Metabolism and aluminum accumulation in Plantago almogravensis and P. algarbiensis in response to low pH and aluminum stress

We investigated the impact of low pH and aluminum on the metabolism and capacity for Al accumulation in shoots of the plantain species Plantago algarbiensis and P. almogravensis. We found that increasing the concentration of Al in the medium increased accumulation of it in the shoots of both plants (although more in P. almogravensis than in P. algarbiensis). The presence of Al in the medium induced proline and saccharide synthesis in P. almogravensis without affecting lipid peroxidation, but increased proline synthesis and lipid peroxidation in P. algarbiensis without affecting the saccharide content. Lipid peroxidation in P. algarbiensis was also enhanced at pH 4.0. The activity of antioxidant enzymes was increased as a response to low pH and Al in both species. Our data indicate that both species can accumulate high levels of Al but they have different sensitivities to low pH and/or the presence of Al in the growth medium.     

Comparison of tolerance of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Vigna radiata</Emphasis> to cadmium

The effect of different cadmium concentrations (6–120 μM) on Hill reaction activity (HRA) of isolated chloroplasts, contents of chlorophylls (Chls) and carotenoids (Cars), and Cd uptake and accumulation in plant organs of Indian mustard (Brassica juncea L. cv. Vitasso) and mung bean [Vigna radiata (L.) Wilczek] were determined. The Cd stress inhibited photochemical activity of isolated chloroplasts of both species and in both tested developmental stages. On the basis of EC₅₀ values, the mung bean showed a higher sensitivity to Cd treatment than Indian mustard. The higher sensitivity of both species was determined in the earlier than in the older developmental stage. The leaves of Cd-treated plants possessed lower contents of Chls and Cars in both species and the negative effect increased with Cd concentration. A difference between species was also found in Cd uptake and accumulation. In both species, Cd was accumulated more in roots than in shoots, with higher accumulation in Indian mustard than in mung bean.     

Micropropagation and conservation of endangered species Plantago algarbiensis and P. almogravensis

Plantago algarbiensis and P. almogravensis are endemic Al tolerant species from the Western-centre of the Algarve region (South of Portugal) and Portuguese Southwest coast, respectively, which are in risk of global extinction. The aim of this work was to establish an efficient protocol to in vitro propagate these species using shoots obtained from in vitro germinated seeds. The best results in terms of multiplication response were afforded in Murashige and Skoog’s (MS) medium supplemented with 6-benzyladenine (8.5 and 9.2 shoots per explant in P. algarbiensis and P. almogravensis, respectively). Shoots of both species showed a great rooting capacity (100 and 80 % for P. algarbiensis and P. almogravensis, respectively) that was not significantly influenced by the concentration of MS macronutrients or auxins. Plants were acclimatized to ex vitro conditions, exhibited normal development (survival rate of 95 and 80 % in P. algarbiensis and P. almogravensis, respectively), and were successfully reintroduced in their natural habitat.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of ten threatened species of <Emphasis Type="Italic">Mammillaria</Emphasis> (Cactaceae)

Mammillaria species are the most numerous within Cactaceae family, and some of them are threatened with extinction as a result of human activities. In this work, results of in vitro propagation are presented for ten Mammillaria species, testing 20 combinations of indole-3-acetic acid (IAA) and kinetin. Best results on shoot formation were obtained using kinetin at two levels: 27.9 and 46.5 μM. All IAA levels tested were able to induce de novo shoot formation in M. bocasana, M. densispina, M. hahniana, M. hutchisoniana, M. orcutii, M. pectinifera, M. perbella, M. picta, M. rhodantha, and M. zephyranthoides. Depending on the IAA level tested, four responding groups were observed concerning their highest shoot-formation number. For all species, the highest average of shoot formation was achieved with 5.7:46.5 or 11.4:46.5 μM IAA/kinetin, yielding 4.8 and 4.7 shoots per explant, respectively, in 60 d. Rooting of regenerated shoots was achieved by leaving the explants in their shoot-induction medium or transferring them to half-strength MS medium. Hardening of regenerated plants was successfully achieved by planting them in peat moss substrate after a desiccation treatment at room temperature for 3 d.     

