羊草受精作用及其胚与胚乳早期发育的观察

利用常规石蜡制片方法研究了羊草受精过程及胚与胚乳的早期发育,其主要结果为：(1)授粉后1h,花粉管破坏1助细胞,释放2精子。精子为眼眉状,难以区分其细胞质鞘;(2)授粉后1～2h,2个精子分别移向卵细胞与极核;(3)授粉后2～3h,精核分别贴附于卵细胞与极核核膜上;(4)授粉后3～10h,精核与卵核融合,并出现雄性核仁,形成合子;(5)授粉后3～4h,精核与极核融合,并出现雄性核仁,形成初生胚乳核,精核与极核的融合比与卵核融合快;(6)传粉后20h,合子分裂,合子的休眠期为10h左右;(7)传粉4h,初生胚乳核分裂,初生胚乳核没有休眠期;(8)羊草双受精作用属于有丝分裂前配子融合类型;(9)胚胎发育属于紫菀型,胚乳发育属于核型胚乳。     

杂交石竹授粉受精及其胚胎发育过程的观察

以‘杂交石竹’为试验材料,利用荧光显微镜观察其授粉后花粉萌发、花粉管生长情况,采用石蜡切片法对其受精及胚胎发育过程进行观察研究。结果表明:(1)授粉后1h母本柱头上少量花粉开始萌发;授粉后4h大量花粉萌发,花粉管生长至柱头中部有胼胝质出现;授粉后6h花粉管生长至子房组织并有少量与胚珠结合;授粉后15h柱头中出现大量胼胝质,花粉管与胚珠结合数增多;授粉后24h胚珠周围出现多条花粉管,其中1条花粉管进入胚珠,部分进入胚囊的花粉管卷曲盘绕生长并产生胼胝质;精细胞与极核的融合主要发生在授粉后17~48h,与卵细胞融合主要于授粉后1~3d。(2)杂交石竹胚发育经过原胚、球形胚、棒状形胚、心形胚、鱼雷形胚和子叶形胚阶段。(3)杂交障碍表现为:只有游离的胚乳核而无胚发育的胚囊、合子未分裂、两极核未融合、球形胚败育。研究表明,杂交石竹存在受精前和受精后障碍,这是导致其结实率低的主要原因。     

小叶杨与美杨种间杂交的胚胎发育和杂种胚珠的离体培养

1.观察了小叶杨(Populus simonii Carr.)和美杨(Populus pyramidalis Borkh.)人工杂交后从花粉粒萌发至胚分化完成的发育过程:1.大多数美杨花粉粒可以在小叶杨的柱头上萌发长出花粉管,花粉管在花柱中正常地生长并通过珠孔进入胚囊,于授粉后4—7天发生双受精作用,形成初生胚乳核和合子。胚乳的发育正常或不正常,后者表现为在游离核阶段的败育或胚乳细胞发育不良。胚在各个发育阶段尤其是球形胚和心形胚阶段,都可能败育,也可以正常发育成为分化完全的有效胚。2.将授粉后19天、22天、26天和29天含有不同发育阶段(心形期、鱼雷期、子叶伸长期)未成熟的杂种胚珠接种于人工培养基上,结果表明:1.M110培养基(1/2 MS+IAA 0.01毫克/升+BA 0.1毫克/升+蔗糖2%)是选用培养基中效果最好的;2.不同发育阶段杂种胚的胚珠可以离体培养成生长正常的小植株。     

甘薯胚胎及果实发育的研究

本文研究甘薯的胚胎发育及果实的形成。授粉后10—30分钟花粉粒在柱头上萌发,2小时花粉管抵达珠孔,5—12小时左右完成双受精。授粉后12小时胚乳核开始第一次有丝分裂;15小时合子开始第一次有丝分裂,18小时形成顶细胞和基细胞;尔后分化成原胚,球形胚,心形胚,鱼雷形胚,成熟胚。在适宜温度下21天左右胚胎发育完成。果为蒴果,其内含有1—4粒种子。授粉后3—4天子房开始膨大形成果实,21—30天蒴果与种子成熟。     

小麦×大麦胚胎发育的研究——Ⅰ.小麦×大麦胚和胚乳发育的观察

本试验以206小麦为母本,二棱大麦旱燕3号和六棱大麦原早1号为父本进进杂交。结果表明,两个组合早期的胚胎发育基本上是正常的,前者成胚率为8.75%,后者为16.8%。胚乳核最早于授粉后3天开始形成细胞。当胚乳细胞充满囊后,很快就转入迅速解体,胚随即停止生长,没有能够进一步分化出器官来。胚乳于授粉15天后,几乎全部败育,胚亦相继夭亡。胚胎发育过程中的不正常现象普遍存在。主要是极核受精过程遭受破坏;合子和初生胚乳核发育停滞;原胚虽能正常发育而胚乳没有形成;胚乳细胞过早形成和迅速解体等。讨论了杂交不孕和胚乳败育的原因。同时,提出了幼胚离体培养的适宜时间。     

