In vitro acclimatization of Curcuma longa under controlled iso-osmotic conditions

In vitro acclimatization has been validated as the successful key to harden the plantlets before transplanting to ex vitro conditions. In the present study, we investigated the potential of different sugar types (glucose, fructose, galactose, sucrose) in regulating morphological, physiological and biochemical strategies, survival percentage and growth performance, and rhizome traits of turmeric under iso-osmotic potential. Leaf greenness (SPAD value) in acclimatized plantlets (4% glucose; −1.355 MPa osmotic potential) of ‘ST018’ was retained and greater than in ‘PB009’ by 1.69-fold, leading to maintain high F_v/F_m (maximum quantum yield of PSII), Φ_PSII (photon yield of PSII) and P_n (net photosynthetic rate) levels, and retained shoot height, leaf length, leaf width, shoot fresh weight and shoot dry weight after one month upon transplanting to ex vitro conditions. In addition, P_n, C_i (intracellular CO₂), g_s (stomatal conductance) and E (transpiration rate) in acclimatized plantlets (6% sucrose; −1.355 MPa osmotic potential) of ‘PB009’ were stabilized as physiological adapted strategies, regulating the shoot and root growth and fresh and dry weights of mini-rhizome. Interestingly, the accumulation of total curcuminoids in mini-rhizome derived from 6% sucrose acclimatized plantlets of ‘ST018’ was greater than in ‘PB009’ by 3.76-fold. The study concludes that in vitro acclimation of turmeric ‘PB009’ and ‘ST018’ using 6% sucrose and 4% glucose, respectively, promoted percent survival, physiological adaptations, and overall growth performances under greenhouse conditions.     

In vitro effectiveness of Curcuma longa and Zingiber officinale extracts on Echinococcus protoscoleces

Hydatid disease is an important economic and human public health problem with a wide geographical distribution. Surgical excision remains the primary treatment and the only hope for complete cure of hydatosis. The most important complications arising from surgical excision, however, is recurrence, which is due to dissemination of protoscolices during the surgery. Pre-surgical inactivation of the contents of the hydatid cyst by injection of scolicidal agent into the cyst has been used as adjunct to surgery in order to overcome the risk of recurrence. In the present study, ethanolic extracts of turmeric (Curcuma longa) and ginger (Zingiber officinale) were tested as scolicidal agent for Echinococcus protoscoleces. Protoscoleces were collected aseptically from sheep livers containing hydatid cysts. Three concentrations (10, 30 and 50 mg/ml) of each extract were investigated and viability of the protoscoleces was tested by 0.1% eosin staining. Ginger extract showed the strongest scolicidal effect (100%) after 20 min at a concentration of 30 mg/ml and 10 min at 50 mg/ml. The maximum scolicidal effect of turmeric was 93.2% after 30 min at a concentration of 50 mg/ml. It is concluded that turmeric and ginger extracts have high scolicidal activity and could be used as effective scolicidal agents against Echinococcus protoscoleces.     

姜黄根茎中姜黄素类成分含量的产地差异及其与环境因子的CCA分析

采用HPLC法对产自四川崇州和犍为、广东四会、广西玉林和博白及金秀、云南马关的姜黄(Curcuma longaL.)根茎中姜黄素类成分含量进行测定,并利用典范对应分析方法(CCA)研究了不同产地姜黄根茎中姜黄素类成分含量与地理-气候因子及根际土壤养分因子间的相关性。结果表明:不同产地根际土壤中有机质、全N、全P和全K含量分别为14.03～32.79、0.39～0.92、0.56～1.55和2.29～9.23 g.kg-1,根际土壤养分含量差异较明显;姜黄多生长在中性偏酸、水肥性能良好的土壤中。姜黄根茎中姜黄素、去甲氧基姜黄素和双去甲氧基姜黄素含量及姜黄素类成分总含量的平均值分别为1.53%、0.42%、0.67%和2.61%;不同产地姜黄根茎中姜黄素类成分含量有显著差异,且同一产地采自不同采样点及不同采样时间的样品姜黄素类成分的含量也有一定差异。姜黄素类成分总含量以广西博白产姜黄根茎最高(4.29%)、广东四会产姜黄根茎最低(1.73%)。CCA分析结果表明:在经度、纬度、海拔、年均气温、极端最高温、极端最低温、年降水量、日照时数和无霜期等地理-气候因子中,年均气温和极端最低温与姜黄素类成分含量极显著正相关;而在pH值及有机质、全N、全P、全K含量等根际土壤养分因子中,有机质含量与姜黄素类成分含量极显著正相关。分析结果显示:影响姜黄根茎中姜黄素类成分含量的主要环境因子是年均气温、极端最低温和根际土壤的有机质含量。     

