Efficient micropropagation of Thunbergia coccinea Wall. and genetic homogeneity assessment through RAPD and ISSR markers.

Thunbergia coccinea Wall. ex D. Don being a rare, ornamental and medicinal plant of India, is needed to propagate for conserving the germplasm and analyzing its phytochemical compounds in the future. A reliable protocol for direct in vitro propagation using nodal shoot meristem of T. coccinea as explant was standardized. The highest number of shoots per explant (22.17 ± 0.54) with maximum shoot length (2.36 ± 0.28) in cm was obtained in Murashige and Skoog (MS) medium supplemented with 9.70 µM of 6-furfurylaminopurine (Kinetin) and 0.053 µM of α-naphthaleneacetic acid (NAA) combination, among all the different plant growth regulators (PGR's) and concentrations tested. The aforesaid PGR's combination was optimum for axillary shoot bud induction and multiplication in T. coccinea. The best rooting was observed on the half-strength MS medium fortified with 2.68 µM NAA with the highest number of roots per shoot (3.75 ± 0.12) and maximum length (5.22 ± 0.32) in cm. All the in vitro raised plantlets were acclimatized in sterile sand and soil mixture (1:1) with a survival rate of 70% on earthen pots under greenhouse conditions. PCR-based RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat) molecular markers were employed to determine the genetic homogeneity amongst the plantlets. Twelve (12) RAPD and nine (9) ISSR primers developed a total of 104 and 91 scorable bands, respectively. The band profiles of micropropagated plantlets were monomorphic to the mother, donor in vivo plant, and similarity values varied from 0.9542-1.000. The dendrogram generated through UPGMA (unweighted pair group method with arithmetic mean) showed 99% similarities amongst all tested plants confirming the genetic uniformity of in vitro raised plants.

Multiple stressors in multiple species: Effects of different RDX soil concentrations and differential water-resourcing on RDX fate, plant health, and plant survival.

Response to simultaneous stressors is an important facet of plant ecology and land management. In a greenhouse trial, we studied how eight plant species responded to single and combined effects of three soil concentrations of the phytotoxic munitions constituent RDX and two levels of water-resourcing. In an outdoor trial, we studied the effects of high RDX soil concentration and two levels of water-resourcing in three plant species. Multiple endpoints related to RDX fate, plant health, and plant survival were evaluated in both trials. Starting RDX concentration was the most frequent factor influencing all endpoints. Water-resourcing also had significant impacts, but in fewer cases. For most endpoints, significant interaction effects between RDX concentration and water-resourcing were observed for some species and treatments. Main and interaction effects were typically variable (significant in one treatment, but not in another; associated with increasing endpoint values for one treatment and/or with decreasing endpoint values in another). This complexity has implications for understanding how RDX and water-availability combine to impact plants, as well as for applications like phytoremediation. As an additional product of these greenhouse and outdoor trials, three plants native or naturalized within the southeastern United States were identified as promising species for further study as in situ phytoremediation resources. Plumbago auriculata exhibited relatively strong and markedly consistent among-treatment mean proportional reductions in soil RDX concentrations (112% and 2.5% of the means of corresponding values observed within other species). Likewise, across all treatments, Salvia coccinea exhibited distinctively low variance in mean leaf chlorophyll content index levels (6.5% of the means of corresponding values observed within other species). Both species also exhibited mean wilting and chlorosis levels that were 66% and 35%, and 67% and 84%, of corresponding values observed in all other plants, respectively. Ruellia caroliniensis exhibited at least 43% higher mean survival across all treatments than any other test species in outdoor trials, despite exhibiting similar RDX uptake and bioconcentration levels.

Alterations in Herbage Yield, Antioxidant Activities, Phytochemical Contents, and Bioactive Compounds of Sabah Snake Grass (Clinacanthus Nutans L.) with Regards to Harvesting Age and Harvesting Frequency.

