Molecular and Biochemical Characterization in Rauvolfia tetraphylla Plantlets Grown from Synthetic Seeds Following In Vitro Cold Storage

Synseed technology is one of the most important applications of plant biotechnology for in vitro conservation and regeneration of medicinal and aromatic plants. In the present investigation, synseeds of Rauvolfia tetraphylla were produced using in vitro-proliferated shoots upon complexation of 3 % sodium alginate and 100 mM CaCl₂. The encapsulated buds were stored at 4, 8, 12, and 16 °C and high conversion was observed in synseeds stored at 4 °C for 4 weeks. The effect of different medium strength on in vitro conversion response of synseed was evaluated and the maximum conversion (80.6 %) into plantlets was recorded on half-strength woody plant medium supplemented with 7.5 μM 6-benzyladenine and 2.5 μM α-naphthalene acetic acid after 8 weeks of culture. Plantlets with well-developed shoot and roots were hardened and successfully transplanted in field condition. After 4 weeks of transfer to ex vitro conditions, the performance of synseed-derived plantlets was evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo-grown plants. Short-term storage of synthetic seeds at low temperature had no negative impact on physiological and biochemical profile of the plants that survived the storage process. Furthermore, clonal fidelity of synseed-derived plantlets was also assessed and compared with mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeats analysis. No changes in molecular profiles were found among the regenerated plantlets and comparable to mother plant, which confirm the genetic stability among clones. This synseed protocol could be useful for in vitro clonal multiplication, conservation, and short-term storage and exchange of germplasm of this antihypertensive drug-producing plant.     

Assessment of genetic fidelity in Rauvolfia serpentina plantlets grown from synthetic (encapsulated) seeds following in vitro storage at 4 °C

An efficient method was developed for plant regeneration and establishment from alginate encapsulated synthetic seeds of Rauvolfia serpentina. Synthetic seeds were produced using in vitro proliferated microshoots upon complexation of 3% sodium alginate prepared in Llyod and McCown woody plant medium (WPM) and 100 mM calcium chloride. Re-growth ability of encapsulated nodal segments was evaluated after storage at 4 °C for 0, 1, 2, 4, 6 and 8 weeks and compared with non-encapsulated buds. Effects of different media viz; Murashige and Skoog medium; Lloyd and McCown woody Plant medium, Gamborg’s B5 medium and Schenk and Hildebrandt medium was also investigated for conversion into plantlets. The maximum frequency of conversion into plantlets from encapsulated nodal segments stored at 4 °C for 4 weeks was achieved on woody plant medium supplement with 5.0 μM BA and 1.0 μM NAA. Rooting in plantlets was achieved in half-strength Murashige and Skoog liquid medium containing 0.5 μM indole-3-acetic acid (IAA) on filter paper bridges. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plant. The genetic fidelity of Rauvolfia clones raised from synthetic seeds following four weeks of storage at 4 °C were assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. All the RAPD and ISSR profiles from generated plantlets were monomorphic and comparable to the mother plant, which confirms the genetic stability among the clones. This synseed protocol could be useful for establishing a particular system for conservation, short-term storage and production of genetically identical and stable plants before it is released for commercial purposes.     

Somatic Embryogenesis and Synthetic Seed Production—a Biotechnological Approach for True-to-Type Propagation and In Vitro Conservation of an Ornamental Bulbaceous Plant Drimiopsis kirkii Baker.

An efficient plant regeneration protocol through indirect somatic embryogenesis pathway via callus had been developed from the leaf explant of an ornamental bulbaceous plant Drimiopsis kirkii. Optimum friable calli were induced on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/l of 2,4-dichlorophenoxyacetic acid and 1.0 mg/l of α-naphthalene acetic acid (NAA). On subculturing the callus on MS medium supplemented with 2.5 mg/l of thidiazuron (TDZ), 73.3 % of the cultures responded with 20.4?±?0.3 somatic embryos (SEs) per 500 mg callus at different stages of development after 6 weeks of culture. The highest response of 86.7 % with 28.3?±?0.5 embryos per 500 mg callus was observed on MS medium supplemented with 2.5 mg/l TDZ and 1.0 mg/l NAA. SEs were encapsulated in calcium alginate beads for the production of synthetic seeds (SSs) and their storability was investigated. The highest SS germination (93.3 %) was observed in 1.0 % sodium alginate followed by 86.7 % germination with 2.5 % sodium alginate. The SSs were stored at three different temperatures (4, 15, and 24?ºC) up to 6 months. The SSs kept at 15 °C showed 64.4 % germinability even after 4 months of storage. Both nonencapsulated and encapsulated SE-derived plants were successfully transferred to soil with 93.3 and 88.3 % survival rate accordingly. Randomly amplified polymorphic DNA (RAPD) analysis revealed that there were no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirmed the most reliable methods, which can be further used for genetic transformation studies as well as for mass propagation of ornamental D. kirkii at a commercial level.     

