Abnormal chromosomes and DNA content in micropropagated rhubarb (<Emphasis Type="Italic">Rheum rhaponticum </Emphasis>L<Emphasis Type="Italic">.</Emphasis>) PC49

Rhubarb (Rheum rhaponticum L.) is a tetraploid (2n = 44) vegetable crop growing in Europe and North America. The results observed 44 chromosomes for both cultivars Timperly Early and PC49, but showing significant differences in DNA contents between them. Abnormal chromosome numbers (2n = 22 and 32) and chromosomal fragments were observed only in micropropagated plants of rhubarb PC49, suggesting somaclonal variation. The results provide further information on understanding morphological variation and increased disease susceptibility of micropropagated rhubarb PC49 in our previous investigation.     

Assessment of genetic stability of <Emphasis Type="Italic">in vitro</Emphasis> grown <Emphasis Type="Italic">Dictyospermum ovalifolium</Emphasis>

In the present study, a polymerase chain reaction (PCR)-based method namely inter simple sequence repeat (ISSR) was employed to assess genetic stability in tissue culture-derived Dictyospermum ovalifolium plantlets. To study genomic stability of micropropagated plants, 14 individuals were randomly tagged among a population of 2500 regenerants and were compared with single donor mother plant. A total of 51 clear and reproducible bands ranging from 200 bp to 2.1 kb were scored corresponding to an average of 3.64 bands per primer. Two of the 51 bands were polymorphic (3.92 %) among 14 individuals, thus indicating the occurrence of low level genomic variation in the micropropagated plants. Cluster analysis indicates that genetic similarity values were 0.978 which allows classification of the plants to distinct groups. Further an attempt was made to reintroduce the micropropagated plants into their natural habitat. Over one thousand six hundred fifty plants were successfully established.     

Characterization of a field-grown transgenic pineapple clone containing the genes <Emphasis Type="Italic">chitinase</Emphasis>, <Emphasis Type="Italic">AP24</Emphasis>, and <Emphasis Type="Italic">bar</Emphasis>

We previously introduced the bar gene, along with chitinase and AP24 genes, into the pineapple genome. The present report focuses on the evaluation of the first vegetative generation of a transgenic clone containing these genes. Three materials were compared: macropropagated controls (non-transformed), micropropagated controls (non-transformed), and micropropagated transformed plants. From each group, 50% of the plants were sprayed with FINALE^® 3 mo after the experiment initiation. The characterization was performed after 1 yr of field growth. FINALE^® killed all non-transgenic plants. Plants that survived the herbicide application showed 2n?=?50 chromosomes in their roots after 1 yr in the field. Micropropagated transformed plants sprayed with FINALE^® did not show phenotype differences from micropropagated transformed plants not sprayed with the herbicide. Between the micropropagated transformed plants sprayed with FINALE^® and the micropropagated control plants, the following differences were observed: modifications in levels of cell wall-linked, free and total phenolics, and total proteins. Moreover, changes of the fruit mass without crown were also recorded. Between the micropropagated transformed plants sprayed with FINALE^® and the macropropagated control plants, levels of chlorophyll b, total chlorophyll pigments, and proteins were different. Furthermore, activities of phenylalanine ammonia-lyase, superoxide dismutase, and glutamine synthetase were dissimilar. The plant height and diameter, and the crown height were also different. Until now, we have evaluated transformed pineapple plants during hardening and field growth. Although some unexpected variations were recorded, we believe they are not relevant enough to justify rejection of transgenesis as an important tool for pineapple genetic improvement.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Sebacina vermifera</Emphasis> increase growth performance at the expense of herbivore resistance in <Emphasis Type="Italic">Nicotiana attenuata</Emphasis>

A Sebacinales species was recovered from a clone library made from a pooled rhizosphere sample of Nicotiana attenuata plants from 14 native populations. Axenic cultures of the related species, Piriformospora indica and Sebacina vermifera, were used to examine their effects on plant performance. Inoculation of N. attenuata seeds with either fungus species stimulated seed germination and increased growth and stalk elongation. S. vermifera inoculated plants flowered earlier, produced more flowers and matured more seed capsules than did non-inoculated plants. Jasmonate treatment during rosette-stage growth, which slows growth and elicits herbivore resistance traits, erased differences in vegetative, but not reproductive performance resulting from S. vermifera inoculation. Total nitrogen and phosphorous contents did not differ between inoculated and control plants, suggesting that the performance benefits of fungal inoculation did not result from improvements in nutritional status. Since the expression of trypsin proteinase inhibitors (TPI), defensive proteins which confer resistance to attack from Manduca sexta larvae, incur significant growth and fitness costs for the plant, we examined the effect of S. vermifera inoculation on herbivore resistance and TPI activity. After 10 days of feeding on S. vermifera-inoculated plants, larval mass was 46% higher and TPI activity was 48% lower than that on non-inoculated plants. These results suggest that Sebacina spp. may interfere with defense signaling and allow plants to increase growth rates at the expense of herbivore resistance mediated by TPIs.     

