Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata

Summary Somatic hybrid plants of Rutaceae were obtained by protoplast fusion between Citrus sinensis Osb. (Trovita orange) and Poncirus trifoliata. Protoplasts isolated from embryogenic cells of C. sinensis and from leaves of P. trifoliata, and the culture of fusion products in the presence of high concentrations of sucrose were essential requirements for the selection of hybrids. Green globular embryoids derived from protoplasts resulted in the regeneration of trifoliate plants. Other morphological characters of these plants were intermediate between both parents. The chromosome number in one of the hybrid plants was 36, which was the sum of C. sinensis (2n=18) and P. trifoliata (2n=18). EcoRI restriction analysis of rDNA confirmed the presence of parental nuclear DNAs in the hybrid.     

Identification of stress-induced genes from the drought tolerant semi-arid legume crop horsegram (Macrotyloma uniflorum (Lam.) Verdc.) through analysis of subtracted expressed sequence tags

Abiotic stresses adversely affect the agricultural productivity worldwide. Horsegram (Macrotyloma uniflorum (Lam.) Verdc.) is a legume crop that can tolerate severe adverse environmental conditions such as drought, salinity and heavy metal contamination. As a first step towards characterization of genes that contribute to combating abiotic stresses, construction and analysis of subtracted cDNA library is reported here. Using this strategy a total of 1050 ESTs were isolated, sequenced, 959 high quality ESTs were obtained and clustered. Further, our analysis revealed that of these 531 sequences are unique and 30% of these have no homology to known proteins in the database. This observation has great relevance since horsegram is a stress-adapted legume crop. Further, to validate the identified differentially expressed genes, expression profiles of selected clones were analyzed using reverse-northern, northern blot analysis and we show that indeed these clones are differentially expressed under various abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance also discussed.     

Refinement of the Citrus Tristeza Virus Resistance Gene (Ctv) Positional Map in Poncirus trifoliata and Generation of Transgenic Grapefruit (Citrus paradisi) Plant Lines with Candidate Resistance Genes in this Region

Citrus tristeza virus (CTV) is a major pathogen of Citrus. A single dominant gene Ctv present in the trifoliate relative of Citrus, Poncirus trifoliata confers broad spectrum resistance against CTV. Refinement of genetic maps has delimited this gene to a 121 kb region, comprising of ten candidate Ctv resistance genes. The ten candidate genes were individually cloned in Agrobacterium based binary vector and transformed into three CTV susceptible grapefruit varieties. Two of the candidate R-genes, R-2 and R-3 are exclusively expressed in transgenic plants and in Poncirus trifoliata, while five other genes are also expressed in non-transformed Citrus controls. Northern blotting with a CTV derived probe for assessment of infection in virus inoculated plants over a span of three growth periods, each comprising of six to eight weeks, indicates either an absence of initiation of infection or it’s slow spread in R-2 plant lines or an initial appearance of infection and it’s subsequent obliteration in some R-1 and R-4 plant lines. Limited genome walk up- and downstream form R-1 gene, based on it’s 100% sequence identity between Poncirus and Citrus, indicates promoter identity of 92% between the two varieties. Further upstream and downstream sequencing indicates the presence of an O-methyl transferase and a Copia like gene respectively in Citrus instead of the amino acid transporter like gene upstream and a sugar transporter like gene downstream in Poncirus. The possibility of recombinations in the resistance locus of Citrus and the need for consistent monitoring for virus infection and gene expression in the transgenic Citrus trees is discussed. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.)

Summary A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.) was developed using epicotyl segments. The segments were infected with Agrobacterium harboring the binary vector pBI121 or pBI101-O12-p1. Both vectors contained the neomycin phosphotransferase II (NPTII) and the -glucuronidase (GUS) genes. In the plasmid pBI101-O12-p1, the GUS gene was directed to the promoter region of ORF12 (rolC) of the Ri plasmid. On a selection medium containing 100 or 200 g/ml kanamycin, adventitious shoots were formed from 21.7–44.6% of the segments. Histochemical GUS assay showed that 55.4–87.7% of the shoots expressed the GUS gene. The stable integration of this gene was also confirmed by polymerase chain reaction (PCR) analysis and by Southern blot analysis. When the pBI101-O12-p1 plasmid was used, the GUS activity was found to be located in phloem cells of leaf, stem and root. More than 100 transformed plants were obtained using this method within 2–3 months.     

