti‑microbial potential. Materials and Methods: Callus culture of Vitex negundo has been established on Murashige and Skoog’s medium supplemented with 2, 4‑D using leaf explants. Growth index (GI) was calculated for the tissues obtained after 2, 4, 6 and 8 weeks, respectively. Calli of 2, 4, 6 and 8 weeks old were used for extraction of total flavonoids (free and bound) using standard method. Flavonoid extract of 6 weeks old callus with maximum GI and maximum flavonoid content was then tested against Staphylococcus aureus, Agrobacterium tumefaciens (bacteria), Candida albicans (yeast) for their anti‑microbial potential. Minimum inhibitory concentration, minimum bactericidal/fungicidal concentrations and total activity were also evaluated. Results: GI was found maximum in 6 weeks old tissue (0.76). Total flavonoids from 6 week old tissue showed maximum zone of inhibition against A. tumefaciens. Kaempferol was identified from total flavonoids of 6 week old callus through, thin layer chromatography (TLC), preparative TLC, Melting point and Infra red spectral studies. Content of kaempferol in 6‑week‑old calli was also estimated using standard Spectrophotometric method, which came out to be 54.25 μg/mg d.wt (dry weight) of calli. Conclusion: The protocol of callus induction of V. negundo opens new vistas that could facilitate phytochemical production, extraction of pharmaceuticals and their screening against different human pathogens from the callus without harvesting the plant itself. Key words: Anti‑microbial potential, callus, flavonoids, Vitex negundo

Shoot culture of Bacopa monnieri: standardization of explant, vessels and bioreactor for growth and antioxidant capacity

Standardization of biomass production in different vessels and bioreactor using explants and media for growth, total phenolic content and antioxidant capacity of shoot culture of Bacopa monnieri is described. Maximum number of shoots per explant, higher explants response irrespective of the type of explants, and higher shoot length was obtained on MS medium containing BAP (2.5mgl(-1)) and IAA (0.01mgl(-1)) with 3% sucrose. This medium was selected by varying BAP concentration and recorded optimal for shoot culture on gelled medium. The condition of 0.5cm explant size and 20 explant/40ml (1 explant/2ml) was optimal for high explant response, number of shoots per explant regenerated and shoots length. Among the different vessels used, maximum growth index was achieved in Growtek bioreactor (10.0) followed by magenta box (9.16), industrial glass jar (7.7) and conical flask (7.2). The cultures grown in conical flask (100ml) were used as control. The total phenolic content and antioxidant capacity of in vitro grown plants was higher to that recorded for in vivo material. Among in vitro regenerated plants, the activity was maximal in the tissues grown in 250ml conical flask. The most critical function for vessels is to support the optimum profusion (growing area for maximum growth) of shoots and for B. monnieri, Growtek bioreactor supported 1980 shoots l(-1) medium as compared to control (938 shoots l(-1)). Growtek bioreactor was considered effective system to produce B. monnieri biomass in culture without loss of antioxidant properties.

Enhancement of Growth and Coumarin Production in Hairy Root Cultures of Witloof Chicory (Cichorium intybus L.cv.Lucknow local) under the Influence of Fungal Elicitors.

Studies were conducted to determine the possible production of coumarins in hairy root cultures of Cichorium intybus L.cv. Lucknow local under the influence of microbial agents. Pythium aphanidermatum and Phytopthora parasitica var. nicotiana were cultured and their mycelial and medium filtrate were used for the elicitation of coumarin production. The media filtrate of P. parasitica at 1.0% v/v added to the MS basal medium led to the maximum growth of hairy roots of C. intybus, which was 1.57-fold higher than the untreated control on the 28th day of culture, along with maximum esculin and esculetin yields which were 4.06- and 3.71-fold higher than the control on the 28th day. The yields of esculin and esculetin in hairy root cultures of chicory strongly correlated with growth. To check the effect of these fungal elicitors on endogenous polyamine metabolism, titers of total endogenous polyamines were analyzed. It was confirmed that the media filtrate of P. parasitica at 1.0% v/v concentration resulted in maximum accumulation of total endogenous polyamines, wherein endogenous spermine titers were found to be maximum as compared to endogenous spermidine and putrescine titers on the 28th day. Total endogenous spermine in the case of 1.0% MF v/v P. parasitica was 1.3-fold higher than that of the control on the 28th day of culture. Maximum growth index with greater length of primary root (17.61+/-0.18 cm) and greater number of secondary and tertiary roots was recorded for the medium filtrate of P. parasitica at 1.0% v/v concentration. This study also provided an insight into the morphological changes in terms of branching patterns, occurring in roots under the influence of these fungal elicitors.

