Genome-Wide Identification of CYP72A Gene Family and Expression Patterns Related to Jasmonic Acid Treatment and Steroidal Saponin Accumulation in Dioscorea zingiberensis.

Dioscorea zingiberensis is a medicinal herb containing a large amount of steroidal saponins, which are the major bioactive compounds and the primary storage form of diosgenin. The CYP72A gene family, belonging to cytochromes P450, exerts indispensable effects on the biosynthesis of numerous bioactive compounds. In this work, a total of 25 CYP72A genes were identified in D. zingiberensis and categorized into two groups according to the homology of protein sequences. The characteristics of their phylogenetic relationship, intron-exon organization, conserved motifs and cis-regulatory elements were performed by bioinformatics methods. The transcriptome data demonstrated that expression patterns of DzCYP72As varied by tissues. Moreover, qRT-PCR results displayed diverse expression profiles of DzCYP72As under different concentrations of jasmonic acid (JA). Likewise, eight metabolites in the biosynthesis pathway of steroidal saponins (four phytosterols, diosgenin, parvifloside, protodeltonin and dioscin) exhibited different contents under different concentrations of JA, and the content of total steroidal saponin was largest at the dose of 100 µmol/L of JA. The redundant analysis showed that 12 DzCYP72As had a strong correlation with specialized metabolites. Those genes were negatively correlated with stigmasterol and cholesterol but positively correlated with six other specialized metabolites. Among all DzCYP72As evaluated, DzCYP72A6, DzCYP72A16 and DzCYP72A17 contributed the most to the variation of specialized metabolites in the biosynthesis pathway of steroidal saponins. This study provides valuable information for further research on the biological functions related to steroidal saponin biosynthesis.

[Preliminary study of Dioscorea bulbifera plantlet microtuber in vitro induction].

OBJECTIVE: In order to provide a theoretical basis for the microtuber factory production and its germplasm resources preservation, in vitro induction of Dioscorea bulbifera plantlet microtubers was studied. METHODS: Through plant tissue culture technique and single factor experiment method, stems with buds of Dioscorea bulbifera plantlets as explants, the effects of various factors such as sugar, inorganic salt, cultivation mode, activated carbon and physiological state of stems with buds on in vitro induction of Dioscorea bulbifera microtubers were studied. RESULTS: The optimal sugar of Dioscorea bulbifera microtuber in vitro induction was 60 g/L sucrose or 90 g/L white sugar. The best inorganic salt concentration of Dioscorea bulbifera microtuber in vitro induction was MS. The best culture method of Dioscorea bulbifera microtuber in vitro induction was solid-liquid double layer culture. Activated carbon had a significant effect on Dioscorea bulbifera microtuber in vitro induction, whose optimum concentration was 0.03%. More mature the stem with buds was, the shorter the time of microtuber formation need. CONCLUSION: This experiment establishes a rapid method of Dioscorea bulbifera microtuber in vitro induction for the first time,which provides a new way for the application of Dioscorea bulbifera microtubers in agricultural production.

A protocol for in vitro production of microtubers in Chinese yam (Dioscorea opposita).

Chinese yam (Dioscorea opposita) is an important tuberous crop owing to its dual use as a food as well as a medicine. Tissue culture techniques allow the rapid multiplication of virus-free plant materials. The use of microtubers offers an attractive alternative to in vitro-grown plantlets for the micropropagation and exchange of healthy Chinese yam materials. A protocol for the in vitro production of Chinese yam microtubers was developed in this study. Though we tested both one-step and two-step procedures, only the two-step procedure showed favorable results for tuberization. Media with 60 g L(-1) sucrose yielded the highest microtuber index. We demonstrate that table sugar was an efficient and economical alternative to analytical grade sucrose for microtuber production. Using an orthogonal experimental design, we determined the optimal growth regulator combination for microtuber induction and development. The microtubers obtained from our protocol sprouted readily both in vitro and in soil.

[Study on inducing embryogenic callus of Dioscorea zingiberensis by orthogonal design].

OBJECTIVE: To select the suitable medium to induce embryogenic callus of Dioscorea zingiberensis. METHODS: Plantlet of Dioscorea zingiberesis in vitro was obtained by using apical meristem as explant. The different parts of the plantlets were cultured to select the best explant used for inducing callus and embryoids. Growing rate and diosgenin content were calculated in orthogonal test to optimize combination of phytohormones for inducing embryogenic callus. RESULTS: The leaves were suitable explants to induce callus and embryoid. The inducing rate of callus and embryoids reached 92.5% and 42.5%, respectively. The optimal medium for inducing embryogenic callus was MS + 6-BA 2.0 mg/L + NAA 0.5 mg/L + 2,4-D 1.0 mg/L. CONCLUSION: The results of this study can be used for effective induction of embryogenic callus of Dioscorea zingiberensis, and lay the foundation for the subsequent research of artificial seeds.