Photolarvicidal effect of curcuminoids from Curcuma longa Linn. against Aedes aegypti larvae

In this study we evaluated the photolarvicidal activity of different curcuminoids forms: Natural Turmeric (NT), Synthetic Curcumin (SC) and Synthetic curcumin formulated with sucrose (SCS) against Aedes aegypti larvae which is the major vector of dengue, chikungunya and Zika viruses. Two strategies for curcuminoids delivery were chosen for the experiments: (i) larvae exposed to aqueous solutions of curcuminoids in the concentration range of 5–25 mg/L (w/v) and (ii) larvae exposed to two different types of curcuminoids carriers (pet food). Phototoxic effects of the curcuminoids were evaluated by counting the dead larvae after sunlight exposure, at irradiance range of 30–60 mW/cm². The characteristic photobleaching time for each curcuminoids in solution was also determined in order to infer the safety of these molecules in the natural environment. Curcuminoids solutions showed high photolarvicidal activity after 30 min of sunlight irradiation, with LC₅₀ values of 20.0, 11.6 and 2.2 mg/L for NT, SC and SCS, respectively. The curcuminoids carriers, despite showing high mortality (about 90%), presented photodynamic activity only after 8 h of sunlight irradiation. The curcuminoids remained in the aqueous solution for up to 3 h; subsequently, the maximum absorption peak of these pigments was completely modified. The characteristic photobleaching time for each sample was 6, 32 and 60 min for NT, SC and SCS. These original and relevant results show that the combination of curcuminoids pigments with sunlight establishes an extremely effective alternative for Ae. aegypti larvae control, ensuring high environmental safety due to the low persistence of these molecules in the presence of light.     

In vitro propagation of Acorus calamus Linn.--a medicinal plant

High frequency in vitro propagation protocol was standardized from rhizome explants of A. calamus. Maximum shoot multiplication frequency was obtained on Murashige and Skoog's media supplemented with 4 mg/l 6-benzyl amino purine and 0.5 mg/l indole-3-acetic acid. Regenerated shoots were rooted in vitro or directly transferred to sterile soil and well developed roots were observed within two weeks. The rooted plants were successfully established in the field.     

In vitro induction, screening and detection of high essential oil yielding somaclones in turmeric (Curcuma longa L.)

Leaf of turmeric contains an essential oil used extensively in perfumery, pharmaceuticals and aromatherapy. Five somaclones were induced in turmeric on MS media with varying amounts of plant growth regulators. All somaclones were subsequently transferred to the field. Essential oil was extracted from leaves of in vitro and ex vitro grown plants and subjected to quantitative and qualitative evaluation. A positive correlation was established between the leaf oil content and oil constituent of in vitro grown and field transferred somaclones. Somaclones (C2, C4, C5) containing 0.16–0.18 % oil in vitro retained normal oil content (0.48–0.5 %) in the field. Similarly in vitro grown somaclones C3 and C7 with 0.36 and 0.25 % oil content retained proportionately increased oil yields of 1 % and 0.76 under ex vitro condition. GC–MS analysis of the oil revealed similar spectrum of constituents both among in vitro and ex vitro grown plants with alpha-phellandrene as major one. Thus the novel method of in vitro screening could be applied for rapid identification of high essential oil yielding turmeric genotypes thereby reducing labour, cost and time required in conventional ex vitro screening of somaclones.     

Influence of magnetic field on the growth,development and rhizome yield of turmeric (Curcuma longa L.)

Plant Cell, Tissue and Organ Culture (PCTOC) - Static magnetic field (SMF) as a priming method (magnetopriming) is used to invigorate plant growth and development culminating in improved...     

姜黄根茎内生真菌区系分析

目的了解姜黄植物根茎中内生真菌群落组成和生态分布规律。方法采用表面消毒法分别于春、冬季从姜黄植物根茎中分离内生真菌,通过形态特征和基于ITS序列的系统发育分析初步确定其分类地位。结果分离获得51株内生真菌(春季33株、冬季18株),初步鉴定其分别归于5个纲、7个目、8个科、8个属(Fusarium,Gibberel-la,Alternaria,Phomopsis,Diaporthe,Nectria,Botryosphaeria,Mucor),其中镰刀菌属(Fusarium,51.0%)和赤霉属(Gibberel-la,17.6%)为优势菌群;春季分离的内生真菌分属于7个属(分离率为82.5%),而冬季的内生真菌仅归入4个属(分离率为45.0%)。结论姜黄植物中内生真菌具有较丰富的物种和系统发育多样性,在类群组成和分布上存在季节性差异,某些内生真菌(Fusariumsp.)具有宿主偏好性。     