Improved Protocol for Somatic Embryogenesis and Calcium Alginate Encapsulation in Anethum graveolens L.: A Medicinal Herb

An improved procedure has been developed for efficient somatic embryogenesis in Anethum graveolens. Green friable embryogenic callus was obtained from hypocotyl segments on medium augmented with 2,4-dichlorophenoxyacetic acid (2,4-D). The highest embryogenic callus induction frequency of 87 % was obtained on Murashige and Skoog (MS) medium containing 1.13 μM 2,4-D. At lower concentration of 2,4-D (0.34 μM) callus turned dark in color and slow growing. Embryogenic cultures (76 %) responded with a mean number of 43 globular and 18 heart stage embryos. Somatic embryo maturation and subsequent conversion into plantlets took place on MS lacking growth regulators. Maximum number of somatic embryos developed on MS medium was 128.3 (per flask) and a plantlet conversion of 82 % was observed. Calcium alginate beads were produced by encapsulating somatic embryos. Highest percent germination (83 %) was observed on 0.8 % agar solidified MS medium with the plantlets acquiring an average length of 2.1 cm. Encapsulated somatic embryos could be stored at 4 °C up to 60 days with a conversion frequency of 49.3 %. Highest protein and proline content has been observed in embryogenic callus with small globular embryos. During morphological differentiation of the somatic embryos, changes in the antioxidant enzymatic system were observed. Superoxide dismutase (SOD) activity increased during initial stages and decreased catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) activities were detected.     

In Vitro Propagation,Encapsulation, and Genetic Fidelity Analysis of Terminalia arjuna: a Cardioprotective Medicinal Tree

The present study described an improved and reproducible in vitro regeneration system for Terminalia arjuna using nodal segment explants obtained from a mature plant. Shoot tips excised from in vitro proliferated shoots were encapsulated in 3 % sodium alginate and 100 mM CaCl₂?2H₂O for the development of synthetic seeds which may be applicable in short-term storage and germplasm exchange of elite genotype. Shoot multiplication was significantly influenced by a number of factors, namely types and concentrations of plant growth regulators, medium composition, repeated transfer of mother explants, subculturing of in vitro regenerated shoot clumps, agar concentrations, and temperature. Maximum numbers of shoots (16.50?±?3.67) were observed on modified Murashige and Skoog (MMS) medium containing 0.5 mg l^?1 of benzylaminopurine (BAP) and 0.1 mg l^?1 of naphthalene acetic acid (NAA). To shortening the regeneration pathway, rooting of micropropagated shoots under in vitro condition was excluded and an experiment on ex vitro rooting was conducted and it was observed that the highest percentage of shoots rooted ex vitro when treated with indole-3-butyric acid (IBA, 250 mg l^?1)?+?2-naphthoxy acetic acid (NOA, 250 mg l^?1) for 5 min. The well-developed ex vitro rooted shoots were acclimatized successfully in soilrite under greenhouse conditions with 80 % survival of plants. Randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants were genetically identical to the mother plant, suggesting the absence of detectable genetic variation in the regenerated plantlets. To the best of our knowledge, this is the first report on synthetic seed production as well as ex vitro rooting and genetic fidelity assessment of micropropagated shoots of T. arjuna.     

Piper nigrum: Micropropagation, Antioxidative enzyme activities, and Chromatographic Fingerprint Analysis for Quality Control