Biochemical changes induced in seeds of <Emphasis Type="Italic">Brassicaceae</Emphasis> wild species during ageing

The aim of the present study was to determine whether the loss of seed germination capacity and vigour in seeds of four wild Brassicaceae species (Brassica repanda, Moricandia arvensis, Rorippa nasturtium-aquaticum and Sinapis alba) during ageing at 45°C and 90% relative humidity was related to changes in lipid peroxidation and membrane integrity. For all of the species, ageing reduced the final germination percentage and increased the length of time required to reach 50% of final germination (T ₅₀). Large differences in longevity were observed among the species. The times required for viability to be reduced to 80 and 50% of maximum germination (P80 and P50) were the lowest for B. repanda, and these values were two times longer for M. arvensis and R. nasturtium-aquaticum and five times longer for S. alba compared with B. repanda. A loss of seed viability was not associated with malondialdehyde accumulation, suggesting that lipid peroxidation did not cause seed deterioration under these conditions. However, the conductivity test effectively detected seed deterioration in these wild Brassicaceae species, and membrane permeability correlated with both germination and vigour loss. This correlation may provide a valuable mean for early detection of seed viability in wild Brassicaceae species.     

High frequency <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> from nodal buds of mature tree

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm⁻³ N⁶-benzyladenine (BA) and 0.5 mg dm⁻³ α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm⁻³ BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm⁻³ of indole-3-butyric acid solution. Successful field establishment and high (80–90 %) survival of plants was observed.     

An efficient in vitro plantlet regeneration of <Emphasis Type="Italic">Cryptocoryne wendtii</Emphasis> and <Emphasis Type="Italic">Cryptocoryne becketti</Emphasis> through shoot tip culture

An efficient micropropagation protocol was established for Cryptocoryne wendtii and Cryptocoryne becketti using shoot tips explants. Multiple shoots were induced from shoot tip explants of both species cultured on agar-gelled as well as liquid MS medium supplemented with 0.5 mg/L BA and 0.2 mg/L IBA (proliferation medium). The multiple shoots of both the species formed on agar-gelled as well as liquid medium were vigorously growing with well-developed roots and leaves after 4 weeks of culture. Highest number of multiple shoots was obtained from shoot tip explants of both the species cultured in liquid proliferation medium after 4 weeks of culture. The shoot tip explants of C. wendtii and C. becketti, that were cultured in liquid proliferation medium (2 weeks) followed by culturing on agar-gelled proliferation medium (2 weeks) also produced the multiple shoots. Shoot tips cultured on agar-gelled medium produced the least number of multiple shoots after 4 weeks of culture. Histological study did not show any abnormalities in the leaves of in vitro plantlets of both the species, cultured in agar-gelled and liquid proliferation medium. The leaves of the in vitro plantlets formed normal mesophyll cells and vascular bundles. More than 95% of the acclimatized plantlets grew vigorously without any morphological abnormalities.     

Micropropagation of mature <Emphasis Type="Italic">Pongamia pinnata</Emphasis> Pierre

Murashige and Skoog’s (MS) basal medium with benzylaminopurine (BA), kinetin (KN), zeatin (Z), and thidiazuron (TDZ) were tested for induction of multiple shoots from mature-tree-derived axillary meristems of Pongamia pinnata. Sprouting of buds was 64% on medium devoid of plant growth regulators (PGR). Incorporation of BA, KN, or Z was ineffective in enhancing sprouting frequency or induction of multiple shoots. Sprouting was completely suppressed in the presence of TDZ. Caulogenic buds appeared in nodal meristems of these explants after withdrawal of TDZ. The number of shoot buds was more on explants precultured in higher concentrations. At higher concentrations of this PGR, a swelling developed at the axil. Multiple shoot primordia appeared and differentiated from this swelling after culturing these explants on MS medium for six passages of 2 wk each. Shoots were harvested and cultured on 0.45 μM TDZ for further proliferation. Primary explants after harvesting of shoots were identified as ‘stump’. Reculturing of stumps on 0.45 μM TDZ produced more shoots. This step was followed for six cycles to obtain additional shoots in each cycle. Shoots maintained on 0.45 μM TDZ elongated and rooted (70%) on growth regulator-free medium. Rooted shoots (65%) survived transfer to a sand/soil mixture. This report describes the protocol for micropropagation of P. pinnata using mature-tree-derived nodal meristems. Recycling of mature stock to produce a stream of useable shoots for subculturing and eventual stabilization is of great value and can possibly be generalized as an isolation protocol especially for woody species. Repeated proliferation of caulogenic buds from the same origin may also find application in rescue of endangered germplasm.     

