Early Sucrose Degradation and the Dominant Sucrose Cleavage Pattern Influence Lycoris sprengeri Bulblet Regeneration In Vitro.

Bulblet formation and development determine the quantitative and qualitative traits, respectively, of bulb yield for most flowering bulbs. For Lycoris species, however, the underlying molecular mechanism remains elusive. Here, clonal bulblets of Lycoris sprengeri (Ls) derived from the same probulb were used as explants to establish efficient and inefficient in vitro regeneration systems by adjusting the 6-benzyladenine (BA) concentrations in media. BA application did not change the biological processes among groups but led to earlier decreases in sucrose and total soluble sugar (TSS) contents. Correlation analyses showed that the BA treatments changed the interaction between carbohydrate and endogenous hormone contents during bulblet regeneration. We found that two sucrose degradation enzyme-related genes, cell wall invertase (CWIN) and sucrose synthase, exhibited exactly opposite expression patterns during the competence stage. In addition, the regeneration system that obtained more bulblets showed significantly higher expression of LsCWIN2 than those that obtained fewer bulblets. Our data demonstrate the essential role of BA in accelerating sucrose degradation and the selection of a dominant sucrose cleavage pattern at the competence stage of in vitro bulblet regeneration. We propose that a relatively active CWIN-catalyzed pathway at the competence stage might promote bulblet regeneration, thus influencing bulb yield.

Profiles of Secondary Metabolites (Phenolic Acids, Carotenoids, Anthocyanins, and Galantamine) and Primary Metabolites (Carbohydrates, Amino Acids, and Organic Acids) during Flower Development in Lycoris radiata.

This study aimed to elucidate the variations in primary and secondary metabolites during Lycorisradiata flower development using high performance liquid chromatography (HPLC) and gas chromatography time-of-flight mass spectrometry (GC-TOFMS). The result showed that seven carotenoids, seven phenolic acids, three anthocyanins, and galantamine were identified in the L. radiata flowers. Most secondary metabolite levels gradually decreased according to the flower developmental stages. A total of 51 metabolites, including amines, sugars, sugar intermediates, sugar alcohols, amino acids, organic acids, phenolic acids, and tricarboxylic acid (TCA) cycle intermediates, were identified and quantified using GC-TOFMS. Among the hydrophilic compounds, most amino acids increased during flower development; in contrast, TCA cycle intermediates and sugars decreased. In particular, glutamine, asparagine, glutamic acid, and aspartic acid, which represent the main inter- and intracellular nitrogen carriers, were positively correlated with the other amino acids and were negatively correlated with the TCA cycle intermediates. Furthermore, quantitation data of the 51 hydrophilic compounds were subjected to partial least-squares discriminant analyses (PLS-DA) to assess significant differences in the metabolites of L. radiata flowers from stages 1 to 4. Therefore, this study will serve as the foundation for a biochemical approach to understand both primary and secondary metabolism in L. radiata flower development.

Changes in carbohydrate metabolism and endogenous hormone regulation during bulblet initiation and development in Lycoris radiata.

BACKGROUND: Lycoris species have great ornamental and medicinal values; however, their low regeneration efficiency seriously restricts their commercial production. Understanding the mechanism of bulblet propagation in this genus, which has remained underexplored to date, could provide a theoretical basis for improving the reproductive efficiency. Therefore, we studied the bulblet initiation and developmental processes in Lycoris radiata. RESULTS: We found that bulblets are formed on the junctions of the innermost layers of scales and the basal plate, and initially present as an axillary bud and gradually develop into a bulblet. We also determined the changes in carbohydrate and endogenous hormone contents during bulblet initiation and development, as well as the expression patterns of genes involved in carbohydrate metabolism and hormone biosynthesis and signaling through transcriptome analysis. Soluble sugars derived from starch degradation in the outer scales are transported to and promote bulblet initiation and development through starch synthesis in the inner scales. This process is mediated by several genes involved in carbohydrate metabolism, especially genes encoding ADP glucose pyrophosphorylase, a crucial starch synthesis enzyme. As for hormones, endogenous IAA, GA, and ABA content showed an increase and decrease during bulblet initiation and development, respectively, which were consistent with the expression patterns of genes involved in IAA, GA, and ABA synthesis and signal transduction. In addition, a decrease in ZR content may be down- and up-regulated by CK biosynthesis and degradation related genes, respectively, with increasing auxin content. Furthermore, expression levels of genes related to BR, JA, and SA biosynthesis were increased, while that of ethylene biosynthesis genes was decreased, which was also consistent with the expression patterns of their signal transduction genes. CONCLUSIONS: The present study provides insights into the effect of carbohydrate metabolism and endogenous hormone regulation on control of L. radiata bulblet initiation and development. Based on the results, we propose several suggestions to improve L. radiata propagation efficiency in production, which will provide directions for future research.

