WRKY11 up-regulated dirigent expression to enhance lignin/lignans accumulation in Lilium regale Wilson during response to Fusarium wilt

Lilium are highly economically valuable ornamental plants that are susceptible to Fusarium wilt caused by Fusarium oxysporum.Lilium regale Wilson,a wild lily native to China,is highly resistant to F.oxysporum.In this study,a WRKY transcription factor,WRKY11,was isolated from L.regale,and its function during the interaction between L.regale and F.oxysporum was characterized.The ectopic expression of LrWRKY11 in tobacco increased the resistance to F oxysporum,moreover,the transcriptome sequencing and UHPLC-MS/MS analysis indicated that the methyl salicylate and methyl jasmonate levels rose in LrWRKY11 transgenic tobacco,meanwhile,the expression of lignin/lignans biosynthesis-related genes including a dirigent(DiR)was up-regulated.The lignin/lignans contents in LrWRKY11-transgenic tobacco also significantly increased compared with the wild-type tobacco.In addition,the resistance of L.regale scales in which LrWRKY11 expression was silenced by RNAi evidently decreased,and additionally,the expression of lignin/lignans biosynthesis-related genes including LrDIR1 was significantly suppressed.Therefore,LrDIR1 and its promoter(PLrDIR1)sequence containing the W-box element were isolated from L.regale.The interaction assay indicated that LrWRKY11 specifically bound to the W-box element in PLrDIR1 and activated LrDIR1 expression.Additionally,β-glucuronidase activity in the transgenic tobacco co-expressing LrWRKY11/PLrDIR1-β-glucuronidase was higher than that in transgenic tobacco expressing PLrDIR1-β-glucuronidase alone.Furthermore,the ectopic expression of LrDIR1 in tobacco enhanced the resistance to F.oxysporum and increased the lignin/lignans accumulation.In brief,this study revealed that LrWRKY11 positively regulated L.regale resistance to F.oxysporum through interaction with salicylic acid/jasmonic acid signaling pathways and modulating LrDIR1 expression to accumulate lignin/lignans.

R2R3-type Lo MYB21 affects jasmonate-regulated development and dehiscence of anthers in lily(Lilium oriental hybrids)

Lilies are widely cultivated for cut flowers,but their large anthers carry a considerable amount of colored pollen that is dispersed easily.Studying the molecular mechanism of anther development and dehiscence could help solve this problem.LoMYB21,encoding a putative R2R3v-myb avian myeloblastosis viral oncogene homolog(MYB)transcription factor,was identified from oriental lilies(Lilium‘Siberia’).Real-time quantitative PCR analysis showed that LoMYB21 was mainly expressed in the anther,filament and stigma and had high expression during the late stages of lily anther development.LoMYB21 had transactivation activity and was located in the nucleus through yeast one-hybrid assays and transient expression in Nicotiana benthamiana.Suppression of LoMYB21 by virus-induced gene silencing(VIGS)in Lilium‘Siberia’led to anther indehiscence and reduced the expression of genes related to Jasmonate acid(JA)biosynthesis and signal transduction.Induction of LoMYB21 in DEX::LoMYB21 transgenic Arabidopsis caused procumbent inflorescences that became infertile,accompanied by higher expression of JA biosynthetic and signaling genes.These results demonstrated that JA content and signaling were abnormal in silenced lily and transgenic LoMYB21 Arabidopsis,which affected anther development.Our study indicated that LoMYB21 could regulate lily anther dehiscence through JA biosynthesis and signaling during the late stages of anther development.

Monitoring Xylem Transport in the Stem of Lilium lancifolium Using Fluorescent Dye 5(6)-Carboxyfluorescein Diacetate

The xylem undergoes physiological changes in response to various environmental conditions during the process of plant growth.To understand these physiological changes,it is extremely important to observe the transport of xylem.In this study,the distribution and structure of vascular bundle in Lilium lancifolium were observed using the method of semithin section.Methods for introducing a fluorescent tracer into the xylem of the stems were evaluated.Then,the transport rule of 5(6)-Carboxyfluorescein diacetate(CFDA)in the xylem of the stem of L.lancifolium was studied by fluorescence dye in live cells tracer technology.The results showed that the vascular bundles of L.lancifolium were scattered in the basic tissue,the peripheral vascular bundles were smaller and densely distributed,and the closer to the center,the larger the volume of vascular bundles and the more sparsely distributed.The vascular bundles of L.lancifolium are limited external tenacity vascular bundles,which are composed of phloem and xylem.The most suitable method for CFDA labeling the xylem of isolated stem segments of L.lancifolium was solution soaking for 24 h.The running speed of CF in the isolated stem was 0.3 cm/h,which was consistent with the running speed of the material in the field.CF could be transported between the xylem and parenchyma cells,indicating that the material transport in the xylem could be through the symplastic pathway.The above results laid a foundation for the study of the xylem transport mechanism and the xylem pathogen disease of lily.

