Effects of Heavy Metals and Saline-alkali on Growth,Physiology and Biochemistry of Orychophragmus violaceus

[Objective] The aim was to study on effects of heavy metals and saline-alkali on growth, physiology and biochemistry of Orychophragmus violaceus. [Method] Taken Orychophragmus violaceus as materials, growth, physiology and biochemistry were explored under stress of saline-alkali and heavy metals (light, moderate and severe saline-alkali, Pb, Pb + Cd, light saline-alkali + Pb, moderate saline-alkali + Pb, severe saline-alkali + Pb, light saline-alkali + Pb + Cd, moderate saline-alkali + Pb + Cd and severe saline-alkali + Pb + Cd) with control group set. [Result] Light stress of saline-alkali had little effect on membrane permeability, as follows: MDA contents in leaves and root systems declined by 25.6% and 9.0% compared with control group; Pb (500 mg/L) stress promoted synthetization of photosynthetic pigments, as follows: chlorophyll a and b and carotenoid increased by 0.86%, 0.69% and 6.25% than those of control group; combined stresses of Pb and Cd destroyed synthetization of photosynthetic pigments, among which carotenoid was more sensitive; under combined stresses of saline-alkali, Pb and Cd, POD and SOD activities, soluble saccharides and Pro content all increased and activities of POD and SOD in root system were both higher than those in leaves. [Conclusion] Orychophragmus violaceus is with resistance against light combined stresses of saline-alkali and Pb (500 mg/L).

Preliminary Studies on the Intergeneric Hybridization Between Brassica napus with OguCMS and Orychophragmus violaceus

In the cross between B. napus with OguCMSOrychophragmus violaceus, pre-fertilizatiocompatibility was observed. An intergeneric hybrid was successfully obtained by usinovary culture. The hybrid status was confirmed by cytology, morphology, fertilityisozyme and miosis analysis. The seed setting rate was 0.0072%. An amphidiploid of thhybrid was also produced by the use of chromosome-doubling method with 0.1% colchicinetreatment. Obtaining this hybrid and its amphidiploid has important theoretical anutilization value.

Transformation of Orychophragmus violaceus Using Agrobacterium tumefaciens And Regeneration of Transgenic Plantsa

In this paper, we reported firstly the transgenic plants of Orychophragmus violateus in the world. Excised cotyledon and hypocotyls of Orychophragmus violaceus were used as explants for genetic transformation. After 2-3 days of co-cultivation with Agrobacterium tumefaciens strain A208SE(PROA93),the hypocotyls and cotyledon were transferred onto selection medium containing 25 mg/L Km and 250 mg/L Ap. 8 weeks later, shoots emerged,then the shoots were excised and transferred onto root medium containing 25 mg/L Km and 100 mg/L Cef. The roots were formed within 4-5 weeks.The whole plants were transplanted into pots and grew well. The frequency of plant regeneration of hypocotyls was about 30%,and that of cotyledon was 51%.The regenerated plants showed high enzymatic activities ofglucuronidase and neomycin phosphotransferase II. Southern blot analysis confirmed that NPT II gene had been stably integrated into the chromosomal genome of Orychophragmus violaceus .the transformation frequency of hypocotyls was 10%,and that of cotyledon was 5.5%.

Genome assembly of the Brassicaceae diploid Orychophragmus violaceus reveals complex whole-genome duplication and evolution of dihydroxy fatty acid metabolism

Orychophragmus violaceus is a Brassicaceae species widely cultivated in China,particularly as a winter cover crop in northern China because of its low-temperature tolerance and low water demand.Recently,O.violaceus has also been cultivated as a potential industrial oilseed crop because of its abundant 24-carbon dihydroxy fatty acids(diOH-FAs),which contribute to superior high-temperature lubricant properties.In this study,we performed de novo assembly of the O.violaceus genome.Whole-genome synteny analysis of the genomes of its relatives demonstrated that O.violaceus is a diploid that has undergone an extrawhole-genome duplication(WGD)after the Brassicaceae-specific a-WGD event,with a basic chromosome number of x=12.Formation of diOH-FAs is hypothesized to have occurred after the WGD event.Based on the genome and the transcriptome data from multiple stages of seed development,we predicted that OvDGAT1-1 and OvDGAT1-2 are candidate genes for the regulation of diOH-FA storage in O.violaceus seeds.These results may greatly facilitate the development of heat-tolerant and eco-friendly plant-based lubricants using O.violaceus seed oil and improve our understanding of the genomic evolution of Brassicaceae.

