Generation and Analysis of Pathogenicity-related Gene Mutants of Colletotrichum gloeosporioides Using a Novel Promoter Trapping System

Agrobactedum tumefac/ens-mediated transformation （ATMT） is an efficient tool for insertional mutagenesis and is used in a wide variety of plants. This paper reports a promoter trapping method to generate mutants in the filamentous fungus, Colletotrichum gloeosporioides, by ATMT insertion of a trapping vector （pCAHPH） that carries a promoterless hygromycin phosphotransferase （hph） gone. Transformants were selected on the media containing 200 ~mL hy^omycin B, and screened for pathogenicity-related gene mdtants. Their pathogenicity-related mutants T-DNA flanking sequences were then cloned and analyzed. Hph genes were amplified from mutant genomic DNA but not from wild-type DNA, indicating that the phenotypic alternations of these mutants were the results of T-DNA inser- tion. T-DNA flanking sequences were obtained using modified themud asymmetric interlaced PCR. Two right-sided flanking sequences were highly homologous to proteins from other species.

Variations in chlorophyll content, stomatal conductance, and photosynthesis in Setaria EMS mutants

Chlorophyll (Chl) content,especially Chl b content,and stomatal conductance (G_s) are the key factors affecting the net photosynthetic rate (P_n).Setaria italica,a diploid C_4 panicoid species with a simple genome and high transformation efficiency,has been widely accepted as a model in photosynthesis and drought-tolerance research.The current study characterized Chl content,G_s,and P_n of 48 Setaria mutants induced by ethyl methanesulfonate.A total of 24,34,and 35 mutants had significant variations in Chl content,G_s,and P_n,respectively.Correlation analysis showed a positive correlation between increased G_s and increased P_n,and a weak correlation between decreased Chl b content and decreased P_n was also found.Remarkably,two mutants behaved with significantly decreased Chl b content but increased P_n compared to Yugu 1.Seven mutants behaved with significantly decreased G_s but did not decrease P_(n )compared to Yugu 1.The current study thus identified various genetic lines,further exploration of which would be beneficial to elucidate the relationship between Chl content,G_s,and P_n and the mechanism underlying why C_4 species are efficient at photosynthesis and water saving.

An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica

Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.

Precore/basal core promoter mutants quantification throughout phases of hepatitis B virus infection by Simpleprobe

AIM:To investigate precore/basal core promoter(PC/BCP) mutants throughout hepatitis B virus(HBV) infection and to determine their relationship to hepatitis B early antigen(HBeA g) titers.METHODS:We enrolled 191 patients in various stages of HBV infection at the Huashan Hospital and the Taizhou Municipal Hospital from 2010 to 2012.None of the patients received antiviral therapy.HBV DNA from serum,was quantified by real-time PCR.The HBV genotype was determined by direct sequencing of the S gene.We used the Simpleprobe ultrasensitivequantitative method to detect PC/BCP mutants in each patient.We compared the strain number,percentage,and the changes in PC/BCP mutants in different phases,and analyzed the relationship between PC/BCP mutants and HBe Ag by multiple linear regression and logistic regression.RESULTS:Patients with HBV infection(n = 191) were assigned to groups by phase:Immune tolerance(IT) = 55,Immune clearance(IC) = 67,Low-replicative(LR) = 49,and HBeA g-negative hepatitis(ENH) = 20.Of the patients(male,112; female,79) enrolled,122 were HBe Ag-positive and 69 were HBe Ag-negative.The median age was 33 years(range:18-78 years).PC and BCP mutation detection rates were 84.82%(162/191) and 96.86%(185/191),respectively.In five HBe Ag-negative cases,we detected double mutation G1896A/G1899 A.The logarithm value of PC mutant quantities(log10 PC) significantly differed in IT,IC,and LR phases,as well as in the ENH phase(F = 49.350,P < 0.001).The logarithm value of BCP mutant quantities(log10 BCP) also differed during the four phases(F = 25.530,P < 0.001).Log10 PC and log10 BCP values were high in the IT and IC phases,decreased in the LR phase,and increased in the ENH phase,although the absolute value at this point remained lower than that in the IT and IC phases.PC mutant quantity per total viral load(PC%) and BCP mutant quantity per total viral load(BCP%) differed between phases(F = 20.040,P < 0.001; F = 10.830,P < 0.001),with PC% and BCP% gradually increasing in successive phases.HBeA g titers negatively correlated with PC%(Spearman's rho =-0.354,P < 0.001) and BCP%(Spearman's rho =-0.395,P < 0.001).The negative correlation between PC% and HBeA g status was significant(B =-5.281,P = 0.001),but there was no such correlation between BCP% and HBeA g status(B =-0.523,P = 0.552).CONCLUSION:PC/BCP mutants become predominant in a dynamic and continuous process.Log10 PC,log10 BCP,PC% and BCP% might be combined to evaluate disease progression.PC% determines HBeA g status.