白桦雌花发育、大孢子发生及胚胎发育的解剖学观察

白桦雌花从开花到雌性器官的成熟需经历1个月左右的时间,解剖学观察表明：四月下旬越冬的雌蕊原基开始了活跃的分裂和分化。子房和柱头开始生长。四月末开花,五月初授粉。此后胚珠开始长大。五月中旬即分化形成珠被,珠心,珠被为单层珠被,胚珠为厚珠心胚珠,胚珠倒生,五月中下旬,珠心内产生大孢子母细胞,一周左右发育为成熟胚囊-七细胞八核胚囊,五月末完成双受精,白桦胚胎发育经过合子,原胚,球形胚,心形胚和鱼雷形胚等时期最后发育成熟,胚乳发育与胚胎同步,即受精的极核进行几次分裂后形成核型胚乳,胚乳核不断增多,在形成心形胚后胚乳细胞形成细胞壁。     

小麦×大麦胚胎发育的研究——Ⅰ.小麦×大麦胚和胚乳发育的观察

本试验以206小麦为母本,二棱大麦旱燕3号和六棱大麦原早1号为父本进进杂交。结果表明,两个组合早期的胚胎发育基本上是正常的,前者成胚率为8.75%,后者为16.8%。胚乳核最早于授粉后3天开始形成细胞。当胚乳细胞充满囊后,很快就转入迅速解体,胚随即停止生长,没有能够进一步分化出器官来。胚乳于授粉15天后,几乎全部败育,胚亦相继夭亡。胚胎发育过程中的不正常现象普遍存在。主要是极核受精过程遭受破坏;合子和初生胚乳核发育停滞;原胚虽能正常发育而胚乳没有形成;胚乳细胞过早形成和迅速解体等。讨论了杂交不孕和胚乳败育的原因。同时,提出了幼胚离体培养的适宜时间。     

星星草受精作用及其胚与胚乳早期发育的观察

利用常规石蜡切片法对星星草[Puccinellia tenuiflora(Griseb．)Scribn．et Merr．]受精过程及胚与胚乳的早期发育进行了观察,主要结论如下：(1)开花后2h,花粉管破坏1个助细胞,释放2个精子,精子呈逗点状。(2)开花后2～3h,2个精子分别移向卵细胞与极核。(3)开花后3～5h,精核分别贴附于卵细胞与极核的核膜上。(4)开花后5～10h,精核与卵核融合,雄性核仁出现,合子形成。(5)开花后5～6h,精核与极核融合,并出现雄性核仁,形成初生胚乳核,精核与极核的融合比与卵核融合要快。(6)开花后20h左右,合子分裂。(7)开花后8h,初生胚乳核。     

黑节草双受精过程及胚胎发育的研究

黑节草从传粉到受精约需１３０ｄ,精子在花粉管中形成,胚囊发育属蓼型胚囊,因反足细胞较早退化,故受精前胚囊多只由卵器和中央细胞组成。精卵核融合时,精核染色质进入卵核后凝集成颗粒状,并在原位与卵核的染色质融合,雌、雄性核仁一直维持至合子的第一次分裂期前。双受精作用正常,属于有丝分裂前配子融合类型,初生胚乳核发生２－３次分裂后逐渐退化消失,胚的发育局限于球形胚阶段。     

普通小麦与球茎大麦杂交后受精及胚胎发育的细胞形态学研究

普通小麦(2n=6x=42)品种及其杂种F_1为母本与四倍体球茎大麦(2n=4x=28)杂交,发现属间杂交的受精过程和胚胎生长发育比小麦自交时缓慢。球茎大麦花粉给小表授粉后4小时精子进入卵接触卵核,6小时初生胚乳核开始形成。杂合子第一次有丝分裂和授粉后22小时胚乳核的分裂中出现染色体落后及微核,授粉后9天胚乳核出现解体现象,10天后幼胚局部细胞也出现解体。13天后胚囊成为空腔,已观察不到细胞轮廓。在北京田间于授粉后9—10日即须将幼胚离体培养。     