The Study on Induction and Proliferation of Tube Bulbs in Lilium brownii

With different parts of bulb scale as explants, the proliferation method of Guizhou Lilium brownii were studied with 3% sodium hypochlorite and MS medium with different concentrations of hormones. The results show that it is feasible to disinfect the bulbs of Lilium brownii with 3% sodium hypoehlorite, moreover, the sodium hypochlorite is very cheap and harmless to researchers, experimental materials and environment. MS ＋ NAA 0.3 mg/L ＋ 6-BA 1.5 mg/L is optimum for the induction of bulbs and, the basal part of Lilium brownie is the optimum explants. After culture for 25 d on the same medium, the tube bulbs could be obtained with the characteristics of high propagation coefficient, strong and new roots. The survival rate is over 90% for transplantation of tube bulbs with diameter between 1-2cm. The method developed in the present study can proliferate abundant Lilium brownii seedling in short time.

Study on the Integration of T-DNA into Lilium longiflorum

[ Objective] The aim of this study is to obtain transgenic Lilium longiflorum Thumb. [ Method] A two-step method of explant and the T-DNA integration technique were employed to transform Lilium longiflorum via Agrobacterium mediated method. [ Result] The best infection effect appeared under the OD600 value of Agrobacterium within 0.6 -0.8, the addition of 250 mg/L AS could increase the transformation efficiency. The optimal concentration of G418 for screening is 50 mg/L. Some putative transgenic plants of Lilium longiflorum with resistance to G418 showed positive in PCR, preliminarily proving that T-DNA gene had integrated into the genome of lily. [ Conclusion] The study may lay a foundation for breeding excellent lily varieties through TDNA integration technique.

Identification and Biological Characteristics of Blank Spot Pathogen from Lilium lancifolium

With the increasing planting area of Lilium lancifolium, the leaf fungal dis-ease of L. lancifolium is becoming more and more serious. In June and July of 2014, the excessive rainfal leads to the serious outbreak of leaf disease of L. lanci-folium. In mid-June, the wilting rate of L. lancifolium in seriously-infected field was even up to 50%-70%. In some fields, the shoots of L. lancifolium even al wilted. The pathogen was isolated from the infected leaves of L. lancifolium. Its pathogenici-ty, spore morphology, 18S rDNA sequence and biological characteristics were stud-ied. The results showed the isolated pathogen was Alternaria alternate. The lethal temperature of mycelial growth was 55 ℃. The optimum pH was 6-7. Among the tested carbon sources and nitrogen sources, the optimum carbon source was mal-tose, and the optimum nitrogen source was yeast extract.

Analysis of Protein and Isoenzyme of LiLum davidii var.unicolor,Lilium Asiatic Hybrids and Their Hybrid F_1

[Objective]The paper was to provide theoretical basis for selection of parental combination and early identification of hybrids.[Method]The soluble protein and peroxidase of LiLum davidii var.unicolor,Lilium Asiatic hybrids and their filial generations were analyzed using polyacrylamide gel electrophoresis technique.[Result]The protein spectrum of filial generation with L.davidii var.unicolor as parent not only appeared the homologous band as parent with darker coloring,but also had new bands compared with parent.Peroxidase zymogram of hybrid F1 mainly displayed incomplete complementary and hybrid type of parent.[Conclusion]Protein spectrum and peroxidase zymogram could be used as the biochemical markers for the identification of hybrids of lily,which could also detect the target traits of plant.

Role of Wall-bound β-Glucanases in Regulating Tip-growth of Lilium longiflorum Pollen Tubes

Glucanases were found in the cell wall of Lilium longiflorum Thunb. pollen tubes grown in vitro . The activity of β_glucanases was, in a certain extent, decreased by nojirimycin, an inhibitor of glucosidase. Pollen germination percentage reduced dramatically when nojirimycin was applied in the culture medium. In case that nojirimycin was added at 0 or 1 h after the onset of incubation, the inhibition rate was 99.6% and 91.4%, respectively. When 3 mmol/L of nojirimycin was applied in the liquid medium at 0, 1, 1.5 and 2 h after the onset of incubation, the growth of pollen tubes was interrupted, which resulted in the morphological change of the pollen tubes such as the newly grown portion of pollen tubes being bent, curved and swollen. Tracing the growth pattern of the individual pollen tube grown in semi_solid medium by video microscopy, the authors demonstrated that pollen tube growth rate was strongly inhibited by nojirimycin at concentrations ranged from 0.003 to 3 mmol/L. Moreover, the cytoplasmic arrangement and the morphology of the pollen tubes were also affected by nojirimycin. The growth inhibition brought about by nojirimycin was reversible. These results indicated that β_glucanases, which degrade 1,3_β_glucan and/or 1,4_β_glucan or 1,3:1,4_β_glucan constructed in the cell wall, are involved in pollen germination and pollen tube growth. It provides new insight into an understanding of the contribution of β_glucanases to the cell wall extensibility and the crucial role of cell wall in regards to the regulation of pollen tube growth.