Field identification of morphological and physiological traits in two special mutants with strong tolerance and high sensitivity to drought stress in upland rice(Oryza sativa L.)

The two mutants idr1-1 and 297-28, which were obtained from the radiation mutation of HD297 and IAPAR9, were used as experimental materials in this study for a 2-year(2012 and 2013) experiment about field drought resistance identification in Beijing, China. Key agronomic traits and water-related physiological indexes were observed and measured, including the leaf anti-dead level(LADL), days to heading, plant height, setting percentage, aboveground biomass, leaf water potential(LWP), net photosynthetic rate(Pn) and transpiration rate. The results showed that the mutant idr1-1 that was under drought stress(DS) conditions for 2 years had the highest LADL grades(1.3 and 2.0) among all the materials, and they were 2–3 grades stronger than the wild-type IAPAR9 with an average that was 21.4% higher for the setting percentage than the wild type. Compared with the IAPAR9 for the 2-year average delay in the days to heading and the reduction rates in the plant height, setting percentage, and aboveground biomass under DS compared with the well-watered(WW) treatment, idr1-1 showed 3.2% less delay and 19.1, 16.4, and 6.1% less reduction, respectively. The idr1-1 in the LWP always exhibited the highest performance among all the materials. The Pn of idr1-1 under severe and mild DS comparing with that under WW was slightly decreased and even slightly increased, respectively, leading to an average reduction rate of only 0.92%, which was 26.93% less than that of IAPAR9. Under the severe DS, idr1-1 still showed the highest value of 16.88 μmol CO2 m–2 s–1 among all the materials and was significantly higher than that of IAPAR9(11.66 μmol CO2 m–2 s–1). Furthermore, only idr1-1 had the increased and the highest transpiration rate values(7.6 and 6.04 mmol H2 O m–2 s–1) under both mild and severe DS compared with the values under WW, when the transpiration rate of all the other materials significantly decreased. By contrast, the 297-28 in terms of the LADL grade under DS was the lowest(7.0), and it was four grades weaker than its wildtype HD297 and even one grade weaker than the drought-sensitive paddy rice SN265. For the 2-year average reduction rates in aboveground biomass and plant heights under DS compared with those under the WW, 297-28 was 31.6 and 31.8% higher than HD297, respectively. Meanwhile, 297-28 showed the worst performance for the LWP, Pn, and transpiration rate. These results suggest that idr1-1 might be a superior drought tolerant mutant of upland rice found in China. It has a strong ability to maintain and even enhance leaf transpiration while maintaining a high plant water potential under DS, thus supporting a high Pn and alleviating the delay in agronomic trait development and yield loss effectively. 297-28 is a much more highly drought-sensitive mutant that is even more sensitive than paddy rice varieties. The two mutants could be used as drought tolerance controls for rice germplasm identification and the drought resistant mechanism studies in the future. idr1-1 is also suitable for breeding drought-tolerant and lodging-resistant high-yield rice varieties.

Chemical mutagenesis and soybean mutants potential for identification of novel genes conferring resistance to soybean cyst nematode

The resistance of soybean(Glycine max(L.) Merr.) to soybean cyst nematode(SCN, Heterodera glycines Ichinohe), which is a devastating pathogen in soybean production and causes a large quantity of annual yield loss worldwide, can shift during the long-term interaction and domestication. It is vital to identify more new resistance genetic sources for identification of novel genes underlying resistance to SCN for management of this pathogen. In the present study, first, two ethane methylsulfonate-mutagenesis soybean M2 populations of PI 437654, which shows a broad resistance to almost all of SCN races, and Zhonghuang 13, which is a soybean cultivar in China conferring strong resistance to lodging, were developed. Many types of morphological phenotypes such as four-and five-leaflet leaves were observed from these two soybean M2 populations. Second, 13 mutants were identified and confirmed to exhibit alteration of resistance to SCN race 4 through the forward genetic screening of 400 mutants of the PI 437654 M2 population, the rate of mutants with alteration of SCNinfection phenotype is 3.25%. Third, these identified mutants were further verified not to show any changes in the genomic sequences of the three known SCN-resistant genes, GmSHMT08, GmSNAP18 and GmSANP11, compared to the wildtype soybean; and all of them were still resistant to SCN race 3 similar to the wild-type soybean. Taken together, we can conclude that the 13 mutants identified in the present study carry the mutations of the new gene(s) which contribute(s) to the resistance to SCN race 4 in PI 437654 and can be potentially used as the genetic soybean sources to further identify the novel SCN-resistant gene(s).

