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).

Morphological and Cytogenetic Analysis on the Advanced Generations of Generic Hybrids between Brassica napus and Orychophragmus violaceu

[Objective] This study aimed to reveal the genetic changes of advanced generation hybrids between Brassica napus and Orychophragmus violaceus. [Method] The morphological characteristics such as the plant shape, branching sites, leaf shape, leaf color, primary branches and secondary branches, as well as the cytological characteristics of the advanced generation hybrids (F8-F10) between B. napus and O. violaceus were observed. [Result] The morphology analysis revealed that the hybrid progeny was more like B. rapa in leaf shape, leaf color, plant shape and early flowering phenotype, whereas more like B. napus in number of secondary branches, silique length and 1 000-seed weight. Analysis on the cytogenetics characteristics showed that these advanced inbred progenies were hypoploids with less than 38 chromosomes; moreover, all the chromosomes from O. violaceus had been lost. Chromosome pairings at meiotic diakinesis of hybrids between the advanced inbred progenies and B. rapa revealed that chromosomes lost in hypoploids possibly belonged to the C genome of B. oleracea. With generations developing, chromosomes number of plants from two populations gradually increased and developed into the number of B. napus (2n=38). [Conclusion] This study will provide reference to reveal the source of chromosome lost in hypoploids and the morphological change of hybrids.

Study on the Flower Development of Testtube Plantletsof Orychophragmus violaceus

In this study, testtube plantlets of O.Violaceus were cultured on MS media and treated with low temperature, phytohormones, etc. The following results were obtained: 1. The cold treatment of 5～7℃ for more than 7 days was needed for flower initiation of the testtube plantlets originated from germinated seeds. 2. When cultured on the MS media supplemented with 2mg/L gibberellin (GA3), the testtube plantlets could initiate flowers without cold treatment. 3. When the MS media was supplemented with a reduced amount of NH4NO3, flower initiation of the testtube plantlets could be accelerated. The results of the study on flower development of O.Violaceus may be useful for the production of testtube flowers of this ornamental plant species.

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.

Does bicarbonate affect the nitrate utilization and photosynthesis of Orychophragmus violaceus?

The effect of bicarbonate(HCO_3^-) on the growth and development of plants varies by species. To better understand inorganic carbon and nitrogen assimilation changes of karst-adaptable plants under different HCO_3^- treatments, we conducted experiments on seedlings and in vitro plantlets of Orychophragmus violaceus(Ov).We found that the vital photosynthesis potential(as measured by net photosynthetic rate, actual photochemical efficiency of photosystem-II, photochemical quenching coefficient, and the instantaneous carbon isotope ratio of3-phosphoglycerate) was consistent under different HCO_3^- treatments of Ov. Bicarbonate's lack of effect on carbon assimilation of Ov may be related to carbonic anhydrase in Ov converting HCO_3^- to H2 O and CO2. In this way, Ov could prevent HCO_3^- ion toxicity and high pH from harming its growth and development under HCO_3^- stress.This study also found that high HCO_3^- concentrations could promote nitrogen assimilation and utilization of Ov through changes in related indexes(foliar nitrogen isotope fractionation ratio, stable nitrogen isotope assimilation ratio, foliar stable nitrogen isotope fractionation, nitrate nitrogen utilization efficiency, and nitrate utilization share)under different HCO_3^- treatments. Bicarbonate has different effects on photosynthesis and on inorganic nitrogen assimilation of Ov, which may be connected tophotosynthesis providing electrons for nitrate/nitrite reduction through the photosynthetic chain.

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%.

诸葛菜Orychophragmus violaceu(sL.)O.E.Schulz.

诸葛菜是十字花科诸葛菜属一年或二年生草本植物,高可达50 cm,无毛;茎直立,基生叶及下部茎生叶大头羽状全裂,顶裂片近圆形或短卵形,侧裂片卵形或三角状卵形,叶柄疏生细柔毛;花紫色、浅红色或褪成白色,花萼筒状,紫色,花瓣宽倒卵形,密生细脉纹,长角果线形,种子卵形至长圆形,黑棕色,4~5月开花,5~6月结果。

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.

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.

盐胁迫对二月兰(Orychophragmus violaceus)种子萌发的影响

为了了解盐渍化土壤条件对二月兰(Orychophragmus violaceus)种子萌发的影响,及种子萌发及萌发过程中可溶性糖、蛋白质、淀粉酶活性的影响,本研究分别用浓度为0.3%、0.6%、0.9%、1.2%、1.5%的NaCl、Na_2SO_4和二者质量比为1:1的混合盐处理二月兰种子,并观测溶性糖、蛋白质、淀粉酶活性。结果表明,二月兰种子的发芽率、发芽势、发芽指数均随着盐浓度增加而下降,且盐浓度愈高对种子萌发抑制作用愈强。二月兰种子耐盐适宜范围为NaCl 0.29%、Na_2SO_4 0.30%、混合盐0.24%,耐盐半致死浓度为NaCl 0.63%、Na_2SO_4 0.65%、混合盐0.60%,耐盐极限浓度为NaCl 1.18%、Na_2SO_41.21%、混合盐1.18%。说明二月兰种子萌发中蛋白质的分解受到一定程度的抑制,较高浓度盐处理强烈抑制了淀粉酶活性,盐处理使可溶性糖含量增加。为以后二月兰在盐渍化条件下的应用提供理论依据。