A reliable in vitro regeneration system for the economical and medicinally important Piper nigrum L. has been established. Callus and shoot regeneration was encouraged from leaf portions on Murashige and Skoog (MS) medium augmented with varied concentrations of plant growth regulators. A higher callus production (90 %) was observed in explants incubated on MS medium incorporated with 1.0 mg?L^?1 6-benzyladenine (BA) along with 0.5 mg?L^?1 gibberellic acid after 4 weeks of culture. Moreover, a callogenic response of 85 % was also recorded for 1.0 mg?L^?1 BA in combination with 0.25 mg?L^?1 α-naphthalene acetic acid (NAA) and 0.25 mg?L^?1 2,4-dichlorophenoxyacetic acid or 0.5 mg?L^?1 indole butyric acid (IBA) along with 0.25 mg?L^?1 NAA and indole acetic acid. Subsequent sub-culturing of callus after 4 weeks of culture onto MS medium supplemented with 1.5 mg?L^?1 thiodiazoran or 1.5 mg?L^?1 IBA induced 100 % shoot response. Rooted plantlets were achieved on medium containing varied concentrations of auxins. The antioxidative enzyme activities [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] revealed that significantly higher SOD was observed in regenerated plantlets than in other tissues. However, POD, CAT, and APX were higher in callus than in other tissues. A high-performance liquid chromatography (HPLC) fingerprint analysis protocol was established for quality control in different in vitro-regenerated tissues of P. nigrum L. During analysis, most of the common peaks represent the active principle “piperine.” The chemical contents, especially piperine, showed variation from callus culture to whole plantlet regeneration. Based on the deviation in chromatographic peaks, the in vitro-regenerated plantlets exhibit a nearly similar piperine profile to acclimated plantlets. The in vitro regeneration system and HPLC fingerprint analysis established here brought a novel approach to the quality control of in vitro plantlets, producing metabolites of interest with substantial applications for the conservation of germplasm.     

High Frequency Shoots Regeneration for Mass Multiplication of Phyllanthus fraternus Webster—An Important Antiviral and Hepatoprotective Plant

An efficient, rapid, and highly reproducible regeneration protocol was successfully developed for Phyllanthus fraternus from the field-derived mature nodal segments. The explants induced multiple shoots on cytokinin containing medium. The highest frequency (99 %) and maximum number of shoots (19.75) were induced on Murashige and Skoog’s (MS) medium supplemented with 2.22 μM 6-benzylaminopurine after 3–4 weeks of culture initiation. The elongated shoots were rooted on MS medium supplemented with indol-3-butyric acid (IBA) or α-naphthalene acetic acid. Pulse treatment of microshoots promoted significant increase in the percentage of rooting and number of root regeneration per shoot. The highest rooting (100 %) and maximum number of roots (8.75) per shoot was obtained when shoots were dipped in IBA solution (0.98 mM) for 5 min and further subcultured on MS basal medium. Plantlets were successfully acclimatized and established in soil. Regenerated plants were grown normally in the field without showing any morphological variations. This cost-effective protocol will help the mass multiplication of P. fraternus for commercial propagation and high biomass production of this valuable medicinal plant.     

Effect of Adenine Sulphate Interaction on Growth and Development of Shoot Regeneration and Inhibition of Shoot Tip Necrosis Under In Vitro Condition in Adult Syzygium cumini L.—a Multipurpose Tree

An efficient method for cloning Syzygium cumini (above 40 years old) through mature nodal segments has been successfully developed and that could be exploited for large-scale production of this valuable multipurpose tree. Nodal segments from mature tree were taken as explants and cultured on MS basal medium with different cytokinins (BA, Kin, AdS). The application of BA proved to be the best responsive cytokinin for the induction of shoot buds and shoots, but the proliferated shoots exhibited slower and stunted growth accompanied with abscission of leaves and shoot tip necrosis (STN). The problem of leaf abscission and STN was considerably reduced by the application of an adjuvant, adenine sulphate (AdS) in the optimal medium which led to the production of a maximum of 14 shoots. Further improvement in shoot bud regeneration and improved growth pattern of the regenerating tissue was obtained on the media comprised of MS?+?BA (10 μM)?+?GA₃ (2.5 μM). A total number of 15 shoots with mean shoot length of 5.9 cm was obtained. The healthy elongated shoots were then rooted on MS basal augmented with NAA (5 μM). The plantlets obtained were healthy and were successfully acclimatized and transferred under field condition with 70 % survival rate.     