The influences of four types of soil on the growth, physiological and biochemical characteristics of Lycoris aurea (L' Her.) Herb.

Based on the characteristics of Lycoris aurea (L. aurea) natural distribution and local soil types, we selected four representative types of soil, including humus soil, sandy soil, garden soil and yellow-brown soil, for conducting the cultivation experiments to investigate key soil factors influencing its growth and development and to select the soil types suitable for cultivating it. We found that there existed significant differences in the contents of mineral elements and the activities of soil enzymes (urease, phosphatase, sucrase and catalase) etc. Among which, the contents of organic matters, alkali-hydrolysable nitrogen, Ca and Mg as well as the activities of soil enzymes in humus soil were the highest ones. In yellow-brown soil, except for Fe, the values of all the other items were the lowest ones. Net photosynthetic rate (Pn), biomass and lycorine content in humus soil were all the highest ones, which were increased by 31.02, 69.39 and 55.79%, respectively, as compared to those of yellow-brown soil. Stepwise multiple regression analysis and path analysis indicated that alkali-hydrolysable nitrogen, and Ca etc. were key soil factors influencing Pn, biomass and lycorine content of L. aurea. Thus, humus soil can be used as medium suitable for artificial cultivation of L. aurea.

[Effect of precursor on growth and accumulation of alkaloids of Lycoris radiata suspension cells].

In order to investigate the effects of phenylalanine, tyrosine and tyramine on the growth of Lycoris radiata suspension cells and the accumulation of alkaloids, the growth quantity of the cells as well as the content of alkaloids in cells were determined, which were treated with above three kinds of precursors alone and phenylalanine combined with tyrosine respectively. The results indicate that the addition of phenylalanine alone and addition of phenylalanine on the basis of tyrosine at high concentration (200 micromol/L) had no significant effect on the growth of Lycoris radiata suspension cells and the content of alkaloids in cells; whereas tyrosine and tyramine promoted the growth of the cells and alkaloids accumulation. Treated with tyrosine at high concentration (200 micromol/L), the content of alkaloids of the cells was 2.56-fold higher than that of the control group, the amounts of lycoramine (3.77 mg/g) and galanthamine (4.46 mg/g) were 6.61-fold and 6.97-fold higher than that of the control group, respectively. When treated with tyramine (200 micromol/L), the amount of alkaloids in Lycoris radiata suspension cells was 2.63-fold higher than that of the control group, and the amounts of lycoramine (4.45 mg/g) and galanthamine (5.14 mg/g) were 9.08-fold and 9.18-fold higher than that of the control group, respectively. The above results demonstrate that adding tyrosine and tyramine in the media significantly promoted the growth of the Lycoris radiata suspension cells and alkaloids accumulation in the cells.

[Tissue culture effect of different size explants of Lycoris aurea].

OBJECTIVE: To solve the puzzle about the right size of the explant of wild medicinal plant Lycoris aurea for tissue culture. METHODS: Three-size explants: 9.0 x 8.0 mm, 6.0 x 5.0 mm, 3.0 x 2.0 mm of endothecium bulb-scale joined by a strip of stem plate and middle-layer bulb-scale joined by a strip of stem plate were cultured on two kinds of the medium: MS +9 mg/L BA +5 mg/ L NAA +0.7% agar +3% cane sugar and MS +5 mg/L BA +9 mg/L NAA +0.7% agar +3% cane sugar, and the culture effects of the three-size explants for Lycoris aurea were studied. RESULTS AND CONCLUSION: The results showed that the explant size on 6.0 x 5.0 mm of the endothecium bulb-scale cultured on the medium: MS +9 mg/L BA +5 mg/L NAA +0.7% agar +3% cane sugar had the advantage of forming adventitious buds and roots, and inhibiting brown samples, and to the middle-layer bulb-scale explant of the same size cultured on the same medium was next. Correlation analysis showed that the culture effect of the three-size explants on one kind of medium was significantly correlated with that of theirs on another kind of medium, which further proved that the result of Duncans multiple range test, i.e. if the effect of an explant size on one kind of medium were better, its effect on another kind of medium would also be better. Both Duncan's multiple range test and correlation analysis indicated that the effect of the size on 6.0 x 5.0 mm explant of the double endothecium bulb-scale joined by a strip of stem plate was better than those of the other sizes on the experimental explants, and to that of 6.0 x 5.0 mm size of the double middle-layer bulb-scale joined by a strip of stem plate was next.