Sabah snake grass or Clinacanthus nutans has drawn public interest having significant economic benefits attributable to the presence of phytochemicals and several interesting bioactive constituents that may differ according to harvesting age and harvesting frequency. The current study was aimed to evaluate the effect of harvesting age and harvesting frequency towards herbal yield, antioxidant activities, phytochemicals synthesis, and bioactive compounds of C. nutans. A factorial randomized completely block design with five replications was used to illustrate the relationship between herbal yield, DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ferric reducing antioxidant power (FRAP) assays, total phenolic and flavonoid content affected by harvesting age (week 8, 12, and 16 after transplanting), and harvesting frequency (harvest 1, 2, and 3). The bioactive compounds by HPLC were also determined to describe the interaction effect between both harvesting age and harvesting frequency. The yield, antioxidant activities, and phytochemical contents were gradually increased as the plant grew, with the highest recorded during week 16. However, the synthesis and activities of phytochemicals were reduced in subsequent harvests despite the increment of the herbal yield. All bioactive compounds were found to be influenced insignificantly and significantly by harvesting age and harvesting frequency, respectively, specifically to shaftoside, iso-orientin, and orientin. Among all constituents, shaftoside was the main compound at various harvesting ages and harvesting frequencies. These results indicated that harvesting at week 16 with 1st harvest frequency might enhance the yield while sustaining the high synthesis of polyphenols and antioxidant activities of C. nutans.

Source of metal contamination in sediment, their ecological risk, and phytoremediation ability of the studied mangrove plants in ship breaking area, Bangladesh.

Samples for sediment and two species of native mangrove plants were collected from seven sampling sites for assessing the level of metal contamination. Results of the studied metals displayed the order of pollution as Fe > Ti > Zr > Rb > Zn > Sr > Pb > Y > Cu > Cr > As accordingly. Geoaccumulation index and contamination factor revealed that the sediment samples were unpolluted to moderately polluted by Zn, Fe, Ti, Rb, Y, and Zr. Ecological risk factor depicted a pollution-free condition in the study areas. PCA, CA, and correlation coefficient indicated that the source of the metals in the environment was anthropogenic. Bioconcentration factor values were found to be below 1 in both plant species. Conversely, transfer factor values for most heavy metals were found to be >1 in both plant species, which reflects the phytoremediation ability of plants.

De novo characterization of the Baphicacanthus cusia(Nees) Bremek transcriptome and analysis of candidate genes involved in indican biosynthesis and metabolism.

Baphicacanthus cusia (Nees) Bremek is an herb widely used for the clinical treatment of colds, fever, and influenza in Traditional Chinese Medicine. The roots, stems and leaves can be used as natural medicine, in which indigo and indirubin are two main active ingredients. In this study, quantification of indigo, indirubin, indican and adenosine among various tissues of B. cusia was conducted using HPLC-DAD. Leaves have significantly higher contents than stems and roots (380.66, 315.15, 20,978.26, 4323.15 µg/g in leaves, 306.36, 71.71, 3,056.78, 139.45 µg/g in stems, and 9.31, 7.82, 170.45, 197.48 µg/g in roots, respectively). De novo transcriptome sequencing of B. cusia was performed for the first time. The sequencing yielded 137,216,248, 122,837,394 and 140,240,688 clean reads from leaves, stems and roots respectively, which were assembled into 51,381 unique sequences. A total of 33,317 unigenes could be annotated using the databases of Nr, Swiss-Prot, KEGG and KOG. These analyses provided a detailed view of the enzymes involved in indican backbone biosynthesis, such as cytochrome P450, UDP-glycosyltransferase, glucosidase and tryptophan synthase. Analysis results showed that tryptophan synthase was the candidate gene involved in the tissue-specific biosynthesis of indican. We also detected sixteen types of simple sequence repeats in RNA-Seq data for use in future molecular mark assisted breeding studies. The results will be helpful in further analysis of B. cusia functional genomics, especially in increasing biosynthesis of indican through biotechnological approaches and metabolic regulation.

Mangrove succession enriches the sediment microbial community in South China.

Sediment microorganisms help create and maintain mangrove ecosystems. Although the changes in vegetation during mangrove forest succession have been well studied, the changes in the sediment microbial community during mangrove succession are poorly understood. To investigate the changes in the sediment microbial community during succession of mangroves at Zhanjiang, South China, we used phospholipid fatty acid (PLFA) analysis and the following chronosequence from primary to climax community: unvegetated shoal; Avicennia marina community; Aegiceras corniculatum community; and Bruguiera gymnorrhiza + Rhizophora stylosa community. The PLFA concentrations of all sediment microbial groups (total microorganisms, fungi, gram-positive bacteria, gram-negative bacteria, and actinomycetes) increased significantly with each stage of mangrove succession. Microbial PLFA concentrations in the sediment were significantly lower in the wet season than in the dry season. Regression and ordination analyses indicated that the changes in the microbial community with mangrove succession were mainly associated with properties of the aboveground vegetation (mainly plant height) and the sediment (mainly sediment organic matter and total nitrogen). The changes in the sediment microbial community can probably be explained by increases in nutrients and microhabitat heterogeneity during mangrove succession.