Modules of co-regulated metabolites in turmeric (Curcuma longa) rhizome suggest the existence of biosynthetic modules in plant specialized metabolism

Turmeric is an excellent example of a plant that produces largenumbers of metabolites from diverse metabolic pathways or networks.It is hypothesized that these metabolic pathways or networkscontain biosynthetic modules, which lead to the formation ofmetabolite modules—groups of metabolites whose productionis co-regulated and biosynthetically linked. To test whethersuch co-regulated metabolite modules do exist in this plant,metabolic profiling analysis was performed on turmeric rhizomesamples that were collected from 16 different growth and developmenttreatments, which had significant impacts on the levels of 249volatile and non-volatile metabolites that were detected. Importantly,one of the many co-regulated metabolite modules that were indeedreadily detected in this analysis contained the three majorcurcuminoids, whereas many other structurally related diarylheptanoidsbelonged to separate metabolite modules, as did groups of terpenoids.The existence of these co-regulated metabolite modules supportedthe hypothesis that the 3-methoxyl groups on the aromatic ringsof the curcuminoids are formed before the formation of the heptanoidbackbone during the biosynthesis of curcumin and also suggestedthe involvement of multiple polyketide synthases with differentsubstrate selectivities in the formation of the array of diarylheptanoidsdetected in turmeric. Similar conclusions about terpenoid biosynthesiscould also be made. Thus, discovery and analysis of metabolitemodules can be a powerful predictive tool in efforts to understandmetabolism in plants. Key words: Biosynthesis, Curcuma longa, curcumin, metabolite module, metabolomics, rhizome, specialized metabolism Received 16 June 2008; Revised 29 September 2008 Accepted 2 October 2008     

The protective effects of Curcuma longa Linn. extract on carbon tetrachloride-induced hepatotoxicity in rats via upregulation of Nrf2

This study was designed to investigate the potentially protective effects of Curcuma longa Linn. extract (CLE) on carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Male Sprague-Dawley rats were pretreated with 50 or 100mg/kg of CLE or 100mg/kg of butylated hydroxytoluene (BHT) for 14 days before CCl4 administration. In addition, the CLE control group was pretreated with 100mg/kg CLE for only 14 days. Three hours after the final treatment, a single dose of CCl4 (20mg/kg) was administrated intraperitoneally to each group. After the completion of this phase of the experiment, food and water were removed 12 h prior to the next step. The rats were then anesthetized by urethane and their blood and liver were collected. It was observed that the aspartate aminotransferase and alanine aminotransferase activities of the serum, and the hepatic malondialdehyde levels had significantly decreased in the CLE group when compared with the CCl4-treated group. The antioxidant activities, such as superoxide dismutase, catalase, and glutathione peroxidase activities, in addition to glutathione content, had increased considerably in the CLE group compared with the CCl4-treated group. Phase II detoxifying enzymes, such as glutathione S-transferase, were found to have significantly increased in the CLE group as opposed to the CCl4-treated group. The content of Nrf2 was determined by Western blot analysis. Pretreated CLE increased the level of nuclear translocated Nrf2, and the Nrf2 then increased the activity of the antioxidant and phase II detoxifying enzymes. These results indicate that CLE has protective effects against CCl4-induced hepatotoxicity in rats, via activities of antioxidant and phase II detoxifying enzymes, and through the activation of nuclear translocated Nrf2.     

Mining and characterization of EST derived microsatellites in Curcuma longa L

Turmeric (Curcuma longa L.) (Family: Zingiberaceae) is a perennial rhizomatous herbaceous plant often used as a spice since time immemorial. Turmeric plants are also widely known for its medicinal applications. Recently EST-derived SSRs (Simple sequence repeats) are a free by-product of the currently expanding EST (Expressed Sequence Tag) databases. SSRs have been widely applied as molecular markers in genetic studies. Development of high throughput method for detection of SSRs has given a new dimension in their use as molecular markers. A software tool SciRoKo was used to mine class I SSR in Curcuma EST database comprising 12953 sequences. A total of 568 non-redundant SSR loci were detected with an average of one SSR per 14.73 Kb of EST. Furthermore, trinucleotide was found to be the most abundant repeat type among 1-6-nucleotide repeat types. It accounted for 41.19% of the total, followed by the mononucleotide (20.07%) and hexanucleotide repeats (15.14%). Among all the repeat motifs, (A/T)n accounted for the highest proportion followed by (AGG)n. These detected SSRs can be greatly used for designing primers that can be used as markers for constructing saturated genetic maps and conducting comparative genomic studies in different Curcuma species.     