In Vitro Propagation and Assessment of the Genetic Fidelity of Musa acuminata (AAA) cv. Vaibalhla Derived from Immature Male Flowers

An efficient in vitro propagation method has been developed for the first time for Musa acuminata (AAA) cv. Vaibalhla, an economically important banana cultivar of Mizoram, India. Immature male flowers were used as explants. Murashige and Skoog’s (MS) medium supplemented with plant growth regulators (PGRs) were used for the regeneration process. Out of different PGR combinations, MS medium supplemented with 2 mg L^?1 6-benzylaminopurine (BAP) + 0.5 mg L^?1 α-naphthalene acetic acid (NAA) was optimal for production of white bud-like structures (WBLS). On this medium, explants produced the highest number of buds per explant (4.30). The highest percentage (77.77) and number (3.51) of shoot formation from each explants was observed in MS medium supplemented with 2 mg L^?1 kinetin + 0.5 mg L^?1 NAA. While MS medium supplemented with a combination of 2 mg L^?1 BAP + 0.5 mg L^?1 NAA showed the maximum shoot length (14.44 cm). Rooting efficiency of the shoots was highest in the MS basal medium without any PGRs. The plantlets were hardened successfully in the greenhouse with 96 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro regenerated plantlets of M. acuminata (AAA) cv. Vaibalhla. Eight RAPD and 8 ISSR primers were successfully used for the analysis from the 40 RAPD and 30 ISSR primers screened initially. The amplified products were monomorphic across all the regenerated plants and were similar to the mother plant. The present standardised protocol will find application in mass production, conservation and genetic transformation studies of this commercially important banana.     

An Improved Micropropagation of Arnebia hispidissima (Lehm.) DC. and Assessment of Genetic Fidelity of Micropropagated Plants Using DNA-Based Molecular Markers

An efficient and improved in vitro propagation method has been developed for Arnebia hispidissima, a medicinally and pharmaceutically important plant species of arid and semiarid regions. Nodal segments (3–4 cm) with two to three nodes obtained from field grown plants were used as explants for shoot proliferation. Murashige and Skoog’s (MS) medium supplemented with cytokinins with or without indole-3-acetic acid (IAA) or naphthalene acetic acid was used for shoot multiplication. Out of different PGRs combinations, MS medium containing 0.5 mg l^?1 6-benzylaminopurine and 0.1 mg l^?1 IAA was optimal for shoot multiplication. On this medium, explants produced the highest number of shoots (47.50?±?0.38). About 90 % of shoots rooted ex vitro on sterile soilrite under the greenhouse condition when the base (2–4 mm) of shoots was treated with 300 mg l^?1 of indole-3-butyric acid for 5 min. The plantlets were hardened successfully in the greenhouse with 85–90 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro-regenerated plants of A. hispidissima. Out of 40 (25 RAPD and 15 ISSR) primers screened, 15 RAPD and 7 ISSR primers produced a total number of 111 (77 RAPD and 34 ISSR) reproducible amplicons. The amplified products were monomorphic across all the micropropagated plants and were similar to the mother plant. To the best of our knowledge, it is the first report on the assessment of the genetic fidelity in micropropagated plants of A. hispidissima.     

Shoot organogenesis from root-derived callus of Rhinacanthus nasutus (L.) Kurz. and assessment of clonal fidelity of micropropagted plants using RAPD analysis

An efficient regeneration system was established for an ethnomedicinal shrub Rhinacanthus nasutus from root-derived callus organogenesis. The root segments were cultured on MS medium supplemented with various concentrations of Kn (1.0–4.0 μM) alone or in combination with IBA (0.2–0.6 μM) or 2, 4-D (0.5–1.5 μM). The optimum frequency (94 %) of callus induction was recorded on MS medium supplemented with 3.0 μM Kn and 0.4 μM IBA. For shoot regeneration from callus, MS medium supplemented with different concentrations (1.0–7.0 μM) of BA or TDZ alone or in combination with NAA (0.2–1.0 μm) was employed. The highest frequency of shoot regeneration (91 %) and mean number of shoots (28.3) were observed on MS medium supplemented with 5.0 μM BA and 0.7 μM NAA. The shoots were excised and cultured on MS medium with 4.0 μM IBA produced 3.4 roots per shoot in 88 % cultures. Of the 65 plants transferred to soil 54 survived (83 %). The plants were transferred to field after successful hardening. RAPD analysis of the regenerated plants showed high similarity with the mother plant.     

