Insights into glucosinolate accumulation and metabolic pathways in Isatis indigotica Fort.

BACKGROUND: Glucosinolates (GSLs) play important roles in defending against exogenous damage and regulating physiological activities in plants. However, GSL accumulation patterns and molecular regulation mechanisms are largely unknown in Isatis indigotica Fort. RESULTS: Ten GSLs were identified in I. indigotica, and the dominant GSLs were epiprogoitrin (EPI) and indole-3-methyl GSL (I3M), followed by progoitrin (PRO) and gluconapin (GNA). The total GSL content was highest (over 20 µmol/g) in reproductive organs, lowest (less than 1.0 µmol/g) in mature organs, and medium in fresh leaves (2.6 µmol/g) and stems (1.5 µmol/g). In the seed germination process, the total GSL content decreased from 27.2 µmol/g (of seeds) to 2.7 µmol/g (on the 120th day) and then increased to 4.0 µmol/g (180th day). However, the content of indole GSL increased rapidly in the first week after germination and fluctuated between 1.13 µmol/g (28th day) and 2.82 µmol/g (150th day). Under the different elicitor treatments, the total GSL content increased significantly, ranging from 2.9-fold (mechanical damage, 3 h) to 10.7-fold (MeJA, 6 h). Moreover, 132 genes were involved in GSL metabolic pathways. Among them, no homologs of AtCYP79F2 and AtMAM3 were identified, leading to a distinctive GSL profile in I. indigotica. Furthermore, most genes involved in the GSL metabolic pathway were derived from tandem duplication, followed by dispersed duplication and segmental duplication. Purifying selection was observed, although some genes underwent relaxed selection. In addition, three tandem-arrayed GSL-OH genes showed different expression patterns, suggesting possible subfunctionalization during evolution. CONCLUSIONS: Ten different GSLs with their accumulation patterns and 132 genes involved in the GSL metabolic pathway were explored, which laid a foundation for the study of GSL metabolism and regulatory mechanisms in I. indigotica.

A SEPALLATA1-like gene of Isatis indigotica Fort. regulates flowering time and specifies floral organs.

An orthologous gene of SEPALLATA1, designated as IiSEP1, was isolated from Isatis indigotica. The genomic DNA of IiSEP1 is 3.1 Kb in length. The full-length cDNA of IiSEP1 is 1481 bp and contains a 756 bp ORF encoding a 251-amino-acid protein. Sequence comparison revealed that IiSEP1 belonged to the MADS-box gene family. IiSEP1 contains 7 exons and 6 introns, showing similar exon-intron structure with Arabidopsis SEP1. Phylogenetic analysis suggested that IiSEP1 belonged to AGL2/SEP subfamily and was likely to be an I. indigotica ortholog of Arabidopsis SEP1. Quantitative real-time PCR showed that IiSEP1 was predominantly expressed in the reproductive organs. Ectopic expression of IiSEP1 in Arabidopsis resulted in early flowering, accompanied with the reduction of inflorescence number and the production of terminal flower on the top of the main stems. Moreover, IiSEP1 overexpressing flowers generated numerous variations in phenotype. The sepals were changed into petal-sepal mosaic structures or displayed carpelloid features, and transparent ovules were formed in internal surface of these sepals. In addition, some flowers were constituted by sepals and pistil, but lacked petals and stamens. Taken together, IiSEP1 might play important roles in reproductive growth of I. indigotica and could affect the morphogenesis of flowers and fruits.

Effects of germination season on life history traits and on transgenerational plasticity in seed dormancy in a cold desert annual.

The maternal environment can influence the intensity of seed dormancy and thus seasonal germination timing and post-germination life history traits. We tested the hypotheses that germination season influences phenotypic expression of post-germination life history traits in the cold desert annual Isatis violascens and that plants from autumn- and spring-germinating seeds produce different proportions of seeds with nondeep and intermediate physiological dormancy (PD). Seeds were sown in summer and flexibility in various life history traits determined for plants that germinated in autumn and in spring. A higher percentage of spring- than of autumn-germinating plants survived the seedling stage, and all surviving plants reproduced. Number of silicles increased with plant size (autumn- > spring-germinating plants), whereas percent dry mass allocated to reproduction was higher in spring- than in autumn-germinating plants. Autumn-germinating plants produced proportionally more seeds with intermediate PD than spring-germinating plants, while spring-germinating plants produced proportionally more seeds with nondeep PD than autumn-germinating plants. Flexibility throughout the life history and transgenerational plasticity in seed dormancy are adaptations of I. violascens to its desert habitat. Our study is the first to demonstrate that autumn- and spring-germinating plants in a species population differ in proportion of seeds produced with different levels of PD.