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.

Competitive interaction of Axonopus compressus and Asystasia gangetica under contrasting sunlight intensity.

Axonopus compressus is one of the native soft grass species in oil palm in Malaysia which can be used as a cover crop. The competitive ability of A. compressus to overcome A. gangetica was studied using multiple-density, multiple-proportion replacements series under a glasshouse and full sunlight conditions in a poly bag for 10 weeks. A. compressus produced more dry weight and leaf area when competing against A. gangetica than in monoculture at both densities in the full sunlight and at high density in the shade. Moreover, the relative yield and relative crowding coefficients also indicated A. compressus is a stronger competitor than A. gangetica at both densities in the full sunlight and high density in the shade. It seemed that A. gangetica plants in the shade did not compete with each other and were more competitive against A. compressus as could influence A. compressus height in the shade. It is concluded that although suppression of A. gangetica by A. compressus occurred under full sunlight, irrespective of plant density, this ability reduced under shade as A. compressus density decreased. The result suggests that A. compressus in high density could be considered as a candidate for cover crops under oil palm canopy.

Adventitious shoot regeneration from leaf explant of dwarf hygro (Hygrophila polysperma (Roxb.) T. Anderson).

Dwarf hygro (Hygrophila polysperma) is an ornamental aquatic plant that changes its leaf colours to pinkish in high light. It is listed as a medicinal plant in medicinal plant lists of Indian states of West Bengal and Karnataka. It is also used as a screening tool for toxicities and a bioindicator to detect and control algae. The study reported in vitro adventitious shoot regeneration from leaf explants cultured on MS medium containing 0.10-1.60 mg/L Kin/TDZ with or without 0.10 mg/L IBA and 500 mg/L Amoklavin to eradicate endogenic bacterial contamination. Direct adventitious shoot regeneration started within one week from both culture mediums followed by late callus induction which was more prominent on TDZ containing media compared to Kin containing media. Addition of 0.10 mg/L IBA with both Kin and TDZ increased shoot regeneration frequency, mean number of shoots per explant, and mean shoot length. Maximum number of 16.33 and 20.55 shoots per explant was obtained on MS medium containing 0.80 + 0.10 mg/L Kin-IBA and 0.10 + 0.10 mg/L TDZ-IBA, respectively. Regenerated shoots were rooted on MS medium containing 0.20-1.00 mg/L IBA followed by successfull acclimatization in aquariums. Regenerated plantlets were also tested in jars containing distilled water that showed the pH 6-9 for the best plant growth and development.

Green nanoemulsion-laden glyphosate isopropylamine formulation in suppressing creeping foxglove (A. gangetica), slender button weed (D. ocimifolia) and buffalo grass (P. conjugatum).

BACKGROUND: Pesticides are developed with carriers to improve their physicochemical properties and, accordingly, the bioefficacy of the applied formulation. For foliar-applied herbicide, generally less than 0.1% of the active ingredient reaching the target site could reduce pesticide performance. Recently, a carrier of nanoemulsion consisting of oil, surfactant and water, with a particle size of less than 200 nm, has been shown to enhance drug permeability for skin penetration in pharmaceutical delivery systems. In the present work, the aim was to formulate a water-soluble herbicide, glyphosate isopropylamine (IPA), using a green nanoemulsion system for a biological activity study against the weeds creeping foxglove, slender button weed and buffalo grass. RESULTS: The nanoemulsion formulations displayed a significantly lower spray deposition on creeping foxglove (2.9-3.5 ng cm(-2) ), slender button weed (2.6-2.9 ng cm(-2) ) and buffalo grass (1.8-2.4 ng cm(-2) ) than Roundup(®) (3.7-5.1 ng cm(-2) ). The visible injury rates of weeds treated with the nanoemulsion formulations were statistically equivalent to those relating to Roundup(®) at 14 days after treatment, with a control range of 86.67-96.67%. CONCLUSION: It was hypothesised that the significant difference in spray deposition with equal injury rates can be attributed to enhanced bioactivity of the nanoemulsion formulations. This initial discovery could be the platform for developing better penetration of agrochemical formulations in the future.

