High-speed homogenization coupled with microwave-assisted extraction followed by liquid chromatography-tandem mass spectrometry for the direct determination of alkaloids and flavonoids in fresh Isatis tinctoria L. hairy root cultures.

A new, simple and efficient analysis method for fresh plant in vitro cultures-namely, high-speed homogenization coupled with microwave-assisted extraction (HSH-MAE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-was developed for simultaneous determination of six alkaloids and eight flavonoids in Isatis tinctoria hairy root cultures (ITHRCs). Compared with traditional methods, the proposed HSH-MAE offers the advantages of easy manipulation, higher efficiency, energy saving, and reduced waste. Cytohistological studies were conducted to clarify the mechanism of HSH-MAE at cellular/tissue levels. Moreover, the established LC-MS/MS method showed excellent linearity, precision, repeatability, and reproducibility. The HSH-MAE-LC-MS/MS method was also successfully applied for screening high-productivity ITHRCs. Overall, this study opened up a new avenue for the direct determination of secondary metabolic profiles from fresh plant in vitro cultures, which is valuable for improving quality control of plant cell/organ cultures and sheds light on the metabolomic analysis of biological samples. Graphical Abstract HSH-MAE-LC-MS/MS opened up a new avenue for the direct determination of alkaloids and flavonoids in fresh Isatis tinctoria hairy root cultures.

Development of a complete set of monosomic alien addition lines between Brassica napus and Isatis indigotica (Chinese woad).

KEY MESSAGE: A complete set of monosomic alien addition lines of Brassica napus with one of the seven chromosomes of Isatis indigotica and the recombinant mitochondria was developed and characterized. Monosomic alien addition lines (MAALs) are valuable for elucidating the genome structure and transferring the useful genes and traits in plant breeding. Isatis indigotica (Chinese woad, 2n = 14, II) in Isatideae tribe of Brassicaceae family has been widely cultivated as a medicinal and dye plant in China. Herein, the intertribal somatic hybrid (2n = 52, AACCII) between B. napus cv. Huashuang 3 (2n = 38, AACC) and I. indigotica produced previously was backcrossed recurrently to parental B. napus, and 32 MAAL plants were isolated. Based on their phenotype, 5S and 45S rDNA loci and chromosome-specific SSR markers, these MAALs were classified into seven groups corresponding to potential seven types of MAALs carrying one of the seven I. indigotica chromosomes. One of the MAALs could be distinguishable by expressing the brown anthers of I. indigotica, other two hosted the chromosome with 5S or 45S rDNA locus, but the remaining four were identifiable by SSR markers. The simultaneous detection of the same SSR maker and gene locus in different MAALs revealed the paralogs on the chromosomes involved. The recombinant mitochondrial genome in MAALs was likely related with their male sterility with carpellody stamens, while the MAAL with normal brown anthers probably carried the restoring gene for the male sterility. The complete set of MAALs should be useful for exploiting the I. indigotica genome and for promoting the introgression of valuable genes to B. napus.

[Identification of cellular morphology and flow cytometry on different tetraploid Isatis indigotica strains].

OBJECTIVE: To identify tetraploid Isatis indigotica strains through morphology and flow cytometry. METHODS: The tissue culture seedlings of tetraploid Isatis indigotica were root-tip squashed and chromosome counted before rooted climatized and transplanted in field. The plants in field were taken as experimental materials. Macroscopic observation was applied to identify by form and structure; Free-hand section was used to observe the length, width and density of stomas; And flow cytometry was applied to identify the ploidy. RESULTS: Compared with diploid plants, tetraploid plants had obvious changes in form and structure. The stomas from the tetraploid were notably longer, and the number of guard cells in chloroplasts was remarkably larger. The experiment materials were proved to be tetraploid by flow cytometry. CONCLUSION: The materials are tetraploid plants. Macroscopic observation, the length of stoma and the number of guard cells in chloroplasts can be taken as aided identification for ploidy of mutagenesis materials. Meanwhile, flow cytometry can be applied to identify the ploidy of Isatis indigotica.

Production and cytogenetic characterization of intertribal somatic hybrids between Brassica napus and Isatis indigotica and backcross progenies.

Intertribal somatic hybrids between Brassica napus (2n = 38, AACC) and a dye and medicinal plant Isatis indigotica (2n = 14, II) were obtained by fusions of mesophyll protoplasts. From a total of 237 calli, only one symmetric hybrid (S2) and five asymmetric hybrids (As1, As4, As6, As7 and As12) were established in the field. These hybrids showed some morphological variations and had very low pollen fertility. Hybrids S2 and As1 possessed 2n = 52 (AACCII), the sum of the parental chromosomes, and As12 had 2n = 66 (possibly AACCIIII). Hybrids As4, As6 and As7 were mixoploids (2n = 48-62). Genomic in situ hybridization analysis revealed that pollen mother cells at diakinesis of As1 contained 26 bivalents comprising 19 from B. napus and 7 from I. indigotica and mainly showed the segregation 26:26 at anaphase I (AI) with 7 I. indigotica chromosomes in each polar group. Four BC(1) plants from As1 after pollinated by B. napus resembled mainly B. napus in morphology but also exhibited some characteristics from I. indigotica. These plants produced some seeds on selfing or pollination by B. napus. They had 2n = 45 (AACCI) and underwent pairing among the I. indigotica chromosomes and/or between the chromosomes of two parents at diakinesis. All hybrids mainly had the AFLP banding patterns from the addition of two parents plus some alterations. B. napus contributed chloroplast genomes in majority of the hybrids but some also had from I. indigotica. Production of B. napus-I. indigotica additions would be of considerable importance for genome analysis and breeding.