[Effects of different K fertilizer and water level on growth and physiological characteristics of Isatis indigotica].

The experiment included three potassium levels (K0 0 g x kg(-1), K1 0.33 g x kg(-1), K2 0.67 g x kg(-1)) and two water gradients (well watered and drought stress), then measured growth indicators, SOD, POD, CAT activities and concents of osmotic regulation substances. To explore the effects of K fertilizer and water on growth and physiological characteristics of Isatis indigotica, providing reference for improving drought resistance of I. indigotica. The result showed drought stress inhibited the growth and decreased the biomass of I. indigotica but K fertilizer can alleviate the drought stress. Compared with K0 treatment, K1, K2 treatment increased the biomass of overground part of by 89. 13% ,60. 87% under drought stress. The corresponding increase in soluble sugar content was 16.67%, 5.00%, and in proline content was 42.41%, 65.62%, respectively. SOD,POD and CAT activities was significantly improved in K1, K2 treatment in comparison with K0 treatment under drought stress, but soluble protein content significantly reduced. The conclusion is that appropriate amount of K fertilizer can increase the activities of antioxidase and the content of osmoregulation substance under drought stress, and improve drought resistance of I. indigotica.

[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.

[Effects of drought stress on growth and water use efficiency of two medicinal plants].

Growth characteristics, stable carbon isotope discrimination (Delta13C), water use efficiency (WUE), and their correlation of Cassia obtusifolia and Isatis indigotica were measured at three soil water levels, i. e., 30%, 50% and 75% of field water holding capacity (FWHC), and at three growth stages. The growth indices of the two medicinal plants at 75% of FWHC were higher than those at 30% and 50% of FWHC, suggesting that the two medicinal plants could obtain high production under sufficient moisture condition. The Delta13C(A) (aboveground biomass-based Delta13C) and Delta13C(T) (total biomass-based Delta13C) decreased, and the WUE(A) (aboveground biomass-based WUE) and WUE(T) (total biomass-based WUE) of C. obtusifolia and I. indigotica increased with the increasing degree of drought stress. The growth indices of the two medicinal plants had little difference in the different water treatments, which indicated that the two medicinal plants were insensitive to drought stress. Water use efficiency of I. indigotica had significant negative relationships with aboveground biomass and total biomass, while that of C. obtusifolia had a significant positive correlation with the root/shoot ratio.

[Effect of different nitrogen forms and concentrations on biomass and alkaloids of isatidis folium].

OBJECTIVE: In order to provide theoretical basis of improving nitrogen utilization efficiency in Isatis indigotica, the biomass and active components in Isatidis Folium under different nitrogen forms and concentrations were analyzed. METHOD: I. indigotica was cultivated by sand culture in greenhouse, and the experiment was designed with orthogonal test L25 (5(6)). As an index to the biomass and indigo, indirubin, the effects on the I. indigotica by three factors [NO3(-) -N, NH4(+) -N, CO(NH2)2] at five different levels were studied. RESULT: There were significant difference of the biomass and active components by different nitrogen forms and concentrations. The effect of amide nitrogen [CO(NH2)2] on biomass of Isatidis Folium was the most apparent, and the effect of ammonium nitrogen on indirubin was more obvious. Considering the biomass and active ingredient, one combination was optimized, which was (NH4)2SO(4)-7.5 mmol x L(-1), KNO(3)-2.5 mmol x L(-1), CO(NH2)(2)- 5 mmol x L(-1). CONCLUSION: It is important to promote the growth in pre-stage of I. indigotica, and cost-effective combination of balanced nitrogen fertilizer could reasonably promote the growth, and improve the contents of active components and individual biomass.

[Plant traits and quality evaluation of different cultivation forms of Isatis].