Enhancement of diosgenin production in Dioscorea zingiberensis cell cultures by oligosaccharides from its endophytic fungus Fusarium oxysporum Dzf17.

The effects of the oligosaccharides from the endophytic fungus Fusarium oxysporum Dzf17 as elicitors on diosgenin production in cell suspension cultures of its host Dioscorea zingiberensis were investigated. Three oligosaccharides, DP4, DP7 and DP10, were purified from the oligosaccharide fractions DP2-5, DP5-8 and DP8-12, respectively, which were prepared from the water-extracted mycelial polysaccharide of the endophytic fungus F. oxysporum Dzf17. When the cell cultures were treated with fraction DP5-8 at 20 mg/L on day 26 and harvested on day 32, the maximum diosgenin yield (2.187 mg/L) was achieved, which was 5.65-fold of control (0.387 mg/L). When oligosaccharides DP4, DP7 and DP10 were individually added to 26-day-old D. zingiberensis cell cultures at concentrations of 2, 4, 6, 8 and 10 mg/L in medium, DP7 at 6 mg/L was found to significantly enhance diosgenin production, with a yield of 3.202 mg/L, which was 8.27-fold of control. When the cell cultures were treated with DP7 twice on days 24 and 26, and harvested on day 30, both diosgenin content and yield were significantly increased and reached the maximums of 1.159 mg/g dw and 4.843 mg/L, both of which were higher than those of single elicitation, and were 9.19- and 12.38-fold of control, respectively.

Effects of polysaccharide elicitors from endophytic Fusarium oxysporium Dzf17 on growth and diosgenin production in cell suspension culture of Dioscorea zingiberensis.

Three polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharide (SPS), were prepared from the endophytic fungus Fusarium oxysporium Dzf17 isolated from the rhizomes of Dioscorea zingiberensis. The effects of the time of addition and polysaccharide concentration on the growth and diosgenin accumulation in cell suspension culture of D. zingiberensis were studied. Among them, WPS was found to be the most effective polysaccharide. When WPS was added to the medium at 20 mg/L on the 25th day of culture, the cell dry weight was increased 1.34-fold, diosgenin content 2.85-fold, and diosgenin yield 3.83-fold in comparison to those of control. EPS and SPS showed moderate and relatively weak enhancement effects on cell growth and diosgenin accumulation, respectively. The dynamics of cell growth and diosgenin accumulation when WPS was added to the medium at 20 mg/L on the 25th day of culture were investigated, and results showed that dry weight of cells reached a maximum value on day 30 but the maximum diosgenin content was achieved on day 31.

Sucrose treatment and explant water content: critical factors to consider in development of successful cryopreservation protocols for shoot tip explants of the tropical species Dioscorea rotundata (YAM).

A study was conducted to determine the optimum methods for conditioning explants to be used in the development of a simple protocol for long-term conservation of the germplasm of Dioscorea rotundata via cryopreservation. Shoot tips from cultures maintained in vitro were exposed to high concentrations of sucrose prior to silica gel-based dehydration and vitrification solution-based cryopreservation protocols. Explant water contents were determined, and ultrastructural studies were also carried out. Initially, culturing explants on medium supplemented with 0.3 M sucrose for 3-5 d considerably reduced tissue water content from about 12.2 g/g dry mass to between 4.8 and 5.5 g/g dry mass before cryoprotection with modified PVS2 (MPVS2) or silica gel dehydration. Ultrastructural studies indicated that cells had deposits of starch in plastids following sucrose treatments. Survival for D. rotundata shoot tips treated with MPVS2 vitrification solution, unloaded with 1.0 M sucrose medium and cooled to -7 degree C, was 16 percent for 15 min treatment and 44 percent for 40 min. After the 40 min MPVS2 treatment the TTZ test indicated 88 percent viability retention of explants cooled to -70 degree C, and 44 percent at -196 degree C. Plantlet development was obtained for -70 degree C-cooled shoot tips, whereas only callus development occurred from tissues exposed to liquid nitrogen. Explant regeneration was not obtained with air-dehydration techniques. It was concluded that vitrification-solution based cryopreservation presently offers the best option for conservation of this Dioscorea species.