Microwave treatment of eight seconds protects cells of Isatis indigotica from enhanced UV-B radiation lesions.

To determine the role of microwaves in the stress resistance of plants to enhanced ultraviolet-B (UV-B) radiation, Isatis indigotica Fort. seeds were subjected to microwave radiation for 8 s (wavelength 125 mm, power density 1.26 mW mm(-2), 2450 MHz). Afterwards they were cultivated in plastic pots in an artificial-glass greenhouse maintained at 25 degrees C, 70% relative humidity, and 400 micromol mol(-1) CO2, under visible-light conditions of 1500 micromol m(-2) s(-1) for 8 h day(-1). When the seedlings were 10 days old, they were subjected to 10.08 kJ m(-2) UV-B (PAR: 220 micromol m(-2) s(-1)) radiation for 8 days. Changes in a number of physiological and biochemical characteristics and in the thermal decomposition enthalpy of biomass were measured and used as indicators of the protective capacity of microwave radiation in this experiment. Our results revealed that microwave pretreatment of seeds enhanced UV-B stress resistance in the seedlings by decreasing the concentration of malondialdehyde (MDA) and increasing the concentration of ascorbic acid (AsA) and UV-B-absorbing compounds, increasing the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), and increasing the energy accumulation of photosynthesis. All these results suggest that microwave radiation enhances plant metabolism and results in increased UV-B stress resistance. This is the first investigation reporting the use of microwave pretreatment to protect the cells of Isatis indigotica from UV-B-induced lesions.

Transcriptomic analysis reveals differential gene expressions for cell growth and functional secondary metabolites in induced autotetraploid of Chinese woad (Isatis indigotica Fort.).

The giant organs and enhanced concentrations of secondary metabolites realized by autopolyploidy are attractive for breeding the respective medicinal and agricultural plants and studying the genetic mechanisms. The traditional medicinal plant Chinese woad (Isatis indigotica Fort., 2n = 2x = 14) is now still largely used for the diseases caused by bacteria and viruses in China. In this study, its autopolyploids (3x, 4x) were produced and characterized together with the 2x donor for their phenotype and transcriptomic alterations by using high-throughput RNA sequencing. With the increase of genome dosage, the giantism in cells and organs was obvious and the photosynthetic rate was higher. The 4x plants showed predominantly the normal meiotic chromosome pairing (bivalents and quadrivalents) and equal segregation and then produced the majority of 4x progeny. The total 70136 All-unigenes were de novo assembled, and 56,482 (80.53%) unigenes were annotated based on BLASTx searches of the public databases. From pair-wise comparisons between transcriptomic data of 2x, 3x, 4x plants, 1856 (2.65%)(2x vs 4x), 693(0.98%)(2x vs 3x), 1045(1.48%)(3x vs 4x) unigenes were detected to differentially expressed genes (DEGs), including both up- and down-regulated ones. These DEGs were mainly involved in cell growth (synthesis of expansin and pectin), cell wall organization, secondary metabolite biosynthesis, response to stress and photosynthetic pathways. The up-regulation of some DEGs for metabolic pathways of functional compounds in the induced autotetraploids substantiates the promising new type of this medicinal plant with the increased biomass and targeted metabolites.

Production and analysis of organic acids in hairy-root cultures of Isatis indigotica Fort. (indigo woad).

Hairy roots were induced from both cotyledon and hypocotyl explants of Isatis indigotica Fort. (indigo woad) through transformation with Agrobaterium rhizogenes strain A4, R1601 and ATCC15834. The results showed that the cotyledons were the preferred explants to hypocotyls and A4 was the most suitable A. rhizogenes strain for the transformation and induction of hairy roots of I. indigotica. High-voltage paper electrophoresis (HVPE) analysis demonstrated the production of mannopine in hairy roots and confirmed the successful transfer of Ri T-DNA (root-inducing transferred DNA) of A. rhizogenes into the I. indigotica genome. Five organic acids, namely CPQ [3-(2-carboxyphenol)-4(3 H )-quinazolinone], syringic acid, salicylic acid, benzoic acid and 2-aminobenzoic acid, which were considered as main antiviral components of I. indigotica, were detected in natural roots, hairy roots and liquid media with high-performance capillary electrophoresis. The results showed CPQ production in hairy roots was significantly higher than that in natural roots. Our results also revealed that all the five organic acids could be excreted from hairy roots into liquid media, and the concentrations of organic acids in the liquid media paralleled those in hairy roots. The hairy roots of I. indigotica grew fast and showed an S-shaped growth curve that reached its apex on the day 24 of culture with a 20-fold increase in fresh weight compared with the starting inoculums. The accumulation of the two organic acids CPQ and syringic acid in liquid media paralleled the growth of hairy roots. MS [Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15, 473-497] medium or half-strength MS medium supplemented with 30 g/l maltose was found to be best for hairy-root culture and accumulation of CPQ.