OBJECTIVE: To lay a foundation for the study on good variety selection of Isatis indigotica, comparison of plant traits and quality evaluation of Isatis germplasm resources from different production area was conducted. METHOD: Field cultivation and randomized block experiment were adopted to compare those plant traits of leaf, root, silique and seed of Isatis from different production area and the content of R,S-goitrin and polysaccharide in the root was determined. RESULT: Different cultivation forms of Isatis had significant difference from each other in leaf, root, silique and seed, content of R,S-goitrin and polysaccharide in the root were also different. R,S-goitrin content in Isatis of Chinese cabbage leaf type ( production area: Yunnan ) was comparative higher, 0.59% , while polysaccharide content in autotetraploid Isatis was comparative higher, 8.68%. CONCLUSION: According to the plant traits, Isatis were classified into three types: Chinese cabbage leaf type, cabbage leaf type and mustard leaf type, of which R,S-goitrin content in Chinese cabbage leaf type (production area: Yunnan) was comparative higher, while polysaccharide content in autotetraploid Isatis was comparative higher.

[Establish efficiently transformation system of the Isatis indigotica].

OBJECTIVE: To build up a method of efficiently transforming Isatis indigotica with the Agrobacterium. METHODS: Two kinds of Agrobocterium: ATCC 15834 and RI1601 were used to treat different fraction of Isatis indigotica. Explored the effect of cocultured, different antibiotic concentration and the media on multiply the hairy roots. RESULTS: The explants with out coculture had had higher survival rate rooting rate and earlier sprout time. After ultrasonic treatment of plant, the indacement rate was two times than that of untreated one; The best antibiotic concentration was 400 mg/L; The proportion of the grow speed of the hairy root in the liquid culture media was 2 - 3 times than that of solid culture media, and 37 times of common roots. CONCLUSION: The method is useful for establishing an efficiently transformatiem system of Isatis indigotica by Agrobacterium.

[Contrast of growth characteristics of different germplasm and correlation between morphologic and individual yield of Isatis indigotica].

OBJECTIVE: To study the growth characteristics of different germplasm and analyze the correlation between morphologic and individual yield of Isatis indigotica. METHOD: The planting samples were collected during the growth period, morphological characteristics and root yield per plant in 13 germplasm of I. indigotica from all over the country were determined by correlation and path analysis. RESULT: The growth characteristics of various I. indigotica germplasm were consistent. The difference of morphological characteristics and root yield with various germplasm were significant. The root yield per plant was positively correlated to the main root length and the number of individual plant leaves. CONCLUSION: The number of leaves per plant and root length are selected for high-yield lines, meanwhile, leaf length and leaf diameter are important factors for breeding high-yield lines as well.

[Effects of NH4(+)-N/NO3(-)-N ratio in applied supplementary fertilizer on nitrogen metabolism, photosynthesis and growth of Isatis indigotica].

OBJECTIVE: To study the effect of NH4(+)-N/NO3(-)-N ratios in the applied supplementary fertilizer on the growth, nitrogen metabolis related enzymes activity and photosynthetic characteristics of Isatis indigotica. METHOD: The sand culture experiment was conducted, and seedling of I. indigotica was fertilized with the mixed nutrition that containing the Hoagland's macro elements and the Aron's micro elements, the additional 63 mmol N was supplementary with the NH4(+)-N/NO3(-)-N ratio of 100:0, 75:25, 50:50, 25:75 and 0:100. RESULT: The biomass of I. indigotica increased at first when the supplementary N of NH4(+)-N/NO3(-)-N ratio changed from 100:0 to 50:50 and decreased afterwards. The maximum value was at 50:50 and the minimum at 100: 0. With increasing the ratio of NO3(-)-N, the activity of nitrate reductase and glutamine synthetase increased and then decreased and the relationship between the activity and the ratio could be described with an approximate parabola curve. The net photosynthetic rate of I. indigotica was the highest at the NH4(+)-N/NO3(-)-N ratio of 75:25 and the lowest at 100:0. CONCLUSION: Increasing the NO3(-)-N ratio properly was beneficial to promote the growth and improve the activity of nitrate reductase and glutamine synthetase and net photosynthetic rate of I. indigotica.