Headspace-SPME of <Emphasis Type="Italic">in vitro</Emphasis> shoot-cultures and micropropagated plants of <Emphasis Type="Italic">Lavandula viridis</Emphasis>

In this work the volatiles emitted from in vitro shoot-cultures and micropropagated plants of Lavandula viridis L’Hér. were characterized and compared with those obtained from the field-grown mother-plant, using headspace solid phase micro-extraction following by capillary gas chromatography coupled to mass spectrometry (HS-SPME-GC/MS). The headspace composition consisted mainly in oxygenated monoterpenes (66.7–79.2 %), where the major constituents emitted by the mature field-grown mother-plant, in vitro shoot-cultures and micropropagated plants were 1,8-cineole (74.0, 51.9 and 57.8 %) and camphor (2.9, 15.3 and 8.7 %), respectively. The headspace of in vitro shoot-cultures and micropropagated plants showed greater amount of α-pinene, camphene, β-pinene, β-selinene and selina-3,7(11)-diene, when compared with the field-grown mother-plant.     

Chlorophyll fluorescence in micropropagated <Emphasis Type="Italic">Rhododendron ponticum</Emphasis> subsp. <Emphasis Type="Italic">baeticum</Emphasis> plants in response to different irradiances

The aim of this study was to investigate acclimation of micropropagated plants of Rhododendron ponticum subsp. baeticum to different irradiances and recovery after exposure to high irradiance. Plants grown under high (HL) or intermediate (IL) irradiances displayed higher values of maximum electron transport rate (ETR_max) and light saturation coefficient (E_k) than plants grown under low irradiance (LL). The capacity of tolerance to photoinhibition (as assessed by the response of photochemical quenching, q_p) varied as follows: HL > IL > LL. Thermal energy dissipation (q_N) was also affected by growth irradiance, with higher saturating values being observed in HL plants. Light-response curves suggested a gradual replacement of q_p by q_N with increasing irradiance. Following exposure to irradiance higher than 1500 μmol m⁻² s⁻¹, a prolonged reduction of the maximal photochemical efficiency of PS 2 (F_v/F_m) was observed in LL plants, indicating the occurrence of chronic photoinhibition. In contrary, the decrease in F_v/F_m was quickly reverted in HL plants, pointing to a reversible photoinhibition.     

Genetic stability, <Emphasis Type="Italic">ex vitro</Emphasis> rooting and gene expression studies in <Emphasis Type="Italic">Hagenia abyssinica</Emphasis>

Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic stability of 80 micropropagated Hagenia abyssinica plants, 40 of axillary origin and 40 of adventitious origin. The shoots were isolated from the same mother tree and micropropagated for over two years. Among the 83 RAPD primers screened, 16 gave reproducible band patterns. These 16 primers produced 115 bands for each plant. One plant from axillary origin showed two unique bands with primer OPC-11. All other plants showed identical band patterns. Generally, there was no significant difference in the shoot multiplication rate between shoots of axillary and adventitious origin. Indole-3-acetic acid (IAA) resulted in better ex vitro rooting compared to indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA). Non-micropropagated plants that were grown in the greenhouse for about one year were better in ex vitro rooting compared to those of juvenile material and mature tree derived micropropagated plants of the same treatment. Adventitious rooting related oxygenase gene (ARRO-1) isolated from apple (Malus domestica) was not expressed in H. abyssinica using a complementary DNA representational difference analysis fragment (cDNA RDA14) as a probe.     