De novo Flower-bud Differentiation in Tissue Culture of Marigold, Violet Orychophragmus and Tobacco

Floral bud differentiation in vitro is affected by many factors, among which the effect of exogenous phytohormones is striking. According to the study of floral gradient, however, it is possible that the function of phytohormones in floral bud regeneration in vitro may be just a 'trigger', i.e. providing a suitable condition for those cells to express the po-

Nuclear reconstitution of demembranated Orychophragmus violaceus sperm in Xenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon- densation and pronuclear formation from demembranated plant (Orychophragmus violaceus)sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.

二月兰形态性状的变异分析

二月兰(Orychophragmus violaceus)是集食用、保健、饲用和观赏于一身的优良地被植物。其遗传背景单一、生长周期短,是发展模式植物的优良材料。为了开发其育种价值,我们对二月兰的形态变异式样进行了初步分析。共选取了北京和南京地区的7个居群的34个表型性状进行观测。通过计算分析发现:株高、茎和花序分枝等性状受环境影响较大,因而其分类价值不大;叶形、毛被、叶缘锯齿等性状的变异在居群内是连续的,所以也不是可靠的分类性状;而花部性状的一些变异,包括花瓣的颜色、大小、形态,基本不受环境影响,且这些变异在居群内是不连续的,所以其分类价值相对较高。对17个数值性状的方差分析和多重比较结果表明,在同一年度内,大部分性状在不同地区间差异不显著,在居群间差异极显著;而在不同年度内,居群间差异不显著。最后,对7个居群和30个表型性状进行了数量分类:Q分析表明北京颐和园、南京紫金山山顶公园和南京中山植物园居群比较相似,而北京圆明园、北京海淀区后八家苗圃、南京情侣园和北京天坛公园居群较相似。R分析揭示了二月兰花部、叶部、反映植株毛被和颜色以及表现植株生长量的性状间均具有一定的相关性。这些研究可为种下等级的划分和进一步的遗传研究以及品种培育提供一定的参数资料。

^(60)Co-γ辐射对NaCl胁迫下二月兰种子萌发的影响

以二月兰(Orychophragmus violaeeus)种子为材料,采用室内培养皿发芽方法,分别统计其在清水和0.5%NaCl中的萌发指标并测量幼苗生长指标,以研究^(60)Co-γ射线对二月兰种子萌发及早期幼苗生长的影响。结果表明,在0~300 Gy剂量范围内,^(60)Co-γ辐射提高了二月兰种子的发芽率,同时降低了种子的发芽势。在水处理条件下,^(60)Co-γ辐射对二月兰幼苗株高、根长、鲜重都有不同程度的促进作用。在0.5%NaCl处理条件下,^(60)Co-γ辐射对二月兰幼苗的株高和鲜重产生了一定的促进作用,对根长的生长则是低浓度促进高浓度抑制。

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.

High efficiency production and genomic in situ hybridization analysis of Brassica aneuploids and homozygous plants

Interspecific and intergeneric hybridizations have been widely used in plant genetics and breeding to construct stocks for genetic analysis and to introduce into crops the desirable traits and genes from their relatives. The intergeneric crosses between Brassica juncea (L.) Czern. & Coss., B. carinata A. Braun and Orychophragmus violaceus (L.) O. E. Schulz were made and the plants produced were subjected to genomic in situ hybridization analysis. The mixoploids from the cross with B. juncea were divided into three groups. The partially fertile mixoploids in the first group (2n = 36—42) mainly contained the somatic cells and pollen mother cells (PMCs) with the 36 chromosomes of B. juncea and additional chromosomes of O. violaceus. The mixoploids (2n = 30—36) in the second and third groups were morphologically quite similar to the mother plants B. juncea and showed nearly normal fertility. The plants in the second group produced the majority of PMCs (2n = 36) with their chromosomes paired and segregated normally, but 1—4 pairs of the O. violaceus chromosomes were included in some PMCs. The plants in the third group produced only PMCs with the 36 B. juncea chromosomes, which were paired and segregated normally. The mixoploids (2n = 29—34) from the cross with B. carinata produced the majority of PMCs (2n = 34) with normal chromosome pairing and segregation, but some plants had some PMCs with 1—3 pairs of chromosomes from O. violaceus and other plants had only PMCs with the B. carinata chromosomes. The Brassica homozygous plants and aneuploids with complete or partial chromo-some complements of Brassica parents and various numbers of O. violaceus chromosomes were derived from these progeny plants. The results in this study provided the molecular cytogenetic evidence for the separation of parental genomes which was previously proposed to occur in the hybridizations of these two genera.