百合属(Lilium)几种植物花粉形态的观察

应用光学显微镜和扫描电子显微镜对百合属(Lilium)植物13个种花粉的形态、表面纹饰、萌发沟等性状的观察结果表明,百合属植物花粉皆为椭球形,具单萌发沟,网状纹饰,而花粉大小、网脊宽度、网眼直径等性状存在差异。在供试的13个种中,麝香百合系品种罗瑞拉(Longiflorum hybrids cv.Lorina)的花粉最大,有斑百合(L.concolor var.buschianum Baker)的花粉最小。花粉形态特征和超微结构对百合属植物的分类有一定价值。

催花保鲜剂对百合(Lilium)绿蕾催花保鲜生理的研究

探讨了百合切花绿蕾经两种催花保鲜剂处理后的效果 ,结果表明 ,催花保鲜剂能促进绿蕾提前开花 ,同时对开花后的百合切花有延缓衰老的作用 ,减少了切花在瓶插期间叶绿素与蛋白质含量的降解 ,提高了切花的品质和商品价值。处理 增加了开放率和花蕾长度 ,处理 增加了花瓣长和花径大小 ,同时在试验过程中揭示了百合切花经催花剂处理后的水分生理变化规律 ,筛选出最佳的催花保鲜剂为处理 。

东方百合(Lilium oritential)组织培养研究

以东方百合"索邦"为研究对象,对东方百合组织培养技术进行研究。结果表明:启始培养所用材料的基部诱导率明显高于上部,鳞茎分化芽的数量明显多于鳞片;MS+0.1 mg.L-1BA+0.1 mg.L-1NAA为最佳鳞茎诱导培养基;MS+0.1 mg.L-1BA+0.3 mg.L-1NAA为最佳鳞茎增殖培养基;用1/2MS+0.1 mg.L-1IBA培养基进行生根培养,取得良好的效果。

Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modem lily cultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the FI hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC1 progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic （LA） hybrids in Lilium. Therefore, the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research. The results showed that： at metaphase I, the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell （PMC）, and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva- lents were disjoined and most univalents were divided. Both the disjoined bivalents （half-bivalents） and the divided univalents （sister chromatids） moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents, were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the Fl LA hybrids and the variation of their BCx progenies, but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.

利用百合(Lilium davidii)根尖细胞的微核细胞率作为环境监测指标的探讨

首次报道了利用百合根尖细胞微核细胞率作为环境监测指标的可行性和优越性。实验证明,百合根尖比蚕豆根尖细胞的微核细胞率更敏感。

辽宁的4种野生百合(Lilium spp.)的核型研究

利用染色体常规压片的方法,对辽宁的4种野生百合进行了核型研究。结果如下:①有斑百合L.con-colorSalisb.var.buschianum(Lodd.)Baker.2n=4m(2SAT)+4st+16t(2SAT);②细叶百合L.pumilumDC.2n=6sm(2SAT)+12st+6t;③朝鲜百合L.amabilePalib.2n=4m(2SAT)+8st+12t(2SAT);④垂花百合L.cernumKom.2n=2m(2SAT)+2sm(2SAT)+12st+8t。它们不仅在染色体相对长度、臂比和次缢痕数目及其分布有区别,而且核型类型也不同。除了细叶百合的核型为3A型,其余的均为稳定的3B型。

Effects of intercropping on rhizosphere soil microorganisms and root exudates of Lanzhou lily(Lilium davidii var.unicolor)