An improved micropropagation of <Emphasis Type="Italic">Eclipta alba</Emphasis> by in vitro priming with chlorocholine chloride

An efficient method of micropropagation for Eclipta alba from young nodal axils of shoot tip explants has been developed by giving special attention to ‘priming’ in vitro plantlets in view of increasing their hardening ability after transplantation ex vitro. Among 3 cytokinins—BAP, kinetin and TDZ, BAP was found most effective in inducing and proliferating adventitious shoots. The highest frequency of responding explants (100%) and maximum number of shoots (23.0) per explant were obtained after 60 days culture on MS medium containing 8.8 μM BAP. Cent percent shoots developed roots directly from shoot base when transferred to growth regulator-free MS medium. For priming E. alba microshoots, 6.3 μM of chlorocholine chloride (CCC) was found most effective. The major changes observed in 30 days old treated shoots were, production of increased number of root, elevation of chlorophyll level in leaves and increase in plant biomass. Furthermore, arrested undesirable shoot elongation made the plants sturdier and more suitable for acclimatization. The primed micropropagated E. alba plants were healthy and survived by higher frequency (100%) in soil in comparison to the non-treated plants (84% survival).     

Propagation,growth, and carbohydrates of <Emphasis Type="Italic">Dendrobium</Emphasis> Second Love (Orchidaceae) <Emphasis Type="Italic">in vitro</Emphasis> as affected by sucrose,light, and dark

In general, plant material grown in vitro has low photosynthetic ability to achieve positive carbon balances. Therefore, a continuous supply of carbohydrates from the culture medium is required, and sucrose has been the most commonly used carbon source. In this paper, we investigate the effects of different sucrose concentrations and the presence and absence of light on the endogenous levels of soluble carbohydrates and starch as well as on the proliferation and growth of Dendrobium Second Love (Orchidaceae) in vitro. The possibility of using etiolated stem segments as a means for micropropagating this hybrid was also verified. The results obtained indicated that the presence and absence of light and the sucrose concentrations used influenced the amounts of soluble carbohydrates and starch and the proliferation of D. Second Love shoots and roots. An increase in sucrose concentration caused a progressive increase in the amounts of total carbohydrates and starch. Under both light conditions, sucrose was the main sugar found in the shoots followed by glucose and fructose. The addition of sucrose to the culture medium up to 2% and 4% was advantageous to the number of shoots produced per explant and the root longitudinal growth in the presence and absence of light, respectively. Shoot and root dry matter and the number of roots formed per explant increased as sucrose concentration was raised up to 6% in both light treatments. The use of dark-grown shoot segments proved to be a useful and reliable alternative for the micropropagation of this hybrid.     

High frequency in vitro propagation of <Emphasis Type="Italic">Trichopus zeylanicus</Emphasis> subsp. <Emphasis Type="Italic">travancoricus</Emphasis> using branch–petiole explants

Trichopus zeylanicus subsp. travancoricus (known as Arogyapacha), an endangered ethnomedicinal plant of the Western Ghats of South India, serves as the major source of the commercial drug Jeevani. The present study established a long-term high frequency in vitro propagation protocol for Arogyapacha. Callus obtained from the branch–petiole explants cultured on Murashige and Skoog (MS) medium with 4.5 μM 2,4-dichlorophenoxyacetic acid upon subculture to medium with different concentrations of 6-benzyladenine (BA) either alone or in combination with an auxin favoured shoot morphogenesis. Medium with 13.3 μM BA alone facilitated high frequency shoot bud (mean of 93.2) formation. Medium with lower concentrations of BA (4.4, 6.6 and 8.8 μM) alone or in combination with lower concentration of α-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) favoured better shoot growth than 13.3 μM BA containing medium, but with reduced number of shoot buds. Subsequent cultures on medium with lower concentrations of BA and also on MS basal media facilitated shoot formation as well as growth of shoots. The shoot regeneration potential showed no decline up to 5 years. Culture of the in vitro-derived whole branch–leaf explants on MS basal medium developed shoots directly from the node. On medium with 19.6 μM IBA, the whole branch–leaf explants induced nodular callus from the node, which developed shoots later. Subsequent cultures on medium with BA exhibited high frequency shoot formation. The transfer of shoots after 10–15 days culture on half-strength MS medium containing 2.7 μM NAA to half-strength basal medium induced a mean of 11.3 roots. Field survival of plantlets relied on the soil mix: a 1:4 ratio of sand and red-soil exhibited the highest plantlets survival (86.6%). RAPD profile of the source plant and plants regenerated from calli after 4 years showed no polymorphism. The established plantlets with morpho-floral features similar to that of the source plants flowered normally and set fruits.     