拟南芥抵抗丁香假单胞杆菌过程中AZI1基因功能研究

AZI1属于脂转移蛋白家族,它在拟南芥抵抗病原菌侵染过程中可能起着传递信号物质的作用。该实验以过表达和T-DNA插入突变体及野生型拟南芥植株为材料,通过RNA印迹、蛋白质免疫印迹和原位免疫组织化学方法,研究了拟南芥壬二酸诱导基因AZI1对丁香假单胞杆菌的抗性功能。结果表明:(1)AZI1基因可以被丁香假单胞杆菌、H2O2和乙烯利诱导,它可能参与水杨酸和乙烯介导的抗菌途径。(2)蛋白质免疫印迹实验结果显示,丁香假单胞杆菌侵染叶片的叶柄渗出液中存在AZI1蛋白及其同源物EARLI1,并能够与其他蛋白质形成复合体,说明AZI1有可能通过维管组织移动到个体的其他部位,与信号分子的转移有关。(3)AZI1及其同源物EARLI1主要在花序茎的木质化部位表达,过表达AZI1基因能够促进木质素的合成,提高拟南芥对丁香假单胞杆菌的抗性。     

Development of a Virus-Induced Gene-Silencing System for Functional Analysis of the RPS2-Dependent Resistance Signalling Pathways in Arabidopsis

Virus-induced gene silencing (VIGS) offers a rapid and high throughput technique platform for the analysis of gene function in plants. Although routinely used in some Solanaceous species, VIGS system has not been well established in Arabidopsis thaliana (L.) Heynh. We have recently reported some factors that potentially influence tobacco rattle virus (TRV)-mediated VIGS of phytoene desaturase (PDS) and actin gene expression in Arabidopsis. In this study, we have further established that the Agrobacterium strain used for agro-inoculation significantly affects the VIGS efficiency. Strain GV3101 was highly effective; C58C1 and LBA4404 were invalid, while EHA105 was plant growth stage-dependent for TRV-induced gene silencing. Furthermore, the VIGS procedure optimised for the PDS gene was applied for the functional analysis of the disease resistance gene RPS2-mediated resistance pathway. Silencing of RPS2 led to loss of resistance to the otherwise avirulence strain of Pseudomonas syringae pv. tomato DC3000 carrying the avirulence gene AvrRpt2. Silencing of RIN4, a RPS2 repressor gene, gave rise to conversion of compatible interaction to incompatible. Silencing of NDR1, RAR1 and HSP90, known to be required for the RPS2-mediated resistance, resulted in loss of the resistance, while silencing of EDS1 and SGT1b, which are not required for the RPS2-mediated resistance, caused no change of the resistance. These results indicate that the optimised procedure for the TRV-based VIGS is a potentially powerful tool for dissecting the signal transduction pathways of disease resistance in Arabidopsis. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at An erratum to this article is available at .     

Calmodulin-like Proteins from <Emphasis Type="Italic">Arabidopsis</Emphasis> and Tomato are Involved in Host Defense Against <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Complex signal transduction pathways underlie the myriad plant responses to attack by pathogens. Ca²⁺ is a universal second messenger in eukaryotes that modulates various signal transduction pathways through stimulus-specific changes in its intracellular concentration. Ca²⁺-binding proteins such as calmodulin (CaM) detect Ca²⁺ signals and regulate downstream targets as part of a coordinated cellular response to a given stimulus. Here we report the characterization of a tomato gene (APR134) encoding a CaM-related protein that is induced in disease-resistant leaves in response to attack by Pseudomonas syringae pv. tomato. We show that suppression of APR134 gene expression in tomato (Solanum lycopersicum), using virus-induced gene silencing (VIGS), compromises the plant’s immune response. We isolated APR134-like genes from Arabidopsis, termed CML42 and CML43, to investigate whether they serve a functionally similar role. Gene expression analysis revealed that CML43 is rapidly induced in disease-resistant Arabidopsis leaves following inoculation with Pseudomonas syringae pv. tomato. Overexpression of CML43 in Arabidopsis accelerated the hypersensitive response. Recombinant APR134, CML42, and CML43 proteins all bind Ca²⁺ in vitro. Collectively, our data support a role for CML43, and APR134 as important mediators of Ca²⁺-dependent signals during the plant immune response to bacterial pathogens. This work was supported by a research grant (WAS) and postgraduate scholarships (DC, SLD) from the Natural Science and Engineering Research Council of Canada, the National Science Foundation (IBN-0109633; GBM), and the Swedish Research Council (SKE).     

