Molecular cloning,characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is the first committed step in MVA pathway for isoprenoid biosynthesis in plants. In this study, a full-length cDNA encoding HMGR was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmHMGR (GenBank Accession No.EU680958). The full-length cDNA of SmHMGR was 2,115 bp containing a 1,695 bp open reading frame (ORF) encoding a polypeptide of 565 amino acids. Bioinformatic analyzes revealed that the deduced SmHMGR had extensive homology with other plant HMGRs contained two transmembrane domains and a catalytic domain. Molecular modeling showed that SmHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. Phylogenetic tree analysis indicated that SmHMGR belongs to the plant HMGR super-family and has the closest relationship with HMGR from Picrorhiza kurrooa. Expression pattern analysis implied that SmHMGR expressed highest in root, followed by stem and leaf. The expression of SmHMGR could be up-regulated by salicylic acid (SA) and methyl jasmonate (MeJA), suggesting that SmHMGR was elicitor-responsive. This work will be helpful to understand more about the role of HMGR involved in the tanshinones biosynthesis at the molecular level.     

Features of the hmg 1 subfamily of genes encoding HMG-CoA reductase in potato

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the -glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R₂ pollen of R₁ progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.     

Regulation of isoflavonoid metabolism in alfalfa

Isoflavonoids are believed to play important roles in plant-microbe interactions. During infection of alfalfa (Medicago sativa) leaves with the fungal pathogen Phoma medicaginis, rapid increases in mRNA levels and enzyme activities of isoflavone reductase, phenylalanine ammonia-lyase, chalcone synthase and other defense genes are observed within 1 to 2 hours. The phytoalexin medicarpin and its antifungal metabolite sativan increase beginning at 4 and 8 hours, respectively, along with other isoflavonoids. In contrast, during colonization of alfalfa roots by the symbiotic mycorrhizal fungus Glomus versiforme, expression of the general phenylpropanoid and flavonoid genes phenylalanine ammonia-lyase and chalcone synthase increases while mRNA levels for the phytoalexin-specific isoflavone reductase decrease. The total isoflavonoid content of colonized roots increases with time and is higher than that of uninoculated roots, but the accumulation of the antifungal medicarpin is somehow suppressed.An isoflavone reductase genomic clone has been isolated, promoter regions have been fused to the reporter gene -glucuronidase, and the promoter-reporter fusions have been transformed into tobacco and alfalfa. Using histological staining, we have studied the developmental and stress-induced expression of this phytoalexin-specific gene in whole plants at a more detailed level than other methods allow. The isoflavone reductase promoter is functional in tobacco, a plant which does not synthesize isoflavonoids. Infection of transgenic alfalfa plants by Phoma causes an increase in -glucuronidase staining, as does elicitation of transgenic alfalfa cell cultures, indicating that this promoter fusion is a good indicator of phytoalexin biosynthesis in alfalfa.Abbreviations CA4H cinnamic acid 4-hydroxylase - CHI chalcone isomerase - CHOMT chalcone O-methyltransferase - CHS chalcone synthase - 4CL 4-coumarate:CoA ligase - COMT caffeic acid O-methyltransferase - FGM malonylated glucoside of formononetin - GUS -glucuronidase - IFOH isoflavone 2-hydroxylase - IFR isoflavone reductase - IFS isoflavone synthase - IOMT isoflavone 4-O-methyltransferase - MGM medicarpin 3-O-glucoside-6-O-malonate - PAL L-phenylalanine ammonia-lyase - PTS pterocarpan synthase - VAM vesicular arbuscular mycorrhizal - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide     