Status and Perspectives on the Researches of Rice Glutelin Mutants

Rice (Oryza sativa L. ) is one of the model plants for genomics research. As the raising of functional rice breeding for special usage, glutelin mutants play a more and more important role in the functional rice breeding as well as eukaryotic gene expression and regulation research materials. For example, the rice cultivar special for the patients suffering from kidney disease and diabetes could be developed from the rice glutelin mutants. In this paper, current researches on characterization, mutation mechanism and breeding usage of various rice glutelin mutants, especially the low glutelin content cultivars, were all discussed with perspectives on the trends of the glutelin mutant researches in the era of post-genomics.

Physiological and molecular characteristics of two ploidy mutants in Myrica rubracv.Dongkui

In this study, two ploidy mutant lines ofMyrica rubra cv. Dongkui （DK） were identiifed and named as DB1 and DB2. The lforal organ, leaf cel structure, ploidy, and number of chromosomes of the two mutants were investigated. Meanwhile, anthocyanin contents at different developmental stages were analyzed, and the Cy-3-glu contents of DB1 and DB2 at the ful ripe stages are signiifcantly higher than that of DK by 27.84 and 23.51%, respectively. Furthermore, 6 RNA libraries at two developmental stages （young fruit stage and ful ripe stage） were built for RNA-Seq. By mapping to the reference database, 28407, 28043, and 28683 genes were detected in the young fruit of DB1, DB2, and DK, respectively, while 28040, 22256, and 27351 genes were detected in the ful ripe stage, respectively. There were 281 differentialy expressed genes between DB1 and DK, with 123 and 158 genes up-regulated and down-regulated, respectively, and 47 differentialy expressed genes between DB2 and DK, of which 8 and 39 genes were up-regulated and down-regulated. Using real-time PCR, the expression levels of the eight functional genes at different developmental stages of the fruit were also analyzed. These comprehensive analyses showed that both mutants are different from DK, which is the result of natural doubling of ploidy, thereby generating a pleiotropic effect. As we known, it is the ifrst report to study the relationship between bayberry ploidy alterations and genes involved in regulation of fruit mutations, which wil help to identify the morphological and cytological characteristics ofM. rubragermplasm, and provide a theoretical basis and technical support for genetic improvement and creation of breeding resources.

In Vitro Selection and Identification of Drought-Tolerant Mutants in Sweetpotato

In vitro selection of drought-tolerant mutants in sweetpotato cv. Lizixiang was studied by using PEG6000 as selection stress. Embryogenic suspension cultures were cultured in MS medium containing 035% PEG6000 and 2 mg L 1 2,4-dichlorophenoxyacetic acid (2,4-D). The results indicated that 30% PEG6000 can be used for the optimal selection stress of drought-tolerance. Embryogenic suspension cultures irradiated with 80 Gy gamma-ray were cultured in MS medium containing 30% PEG6000 and 2 mg L 1 2,4-D and 20 drought-tolerant cell aggregates were obtained. These cell aggregates were transferred to solid MS me- dium supplemented with 2 mg L 1 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 1 mg L 1 abscisic acid (ABA), resulting in the germination of somatic embryos. In this study a total of 18 regenerated plants were obtained. The regenerated plants were transplanted in a greenhouse and 11 lines were formed. The analysis on drought treatment of seedlings, water retaining capacity of leaves and coefficient of drought-tolerance showed that 3 lines had significant drought-tolerance in comparison with the control plants.

Deciphering Rice Lesion Mimic Mutants to Understand Molecular Network Governing Plant Immunity and Growth

Plant lesion mimic mutants(lmms)generally possess autoimmunity and hypersensitive response(HR)-like cell death in the absence of biotic or abiotic stress.They have attracted much attention because they are useful tools for deciphering the interaction between defense signaling and growth.Recent studies have identified more than 30 lmms involved in the plant immune response and cell death in rice.Genes underlying these lmms,coding for diverse types of proteins,mainly regulate transcription,protein translation and modification,vesicular trafficking and catalyzation of metabolism.Here,we presented an overview of the most recent advances on the study of lmms in rice and proposed a perspective on potential utilization of LMM genes in agriculture.