Physiological responses of Plantago algarbiensis and P. almogravensis shoots and plantlets to low pH and aluminum stress

We investigated the impact of low pH and aluminum (Al) stress on the growth, nutrients concentration, chlorophyll a fluorescence, photosynthetic pigment contents, proline and carbohydrate accumulation in shoots and plantlets (leaves and roots) of Plantago almogravensis and P. algarbiensis. Both species accumulated considerable and similar amounts of Al in their tissues, mainly in the roots. The presence of Al caused a significant reduction on root elongation in P. algarbiensis. Low pH and Al induced significant changes on nutrient accumulation, but no significant alterations on the maximum efficiency of PSII (F _v/F _m), quantum yield of PSII photochemistry (?_PSII), quantum yield of regulated energy dissipation (?_NPQ) and quantum yield of non-regulated energy dissipation (?_NO) were detected in both species in response to these stresses. However, Al increased significantly the non-photochemical quenching and the chlorophyll b content and decreased the PSII excitation pressure (1 ? q _p) in P. almogravensis leaves. Both stress treatments induced carbohydrate accumulation in the shoots and roots of this species, but not in leaves. In P. algarbiensis, low pH and Al decreased the photosynthetic pigment contents in the shoots, whereas Al stimulated the carbohydrate accumulation in the leaves. Although our data showed that both species are tolerant to Al³⁺ and H⁺, P. almogravensis appeared to be more adapted to maintain cellular physiology and growth under those conditions.     

Differences in Al tolerance between Plantago algarbiensis and P. almogravensis reflect their ability to respond to oxidative stress

We evaluated the impact of low pH and aluminum (Al) on the leaves and roots of Plantago almogravensis Franco and Plantago algarbiensis Samp., focusing on energy partitioning in photosystem II, H₂O₂ levels, lipid peroxidation, electrolyte leakage (EL), protein oxidation, total soluble protein content and antioxidant enzyme activities. In both species, Al triggered more changes in oxidative metabolism than low pH alone, particularly in the roots. We found that Al increased the levels of H₂O₂ in P. algarbiensis roots, but reduced the levels of H₂O₂ in P. almogravensis leaves and roots. Neither low pH nor Al affected the spatial heterogeneity of chlorophyll fluorescence, the maximum photochemical efficiency of PSII (F_v/F_m), the actual quantum efficiency of PSII (?_PSII) or the quantum yields of regulated (?_NPQ) and nonregulated (?_NO) energy dissipation, and there was no significant change in total soluble protein content and EL. In P. algarbiensis, Al increased the carbonyl content and the activities of superoxide dismutase (SOD) and catalase (CAT) in the roots, and also CAT, ascorbate peroxidase and guaiacol peroxidase activities in the leaves. In P. almogravensis, Al reduced the level of malondialdehyde in the roots as well as SOD activity in the leaves and roots. We found that P. almogravensis plantlets could manage the oxidative stress caused by low pH and Al, whereas the P. algarbiensis antioxidant system was unable to suppress Al toxicity completely, leading to the accumulation of H₂O₂ and consequential protein oxidation in the roots.     

Mycotoxin production by different ochratoxigenic <Emphasis Type="Italic">Aspergillus</Emphasis> and <Emphasis Type="Italic">Penicillium</Emphasis> species on coffee- and wheat-based media