Functional analysis of the <Emphasis Type="Italic">Malus domestica MdHMGR2</Emphasis> gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The first rate-limiting enzyme of the mevalonate pathway during isoprenoid biosynthesis is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In this study, the expression pattern of the MdHMGR2 gene in Malus domestica suggests that MdHMGR2 was expressed in a tissue-specific manner and was significantly induced by ethephon (ETH), indoleacetic acid (IAA), methyl jasmonate (MeJA), and salicylic acid (SA). The MdHMGR2 promoter was isolated, sequenced, and analyzed through bioinformatics tools, and the results suggest the presence of various putative cis-acting elements responsive to different hormones. Activity of β-glucuronidase (GUS) driven by the full length MdHMGR2 promoter and its 5′deletion fragments was detected in transgenic Arabidopsis thaliana. A strong GUS activity was observed in seedlings, roots, newly growing true leaves, anthers, and stigmas in transgenic Arabidopsis containing the full MdHMGR2 promoter. The results indicate that a region from -1050 to -827 was crucial for promoter activity. In addition, the MdHMGR2 promoter was induced in response to ETH, IAA, MeJA, and SA. The analysis suggests that an ethylene-responsive element in the region from -1050 to -1005 was required for the ethylene inducibility.     

Stable transformation of the food yam Dioscorea alata L. by particle bombardment

Summary A biolistic particle gun was used to deliver genetic material into intact yam cells. Cultured suspension cells of D. alata were bombarded with microprojectiles coated with pBI221.2 DNA and histochemical assays were carried out to show transient GUS expression in bombarded cells. Stably transformed D. alata cells were recovered from cultured cells after bombardment with microprojectiles coated with pRT99gus harbouring both the nptII and uidA genes. Bombarded cells were selected on a medium containing geneticin (G418). Two months after bombardment, calli resistant to G418 were assayed for GUS expression. There was a 100% correlation between resistance to G418 and GUS expression. From these calli, four cell lines were established and GUS activity in each line was determined fluorometrically. The use of a specific GUS inhibitor showed that the GUS activity was due to the introduced uidA gene rather than to any intrinsic GUS-like activity originating from the plant. Incorporation of the introduced DNA into the plant genomic DNA was confirmed by Southern analysis.Abbreviations GUS -glucuronidase - MU 4-Methylumbelliferone - MUG 4-Methylumbelliferyl--D-glucuronide - PVP Polyvinylpyrrolidone - SDS Sodium dodecyl sulphate - TAE Tris-acetate-EDTA buffer - X-Gluc 5-Bromo-4-chloro-3-indolyl--D-glucuronide     

Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible

The rice (Oryza sativa) phytoalexins, momilactones and oryzalexins, are synthesized by the isoprenoid pathway. An early step in this pathway, one that is rate-limiting in mammalian systems, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). A gene that encodes this enzyme has been isolated from rice, and found to contain an open reading frame of 1527 bases. The encoded protein sequence of the rice HMGR appears to be conserved with respect to other HMGR proteins, and 1 or 2 membrane-spanning domains characteristic of plant HMGRs are predicted by a hydropathy plot of the amino acid sequence. The protein is truncated at its 5 end, and shows reduced sequence conservation in this region as compared to other plant sequences. The rice genome contains a small family of HMGR genes. The isolated gene, HMGR I, is expressed at low levels in both vegetative and floral organs of rice plants. It is not induced in plants by wounding, but is strongly and rapidly induced in suspension cells by a fungal cell wall elicitor from the pathogenMagnaporthe grisea, causal agent of rice blast disease. This suggests that HMGR I may be important in the induction of rice phytoalexin biosynthesis in response to pathogen attack, and therefore may play a key role as a component of the inducible defense mechanism in rice.     

Solubilization and resolution of the membrane-bound nitrite reductase from Paracoccus halodenitrificans into nitrite and nitric oxide reductases

Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-cholamidopropyldimethylammonio)-1-(2-hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.Abbreviations PMS phenazine methosulfate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - CHAPSO 3-(3-cholamidopropyl-dimethylammonio)-1-(2-hydroxy-1-propanesulfonate) - NH buffer 150 mM NaCl-50 mM - HEPES pH 7.5; octylglucoside, octyl--d glucopyranoside - NIR intrite reductase (nitrite to nitric oxide) - NOR nitric oxide reductase (nitric oxide to nitrous oxide)     