Role of TDZ in the Quick Regeneration of Multiple Shoots from Nodal Explant of Vitex trifolia L.—an Important Medicinal Plant

The effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Vitex trifolia. Multiple shoots were induced in nodal explants obtained from a mature tree on Murashige and Skoog (MS) medium supplemented with TDZ in various concentrations (0.5, 1.0, 2.5, 5.0, 7.5, or 10.0???M). Prolonged exposure of the culture to TDZ had an adverse affect. To avoid this, the cultures were transferred to TDZ-free MS medium or MS medium fortified with various concentrations of 6-benzyladenine (BA) alone or in combination with ??-naphthalene acetic acid (NAA) to enhance multiplication, proliferation, and elongation of induced shoots. Optimum shoot multiplication and elongation was achieved when TDZ-exposed explants were repeatedly subcultured on MS media containing a combination of 1.0???M BA and 0.5???M NAA. The highest shoot regeneration frequency (90?%) and maximum number (22.3?±?0.2) of shoots per explant with shoot length of (5.2?±?0.2?cm) was recorded on MS medium fortified with 5.0???M TDZ. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5???M NAA. Properly rooted plantlets were successfully hardened off and acclimatized in thermocol cups containing sterile Soilrite. These plantlets were then transferred to pots containing different potting substrate; percentage survival of the plantlets was highest in vermiculite/garden soil mixture (1:1) and successfully transfer to greenhouse under sunlight.     

Preconditioning of Axillary Buds in Thidiazuron-Supplemented Liquid Media Improves In Vitro Shoot Multiplication in <Emphasis Type="Italic">Nyctanthes arbor-tristis</Emphasis> L.

An efficient tissue culture technology has been designed for mass multiplication of Nyctanthes arbor-tristis L. by preculturing nodal explants in thidiazuron (TDZ)-supplemented liquid Murashige and Skoog (MS) media. Direct inoculation of nodal segments on semi-solid MS medium augmented with various concentrations of TDZ (0.1 to 0.9 μM) produced shoots but with low regeneration response and few shoots per explant. Hence, nodal explants were pretreated with greater concentrations of TDZ (5 to 100 μM) in liquid MS media for different durations (4, 8, 12, and 16 days) with the aim of improving shoot regeneration response from cultured explants. After pretreatment, explants were transferred to agar-solidified hormone-free MS medium. Best response in terms of percent regeneration (94%), number of shoots per explant (20.00 ± 1.15), and greatest shoot length (7.23 ± 0.83 cm) were obtained with nodal segments pretreated in75 μM TDZ for 8 days. Similarly, root induction was obtained from pulse-treated microshoots for 24 h with 200 μM indole-3-butyric acid (IBA) followed by their transfer to 1/2 MS medium which produced an average of 5.50 ± 0.92 roots per microshoot. The rooted plantlets were transplanted to soil with 80% success rate.     

An Efficient and Reproducible Method for in vitro Clonal Multiplication of Rauvolfia tetraphylla L. and Evaluation of Genetic Stability using DNA-Based Markers

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or ??-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90?%) and maximum number of shoot (35.4?±?2.3) per explant were observed on WPM medium supplemented with 7.5???M BA, 2.5???M NAA, and 30?g/l sucrose at pH?5.8. Well-developed shoots, 4?C5?cm in length, were successfully rooted ex vitro at 90?% by a 30-min pulse treatment with 150???M IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90?% when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.     

Plant Regeneration Through Callus Organogenesis and True-to-Type Conformity of Plants by RAPD Analysis in Desmodium gangeticum (Linn.) DC.

An efficient plant regeneration protocol was established for an endangered ethnomedicinal plant Desmodium gangeticum (Linn.) DC. Morphogenic calli were produced from 96 % of the cultures comprising the immature leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.0 mg?l^?1) in combination with 6-benzylaminopurine (BA; 0.8 mg?l^?1). For callus regeneration, various concentrations of BA (1.0–5.0 mg?l^?1) or thidiazuron (TDZ; 1.0–5.0 mg?l^?1) alone or in combination with indole-3-acetic acid (IAA; 0.2–1.0 mg?l^?1) were used. Highest response of shoot regeneration was observed on MS medium fortified with TDZ (4.0 mg?l^?1) and IAA (0.5 mg?l^?1) combination. Here, 100 % cultures responded with an average number of 22.3 shoots per gram calli. Inclusion of indole-3-butyric acid in half MS medium favored rooting of recovered shoots. Out of 45 rooted plants transferred to soil, 40 survived. Total DNA was extracted from the leaves of the acclimatized plants of D. gangeticum. Analysis of random amplified polymorphic DNA using 13 arbitrary decanucleotide primers showed the genetic homogeneity in all the ten plants regenerated from callus with parental plant, suggesting that shoot regeneration from callus could be used for the true-to-type multiplication of this plant.     

Optimization of Adventitious Root Culture for Production of Biomass and Secondary Metabolites in Prunella vulgaris L.