Short-term and long-term time course studies of turmeric (Curcuma longa L.) microrhizome development in vitro

Turmeric (Curcuma longa L.) plantlets were cultured in MS (Murashige and Skoog Physiol Plant 15:473–497, 1962) liquid medium with 6% sucrose. Microrhizome development was observed in the presence of methyl jasmonate (MeJa) (0, 5 and 16 μM) and benzyladenine (BA) (0, 0.32 and 1 μM). Leaf, root, rhizome growth, and sugar use were measured weekly for 6 weeks in small vessels (180 ml) and four times in 23 weeks in larger vessels (2.5 l). MeJa reduced leaf, root, and rhizome biomass. BA had a positive effect on biomass accumulation. Microrhizome mass increased at a linear rate during 6 weeks of culture while roots and leaves accumulated biomass at an exponential rate. Sugar use correlated nearly directly to whole plant dry weight (DW) in the short and long-term experiments. Microrhizomes became a larger fraction of whole plant DW as plantlets aged. After 6 weeks, about 1.8 g of microrhizome DW per liter of media had been produced (in both time courses), and after 23 weeks, about 26 g of microrhizome DW per liter of media had been produced. Secondary rhizomes were first observed at 14 weeks, and most plants had them by 23 weeks. A method for rhizome production in a long-term culture system was described. The linear relationship between DW and sugar use will be useful in the eventual development of a model for sugar use to biomass to secondary metabolite production. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rhizobacterial-mediated induction of defense enzymes to enhance the resistance of turmeric (Curcuma longa L) to Pythium aphanidermatum causing rhizome rot

Rhizobacteria isolated from the rhizosphere soil were evaluated for their ability to control rhizome rot in turmeric (Curcuma longa L). These isolates were characterised as Pseudomonas fluorescens and Bacillus subtilis. Under in vitro condition, two isolates, namely P. chlororaphis (PcPA23) and B. subtilis (BsCBE4), showed maximum inhibition of mycelial growth of Pythium aphanidermatum, were found effective in reducing rhizome rot of turmeric both under greenhouse and field conditions and increased the plant growth and rhizome yield. Both the isolates were further tested for its ability to induce production of defense-related enzymes and chemicals in plants. Increased activities of phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, chitinase and β-1,3-glucanase were observed in PcPA23 and BsCBE4 pre-treated turmeric plants challenged with P. aphanidermatum. Moreover, higher accumulation of phenolics was noticed in plants pre-treated with PcPA23 and BsCBE4 challenged with P. aphanidermatum. Thus, the present study shows that in addition to direct antagonism and plant growth promotion, induction of defense-related enzymes involved in the phenyl propanoid pathway collectively contributed to enhance resistance against invasion of Pythium in turmeric.     

In vitro multiplication and microrhizome induction in Curcuma aromatica Salisb.

Shoot multiplication and plant regeneration was achieved from freshly sprouted shoots of Curcuma aromatica on Murashige and Skoog's medium supplemented with BA alone (1–7 mgL^–1) or a combination of BA(1–5 mgL^–1) and Kn (0.5–1 mgL^–1). A concentration of 5 mgL^–1 BA was optimum for shoot multiplication and rooting of shoots. The regenerated plants grew profusely on transfer to liquid medium.In vitro raised plants were successfully established in the field. Microrhizomes were induced at the base of the in vitro derived shoots upon transfer to medium containingvarious combinations and concentrations of sucrose and BA and grown under varying photoperiods. MS basal medium with 5 mgL^–1 BA, 60 gL^–1 sucrose and an8 h photoperiod was optimum for induction ofmicrorhizomes within 30 days of culture. Harvestedmicrorhizomes stored in moist sand in poly-bagssprouted after 2 months of storage at roomtemperature. For in vitro storage, microrhizomeswere grown in medium containing 0.1 mgL^–1 BA.Microrhizome formation was found to be controlled bythe concentrations of BA and sucrose as well asphotoperiod during culture.     

In vitro propagation ofAtractylodes lancea

Shoot cultures ofAtractylodes lancea DC. (Compositae) have been established by inoculating the flower bud on Linsmaier-Skoog's medium supplemented with 10⁻⁵ M naphthaleneacetic acid and 10⁻⁵ M 6-benzyl-aminopurine. Shoots were multiplied on a medium containing 10⁻⁶ M 6-benzylaminopurine. Propagated shoots rooted on a medium devoid of any plant growth regulators were transferred to potting soil and finally to the field.     