Transformation of Solanum brevidens using Agrobacterium tumefaciens

Leaf pieces of in vitro-cultured plantlets of the wild potato species Solanum brevidens Phil. were cocultivated with Agrobacterium tumefaciens that contained nptII and uidA genes on the disarmed plasmid pBI121. Independent transgenic shoots were regenerated from solidified and liquid medium that contained 50 mg l^–1 kanamycin. Two Agrobacterium strains were investigated for transformation efficiency. GV2260, which contained p35SGUSINT, resulted in a 11% transformation frequency, compared with 1% using LBA4404. Transformation rates were 7% in liquid culture and 3% on solidified medium. All kanamycinresistant, putatively transformed plantlets were confirmed positive by histochemical GUS assays. GUS activity in 22 independently transformed plants was quantified by fluorometric assay. Southern analysis of randomly selected transgenic plants showed that each transgenic plant contained at least one copy of the uidA gene.Abbreviations GUS ß-glucuronidase - MS Murashige-Skoog medium - BA 6-benzylaminopurine - 2ip 6-(, -dimethylallylamino)purine - IAA indole-3-acetic acid - GA₃ gibberellic acid - npt II neomycin phosphotransferase II - NOS nopaline synthase - MUG 4-methyl umbelliferyl glucuronide - MU 7-hydroxy-4-methylcoumarin - X-gluc 5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid     

The promoter of aBrassica napus polygalacturonase gene directs pollen expression ofβ-glucuronidase in transgenicBrassica plants

A 647-bp 5-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to the-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.Abbreviation GUS -Glucuronidase     

Expression and inheritance of foreign genes in transgenic peanut plants generated byAgrobacterium-mediated transformation

To evaluate and characterize the stability of traits transferred viaAgrobacterium transformation, foreign gene expression must be examined in sexually derived progeny. The objective of this study was to analyze three transgenic peanut plants, 1-10, 12-1, and 17-1, for the inheritance and expression of their foreign genes. Segregation ratios for the introduced genes in T₂ plants gave either 100% or 3:1 expression of the -glucuronidase (GUS) gene, demonstrating recovery of both homozygous and heterozygous T₁ plants. Fluorometric GUS assay in T₁ and T₂ generations of all three plants showed that the GUS gene was stably expressed in the progeny. DNA analyses showed 100% concordance between the presence of the foreign gene and enzyme activity. Our results demonstrate that transgenes in peanut introduced byAgrobacterium can be inherited in a Mendelian manner.Abbreviations GUS -Glucuronidase - MS Murashige and Skoog - MU 4-Methylumbelliferone - NPTII Neomycin phosphotransferase II     

Import of polyphenol oxidase by chloroplasts is enhanced by methyl jasmonate

Polyphenol oxidase (PPO; EC 1.10.3.2 or EC 1.14.18.1) takes part in the response of tomato plants (Lycopersicon esculentum Mill.) to wounding and herbivore attack, mediated by the octadecanoid wound-signaling pathway. Wounding and methyl jasmonate (MeJA) induce expression of ppo genes and markedly increase the level of the enzyme. We report that pretreatment with MeJA also markedly increased the ability of isolated tomato chloroplasts to import and process PPO precursors (pPPO). Pea (Pisum sativum L.) chloroplasts showed no such response. Wounding or ethylene alone was ineffective but ethylene was synergistic with MeJA. Treatment with MeJA conferred a strong binding of pPPO, or its processing intermediate, to thylakoids and subsequent translocation into the lumen and processing to the mature protein. The effect on PPO import and translocation was evident after 8–16 h exposure to MeJA. Membrane-bound pPPO was cross-linked to a proteinaceous component of the thylakoid translocation apparatus, apparently induced by MeJA. The import and processing of other nuclear-encoded thylakoid proteins were not affected by MeJA in tomato. A 90-kDa protein that co-fractionated with thylakoids was induced along with the increase in competence for PPO import, and was identified as the proteinase-inhibitor multicystatin. It is concluded that the 90-kDa protein observed is part of the MeJA-induced defense response of tomato, not a component of the thylakoid translocation apparatus.Abbreviations Chl Chlorophyll - i and p Prefixes used to denote the intermediate and precursor forms of a protein, respectively - JA Jasmonic acid - LSU Large subunit of Rubisco - MeJA Methyl jasmonate - OE23 and OE33 23- and 33-kDa subunits of the oxygen-evolving complex of PSII - PC Plastocyanin - pPPO (iPPO, PPO) Precursor (intermediate, mature) form of polyphenol oxidase     