Adventitious root cultures of Prunella vulgaris L. were established in shaking flask system for the production of biomass and secondary metabolites. Adventitious root cultures were induced from callus cultures obtained from leaf explants on solid Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0 mg l^?1) and naphthalene acetic acid (NAA; 1.5 mg l^?1). Thereafter, 0.49 g inoculum was transferred to liquid MS medium supplemented with different concentrations of NAA (0.5–2.0 mg l^?1). Growth kinetics of adventitious roots was recorded with an interval of 7 days for 49 days period. Highest biomass accumulation (2.13 g/l) was observed in liquid medium containing 1.0 mg l^?1 NAA after 21 days of inoculation. However, other concentrations of NAA also showed similar accumulation pattern but the biomass gradually decreases after 49 days of inoculation. Adventitious roots were collected and dried for investigation of total phenolics (TP), total flavonoids (TF), and antioxidant activities. Higher TPC (0.995 GAE mg/g-DRB) and TFC (6.615 RE mg/g-DRB) were observed in 0.5 mg l^?1 NAA treated cultures. In contrast, higher antioxidant activity (83.53 %) was observed 1.5 mg l^?1 NAA treated cultures. These results are helpful in up scaling of root cultures into bioreactor for secondary metabolites production.     

Shoot Multiplication and Callus Induction of Labisia pumila var. alata as Influenced by Different Plant Growth Regulators Treatments and Its Polyphenolic Activities Compared with the Wild Plant

This study aims to investigate whether the in vitro-cultured L. pumila var. alata has higher antioxidant activity than its wild plant. An 8-week-old L. pumila var. alata nodal segment and leaf explants were cultured onto Murashige and Skoog (MS) medium supplemented with various cytokinins (zeatin, kinetin, and 6-benzylaminopurine (BAP)) for shoot multiplication and auxins (2,4-dichlorophenoxyacetic acid (2,4-D) and picloram) for callus induction, respectively. The results showed that 2 mg/L zeatin produced the optimal results for shoot and leaf development, and 0.5 mg/L 2,4-D produced the highest callus induction results (60%). After this, 0.5 mg/L 2,4-D was combined with 0.25 mg/L cytokinins and supplemented to the MS medium. The optimal results for callus induction (100%) with yellowish to greenish and compact texture were obtained using 0.5 mg/L 2,4-D combined with 0.25 mg/L zeatin. Leaves obtained from in vitro plantlets and wild plants as well as callus were extracted and analyzed for their antioxidant activities (DPPH and FRAP methods) and polyphenolic properties (total flavonoid and total phenolic content). When compared with leaf extracts of in vitro plantlets and wild plants of L. pumila var. alata, the callus extract displayed significantly higher antioxidant activities and total phenolic and flavonoid content. Hence, callus culture potentially can be adapted for antioxidant and polyphenolic production to satisfy pharmaceutical and nutraceutical needs while conserving wild L. pumila var. alata.     

In Vitro Plant Propagation of Catharanthus roseus and Assessment of Genetic Fidelity of Micropropagated Plants by RAPD Marker Assay

An investigation was carried out to develop an efficient micropropagation protocol for Catharanthus roseus. Experiments were conducted to optimize suitable media for in vitro shoot multiplication and root induction. Out of the different media compared for in vitro shoot multiplication, Murashige and Skoog (MS) medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.2 mg/l α-naphthaleneacetic acid showed better response in terms of the emergence of shoots from axillary buds as well as proliferation and multiplication of shoots. The shoots when placed on half strength of MS medium having 1 mg/l indole 3-butyric acid and 0.25 % charcoal showed cent percent root induction with maximum number of roots per shoot (4.2) as well as maximum root length (1.72 cm). Further, clonal fidelity of the in vitro-raised plants was carried out using randomly amplified polymorphic DNA marker and results indicated that all the tissue culture-derived plants are true-to-type and there were no somaclonal variations among these plants.     

Optimization of Factors Influencing Microinjection Method for Agrobacterium tumefaciens-Mediated Transformation of Tomato

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD₆₀₀?=?0.2–1.0). The germinated seeds were cocultivated in the MS medium fortified with (0–200 mM) acetosyringone and minimal concentrations of (0–20 mg?L^?1) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD₆₀₀?=?0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg?L^?1 thidiazuron, 1.5 mg?L^?1 indole-3-butyric acid, 30 mg?L^?1 kanamycin, and 0–1.5 mg?L^?1 adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.     

Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR

High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 μM BA. The presence of 4.0 μM TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 μM and 2.0, 4.0 μM respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 μM IBA or 4.0 μM NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 μM IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative ¹H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p ≤ 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.