Study on Screening of TaGA2ox1 Mutants in Wheat by Ion Beam Irradiation

As a kind of mutagen, ion beam irradiation can create abundant biological mutations. A population of about 2000 lines was generated by irradiating dry wheat seeds of XiaoYan 81 with low-energy nitrogen ion beams. The traits of the plant, such as height, spike type, fertility, stem color and awn length, were investigated. The mutation rate in terms of the plant height in M2 was 2.9%. Eighteen deletion mutants of TaGA2ox1 were obtained. Associate analysis showed that TaGA2ox1 was closely related to the plant height. Most of the TaGA2ox1-deleted mutants were higher than the control, suggesting that the biological function of TaGA2ox1 is similar to its homologues in other plants. These results demonstrate that ion beam irradiation is an efficient tool in the construction of a mutant library for wheat.

Characterization of dwarf mutants and molecular mapping of a dwarf locus in soybean

Plant height is one of the most important traits in soybean. The semi-dwarf soybean cultivars could improve the ability of lodging resistance to obtain higher yield. To broaden the dwarfism germplasm resources in soybean, 44 dwarf mutants were identified from a gamma rays mutagenized M-2 population. Two of these mutants, Gmdwf1（Glycine max dwarf 1） and Gmdwf2（Glycine max dwarf 2）, were investigated in this study. Genetic analysis showed that both mutants were inherited in a recessive manner and their mutated regions were delimited to a 2.610-Mb region on chromosome 1 by preliminary mapping. Further fine mapping study proved that the two mutants had a common deletion region of 1.552 Mb in the target region, which was located in a novel locus site without being reported previously. The dwarfism of Gmdwf1 could not be rescued by gibberellin（GA） and brassinolide（BR） treatments, which indicated that the biosynthesis of these hormones was not deficient in Gmdwf1.

Preliminary Study on the Mechanism of Flower Color Variation in White Mutants of Anthurium andeaeanum

[Objectives] This study was conducted to explore the physiological mechanism of flower color variation in the white mutants of Anthurium andeaeanum. [Methods] The seven white mutants of 'Alabama' and 'Turenza' were used as materials to analyze the pigment types, flavonoid types and content and anthocyanin content in the wild type and mutants. [Results] The white spathe mainly contained flavonoids, flavonols, dihydroflavonols and dihydroflavonols; the white mutants of 'Alabama' had a higher total flavonoid content than the wild type, while the white mutants of 'Turenza' showed an opposite trend; and the spathe of the wild type had the highest anthocyanin content, and the pink part of the two-color mutant or the spathe of the binary color mutant contained trace anthocyanins, while no anthocyanins were detected in the white part of the mutants. [Conclusions] The main cause of the white mutants of A. andeaeanum is related to anthocyanin metabolism.

Changes in Chlorophyll Fluorescence of Rice Mutants Induced by High Hydrostatic Pressure

Three mutants of rice (Oryza sativa L. ), Mutant 1, Mutant 2 and Mutant 3, which were selected by high hydrostatic pressure (75 MPa), and their parent Yuexiangzhan were used to study the changes in chlorophyll fluorescence during different growth stages. In all the three mutants, the function of PSⅡ was improved, F_v/F_m ratio of mutants increased compared to their parent at tillering and heading stage, and ΦPSⅡ also improved except for Mutant 2 at heading stage. Similar to their parent, the mutants exhibited slight photoinhibition at noon and almost complete recovery to initial levels of 6:00 after 18:00 at heading stage. At milking stage, the photoinhibition in the mutants was obvious, and recovered rapidly compared to the parent. Yields of individual plant and grain/straw ratio were also higher in three mutants than the parent. Results indicated that characteristics of chlorophyll fluorescence in leaves of mutants and their photoinhibition in the field had changed. It is suggested that high hydrostatic pressure induction could be applied as a new effective approach in high-yield rice breeding in the future.

Functional Analysis for Rolling Leaf of Somaclonal Mutants in Rice (Oryza sativa L.)