Interaction of silicon and cadmium in <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

The objective of this study was to determine the effect of silicon (Si) and cadmium (Cd) on root and shoot growth and Cd uptake in two hydroponically cultivated Brassica species (B. juncea (L.) Czern. cv. Vitasso and B. napus L. cv. Atlantic). Both species are potentially usable for phytoextraction. Inhibitory effects of Cd on root elongation were diminished by the impact of Si. Primary roots elongation in the presence of Cd + Si compared with Cd was stronger and the number of lateral roots was lower in B. juncea than in B. napus. Cd content per plant was higher in B. napus roots and shoots compared with B. juncea. Suberin lamellae were formed closer to the root apex in Cd + Si than in Cd treated plants and this effect was stronger in B. napus than in B. juncea. Accelerated maturation of endodermis was associated with reduced Cd uptake. Cd decreased the content of chlorophylls and carotenoids in both species, but Si addition positively influenced the content of photosynthetic pigments which was higher in B. napus than in B. juncea. Si enhanced more substantially translocation of Cd into the shoot of B. napus than of B. juncea. Based on our results B. napus seems to be more suitable for Cd phytoextraction than B. juncea because these plants produce more biomass and accumulate higher amount of Cd. The protective effect of Si on Cd treated Brassica plants could be attributed to more extensive development of suberin lamellae in endodermis.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of endangered <Emphasis Type="Italic"> Rhododendron ponticum</Emphasis> L. subsp. <Emphasis Type="Italic">baeticum</Emphasis> (Boissier &; Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Andersons modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.     

Factors affecting <Emphasis Type="Italic">in vitro</Emphasis> propagation and field establishment of <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

The effect of plant growth regulators (PGRs), gelling agents, sucrose and heat shock on shoot multiplication, shoot growth, rooting and subsequent survival of Chlorophytum borivilianum Sant. et Fernand was evaluated. Benzyladenine (BA) was found to be better cytokinin over kinetin (KIN) for shoot multiplication. Sucrose concentrations from 116–290 mM in the basal medium (BM) promoted shoot multiplication. Heat shock (50 °C, 1 h) also promoted shoot multiplication at these sucrose concentrations on both BM medium and BM supplemented with 5.0 μM BA. Beneficial effect of sucrose was also observed on rooting of shoots on BM as well as BM supplemented with 5.0 μM indole-3-butyric acid (IBA). Phytagel as a gelling agent was found to be more effective for shoot proliferation and growth compared to agar. Amongst various soil mixtures tested, higher survival of plants was observed in soil containing Vermicompost. It was interesting to note that a maximum plant survival (> 95 %) was observed when plants were directly transferred to net-house (irradiance reduced to 50 % with green net, without humidity and temperature control) than poly-house (with humidity and temperature control). Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis of regenerated plants showed genetic similarity to mother plant.     

Overexpression of the <Emphasis Type="Italic">Activated Disease Resistance 1-like1</Emphasis> (<Emphasis Type="Italic">ADR1-L1</Emphasis>) Gene Results in a Dwarf Phenotype and Activation of Defense-Related Gene Expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The protein encoded by the activated disease resistance 1-like1 (ADR1-L1) gene (locus name, At4g33300) belongs to the activated disease resistance 1 (ADR1) family of coiled-coil nucleotide-binding site leucine-rich repeat-type disease resistance proteins. This family contains four proteins and they have specific features in their amino acid sequences. It has been reported that ADR1 protein belongs to the ADR1 family, which is related to not only defense response but also drought tolerance. We found that transgenic plants overexpressing the ADR1-L1 gene showed a dwarf phenotype and morphological change in leaves. The expression levels of defense-related genes and the resistance to Pseudomonas syringae pv. tomato DC3000 were increased in transgenic plants. However, enhancement of drought tolerance and activation of abiotic response genes were not observed. When the growth temperature was changed from 22°C to 28°C, the expression of defense-related genes and the enhancement of resistance to a bacterial pathogen were suppressed and the dwarf phenotype and morphological change of leaves recovered.     

Asexual propagations of introduced exotic macrophytes <Emphasis Type="Italic">Elodea nuttallii</Emphasis>, <Emphasis Type="Italic">Myriophyllum aquaticum</Emphasis>, and <Emphasis Type="Italic">M. propinquum</Emphasis> are improved by nutrient-rich sediments in China