Cryopreservation of asian Dioscorea bulbifera l. and D. alata l. by vitrification: importance of plant growth regulators.

The aim of this study was to develop cryopreservation protocols for Asian races of Dioscorea bulbifera and D. alata with high survival and plant regeneration after cryopreservation. Using a vitrification procedure, survival of shoot tips postcryopreservation of up to 89% in D. bulbifera and up to 82% in D. alata were recorded when excised shoot tips were pretreated overnight with 0.3 M sucrose in MS medium, followed by loading with 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degrees C, exposure to PVS2 solution for 90 min at 0 degrees C, immersion in liquid nitrogen for 1 h, rewarming at 40 degrees C for 2 min, unloading in medium with 1.2 M sucrose for 20 min and culturing on growth recovery medium. During growth recovery, 58% shoot regeneration was obtained in D. bulbifera when cryopreserved shoot tips were initially cultured for 40 days on MS medium with 1.5 mg/L BAP, 0.15 mg/L NAA and 0.2 mg/L GA3 followed by culturing on a medium with 0.05 mg/L BAP and 0.15 mg/ L NAA. However, a maximum of 39% shoot regeneration was recorded in D. alata when cryopreserved shoot tips were initially cultured for 40 days on medium M2 (MS containing 1/5 NH4NO3 and 40 g/L sucrose) supplemented with 1.0 mg/L BAP, 1.0 mg/L zeatin, 0.15 mg/L IAA and 0.2 mg/L GA3. Subsequently, the regenerating shoots were cultured for 30 days on medium M2 with 1.0 mg/L BAP, 0.3 mg/L zeatin, 0.02 mg/L NAA and 0.2 mg/L GA3 followed by culturing for another 30 days on medium with 0.5 mg/L BAP, 0.02 mg/L NAA and 0.2 mg/L GA3. Finally, transfer onto medium with 0.05 mg/L BAP and 0.15 mg/L NAA stimulated production of fully grown plantlets. Alteration of post-thaw culture media with plant growth regulators and their application at various stages of growth recovery was crucial for regeneration of shoot tips and formation of plantlets in D. alata.

Cryopreservation of plantlet nodes of Dioscorea opposita Thunb. using a vitrification method.

A cryopreservation method by vitrification was developed for long-term storage of Dioscorea opposita Thunb., a valuable native medicinal plant species in Henan Province of China. The cryopreservation protocol was established with cultivar B and evaluated with another four cultivars, Tiegun, 47, Taigu and Huaiqing 1. The results showed that nodes with a bud excised from 60 d plantlets were desirable for the cryopreservation. The optimum procedure was established as: 1) the plantlets were cultured on the Murashige and Skoog (MS) medium supplemented with 2 mg L(-1) KT and 0.02 mg L(-1) NAA at 4 degree C for 7 d before nodes with length of 1-1.5 cm were excised; 2) the nodes were precultured at 4 degree C for 7 d on the MS supplemented with 10 percent dimethyl sulfoxide (DMSO) followed by loading with 60 percent Dioscorea vitrification solution 1 (DVS1): 22 percent (w/v) glycerol + 13 percent (w/v) ethylene glycol + 13 percent (w/v) polyethylene glycol + 10 percent (w/v) DMSO for 60 min at 0 degree C and dehydrated with 100 percent DVS1 for 60 min at 0 degree C; 3) the nodes were then immersed into liquid nitrogen (LN) directly and conserved for 180 d; 4) after rapid thawing in a water-bath at 37 degree C, the nodes were rinsed four times with MS medium supplemented with 5 percent sucrose, then transferred to the MS medium supplemented with 2 mg L(-1) kinetin (KT) and 0.02 mg L(-1) NAA for regeneration. In the present research the regeneration rate of cv. B was about 77.1 percent, those of cvs. Tiegun and Huaiqing 1 were 67.2 percent and 54.0 percent respectively, while cvs. Taigu and 47 were about 40 percent. There were no visual changes observed between the plantlets regenerated from nodes with and without cryopreservation in terms of the morphology indices, indicating that the method established could be applicable to D. opposita with optimized protocol.

[Application of orthogonal design in tissue culture of Dioscorea opposite].