Both yield and quality of Lanzhou lily(Lilium davidii var. unicolor) are seriously affected by continuous cropping. We attempted to understand the effects of intercropping on the obstacles associated with continuous cropping of Lanzhou lily(Lilium davidii var. unicolor). The changes of rhizosphere microbial biomass and diversity in interplanting and monoculturing systems were studied by using the Illumina Hi Seq sequencing technique. The contents and composition of lily root exudates were measured by gas chromatography–mass spectrometer(GC–MS). The intercropping results of Lanzhou lily showed:(1) There was no difference in the composition of the rhizosphere soil microbes at the phylum level, but the relative abundance of the microbes decreased; and the relative abundance of harmful fungi such as Fusarium sp. increased. The relative abundance of Pleosporales sp. and other beneficial bacteria were reduced. After OTU(operational taxonomic unit)clustering, there were some beneficial bacteria, such as Chaetomium sp., in the lily rhizosphere soil in the interplanting system that had not existed in the single-cropping system. We did not find harmful bacteria that had existed in the single-cropping systm in the rhizosphere soil of interplanting system. The above results indicated that the changes of relative abundance of soil fungi and bacteria in lily rhizosphere soil was not conducive to improving the ecological structure of rhizosphere soil microbes. At the same time, the microbial composition change is very complex—beneficial and yet inadequate at the same time.(2) Root exudates provide a matrix for the growth of microorganisms. Combined with the detection of root exudates, the decrease in the composition of the root exudates of the lily was probably the reason for the decrease of the relative abundance of microbes after intercropping. At the same time, the decrease of the relative content of phenolic compounds, which inhibit the growth of microorganisms, did not increase the relative content of rhizosphere soil microorganisms. Changes in amino acids and total sugars may be responsible for the growth of Fusarium sp.. The results showed that the intercropping pattern did not noticeably alleviate the obstacle to continuous cropping of Lanzhou lily, and the change of microbial biomass and diversity was even unfavorable. However, the emergence of some beneficial bacteria, the disappearance of harmful fungi, and other changes with intercropping are in favor of alleviation of obstacles to continuous cropping of Lanzhou lily.

山丹（lilium pumilum）的组织培养和再生鳞茎的形态解剖学研究

取山丹鳞片，在附加不同激素水平的ＭＳ或ＬＳ培养基上培养，筛选出最适合山丹快速繁殖的培养基（ＭＳ＋ＢＡ＿０．ｓ＋ＮＡＡ＿０．５）．本文观察了再生鳞茎的外部形态，用石蜡切片法研究了愈伤组织和再生鳞茎的解剖结构。结果表明：再生鳞茎在形成后３０ｄ内形态结构正常，但随着培养时间的延长，鳞片上部发育成脆弱的条形叶；叶内细胞一部分解体，在叶肉内形成通气组织。

γ-射线和秋水仙素对兰州百合(Lilium davidii var.Willmottiae)体细胞诱变的效应

本文报道了γ-射线和秋水仙素对百合体细胞诱变后的生物学效应和形态变异,并为诱变处理提供较适宜的剂量和浓度。结果指出,诱发幼苗有利变异的有效处理组合是剂量为200—400伦(剂量率100伦╱分)的γ-射线和浓度为2.0mg/l的秋水仙素。试管苗叶色、叶宽,叶长、鳞茎大小、根量和生长势等性状表现变异,在组培条件下这些变异能保持稳定,田间鉴定正在进行中。

157百合(Lilium cr. Gran sasso)染色体核型研究

本文对157百合（LIIiumcrGramSasso）进行了染色体核型研究，结果表明:157百合体细胞染色体数为2n=2x=24；染色体组成为R（2n）=2x=2m（SAT）+2m+2Sm（SAT）+16st+2t；在第1，2对染色体上有随体；其核型分类属于3B型。

A critical review on chemical constituents and pharmacological effects of Lilium

Genus Lilium is famous for edible and medicinal function which is related to its chemical constituents and pharmacological effects.Chemical researches showed that genus Lilium genus mainly contains steroidal saponins,polysaccharides,alkaloids and flavonoids.Pharmacological effects of Lilium include anti-tumor,hypoglycemic,antibacterial,anti-oxidation,anti-depression and anti-inflammatory.This paper summarized chemical constituents and pharmacological effects of Lilium.

Physiology Mechanism of Metabolisms in the Middle Scales of Lilium davidii var. unicolor Bulbs Stored at Low Temperature for Dormancy-Release

Changes in carbohydrate, phenols, free amino acids and endogenous hormones in middle scales of Lilium davidii var.unicolor bulbs during the 101 days storage at 2, 6 and 10°C in moist sawdust were studied. The results indicated that asthe storage time increased and the storage temperature delined, contents of starch and abscisic acid (ABA) decreasedmarkedly, and the level of total soluble sugar, phenols, gibberellic acid (GA3) and indole acetic acid (IAA) increasedsharply. In the first 34 days, the contents of the above substances changed most actively. Correlation analysis revealedthat the increase in content of total soluble sugar and phenols was respectively caused by activation of amylase andphenylalanine ammonia-lyase (PAL). During the storage, the content of free amino acids decreased, and the level ofarginine was the highest and changed most remarkably. According to the results of stepwise regression analysis and pathanalysis, we could primarily conclude that the metabolism of carbohydrates, phenols and free amino acids in the middlescales may be mainly regulated by endogenous GA3.