In vitro propagation ofWrightia tinctoria

In vitro method has been developed for propagation ofWrightia tinctoria R.Br. using cotyledonary node segments. Murashige and Skoog's (MS) medium supplemented with 5.0 mg dm^?3 of 6-benzylaminopurine (BAP) and 0.01 mg dm^?3 of naphthaleneacetic acid (NAA) induced up to eight shoots per explant with an average shoot length of 1.4 cm in 21 d. Three fold multiplication rate was achieved during every subculture of regenerated shoots on the same medium producing an average of 230 shoots per node within 84 d. Reduction in BAP concentration from 5.0 to 1.0 mg dm^?3 during subculture promoted shoot length without affecting the rate of multiplication. The differentiated shoots could be rooted by a dip treatment into preautoclaved indole-3-butyric acid (IBA-500 mg dm^?3 for 5 min) followed by their implantation onto MS medium containing 1/4 salts. Rooting was observed within 8–10 d in approximately 80% of shoots inoculated after IBA treatment. 15 d after rooting, the plantlets were transferred to culture bottles containing soil-Soilrite^TM (1∶1) and liquid nutrient solution comprising 1/4 MS salts. After their partial hardening in these bottles for 10 d they were transferred to pots containing soil-Soilrite (1∶1) mixture with 60% transplantation success. Methods are being standardized to improve the rate of survival and large scale field transfer.     

In vitro propagation of cashewnut

In vitro plant propagation was developed for seedling shoot tips, leaf axils, and cotyledonary nodes of cashew, Anacardium occidentale. Factors affecting multiplication rate included age of explant source, explant type, medium composition, light requirements, and transfer frequency. Cotyledonary nodes produced more buds than other explant types. Nodes had a 90% viability when transferred daily to fresh medium containing activated charcoal for 7 d while exposed to continuous dark. Cultures were then exposed to low light illumination with weekly transfers. The phytohormone composition producing the most buds was 2.32 M kinetin, 9.12 M zeatin and 4.40 M BA. The highest frequency of rooted shoots was obtained by treating shoots with the bacterium, Agrobacterium rhizogenes. Plants also were recovered by induction of roots using auxin treatment on propagated shoots.Abbreviations Kn Kinetin - Zn Zeatin - BA N6-Benzyladenine - 2iP (2-Isopentenyl) adenine - BPA n-Benzyl -9 (2-tetrahydro-pyrany l) adenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - TIBA 2,3,5-Tri-iodo-benzoic acid     

Curcuma longa as feed additive in broiler birds and its patho-physiological effects

Broiler birds (Vencob chicken of 3 days old) when given feed mixed with powdered rhizome of Curcuma longa (CL; @ 1 g/kg) for 42 days of age, showed significant decrease in serum uric acid and albumin as compared to control, whereas significant increase was recorded in the level of serum total protein and globulin. Level of serum glucose, alkaline phosphatase, aspartate amino transferase and calcium showed no significant variation between the two groups. Micronutrient assay revealed significantly higher level of manganese, zinc, iron and copper in treated group as compared to control group. HA/HI test revealed better humoral response against RD vaccine in CL administered birds. Haematological study showed significantly higher haemoglobin and absolute neutrophil count in treated group. Addition of CL as feed additive also resulted in better growth rate, feed consumption and F:C efficiency in the treated birds. Thus, it could be concluded that powdered CL might be a useful feed additive, since it enhanced the F:C efficiency and had nephroprotective properties.     

In vitro propagation of the endangered orchid,Geodorum densiflorum (Lam.) Schltr. through rhizome section culture

Micropropagation of the endangered terrestrial orchid, Geodorum densiflorum (Lam.) Schltr. was achieved through rhizome section culture from in vitro seed- derived rhizomes. Rhizome sections were cultured on Murashige and Skoog (MS) and Knudson C(KC) media supplemented with various growth regulators and 0.1% activated charcoal. The rhizome sections responded on MS medium. Naphthaleneacetic acid (NAA) at 2.0 μM stimulated rhizome growth. However, benzyladenine (BA) at 5.0 μM induced multiple shoots within four weeks of culture and inhibited rhizome growth. The regenerated shoots rooted on MS only or with NAA at 1.0 μM. Well-developed plantlets were transferred to community pots and then to a greenhouse where the plants have been acclimatised. This revised version was published online in June 2006 with corrections to the Cover Date.