An arsenic-accumulating, hypertolerant brassica, Isatis capadocica.

Isatis capadocica, a brassica collected from Iranian arsenic-contaminated mine spoils and control populations, was examined to determine arsenate tolerance, metabolism and accumulation. I. cappadocica exhibited arsenate hypertolerance in both mine and nonmine populations, actively growing at concentrations of > 1 mm arsenate in hydroponic solution. I. cappadocica had an ability to accumulate high concentrations of arsenic in its shoots, in excess of 100 mg kg(-1) DW, with a shoot : root transfer ratio of > 1. The ability to accumulate arsenic was exhibited in both hydroponics and contaminated soils. Tolerance in this species was not achieved through suppression of high-affinity phosphate/arsenate root transport, in contrast to other monocotyledons and dicotyledons. A high percentage (> 50%) of arsenic in the tissues was phytochelatin complexed; however, it is argued that this is a constitutive, rather than an adaptive, mechanism of tolerance.

[Initial RAPD analysis of Isatis indigotica somatic clones' resistance to root rot disease].

OBJECTIVE: To make initial identification on Isatis Indigotica Somatic Clones' resistant mutant. METHODS: The resistant plants were selected under the treatment of root-rot disease crude toxin. The DNA of the same clones and their parents were analysed by RAPD technology. RESULTS: We got initial resistance of the plants and selected 12 primers of which most were identical but the less were different. The genetic background of the somatic clones was identical but the individuals were mutated on the DNA level. CONCLUSION: It provides experimental foundation for further study on separating resistant mutant and resistant gene.

[Effects of sowing date on morphologic characteristics, yield and quality of Radix isatidis].

OBJECTIVE: To study the effects of sowing date on morphologic characteristics, yield and quality of Radix Isatidis, in order to provide a basis for selection of the suitable sowing date. METHOD: The experiment was carried out in a randomized complete block design arranged in split plot, the phonological period of Isatis indigotica was investigated and the morphological indexes, yield and quality at harvest were measured and determined by correlation and variance analysis. RESULT: The different sowing dates showed a great effect on phonological period of I. indigotica. The biological characteristics, yield and active components of Radix Isatidis were negatively correlated with the sowing date, and the difference of morphological characteristics, yield and active compositions with sowing date was significant. CONCLUSION: The suitable sowing date of I. indigotica was during the period of time from late March to beginning of the April in Fuyang area, and considering the factors of rational rotation, the time cloud delayed until late May to the beginning of June.

[Effect of water stress on growth and accumulation of active components of Isatis indigotica].

OBJECTIVE: To study the effect of water stress on the growth and accumulation of active components of Isatis indigotica. METHOD: By pot culture experiment,the characteristic indexes of water requirement, chlorophyll content, transpiration rate, photosynthesis, biomass and effective ingredient indirubin were studied under normal and water stress conditions, respectively. RESULT: With the increase of the extent and duration of water stress, the quantity of water requirement, chlorophyll content, respiration rate, photosynthesis and biomass of I. indigotica were decreased, while water use efficiency increased. CONCLUSION: It is a critical period of water requirement in July, high water content in soil and extreme water stress are not propitious to the accumulation of indirubin, appropriate water stress can stimulate indirubin accumulatio. The harvest of leaves of I. indigotica is optimal in July, while the harvest of root is appropriate in October. Yield and quantity of I. indigotica are improved when the field capacity is between 45% and 70%.

Production and characterization of intertribal somatic hybrids of Raphanus sativus and Brassica rapa with dye and medicinal plant Isatis indigotica.