An increasing number of recent studies indicate that multiple interacting factors can affect the invasion of plants. However, few studies have focused on asexual propagation and the interaction of propagation with environmental factors that regulate the invasive potential of introduced exotic species in aquatic habitats. This study was designed to investigate the differences in asexual propagation between introduced exotic and non-invasive native aquatic macrophytes in nutrient-poor and nutrient-rich sediments and to test the hypothesis that differences in asexual propagation (stem fragment production) and propagule establishment between introduced exotic and non-invasive native macrophytes are driven by sediment nutrient levels. Three exotic aquatic macrophytes (Elodea nuttallii, Myriophyllum aquaticum, and M. propinquum) recently introduced to China and their non-invasive native counterparts (Hydrilla verticillata, M. oguraense, and M. ussuriense) were used for comparison in nutrient-poor (TN 0.59 and TP 0.03 mg g⁻¹) and nutrient-rich (TN 2.35 and TP 0.10 mg g⁻¹) sediments. After 8 weeks of growth, the exotic species tended to produce more total biomass, branch biomass and apical shoots and have higher relative growth rate (RGR) than their native counterparts in nutrient-rich sediment. Rooting efficiency and root growth of exotic fragments were higher than that of native counterparts in nutrient-rich sediment, although the survival rates of fragments did not differ between native and exotic species. In addition, superior traits (rooting efficiency and root growth) of exotic species were also observed in nutrient-poor sediment, but to a lesser degree than in nutrient-rich sediment. These results suggest that asexual propagation of these three introduced exotic macrophytes is more effective in nutrient-rich sediment than in nutrient-poor sediment in China.     

Induction of phytochelatins and antioxidant defence system in <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Vigna radiata</Emphasis> in response to chromium treatments

Growth performance, chromium (Cr) accumulation potential and induction of antioxidative defence system and phytochelatins (PCs) were studied in hydroponically grown Brassica juncea (Indian mustard) and Vigna radiata (mungbean) at various levels of Cr treatments (0, 50, 100, 200 μM Cr). B. juncea accumulated twofolds and threefolds higher Cr in root and shoot, respectively than in V. radiata. Compared to B. juncea, V. radiata was found to be particularly sensitive to Cr as observed by the severity and development of Cr toxicity symptoms and decreased growth. Induction of PC and enzymes of antioxidant defence system were monitored as plant’s primary and secondary metal detoxifying responses, respectively. There was induction of PC and enzymes of antioxidant defence system in both the plants. PCs were induced significantly in roots and shoot of both the plants at all the levels of Cr treatments. Significantly higher activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) were observed in shoot of B. juncea than V. radiata at all the levels of Cr treatments. Induction of PCs along with antioxidant defence system in response to Cr stress suggests the cumulative role of PCs and antioxidants in conferring tolerance against accumulated Cr in B. juncea, and thereby signifies the suitability of this plant as one of the potential remediators of Cr.     

Genetic and environmental control of the <Emphasis Type="Italic">Verticillium</Emphasis> syndrome in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Verticillium spp. are major pathogens of dicotyledonous plants such as cotton, tomato, olive or oilseed rape. Verticillium symptoms are often ambiguous and influenced by development and environment. The aim of the present study was to define disease and resistance traits of the complex Verticillium longisporum syndrome in Arabidopsis thaliana (L.) Heynh. A genetic approach was used to determine genetic, developmental and environmental factors controlling specific disease and resistance traits and to study their interrelations.     

Improved clonal propagation of <Emphasis Type="Italic">Spilanthes</Emphasis><Emphasis Type="Italic">acmella</Emphasis> Murr. for production of scopoletin

A reproducible protocol for clonal propagation of Spilanthes acmella has been established. Routinely, the cultures were established in spring (January–April) season because of the highest aseptic culture establishment and high frequency shoot proliferation. Incorporation of 5 μM N⁶-benzyladenine (BA) to Murashige and Skoog (MS) basal medium showed 100% bud-break and promoted multiple shoot proliferation in cultures. Interestingly, a higher concentration of BA (7–15 μM) promoted stunted shoots with pale leaves while a lower concentration (1–3 μM) resulted in shoots with long internodes and excessive adventitious root proliferation from all over their surface. For recurrent shoot multiplication, single node segments from in vitro-developed shoots were excised and cultured on MS + BA (5 μM) medium where 20.3-fold shoot multiplication was achieved every 5 weeks. Finally, these shoots were successfully rooted on half-strength MS medium (major salts reduced to half-strength) with 50 g l⁻¹ sucrose, with a frequency of 100%. Transplantation survival of micropropagated plants was 88.9%. Additionally, accumulation of scopoletin, a phytoalexin, was revealed for the first time in the uninfected leaves of Spilanthes. Further, the quantitative estimation by HPLC with a fluorescence detector showed that the amounts of scopoletin content (0.10 μg g⁻¹ DW) in the leaves of micropropagated plants are comparable to those of field-grown mother plants. The study thus signifies the effectiveness of in vitro methodology for true-to-type plant regeneration of Spilanthes and their later utility for biosynthesis and constant production of scopoletin throughout the year.     