This study was carried out to facilitate the functional analysis of rice genes. Some 297 insertion plants (1.7%) of the entire lines with the endogenous retrotransposon Tos17 were produced. Phenotypes of these plants in the S2 genera-tion were observed in the field according to different leaf types. Rolling leaf mutants showed thinner sclerenchyma-tous cells, defective arrangement of vascular bundles, and well-formed bulliform cells as compared to the parental cultivar. Two new copies of Tos17 were detected in the rolling leaf type. In the new leaf type, the copy number and activation of Tos12, 15 did not appear as ‘Ilpum’. Flanking sequence tag (FST) analysis of Tos17 in the rolling leaf mutant indicated that new copies of Tos17 were transposed on chromosomes 11 and 12. Annotated homologues of the tagging genes on chromosome 11 were arabinoxylan rabinofuranohydrolase isoenzyme AXAH-I and II. The tagging gene in chromosome 12 was highly correlated with 6 kinds of genes including a transcript regulated factor and a rough sheath 2-like protein. This rolling leaf and flanking sequence data will stimulate the functional analysis of rice genes.

Expression Profiles and Protein Complexes of Starch Biosynthetic Enzymes from White-Core and Waxy Mutants Induced from High Amylose Indica Rice

Physicochemical properties of endosperm starches in milled rice determine its cooking and eating quality.Amylose is synthesized by granule-bound starch synthase I(GBSSI),whilst amylopectin is synthesized by the synergistic activities of starch synthases(SSs),branching enzymes(BEs)and debranching enzymes(DBEs).However,the complexes formed by starch biosynthetic enzymes are not well characterized.Gene expression profiles and protein complexes were determined in white-core(GM645)and waxy(GM077)mutants derived from a high amylose indica rice Guangluai 4(GLA4).In GM645,genes including AGPS1,GBSSI,SSIIa,BEI,BEIIa,BEIIb,PUL,ISA1 and SP were significantly downregulated during seed development.In GM077,the expression levels of AGPL2,AGPS1,AGPS2b,SSIIIa,BEI,PUL and ISA1 were significantly upregulated.Co-immunoprecipitation assays revealed interactions of SSs-BEs,SSs-PUL and BEs-PUL in developing seeds.However,weak SSI-SSIIa interaction was detected in GM077,whilst SSI-PUL interaction was absent.Weak interaction signals for SSI-SSIIa,SSIIa-BEI,SSIIa-BEIIb,BEI-BEIIb and SSI-BEI were also observed in GM645.These results suggest that the protein-protein interactions for starch biosynthesis are modified in mutants,which provides insight into the mechanisms of starch biosynthesis,particularly in indica rice.

Mutagenesis and selection of high efficiency hydrogen-producing mutants by ultraviolet radiation

Hydrogen is an ideal, clean and sustainable energy source for the future because of its high conversion and nonpolluting nature. Biohydrogen production by dark-fermentation appears to have a great potential to be developed for practical application. However, one limiting factor affecting the development of hydrogen-production industrialization is that the hydrogen-producing capacity of bacteria is lower, so how to increase bacteria’s hydrogen-producing ability will be an urgent issue. In this experiment, 2 mutants, namely UV3 and UV7, were obtained by ultra-violet radiation. They grew and produced hydrogen efficiently on iron-containing medium. The hydrogen evolution of UV3 and UV7 were 2 356.68 ml/L and 2 219.62 ml/L at a glucose concentration of 10 g/L, respectively. With wild parent strain Ethanoligenens sp. ZGX4, the hydrogen evolution was 1 806.02 ml/L under the same conditions. Mutants’ hydrogen-producing capacities were about 29.71% and 22.22% higher than that of wild parent strain ZGX4. The maximum H2 production rate by mutants UV3 and UV7 were estimated to be 32.57 mmol H2/g cell h and 31.19 mmol H2/g cell h, respectively, which were 38.18% and 34.78% higher than the control （23.57 mmol H2/g cell h）. The abundant products of UV3 and UV7 were ethanol and acetic, which accounted for 95%-98% of total soluble microbial products. In each case, mutant strains UV3 and UV7 evolved hydrogen at a higher rate than the wild type, showing a possible potential for commercial hydrogen production. Another mutant named UV20’ was also gained whose main end metabolites were butyric acid and acetic acid. This would provide researched material for a discussion of metabolic pathways of hydrogen-producing bacteria.

pH-Dependence of Manganese (II) Oxidation Reaction by Novel Wild-Type and Mutants Recombinant Phlebia radiata Manganese Peroxidase 3 (rPr-MnP3) Enzymes