The Synthesis and Cytotoxic Activity of D-Ribofuranosides and 2-Deoxy-D-Ribofuranosides of Substituted Bis(indolyl)furan, Bis(indolyl)pyrrole, and Indolo[2,3-a]carbazole Derivatives

1-(2,3,5-Tri-O-acetyl)--D-ribofuranosyl indole, the key compound in the synthesis of glycosides with the bis(indole) aglycone, was obtained for the first time by the indoline–indole method. There were synthesized 3-(1-methylindol-3-yl)-4-(1-glycosylindol-3-yl)furan(or pyrrole)-2,5-diones containing the residue of -D-ribofuranose or 2-deoxy--D-ribofuranose and analogous glycosides of indolo[2,3-a]furano(or pyrrolo)[3,4-]carbazol-5,7-diones, which are structurally relative to the antitumor antibiotic rebeccamycin. Their cytotoxicities toward a number of human tumor cell lines were studied in vitro, and the carbazole N-glycosides were shown to be more active than the bis(indole) glycosides. At the same time, the ribofuranosides were found to be less active than the corresponding ribopyranosides synthesized previously.     

Surrogate transformation of perennial ryegrass, Lolium perenne, using genetically modified Acremonium endophyte.

Summary Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/g DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The -glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl -D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum.     

The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins

A radioimmunoassay, combined with high-performance liquid chromatography, has been used to analyse the zeatin-type cytokinins of potato (Solanum tuberosum L. cv. Majestic) tubers and tuber buds throughout growth and storage. During tuber growth, zeatin riboside was the predominant cytokinin detected in all tissues. Immediately after harvest, the total cytokinin concentration fell dramatically in the storage tissue, largely as a consequence of the disappearance of zeatin riboside. During storage, levels of cytokinins in the storage tissue remained relatively constant, but increased in the tuber buds. In the buds of tubers stored at 2°C there was a 20-to 50-fold increase in total cytokinin over six weeks, coinciding with the natural break of innate dormancy. At 10°C the rise in the level of bud cytokinins was slower, correlating with the longer duration of innate dormancy. Injecting unlabelled cytokinins into tubers in amounts known to induce sprouting gave rise to increases in cytokinin concentrations in the buds of the same order as the increase associated with the natural break of dormancy. Metabolism of injected cytokinins was greater in non-dormant than in dormant tubers. The roles of cytokinin concentration and the sensitivity of the buds to cytokinin in the control of dormancy are discussed.Abbreviations CK cytokinin - FW fresh weight - HPLC high-performance liquid chromatography - RIA radioimmunoassay - tio⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purine=zeatin - tio⁶adeglc⁹ 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-glucopyranosyl purine=zeatin-9-glucoside - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosyl purine=zeatin riboside - tio⁶ado-[³H]-diol a radioactive derivative of zeatin riboside, synthesised by periodate-oxidation followed by [³H]NaBH₄-reduction - tio⁶AMP 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-5-phosphoribofuranosyl purine=zeatin riboside 5-monophosphate - t(ioglc⁴)⁶ade 6-(4-O--D-glucopyranosyl-3-methylbut-trans-2-enylamino)-purine=zeatin-O-glucoside     

Effect of α(2–6)-linked sialic acid and α(1–3)-linked fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA)

The effects of branching and substitution of branches by sialic acid and fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA) were examined. Asialo bi-, tri-and tetra-antennary glycans were all retarded but to different extents on a long column of L-PHA-agarose. Asialo tri- and tetra-antennary glycans containing the pentasaccharide unit Gal1-4GlcNAc1-2[Gal1-4GlcNAc1-6]Man were strongly retarded, whereas asialo bi- and tri-antennary glycans lacking the Gal1-4GlcNAc1-6 branch were only weakly retarded. In all instances the interaction with the lectin was completely abolished when either (2–6)-linkedN-acetylneuraminic acid or (1–3)-linked fucose was present at the galactose orN-acetylglucosamine residue of the Gal1-4GlcNAc1-6Man1-6 branch, respectively. The same substitutions on the Gal1-4GlcNAc1-6Man1-6 branch decreased but did not abolish the affinity of the lectin for the glycans. The presence of NeuAc2-6 and Fuc1-3 on the other two branches did not interfere with the binding of the glycans to L-PHA. Furthermore, it appeared that the presence of the Man1-4GlcNAc unit is requried for interaction with the lectin. In order to obtain reliable information on the relative occurrence of tri- and tetra-antennary glycopeptides, this study shows that it is essential to desialylate and to defucosylate the glycans prior to application to L-PHA-agarose.Abbreviations L-PHA leukoagglutinating phytohemagglutinin - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - GP glycopeptide - OS oligosaccharide - HPLC high-performance liquid chromatography - FNR fraction not retarded - FR fraction retarded suffixes MS, BS and TS indicate mono-, bi- and trisialyl derivatives respectively; suffix MF indicates monofucosyl derivatives.structures of the substratesOS2, OS3, OS3, OS4, GP2, GP3, GP4, GP4-MF, OS2(3) andOS2(-) are presented in Fig. 2     