Differences in lead tolerance between Kandelia obovata and Acanthus ilicifolius seedlings under varying treatment times.

The effects of short-term (1 day) and long-term (49 days) of lead (Pb) stress on growth and physiological responses in the leaves and roots of two mangrove plants, Kandelia obovata and Acanthus ilicifolius, were compared. The growth of both species was affected by Pb at Day 49, whereas the root to shoot ratio of K. obovata remained unchanged. Compared with A. ilicifolius, less Pb accumulated in leaves of K. obovata, which indicates that this species is a typical Pb-excluder. Significant linear relationships were observed between the Pb concentrations in the roots and leaves and the Pb treatment concentrations in the sediments in A. ilicifolius but not in K. obovata. The proline concentration increased in both mangrove species at Day 49, especially in A. ilicifolius, but no changes were observed at Day 1. The tolerant species K. obovata tended to acclimate to metal stress by restricting the translocation of toxic metals and by increasing and/or maintaining high superoxide dismutase (SOD) activity, minimizing lipid peroxidation, and exhibiting prolonged unaltered growth (49 days) under Pb treatment. The non-tolerant species, A. ilicifolius, did not acclimate to metal stress, its leaves were seriously damaged with significant increased MDA content, and its SOD activity was decreased. An increase of endogenous jasmonic acid concentration was observed only in K. obovata, both at Day 1 and at Day 49, which suggests that this hormone plays an important role in metal tolerance under short-term and long-term metal treatment.

Remediation of saline soil from shrimp farms by three different plants including soybean (Glycine max (L.) Merr.).

Shrimp farm activity can elevate in-situ soil salinity that in turn may affect any subsequent crop production if land usage changes. The utility of three different plants viz. soybean (Glycine max (L.) Merr.), narrow leaf cat-tail (Typha angustifolia L.) and sea holly (Acanthus ebracteatus Vahl) for phytoremediation of saline soil derived from former shrimp farm activity was investigated. The latter two species have been categorized as halophytes. In experiments of 16 days' duration and using sodium chloride concentrations (50-70 mg g(-1) dry weight) similar to those found in the benthic material of shrimp farms in Nakhon Pathom Province, central Thailand, the bioconcentration factors of sodium chloride (BCF; g soil dry weight g(-1) plant dry weight) in soybean (2240-4840) were found to be significantly higher than those found for narrow leaf cat-tail (16-20) and sea holly (15-17) at p < 0.05. The translocation of sodium chloride from root to shoot was noted in all plant species investigated, as well as wilting and defoliation due to the effects of sodium chloride. Approximately 90 %, 70 % and 60 % removal of sodium chloride in root zone soil was observed after growing soybean, narrow leaf cat-tail and sea holly, respectively. Soybean plants thus showed the greatest ability to decrease soil salinity, with measured root zone soil conductivity levels falling from 16.4-18 dS m(-1) (characteristic of strongly saline soils) to 1.5- 2.1 dS m(-1) (weakly saline). Although an important economic crop, soybean may also have potential in soil remediation.

An efficient in vitro regeneration protocol for a natural dye yielding plant, Strobilanthes flaccidifolious Nees., from nodal explants.

Adventitious shoot buds formation from axillary buds of nodal segments of S. flaccidifolious was achieved on MS medium containing sucrose (3%, w/v), and a-naphthalene acetic acid (NAA; 3 microM) and benzyl adenine (3 microM) in combination. The nodal segments were primed on 'Growtak Sieve' for 48 h on MS medium containing sucrose (2%), polyvinyl pyrollidone (200 mgL(-1)) as antioxidant. About 80% of primed nodal segments responded positively and formed approximately 12 adventitious shoot buds per explants from explants collected during October-November months of every year. The shoot buds converted into plantlets on MS medium containing sucrose (3%) and kinetin (3 microM) where approximately 7 micro shoots developed per subculture after 8 weeks of culture. The regenerated micro shoots induced average 14 roots/plant on medium containing NAA (3 microM). The regenerates were hardened for 6-7 weeks on medium with 1/2MS salt solution and sucrose (2%) under normal laboratory condition before transferring to potting mix. About 70% transplants survived after two months of transfer.