Cold responsive EARLI1 type HyPRPs improve freezing survival of yeast cells and form higher order complexes in plants

Plants have large families of proteins sharing a conserved eight-cysteine-motif (8CM) domain. The biological functions of these proteins are largely unknown. EARLI1 is a cold responsive Arabidopsis gene that encodes a hybrid proline-rich protein (HyPRP) with a three-domain architecture: a putative signal peptide at the N-terminus, a proline-rich domain (PRD) in the middle, and an 8CM domain at the C-terminus. We report here that yeast cells expressing different EARLI1 genes had significantly higher rates of freezing survival than empty-vector transformed controls. Arabidopsis plants with knocked down EARLI1 genes had an increased tendency for freezing-induced cellular damage. EARLI1-GFP fluorescence in transgenic plants and immunoblot analyses using protoplasts suggested cell wall localization for EARLI1 proteins. Immunoblot analyses showed that EARLI1 proteins form higher order complexes in plants, and that the PRD is a soluble and the 8CM an insoluble protein domain. We propose that EARLI1 proteins have a bimodular architecture in which the PRD may interact with the cell wall and the 8CM domain with the plasma membrane to protect the cells during freezing stress.     

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes,in <Emphasis Type="Italic">Arabidopsis</Emphasis> infected with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Identification and expression of the Pseudomonas syringae pv. aptata hrpZPsa gene which encodes an harpin elicitor

A sequence homologous to an internal fragment 0.75 kb BstXI of the Pseudomonas syringae pv. syringae hrpZ gene was identified in Pseudomonas syringae pv. aptata NCPPB 2664, the causal agent of bacterial blight in sugar beet, lettuce and other plants, and in E. coli DH10B (pCCP1069) containing the P. syringae pv. aptata hrp gene cluster. PCR with oligonucleotides, based on the hrpZ_Pss gene and used as primers with the total genomic DNA of P. syringae pv. aptata, amplified a 1 kb fragment that hybridized with the probe in highly stringent conditions. The amplicon was cloned into the pGEM-T® plasmid vector, amplified in E. coli DH5 and sequenced. The sequence showed 95%, 83% and 61% identity with those of hrpZ_Pss, hrpZ_Psg and hrpZ_Pst genes encoding the harpins of the P. syringae pv. syringae, glycinea and tomato, respectively. The amplicon was cloned into the pMAL® expression system. The expressed protein, fused with maltose-binding protein, was cleaved with a specific protease factor Xa, and purified using affinity chromatography. On the basis of the amino acid sequence and its ability to induce HR in tobacco leaves, it was identified as a P. syringae pv. aptata harpin.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

New disease resistance genes in soybean against Pseudomonas syringae pv glycinea: evidence that one of them interacts with a bacterial elicitor

Summary Soybean [Glycine max (L.) Merr.] cultivars Flambeau and Merit differed in their resistance to Pseudomonas syringae pv glycinea (Psg) race 4, carrying each of four different avirulence (avr) genes cloned from Psg or the related bacterium, Pseudomonas syringae pv tomato. Segregation data for F₂ and F₃ progeny of Flambeau x Merit crosses indicated that single dominant and nonallelic genes account for resistance to Psg race 4, carrying avirulence genes avrA, avrB, avrC, or avrD. Segregants were also recovered that carried all four or none of the disease resistance genes. One of the disease resistance genes (Rpg1, complementing bacterial avirulence gene B) had been described previously, but the other three genes — designated Rpg2, Rpg3, and Rpg4 — had not here to fore been defined. Rpg3 and Rpg4 are linked (40.5 ± 3.2 recombination units). Rpg4 complements avrD, cloned from Pseudomonas syringae pv tomato, but a functional copy of this avirulence gene has not thus far been observed in Pseudomonas syringae pv glycinea. Resistance gene Rpg4 therefore may account in part for the resistance of soybean to Pseudomonas syringae pv tomato and other pathogens harboring avrD.     

Cloning of genes required for hypersensitivity and pathogenicity inPseudomonas syringae pv.aptata

A genomic library ofPseudomonas syringae pv.aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kbEcoRI fragment of the cosmid pHIR11, containing thehrp (hypersensitiveresponse andpathogenicity) gene cluster of the closely related bacteriumPseudomonas syringae pv.syringae strain 61, was used as a probe to identify a homologoushrp gene cluster inP. syringae pv.aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium,Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis ofEcoRI-digested genomic DNA ofP. syringae pv.aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome ofP. syringae pv.aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kbBglII fragment of pHIR11. These results indicate thatP. syringae pv.aptata harbourshrp genes that are similar to, but arranged differently from, homologoushrp genes ofP. syringae pv.syringae.Abbreviations HR hypersensitive response - Hrp mutant unable to induce HR and pathogenicity - Psa Pseudomonas syringae pv.aptata - Pss Pseudomonas syringae pv.syringae - Ea Erwinia amylovora