Synthesis of an H type 2 and a Y (Ley) glycoside from thioglycoside intermediates

The trisaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-acetamido-2-deoxy-4-O-[2-O-(-l-fucopyranosyl)--d-galactopyranosyl]--d-glucopyranoside 1 and the tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-acetamido-2-deoxy-3-O-(-l-fucopyranosyl)-4-O-[2-O-(-l-fucopyranosyl)--d-galactopyranosyl]--d-glucopyranoside 2 were synthesized. Thioglycosides, suitably protected, activated directly with methyl trifluoromethanesulfonate or dimethyl(methylthio)sulfonium tetrafluoroborate or activated after bromine treatment with halophilic reagents, were used as glycosyl donors in the construction of the glycosidic linkages.Abbreviations DMTSB dimethyl(methylthio)sulfonium tetrafluoroborate - Phth phthaloyl - MBn p-methoxybenzyl - ClBn p-chlorobenzyl     

Analysis of the essential DNA region for OsEBP-89 promoter in response to methyl jasmonic acid

In rice, the characterization of OsEBP-89 is inducible by various stress-or hormone-stimuli, including ethylene, abscisic acid (ABA), jasmonate acid (JA), drought and cold. Here, we report the investigation of essential DNA region within OsEBP-89 promoter for methyl jasmonic acid (MeJA) induction. PLACE analysis indicates that this promoter sequence contains multiple potential elements in response to various stimuli. First, we fused this promoter with GUS gene and analyzed its expression under MeJA treatment through Agrobacterium infiltration mediating transient expression in tobacco leaves. Our results revealed that this chimeric gene could be inducible by MeJA in tobacco leaves. To further determine the crucial sequences responsible for MeJA induction, we generated a series of deletion promoters which were fused with GUS reporter gene respectively. The results of transient expression of GUS gene driven by these mutant promoters show that the essential region for MeJA induction is positioned in the region between −1200 and −800 in OsEBP-89 promoter containing a G-box (−1127), which is distinct from the essential region containing ERE (−562) for ACC induction. In all, our finding is helpful in understanding the molecular mechanism of OsEBP-89 expression under different stimuli. OsEBP-89, essential DNA region, methyl jasmonic acid, transient assay, promoter, tobacco leaves Contributed equally to this work Supported by the National Basic Research Program of China (Grant No. 2006CB101700) and the National Natural Science Foundation of China (Grant Nos. 30671135, 30525034 and 30730060)     

Transformation of peas

Summary The lateral cotyledonary meristems present in germinating seed were inoculated with a non-oncogenic strain of A. tumefaciens carrying a gene conferring kanamycin resistance as a selectable marker and a -glucuronidase sequence as a reporter gene. Kanamycin resistant plants were derived from the meristems and shown to be transformed on the basis of Southern blots, polymerase chain reaction analysis and tests for -glucuronidase activity. The plants were fertile and tests of their progeny confirmed the transmission of integrated sequences through a sexual generation. This transformation method has the merit of an unlimited supply of material for inoculation and a relatively short time scale from inoculation to the production of rooted plants.Abbreviations BAP benzyl amino purine - GA3 gibberellic acid - GUS =glucuronidase - IBA indole butyric acid - MS Murashige and Skoog medium - NAA naphthalene acetic acid - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - TO first generation transformants - T1 second generation transformants