Effect of medium composition and light on root and rhinacanthin formation in Rhinacanthus nasutus cultures.

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) has long been used in Thai traditional medicine for treatment of tinea versicolor, ringworm, pruritic rash, and abscess. The active constituents are known as a group of naphthoquinone esters, rhinacanthins. This work focused on establishment of R. nasutus root cultures and determination of rhinacanthin production. Induction of R. nasutus root formation was accomplished on solid Gamborg's B5 (B5) medium, supplied with 0.1 mg/L indole-3-butyric acid (IBA) and 20 g/L sucrose. The effects of explants (whole leaf explants and four-side excised leaf explants), light and medium composition on root and rhinacanthin formation were investigated. The root formation from the whole leaf explants was 10 times higher than that from the four-side excised leaf explants. In addition, light possessed an inhibitory effect on the root and rhinacanthin formation of R. nasutus. Medium manipulation found that Murashige and Skoog (MS) medium supplied with 3 mg/L IBA and 30 g/L sucrose was the most suitable for induction of the root formation. Unfortunately, the obtained root cultures produced only rhinacanthin-C in very low amount, 0.026 mg/g dry weight (DW), when they were transferred into the same MS liquid medium. With semisolid medium (4 g/L agar) of the same MS composition, however, the root cultures appeared to produce higher content of rhinacanthin-C, -D and -N (3.45, 0.07 and 0.07 mg/g DW, respectively). Our finding suggests that culturing in semisolid medium is capable of improving of rhinacanthin production in R. nasutus root cultures.

[Effects of exogenous salicylic acid on protein expression level in Baphicacanthus cusia (Nees) Bremek leaves].

By using two-dimensional eletrophoresis method, this paper studied the protein expression level in Baphicacanthus cusia (Nees) Bremek leaves after sprayed with exogenous salicylic acid (SA). A total of significantly different 20 protein spots were obtained, among which, eight protein spots were indentified, being of ATP synthase, alpha tubulin, cell division protein, glyceraldehydephosphate dehydrogenase, and ACC oxidase, respectively. The expression abundance of all identified proteins was up-regulated, except for ACC oxidase which was down-regulated. Therefore, exogenous SA could affect the protein expression level in B. cusia leaves, and improve the plant resistance to environment stress and self-restoration capability.

[RAPD analysis on the germplasm resources of Baphicacanthus cusia].

OBJECTIVE: To explore the genetic diversity of germplasm resources of Baphicacanthus cusia on molecular leve. METHODS: The molecular biological technique-random amplified polymorphic DNA (RAPD) were used. RESULTS: 104 random decamer primers were screened for RAPD fragments of Baphicacanthus cusia. 422 DNA bands were amplified by 12 primers, 55.69% products were found to be polymorphic. Base on UPGMA cluster analysis, a DNA molecular dendrogram was established to discuss the genetic diversity of the germplasm resources of Baphicacanthus cusia. The genetic differences are related to morphological differences on a certain extant, but not to geographic regions. CONCLUSION: There actually existed much genetic diversity on molecular level among different natural populations of Baphicacanthus cusia.

Genetic variation in the response of the weed Ruellia nudiflora (Acanthaceae) to arbuscular mycorrhizal fungi.

The main goal of this work was to test for plant genetic variation in the phenotypic plasticity response of the weed Ruellia nudiflora to arbuscular mycorrhizal (AM) fungi inoculation. We collected plants in the field, kept them under homogeneous conditions inside a nursery, and then collected seeds from these parent plants to generate five inbred lines (i.e., genetic families). Half of the plants of each inbred line were inoculated with AM fungi while the other half were not (controls); a fully crossed experimental design was then used to test for the effects of treatment (with or without AM fungi inoculation) and inbred line (genetic family). For each plant, we recorded the number of leaves produced and the number of days it survived during a 2-month period. Results showed a strong positive treatment effect (plastic response to AM fungi inoculation) for leaf production and survival. Moreover, in terms of survival, the treatment effect differed between genetic families (significant genetic family by treatment interaction). These findings indicate that the positive effect of AM fungi on plant survival (and potentially also growth) differs across plant genotypes and that such condition may contribute to R. nudiflora's capacity to colonize new environments.

Local adaptation of Ruellia nudiflora (Acanthaceae) to biotic counterparts: complex scenarios revealed when two herbivore guilds are considered.