The goal of this study was to determine whether mutation of the Mn-binding site of wild-type recombinant Phlebia radiata manganese peroxidase 3 affected the pH-dependence kinetic parameters. pH range investigated was 2.5 – 12.0. The catalytic efficiency of the mutant enzymes at high and low pH in comparison to the wild-type was investigated using standard rPr-MnP3 protocol. Wild-type recombinant Phlebia radiata MnP3 enzyme showed optimal activity with Mn (II) as substrate at pH 5.0 and remained moderately active (approximately 40%) in the pH range of 6.0 - 9.0. The rPr-MnP3 mutants’ maximum activity ranged between 5.5 and 8.0. Wild-type and mutants rPr-MnP3 enzymes exhibited a similar pH profile with optimum pH of 3.0 for ABTS oxidation. Mutation has severely decreased the catalytic efficiency for Mn (II) oxidation at pH 5.0. The rPr-MnP3 enzymes showed enhanced affinity for Mn (II) at alkaline pH and a more alkaline range for catalysis than ever reported for any Manganese Peroxidase. This study reveals that at higher pH, rPr-MnP3 can function with alternative ligands in the Mn (II) site and does not have an absolutely obligate requirement for an all carboxylate ligand set. These results further strongly confirm that Mn2+ binding site is the only productive catalytic site for Mn (II) oxidation.

Biocontrol potential of Trichoderma asperellum mutants T39 and T45 and their growth promotion of poplar seedlings

This study investigates the biocontrol potential of Trichoderma asperellum mutants against Rhizoctonia solani, Alternaria alternata, and Fusarium oxysporum and growth promotion of Populus davidiana 9 P. alba var.pyramidalis(Pd Pap poplar) seedlings. A T-DNA insertion mutant library of T. asperellum was constructed using Agrobacterium tumefaciens-mediated transformation.Sixty-five positive transformants(T1–T65) were obtained.Growth rates of the mutants T39 and T45 were the same,39.68% faster than the WT. In toxin tolerance tests, only T39 had greater tolerance to A. alternata fermentation broth than the WT, but mutant T45 had the same tolerance as the WT to all fermentation broths. Furthermore, T39 and T45 had a greater antagonistic ability than the WT strain against R. solani and A. alternata. The inhibition rate of the mutants T39 and T45 against A. alternata are 73.92% and80.76%, respectively, and 63.51% and 63.74%, respectively. Furthermore, the three strains increased the activities of superoxide dismutases, peroxidase, catalase(CAT)and phenylalanine ammonia lyase(PAL) in Pd Pap seedling leaves. CAT and PAL activity in the Pd Pap seedling leaveswas 11.25 and 5.50 times higher, respectively, in the presence of T39 than in the control group and 12 and 6.35 times higher, respectively, in the presence of T45 than in the control group. All three strains promoted seedling growth and the root and stem development, especially mutant T45. Mutants T39 and T45 reduced the incidence of pathogenic fungi in poplar and stimulated poplar seedling growth.

Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture

Tissue culture has been widely used for mass propagation of Phalaenopsis. However, somaclonal variation occurred during micropropagation process posed a severe problem by affecting product quality. In this study, wild type and peloric flower buds of Phalaenopsis hybrids derived from flower stalk nodal culture were used for cDNA-RAPD and cDNA suppression subtractive hybridization analyses in order to study their genetic difference in terms of expressed sequence tags. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced. These ESTs sequences can be grouped into several functional categories involved in different cellular processes including metabolism, signal transduction, transcription, cell growth and division, protein synthesis, and protein localization, and into a subcat- egory of proteins with unknown function. Cymbidium mosaic virus transcript was surprisingly found expressed fre- quently in the peloric mutant of P. Little Mary. Real-time RT-PCR analysis on selected ESTs showed that in mutant flower buds, a bZIP transcription factor (TGA1a-like protein) was down-regulated, while up-regulated genes include auxin-regulated protein kinase, cyclophilin, and TCP-like genes. A retroelement clone was also preferentially expressed in the peloric mutant flowers. On the other hand, ESTs involved in DNA methylation, chromatin remodeling and post- transcriptional regulation, such as DNA methyltransferase, histone acetyltransferase, ERECTA, and DEAD/DEAH RNA helicase, were enriched in normal flower buds than the mutants. The enriched transcripts in the wild type indicate the down regulation of these transcripts in the mutants, and vice versa. The potential roles of the analyzed transcripts in the development of Phalaenopsis flowers are discussed.