Enhancement of growth and coumarin production in hairy root cultures of witloof chicory ( Cichorium intybus L.cv.Lucknow local) under the influence of fungal elicitors

Studies were conducted to determine the possible production of coumarins in hairy root cultures of Cichorium intybus L.cv.Lucknow local under the influence of microbial agents. Pythium aphanidermatum and Phytopthora parasitica var. nicotiana were cultured and their mycelial and medium filtrate were used for the elicitation of coumarin production. The media filtrate of P. parasitica at 1.0% v/v added to the MS basal medium led to the maximum growth of hairy roots of C. intybus, which was 1.57-fold higher than the untreated control on the 28th day of culture, along with maximum esculin and esculetin yields which were 4.06- and 3.71-fold higher than the control on the 28th day. The yields of esculin and esculetin in hairy root cultures of chicory strongly correlated with growth. To check the effect of these fungal elicitors on endogenous polyamine metabolism, titers of total endogenous polyamines were analyzed. It was confirmed that the media filtrate of P. parasitica at 1.0% v/v concentration resulted in maximum accumulation of total endogenous polyamines, wherein endogenous spermine titers were found to be maximum as compared to endogenous spermidine and putrescine titers on the 28th day. Total endogenous spermine in the case of 1.0% MF v/v P. parasitica was 1.3-fold higher than that of the control on the 28th day of culture. Maximum growth index with greater length of primary root (17.61±0.18 cm) and greater number of secondary and tertiary roots was recorded for the medium filtrate of P. parasitica at 1.0% v/v concentration. This study also provided an insight into the morphological changes in terms of branching patterns, occurring in roots under the influence of these fungal elicitors.

Plantlet regeneration and bulbil formation in vitro from leaf and stem explants of Curculigo orchioides, an endangered medicinal plant

A method has been developed for rapid multiplication of Curculigo orchioides (Hypoxidaceae) through direct organogenesis and bulbil formation in vitro. Leaf and underground stem explants produced maximum number of shoots (four and 10 per explant, respectively) on B5 medium supplemented with 4.4 μM BAP. Higher concentrations of BAP (≥22 μM) in the medium completely inhibited the growth and shoot proliferation from the leaf explants. Stem-disc-derived callus produced numerous bulbils on the medium containing BAP (8.8–35.2 μM) and sucrose (0.12–0.23 M). Maximum number of bulbils (10 per explant, 0.19 g fresh weight per bulbil) were recorded on the medium supplemented with 35.2 μM BAP and 0.18 M sucrose. Isolated bulbils increased in size (maximum growth index 36.4) and produced secondary bulbils on transfer to medium of the same composition. A maximum number of secondary bulbils (six per bulbil) were produced on the medium containing 35.2 μM BAP and 0.23 M sucrose. Growth of in vitro produced bulbils into plantlets and rhizogenesis from isolated shoots was achieved on the medium devoid of plant growth regulators. Small plantlets developed in vitro were successfully (90% survival) transferred to the soil. Plantlets sprouted in the next season (June 1997) after over-wintering.

Isolator component responsible for inhibition of Mycobacterium avium-M. intracellulare in BACTEC 12B medium.

We previously reported an inhibitory effect on Mycobacterium avium-M. intracellulare (MAI) when blood collected and processed with the Isolator system was placed in BACTEC 12B bottles for radiometric monitoring. We sought to identify the specific component(s) of the Isolator lysis-anticoagulant reagent (LAR) responsible for the inhibitory effect. We added the three components of the LAR, saponin, polyanetholesulfonate, and polypropylene glycol (PPG), to triplicate sets of BACTEC bottles separately and in various combinations. These bottles were then seeded with 10(2) CFU of MAI. The growth index (GI) was observed over a 42-day period and was compared with the GI for a growth control bottle set with no reagents and with the GI for a bottle set containing an equivalent volume of LAR. Growth in all growth control bottles and those bottles containing no PPG reached the maximum GI (GI = 999) within 8 to 10 days. Growth in bottles containing PPG never reached the maximum GI before day 14. In addition, the GIs of one bottle containing all three reagent components as well as all of the bottles containing actual Isolator LAR failed to reach the maximum within 42 days. Our data suggest that PPG is the main cause of the inhibitory effect, but that a secondary synergistic interaction between all three of the reagents may also be present.