This study evaluated whether the herb Ruellia nudiflora is locally adapted to a specialist insect seed predator (SP) and insect folivores, and if plant local adaptation (LA) to the former is more likely. A reciprocal transplant experiment was conducted using three sites in Yucatan (Mexico) (n = 864 plants). A third of the plants of each origin were placed at each site, and we recorded the following during a 9-month period: fruit number, leaf damage, and fruits attacked by SP. Results indicated lack of plant LA for all the variables measured. Instead, seed predation was c. 100% greater for native plants at one study site, suggesting insect LA or plant maladaptation; folivory was homogeneous across sites/origins. Based on these results, we discuss differences in the potential each herbivore guild has to promote plant LA, as well as divergent evolutionary outcomes of plant-herbivore interactions across sites.

Climate alters response of an endemic island plant to removal of invasive herbivores.

Islands experience higher rates of species extinction than mainland ecosystems, with biological invasions among the leading causes; they also serve as important model systems for testing ideas in basic and applied ecology. Invasive removal programs on islands are conservation efforts that can also be viewed as powerful manipulative experiments, but few data are available to evaluate their effects. We collected demographic and herbivore damage data for Castilleja mollis Pennell, an endangered plant endemic to Santa Rosa Island, California, over a 12-year period before, during, and after the implementation of control for introduced cattle, deer, and elk. We used these long-term data to explore mechanisms underlying herbivore effects, assess the results of herbivore reduction at the scales of both individual plants and populations, and determine how temporal variability in herbivory and plant demography influenced responses to herbivore removals. For individual plants, herbivore effects mediated by disturbance were greater than those of grazing. Deer and elk scraping of the ground substantially increased plant mortality and dormancy and reduced flowering and growth. Stem damage from browsing did not affect survivorship but significantly reduced plant growth and flower production. Herbivore control successfully lowered damage rates, which declined steeply between 1997 and 2000 and have remained relatively low. Castilleja mollis abundances rose sharply after 1997, suggesting a positive effect of herbivore control, but then began to decline steadily again after 2003. The recent decline appears to be driven by higher mean growing season temperatures; interestingly, not only reductions in scraping damage but a period of cooler conditions were significant in explaining increases in C. mollis populations between 1997 and 2002. Our results demonstrate strong effects of introduced herbivores on both plant demography and population dynamics and show that climate-driven variation may counteract and mask positive responses to herbivore removal. Regional mean temperatures already have risen significantly over the last 50 years, suggesting that climate change could increasingly swamp the effects of management targeted at other environmental problems. Similar interactions between climate and invasive species will play an even greater role in future management, with long-term data sets like this critical to quantifying such effects.

Biomolecular changes during in vitro organogenesis of Asteracantha longifolia (L.) Nees--a medicinal herb.

High frequency plant regeneration in A. longifolia (L.) was achieved from leaf explant implanted on MS basal medium supplemented with NAA (0.5 mg/l) + BA (2.0 mg/l) through intervening callus phase. Well-developed shoots (>3cm) were successfully rooted on MS medium supplemented with NAA (0.1 mg/l). Protein and total soluble sugar contents were maximum during organogenesis and multiple shoot induction phase compared with non-organogenic callus and root induction phase. Esterase and catalase activities were maximum during organogenic differentiation, while activities were minimum at non-differentiated callus stages. Peroxidase activities were higher during rhizogenesis. Contradiction to peroxidase activity, acid phosphatase activities were high during organogenesis and declined during rhizogenesis. SDS-PAGE analysis of total soluble proteins revealed expression of non-organogenic callus (97.9 kDa), organogenic callus (77.2, 74.1, 21.9 kDa), multiple shoot induction phase (106.6, 26.9, 11.6 kDa) and root induction phase (15.9 kDa) specific polypeptides. Esterase zymogram revealed one band (Rm 0.204) appeared in both organogenic callus and multiple shoot induction phase. Peroxidase zymogram detected two stage specific bands, one band (Rm 0.42) was specific to root induction phase, while another (Rm 0.761) was specific to multiple shoot induction. Catalase and acid phosphatase zymogram resolved one band (Rm 0.752 and 0.435, respectively) in differentiated stages including both multiple shoot induction phase and root induction phase, but absent in undifferentiated phases.