The wheat gene <Emphasis Type="Italic">TaST</Emphasis> can increase the salt tolerance of transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

On the basis of the results of gene chip analysis of the salt-tolerant wheat mutant RH8706-49 under conditions of salt stress, we identified and cloned an unknown salt-induced gene TaST (Triticum aestivum salt-tolerant). Real-time quantitative PCR analysis showed that the expression of the gene was induced by salt stress. Transgenic Arabidopsis plants overexpressing the TaST gene showed higher salt tolerance than the wild-type controls. Subcellular localization studies revealed that the protein encoded by this gene was in the nucleus. In comparison with wild-type controls, transgenic Arabidopsis plants accumulated more Ca²⁺, soluble sugar, and proline and less Na⁺ under salt stress. Real-time quantitative PCR analysis showed that Arabidopsis plants overexpressing TaST also showed increased expression of many stress-related genes. All these findings indicated that TaST can enhance the salt tolerance of transgenic Arabidopsis plants.     

Morphological and physiological responses of <Emphasis Type="Italic">Vitex negundo</Emphasis> L. var. <Emphasis Type="Italic">heterophylla</Emphasis> (Franch.) Rehd. to drought stress

Water is a main factor limiting plant growth. Integrative responses of leaf traits and whole plant growth to drought will provide implications to vegetation restoration. This study investigated the drought responses of Vitex negundo L. var. heterophylla (Franch.) Rehd. with a focus on leaf morphology and physiology, seedling growth and biomass partitioning. Potted 1-year-old seedlings were subjected to four water supply regimes [75, 55, 35 and 15% field capacity (FC)], served as control, mild water stress, moderate water stress and severe water stress. Leaf morphological traits varied to reduce the distance of water transfer under water stress and leaflets were dispersed with drought. Net photosynthetic rate decreased significantly under water stress: stomatal closure was the dominant limitation at mild and moderate drought, while metabolic impairment was dominant at severe drought. The physiological impairment at severe drought could also be detected from the relative lower water use efficiency and non-photochemical quenching to moderate water stress. Total biomass of well-watered plants was more than twice that at moderate water deficit and nearly ten times that at severe water deficit. In summary, V. negundo var. heterophylla had adaptation mechanism to water deficit even in the most serious condition, but different strategies were adopted. Seedlings invested more photosynthate to roots at mild and moderate drought while more photosynthate to leaves at severe drought. A nearly stagnant seedling growth and a sharp decline of total biomass were the survival strategy at severe water stress, which was not favorable to vegetation restoration. Water supply above 15% FC is recommended for the seedlings to vegetation restoration.     

In-vitro development of ovules obtained after pollination of cotton (<Emphasis Type="Italic">Gossypium</Emphasis> spp) flowers with pollen from okra (<Emphasis Type="Italic">Abelmoschus esculentus L. Moench)</Emphasis>

The in vitro response of ovules obtained after pollination of cotton flowers with pollen from Abelmoschus esculentus was studied. For this, 492 cotton flowers from five G. hirsutum varieties, four G. barbadense varieties and 10 F1 interspecific hybrids, were pollinated with pollen from A. esculentus and 5,069 ovules were cultured in vitro. From the cultured ovules, 69 embryos were isolated and 16 of them grew into plants. However, only three of them survived after transplantation. Finally, one plant which originated from the interspecific cross (B403 × Acala Sindos) × A. esculentus reached maturity. The mature plant (Pa0) had no morphological traits from A. esculentus. On the contrary, traits from both cotton species were observed. The flowcytometric analysis of the Pa0 plant indicated that it was hypoaneuploid. Root tip chromosome counts of its offsprings revealed a progressive chromosome increase from the Pa1 to Pa4 generation. Plants with 52 chromosomes or hypoaneuploids with a lower level of chromosomes (46–51) could be isolated from the Pa4 generation. These plants exhibited morphological traits from both cotton species and they were fertile. No signs of A. esculentus morphological characteristics were observed in these plants. It was concluded that aneuploid partial interspecific cotton plants could be produced after pollination of cotton interspecific hybrids with pollen from A. esculentus and application of an in-ovule embryo rescue technique.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.