The genome of Orychophragmus violaceus provides genomic insights into the evolution of Brassicaceaepolyploidizationandits distinct traits

Orychophragmus violaceus,referred to as‘‘eryuelan’’(February orchid)in China,is an early-flowering ornamental plant.The high oil content and abundance of unsaturated fatty acids in O.violaceus seeds make it a potential high-quality oilseed crop.Here,we generated a whole-genome assembly for O.violaceus using Nanopore and Hi-C sequencing technologies.The assembled genome of O.violaceus was~1.3 Gb in size,with 12 pairs of chromosomes.Through investigation of ancestral genome evolution,we determined that the genome of O.violaceus experienced a tetraploidization event from a diploid progenitor with the translocated proto-Calepineae karyotype.Comparisons between the reconstructed subgenomes of O.violaceus identified indicators of subgenome dominance,indicating that subgenomes likely originated via allotetraploidy.O.violaceus was phylogenetically close to the Brassica genus,and tetraploidy in O.violaceus occurred approximately 8.57 million years ago,close in time to the whole-genome triplication of Brassica that likely arose via an intermediate tetraploid lineage.However,the tetraploidization in Orychophragmus was independent of the hexaploidization in Brassica,as evidenced by the results from detailed phylogenetic analyses and comparisons of the break and fusion points of ancestral genomic blocks.Moreover,identification of multi-copy genes regulating the production of high-quality oil highlighted the contributions of both tetraploidization and tandem duplication to functional innovation in O.violaceus.These findings provide novel insights into the polyploidization evolution of plant species and will promote both functional genomic studies and domestication/breeding efforts in O.violaceus.

PEG－mediated Gene Transfer into Orychophragmus Violaceus Cotyledon Protoplast and Regeneration of Transgenic Plants

Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast using transient expression system, which showed 25-30 μg of pasmid, 15% PEG and a pH value of 8.0 as the optimal parameters contributing to the highest expression level. Using these parameters, cotyledon protoplasts were isolated, treated with bacterial plasmid DNA (pBI222 with HPT as selective marker) and PEG, and cultured at a density of 5×10 4/ml.After 10-15 days,they were selected by adding 25 μg/ml hygromycine. One month later, a few calli were observed, which were then transferred onto a solid medium with 50-100 μg/ml hygromycine for proliferation. Later they were transferred successively onto differentiation and rooting media and finally hygromycineresistant whole plants were obtaincd. The plants grew well in pots and a regeneration rate of 5 ×10(-5) was achieved. Then,excised leaves of the transgenic plants were used as explants for Southern blot analysis, which confirmed the stable integration of HPT gene into the chromosomal genome of Orychophragmus violaceus The transformation frequency was 10-5.

Foliar modifications induced by inhibition of polar transport of auxin

The effects of auxin polar transport inhibitors, 9-hydroxy-fluorene-9-carboxylic acid (HFCA); 2, 3, 5-triiodobenzoic acid (TIBA) and trans-cinnamic acid (CA)on leaf pattern formation were investigated with shoots formed from cultured leaf explants of tobacco and cultured pedicel explants of Orychophragmus violaceus, and the seedlings of tobacco and Brassica chinensis. Although the effective concentration varies with the inhibitors used, all of the inhibitors induced the formation of trumpet-shaped and/or fused leaves. The frequency of trumpet-shaped leaf formation was related to the concentration of inhibitors in the medium.Histological observation of tobacco seedlings showed that there was only one main vascular bundle and several minor vascular bundles in normal leaves of the control, but there were several vascular bundles of more or less the same size in the trumpet-shaped leaves of treated ones.These results indicated that auxin polar transport played an important role on bilateral symmetry of leaf growth.