Alkaloid accumulation in different parts and ages of Lycoris chinensis.

The galanthamine, lycorine, and lycoramine content of Lycoris chinensis was researched during development from young to old plants, i.e. in seeds, ten-day-old seedlings, three-month-old seedlings, one-year-old seedlings, and perennial seedlings. Notably the alkaloid level reduced to its lowest content 10 days after seed germinating. Then the accumulation of galanthamine tended to increase with age, reaching a higher value in perennial seedlings. The production pattern of lycorine and lycoramine was found similar to that of galanthamine. Different plant organs were also evaluated for their galanthamine, lycorine, and lycoramine contents. Mature seeds had the highest content of galanthamine (671.33 microg/g DW). Kernels, seed capsules, and root-hairs were the main repository sites for galanthamine, lycorine, and lycoramine. The leaves were the least productive organs.

Effect of abiotic and biotic elicitors on growth and alkaloid accumulation of Lycoris chinensis seedlings.

Three-month-old seedlings of Lycoris chinensis were treated with biotic and abiotic elicitors: yeast elicitor (YE), methyl jasmonate (MJ), salicylic acid (SA), and sodium nitroprusside as NO donator (NO). We have shown that the addition of MJ and NO promotes the accumulation of galanthamine in these seedlings. The effect of these elicitors on the growth of the seedlings, as well as on the amount of the alkaloids accumulated in the seedlings was studied. The results showed that, in general, high doses of MJ and SA had a negative effect on the growth of the seedlings, while appropriate doses of NO and YE had a positive effect on the growth of the seedlings. It was remarkable that the addition of MJ, NO, and YE can promote galanthamine accumulation in seedlings. The accumulation was higher in treatments at higher concentrations of NO (100 microM), where the release of galanthamine was 1.72-fold higher than that of the control at the 10th day of culture. The highest values of lycorine were obtained in seedlings treated with YE at a concentration of 0.01 g/l and by the 10th day of culture; the level was 1.38 times of the control.

[The analysis of differential expression and cloning of genes related to raised secondary lateral veins mutant of Lycoris aurea].

Lycoris aurea exhibits parallel venation, the main vein with many lateral veins in a longitudinal parallel arrangement. There are secondary lateral veins (SLV) between each longitudinal veins. In general, SLVs are not remarkable. In this paper, the material was one kind of Lycoris aurea mutant called Raised Secondary Lateral Veins mutant (RSLV), because many Raised Secondary Lateral Veins are in abaxial surface of its leaves. Its growing potential is weaker than that of wild type and its blades are very thin. Moreover, the stamens of RSLV degenerate completely. Two cDNA libraries were constructed from RSLV mutant and wild type (WT) leaves. From the libraries, 3,122 ESTs, which are longer than 100 bp each after vector sequence removed, were acquired by single-pass sequencing from the 5'end. Following a multistep selection, 512 70-mer oligo-DNA probes were designed for attachment on the microarray slide based on the ESTs. The gene expression profile of RSLV mutant and WT leaves was compared through the microarray at transcriptional level. The microarray experiment results were further confirmed by Quantitative Real-Time PCR (QRT-PCR). We identified 5 genes whose expressions changed more than 2-fold between RSLV mutant and WT leaves. They encode phloem protein 2 (PP2), ferritin, pectin methyl esterase (PME), chlorophyll a/b binding protein (CAB protein) and pyruvate decarboxylase (PDC), respectively. Furthermore, the full-length cDNA sequences of the 5 genes were separately obtained from RSLV and WT by RACE. The relationship between differential expressions of the genes and the formation of the RSLV mutant phenotype were discussed.