Ochratoxin A (OTA) is one of the most widespread mycotoxins, and is produced by several Aspergillus or Penicillium species. Human exposure to OTA is mainly by intake of contaminated food, with cereal products, followed by coffee and red wine as the main sources of OTA. In this study, the OTA production of four ochratoxigenic fungi (two Aspergillus and two Penicillium species) was investigated in four different media, i.e. wheat and coffee model media as food-based media and two standard laboratory media (malt extract glucose agar, MEA and yeast extract sucrose agar, YES). Colony growth was documented and OTA concentrations in cultures were determined at day 2, 4 and 8 of incubation at 25°C by high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). OTA production clearly depended upon time of incubation, fungal species, and medium composition. On coffee based medium, moderate OTA levels were produced by A. ochraceus BFE635 (9.8 μg/g) and by A. niger BFE632 (10.6 μg/g) on day 8 of incubation. In wheat-based medium, these strains produced much more OTA than in coffee. The highest OTA concentration (83.8 μg/g on day 8) was formed by A. ochraceus BFE635 followed by the other Aspergillus niger BFE632 (49 μg/g). Lower OTA levels were produced by P. verrucosum BFE550 and P. nordicum BFE487, in both wheat and in YES medium, whilst OTA was hardly detectable in coffee and in MEA in case of P. nordicum. Colony growth of the tested strains on different media was not indicative of OTA production. Guttation droplets developed on wheat-based medium with the Aspergillus strains within a week, and this phenomenon coincided with the high OTA amounts formed by these species. Results from this study add to our knowledge on the behaviour of ochratoxigenic fungal species when cultured on food based media.     

Cold storage and cryopreservation of hairy root cultures of medicinal plant <Emphasis Type="Italic">Eruca sativa</Emphasis> Mill., <Emphasis Type="Italic">Astragalus membranaceus</Emphasis> and <Emphasis Type="Italic">Gentiana macrophylla</Emphasis> Pall.

To explore the possibility of an effectively long-term preservation of the germplasm of the HR lines of medicinal plant Astragalus membranaceus, Gentiana macrophylla Pall., and Eruca sativa Mill., both cold storage and cryopreservation approaches were attempted and compared. After 5-month cold storage on half strength Murashige and Skoog (1962) (1/2 MS) agar medium (AM), up to 82.9, 75.7, and 100% of the A. membranaceus, G. macrophylla and E. sativa hairy roots (HRs) recovered growth, respectively. The survival rates of A. membranaceus and G. macrophylla HRs significantly decreased, whereas that of E. sativa HR was unchanged with the addition of increased levels of exogenous abscisic acid (ABA) during cold storage. Using the encapsulation–vitrification (EV) method for cryopreservation, the G. macrophylla HRs died, whereas up to 6 and 73% of the A. membranaceus and E. sativa HRs survived, respectively. The HR lines evaluated with both methods showed no significant differences in morphology and growth rate compared with controls that were not subjected to preservation methods. These results suggest that cold storage is a more suitable alternative for the HR lines of the three studied plant species and that specificity of plant species have profound effects on the effectiveness of preservation.     

High-frequency<Emphasis Type="Italic">in vitro</Emphasis> flowering in six species of<Emphasis Type="Italic">ceropegia</Emphasis>

High-frequencyin vitro flowering is reported here fromin vitro regenerated shoots ofin vitro-raised seedlings of rare and endemicCeropegia lawii, Ceropegia maccannii, Ceropegia oculata, andCeropegia sahyadrica, as well as the widely distributedCeropegia bulbosa var.bulbosa andCeropegia hirsuta. In our first set of experiments, the MS medium contained 87 mM sucrose and was supplemented with varying concentrations of BAP (4.4 to 26.6 μM). For the second set of trials, varying concentrations of sucrose (87 to 233 mM) were tested in MS media containing a constant 4.4 p.M BAP. Sub-cultured apical as well as axillary buds flowered with similar frequencies after 30 d of incubation. For all six species, the highest percentage of flowering shoots was obtained with either 26.6 μM BAP or 175 mM sucrose. Although smaller in size, theirin vitro flowers were morphologically comparable within wVo-derived flowers. Variations among species were noted for the number of flower buds per shoot and the percentage of flower formation. Because all six species showed similar responses in both experiments, we can suggest that this protocol is applicable across the wide range ofCeropegia species.     