OBJECTIVE: To study the effects of different factors on buds and microtuber. These factors included plant growth substances and sucrose. METHOD: stems were selected as explants. The effects of three kinds of factors were studied by orthogonal design method including sucrose, 6-BA, NAA on the buds and microtuber induction. The data were analyzed with range analysis and vadance analysis. RESULT AND CONDUSION: The optimal media to induce many buds from stems were MS + 6-BA 1 mg x L(-1) + NAA 1 mg x L(-1) + sucrose 3%, the effect of the three factors was in sequence of sucrose >6-BA > NAA. The optimal media to induce microtuber from stems were MS+6-BA 1.5 mg x V1 +NAA 1.5 mg x L(-1) + sucrose 5%, the effect of the three factors was in sequence of sucrose >6-BA > NAA.

Regeneration of Dioscorea floribunda plants from cryopreserved encapsulated shoot tips: effect of plant growth regulators.

The encapsulation-dehydration protocol for the cryopreservation of in vitro shoot tips of Dioscorea floribunda was optimized. Maximum survival of 87% was obtained when overnight pretreatment with 0.3 M sucrose was followed by encapsulation, preculture in 0.75 M sucrose for 4 d, dehydration in a laminar air flow for 5.5 h, quenching in liquid nitrogen and thawing at 40 degrees C. During recovery growth, 29% shoot formation was obtained when cryopreserved shoot tips were initially cultured for 25 d on a medium with 1.5 mg per liter (-1) BAP, 0.2 mg per liter(-1) NAA and 0.2 mg per liter(-1) GA3 followed by culturing for 15 d on a medium with reduced BAP (1 mg per liter(-1)) but increased NAA (0.5 mg per liter(-1)) and GA3 (0.3 mg per liter(-1)). Finally, transfer on to a medium with further reduced doses of BAP (0.05 mg per liter(-1)) and NAA (0.15 mg per liter(-1)) but without GA3 stimulated production of fully grown plantlets. All plants regenerated without callus formation. Modification of post-thaw culture media with plant growth regulators was essential for regrowth of shoot tips to plantlets.

Responses of antioxidant potentials in Dioscorea rotundata Poir. following paclobutrazol drenching.

The effect of paclobutrazol (PBZ) treatments on the antioxidant metabolism of white yam (Dioscorea rotundata Poir.) was investigated in the present study. PBZ @ 15 mg l(-1) plant(-1) was given to plants by soil drenching, 30, 60, and 90 days after planting (DAP). The non-enzymatic antioxidant contents like ascorbic acid (AA), reduced glutathione (GSH) and alpha-tocopherol (alpha-toc), activities of antioxidant enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), polyphenol oxidase (PPO) and catalase (CAT) were extracted and assayed on 100 DAP from leaf, stem and tubers of both control and PBZ treated plants. It was found that PBZ has a profound effect on the antioxidant metabolism and caused an enhancement in both non-enzymatic and enzymatic antioxidant potentials under treatments in white yam. Our results have good significance, as this increase the innate antioxidant potential of this food crop, which is helpful to satisfy the needs of antioxidants in diet and thereby make it an economically important food crop.

Changes in biochemical constituents and induction of early sprouting by triadimefon treatment in white yam (Dioscorea rotundata Poir.) tubers during storage.

The ability of triadimefon (TDM), a triazolic fungicide, to alter the biochemical constituents and thereby minimizing the days required for sprouting in white yam (Dioscorea rotundata Poir.) tubers during storage under (30+/-2) degrees C in the dark, was studied. TDM at 20 mg/L was given to tubers by dipping the tubers in treatment solution containing 20 mg/L TDM on 10, 25 and 40 d after storage (DAS). Starch, sugars, protein, amino acid contents as well as protease and alpha-amylase activities were estimated on 15, 30 and 45 DAS from two physiological regions viz., apical and basal regions of the tubers. In normal conditions (control) sprouting occurred on 70 to 80 DAS. The starch content decreased, while protein, amino acid, sugar contents and protease and alpha-amylase activities were increased due to TDM treatment and led to early sprouting.

Abiotic metal stress enhances diosgenin yield in Dioscorea bulbifera L. cultures.

Lower concentrations of CuSO(4) (25-75 microM) in the MS medium supplemented with 0.1 mg l(-1) IAA+5.0 mg l(-1) Kn+500 mg l(-1) CH+10 mg l(-1) Cyst hyd enhanced the growth of regenerants of Dioscorea bulbifera L. CuSO(4) (75 microM) induced an appreciable diosgenin yield in the regenerants compared to those obtained on media without Cu. The presence of Cu thus seems to stimulate diosgenin production. The regenerants also differentiated bulbils on lower concentrations of Cu. At CuSO(4) (100 microM), however, cultures showed poor growth as well as a low diosgenin yield. Increased proline and protein contents were recorded in cultures grown on Cu-enriched media.