Intertribal somatic hybrids of Raphanus sativus (2n = 18, RR) and Brassica rapa spp. chinensis (2n = 20, AA) with the dye and medicinal plant Isatis indigotica (2n = 14, I I) were firstly obtained by polyethylene glycol-induced symmetric fusions of mesophyll protoplasts. One mature hybrid with R. sativus established in field had intermediate morphology but was totally sterile. It had the expected chromosome number (2n = 32, RRI I) and parental chromosomes were distinguished by genomic in situ hybridization (GISH) analysis, and these chromosomes were paired as 16 bivalents in pollen mother cells (PMCs) at diakinesis and mainly segregated equally as 16:16 at anaphase I (A I), but the meiotic disturbance in second division was obvious. Five mature hybrids with B. rapa established in field were morphologically intermediate but showed some differences in phenotypic traits and fertility, two were partially fertile. Cytological and GISH investigations revealed that these hybrids had 2n = 48 with AAIIII complement and their PMCs showed normal pairing of 24 bivalents and mainly equal segregation 24:24, but meiotic abnormalities of lagging chromosomes and micronuclei appeared frequently during second divisions. AFLP analysis showed that all of these hybrids had mainly the DNA banding pattern from the addition of two parents plus some alterations. Some hybrids should be used for the genetic improvement of crops and the dye and medicinal plant.

[Effects of various planting densities on dynamic growth rhythm and root yield of Isatis indigotica].

OBJECTIVE: To study the effects of various planting densities on dynamic growth and root yield of Isatis indigotica. METHOD: The planting samples were collected to measure the growth period of each organ. RESULT AND CONCLUSION: Under different planting densities, both main root length and breadth exhibited a trend of "fast-slow" by stages. However, the number of individual plant leaves were showed a trend of "slow-fast" on growth period. Meanwhile, the leaf length and breadth were exhibited a trend of "increase-decrease". The increase of dry leaf, dry root and whole plant dry matter was faster during the period of 65-76 days after seeding. The differences of root and leaf yields under various densities were significant. Planting densities has a great effect on yield of root. It must be shown that there was a positive development between the individual plant and colony. According to the dry matter of root and leaf, treatment B (i.e., 7 cm x 25 cm) was good choice.

[Effects of formula fertilization to yield and content on polysaccharide of Isatis indigotica].

OBJECTIVE: To select the optimum formula fertilization of Isatis indigotica through analyzing the yield and contents of polysaccharide of Radix Isatis for different treatments. METHOD: An orthogonal experiment design on the basis of three factors and four levels was applied for studying the effect of formula fertilization on yield. The contents of polysaccharides were determined with phenol-witriolic colorimetry. RESULT: The optimum formula fertilization of Radix Isatis was carbamide 869.0 kg x hm(-2), superphosphate 1 428.6 kg x hm(-2) and potassium sulfate 0 kg x hm(-2). CONCLUSION: Superphosphate can observably influence the yields of Radix Inditis. while carbamide influence the contents of polysaccharide of Radix Inditis.

[Transformation of bar gene to tetraploid of Isatis indigotica].

The transgenic tetraploid of Isatis indigotica mediated by Agrobacterium tumefaciens was obtained. To transfer the plant binary expression vector pCAMBIA 3300 carrying bar gene, the Agrobacterium tumefaciens strain EHA 105 was used as engineering bacterium. The results of PCR indicated that the bar gene had been transferred into and merged with the genome of Isatis indigotica. This study will make foundation for improvement of other characters of this species with genetic engineering.

[Genetic transformation of autotetraploid Isatis indigotica fort. induced by Ri T-DNA and plant regeneration].

OBJECTIVE: To establish an effective system for genetic transformation of autotetraploid Isatis indigotica by Agrobacterium rhizogenes. METHOD: Agrobacterium rhizogenes strains R1601, ATCC15834 and A4 were employed to induce hairy roots from autotetraploid Isatis indigotica, and the obtained hairy roots were developed into regenerated plants on solid MS media with different Kinds of plant growth regulators. RESULT: Root induction differed obviously. By three Agrobacterium strains. Hairy roots grew rapidly on solid MS medium without plant growth regulators and showed the typical hairy root phenotype: profuse branching, high density of root hairs and plagiotropism. And the transformation of Ri T-DNA was confirmed by opine analysis. The biomass of hairy roots increased nearly 35 times regulator-free MS after suspended in plant growth two weeks. On solid MS medium with BA, adventitious buds were differentiated directly from hairy roots without callus formation. All of the adventitious buds were rooted on root induction medium and developed into regenerated plants. Opine analysis indicated the integration of Ri T-DNA in the transformed plants. CONCLUSION: Agrobacterium rhizogene can induce hairy roots from autotetraploid Isatis indigotica, and the obtained hairy roots can develop into regenerated plants.