Effect of salt stress on physiological and antioxidative responses in two species of <Emphasis Type="Italic">Salicornia</Emphasis> (<Emphasis Type="Italic">S. persica</Emphasis> and <Emphasis Type="Italic">S. europaea</Emphasis>)

The effects of salt stress on growth parameters, free proline content, ion accumulation, lipid peroxidation, and several antioxidative enzymes activities were investigated in S. persica and S. europaea. The seedlings were grown for 2 months in half-strength Hoagland solution and treated with different concentrations of NaCl (0, 85, 170, 340, and 510 mM) for 21 days. The fresh and dry weights of both species increased significantly at 85 and 170 mM NaCl and decreased at higher concentrations. Salinity increased proline content in both the species as compared to that of control. Sodium (Na⁺) content in roots and shoots increased, whereas K⁺ and P_i content in both organs decreased. At all NaCl concentrations, the total amounts of Na⁺ and K⁺ were higher in shoots than in roots. Malondialdehyde (MDA) content declined at moderate NaCl concentrations (85 and 170 mM) and increased at higher levels. With increased salinity, superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) activities also increased gradually in both species. In addition, it seems that GPX, CAT, and SOD activities play an essential protective role in the scavenging reactive oxygen species (ROS) in both species. Native polyacrylamide gel electrophoresis (PAGE) indicated different isoform profiles between S. persica and S. europaea concerning antioxidant enzymes. These results showed that S. persica exhibits a better protection mechanism against oxidative damage and it is more salt-tolerant than S. europaea possibly by maintaining and/or increasing growth parameters, ion accumulation, and antioxidant enzyme activities.     

Secondary metabolism in micropropagated <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L. grown in non-aerated liquid medium

Hypericum perforatum L. is a medicinal plant that has been extensively studied because of its bioactive properties. The objective of this study was to establish a system that could lower the cost of in vitro propagation by using liquid medium, as well as to evaluate the secondary metabolism in the systems tested. Nodal segments of H. perforatum were obtained from in vitro shoots and grown in three liquid culture systems: total immersion (TI), partial immersion (PI), and paper bridge support (PB). Semi-solid medium (3 g L⁻¹ Phytagel™) was used as control (SS). The organogenic responses were evaluated, and phenolic compounds, hypericin, and the activity of polyphenol oxidases (PPO) and peroxidases (POX) were quantified. After 80 days of culture, induction and proliferation of adventitious shoots were similar in the PI and SS systems (65.3 and 71.3 shoots, respectively), whereas PB resulted in the fewest shoots per explant (29.5 shoots). Longer shoots were obtained under the PI conditions. Hyperhydricity was observed in the shoots from the TI system. Browning was visible in shoots from the TI and PB systems. The highest concentrations of phenolic compounds and hypericin were observed in shoots derived from PI and PB, at 80 days of culture. POX activity was higher in shoots cultured in PI at 40 days, whereas PPO was significantly more active at 80 days of culture. Likely, POX was more related to shoot growth, whereas PPO played a later role in response to the culture environment and medium stress.     

Effects of sucrose and pH levels on in vitro shoot regeneration from leaf explants of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> and accumulation of bacoside A in regenerated shoots

Brahmi (Bacopa monnieri) is an important medicinal plant mainly used for the treatment of neurological disorders and depression. Recent investigations revealed that bacoside A is major chemical component shown to be responsible for memory facilitating action of brahmi. The current investigation was carried out to assess the potential for increasing biomass and the concentration of bacoside A in the in vitro regenerated shoots by varying sucrose and pH levels of shoot regeneration medium. The leaf explants were cultured on the Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ kinetin (KN) and with varying concentrations of sucrose (0, 1, 2, 3, 4, 5 and 6% at pH 5.8) and pH (4.5, 5.0, 5.5, 6.0 and 6.5 with 2% sucrose) with the objective of verifying the effects of sucrose and pH level on shoot regeneration and to verify the accumulation of bacoside A in the regenerated shoots. The shoot biomass increased (150.50 ± 2.84 shoots per explant, fresh wt 6.31 ± 0.12 g and dry wt 250 ± 5.00 mg) on the medium supplemented with 2% sucrose and pH which was set at 4.5. The results of HPLC analysis indicate that increase in sucrose concentration (0, 1, 2, 3, 4, 5 and 6% at pH 5.8) lead to decrease in the bacoside A content (39.51, 22.43, 13.05, 12.17, 10.73, 9.56 and 8.93 mg g⁻¹ dry wt, respectively) in regenerated shoots. These findings provide evidence that stressful condition of inadequate supply of carbon elevated synthesis of bacoside A in brahmi shoots. However, 2% sucrose is found suitable for biomass accumulation. Therefore, medium supplemented with 2% sucrose and pH set at 4.5 was found suitable for both biomass (6.31 ± 0.12 g fresh wt and 250 ± 5.00 mg dry wt) and bacoside A accumulation (13.09 mg g⁻¹ dry wt).