Molecular markers flanking citrus tristeza virus resistance gene from Poncirus trifoliata (L.) Raf.

 Two segregating populations for citrus tristeza virus (CTV) resistance derived from Poncirus trifoliata var ‘Flying Dragon’ by self-pollination and pollination to Citrus medica L. var ethrog ‘Arizona’ were inoculated with a common CTV isolate. The presence of virus was checked by the Double Antibody Sandwich Enzyme-Linked Assay and Direct Tissue Blot Inmunoassay at 3, 6, and 12 months after inoculation. Seven RAPDs were found linked to the CTV resistance gene by bulked segregant analysis. The closest linked RAPDs were cloned to obtain linked codominant RFLPs and to increase the precision of the genetic distance estimation. The CTV resistance gene seems to be located between cW18 and cK16. Differences in genetic distances among progenies are large and can be explained by genome-wide reduction in the recombination of progeny derived from male versus female gametes. Received: 5 June 1996 / Accepted: 26 July 1996     

枳PPF-1同源基因的克隆及分析

以枳[Poncirus trifoliata(L.)Raf.]实生苗为材料,采用RT-PCR及RACE技术,获得了PPF-1(开花、衰老相关基因)同源基因的cDNA全长序列PtPPF-1,该cDNA全长1 493 bp,编码452个氨基酸,与豌豆PPF-1、拟南芥ALB3、水稻PPF-1及葡萄一未命名基因的氨基酸序列同源性较高,说明PPF-1基因在进化过程中可能变异较小。半定量RT-PCR分析表明PtPPF-1在叶片中表达量最高,顶芽和茎中相似,在根中未检测到,表明该基因功能与叶绿体有关。     

Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus

Precocious trifoliate orange (Poncirus trifoliata [L.] Raf), an extremely early flowering mutant of P. trifoliata, is an attractive model for functional genomics research in Citrus. A procedure for efficient regeneration and transformation of this genotype was developed by using green fluorescent protein (GFP) gene as visual marker and etiolated stem segments as explants. In vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic shoots and escapes. Transformation efficiency was 20.7% and the transformants were identified by polymerase chain reaction (PCR) and Southern blot analysis. Moreover, the transgenic lines expressed variable amounts of the GFP gene as revealed by real-time PCR analysis. Fifteen transgenic plants flowered 18 months after transfer to the greenhouse and six of them set fruits. GFP expression was also observed in the transgenic flowers and fruits. To test the utility of this system for functional genomics studies, an Arabidopsis thaliana MAC12.2 gene with the potential to produce seedless fruits was introduced into this genotype, and the traits of the transgenic fruits were characterized. The successful transformation of this perennial woody genotype with extremely short juvenility will allow us to test the function of cloned genes in citrus, the improvement of which is hindered by a long juvenility period.     

The Quantitative Growth of Young Seedlings of the Citrus Rootstocks Citrus sinensis (L.) Osbeck, Poncirus trifoliata (L.) Raf. and their hybrid, Carrizo citrange

An 18 months' growth analysis of three citrus rootstocks revealedconsiderable differences in absolute and relative growth-ratesand in the allometric growth of leaf, stem, thorn, and root. Over the whole period the order of the average absolute growth-rateswas citrange > sinensis > trifoliata, and of the relativegrowth-rates citrange > sinensis = trifoliata. Although sinensisachieved the highest R.G.R. this was maintained over a shortperiod only. Sinensis had relatively most leaf, trifoliata moststem, and citrange most root. The deciduousness of trifoliataproduced only an intermittent deviation in the allometric line,the leaf proportion returning to the average course within afew weeks of the start of spring growth. The time course of these changes was not sufficiently regularfor phases of maximum growth to be characterized. During thewinter period of minimum growth there was a loss in weight ofthe roots (in sinensis, and trifoliata) and stem (citrange).     

Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.)

Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23000–28000 and isoelectric points 10.3–10.4) were purified from nonembryogenic Citrus sinensis L. Osbeck cv. Valencia callus tissue. The basic chitinase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitinase. While BCLVC acted as a lysozyme at pH 4.5 and low ionic strength (0.03) it acted as a chitinase/chitosanase at high ionic strengths (0.2) with a pH optimum of ca. 5. The lysozyme activity of BCLVC was inhibited by histamine, imidazole, histidine and the N-acetyl-d-glucosamine oligosaccharide (GlcNAc)₃. The basic chitinase from cv. Valencia callus, BCVC-2, had an N-terminal amino acid sequence similar to tomato and tobacco AP24 proteins. The sequences of the other five chitinases were N-terminal blocked. Whereas BCLVC was capable of hydrolyzing 13.8–100% acetylated chitosans and (GlcNAc)_4–6 oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes expressed varying degrees of hydrolytic capabilities. Experiments with (GlcNAc)_2–6 suggest that BCLVC hydrolysis occurs in largely tetrasaccharide units whereas hydrolysis by the other chitinases occurs in disaccharide units. Cross-reactivities of the purified proteins with antibodies for a potato leaf chitinase (Ab_PLC), BCLVC, BCVC-3, and tomato AP24 indicate that these are separate and distinct proteins.Mention of a trademark, warranty, propriety, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.Abbreviations Ab antibody - BCLVC basic chitinase/lysozyme cv. Valencia callus - BCVC basic chitinase cv. Valencia callus - CE capillary electrophoresis - CM-chitin-RBV carboxymethyl-chitin-remazol brilliant violet - GlcNAc N-acetyl-d-glucosamine - HEWL hen egg-white lysozyme - M_r relativemolecular mass - pI isoelectric point - PLC potato leaf chitinase - PR pathogenesis-related - SEC size exclusion chromatography We thank Mr. M. Burkhart, Ms. T.-T. Ho, and Ms. M. Doherty for their valuable technical assistance. A portion of the funding for this work was made available from the Citrus Production Research Marketing Order by the Division of Marketing and Development, Florida Department of Agriculture and Consumer Services, Bob Crawford, Commissioner.     

Ultrastructure and histochemistry of Citrus limon (L.) stigma

Citrus limon has a wet stigma which can be divided in two zones: a glandular superficial one formed by papillae, and a non-glandular one formed by parenchymatic cells. The stigmatic exudate is produced by the papillae after the latter have reached their ultimate size. The papillae of the mature pistil are of varying size and composition. Both the unicellular and multicellular ones are present. The cells at the base of the papillae are rich in cytoplasm, whereas the tip cells are vacuolated. Histochemical analysis has shown that the exudate of Citrus is composed of lipids, polysaccharides, and proteins. Our results indicate that the lipidic component is produced and secreted first, followed by production and secretion of the polysaccharidic component. The lipidic component of the exudate is produced in the basal papillae cells and accumulates as droplets in dilated parts of the smooth endoplasmic reticulum (SER). Subsequently the lipid droplets are transported to the plasma membrane, and transferred by the latter into the cell walls. Then the exudate component is accumulated in the intercellular spaces and in the middle lamellar regions of the walls. Subsequently, the polysaccharidic component of the exudate is produced and secreted by the tip cells of the papillae.Abbreviations RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum     

High frequency shoot regeneration from trifoliate orange (Poncirus trifoliata L. Raf.) using the thin cell layer method

A method for a high frequency and direct in vitro bud regeneration of a woody species, the trifoliate orange (Poncirus trifoliata L. Raf), was designed. Transverse thin cell layer (tTCL) explants excised from the stem internodes of 1-year-old young plants of P. trifoliata regenerated bud in vitro on a medium containing 6-benzylaminopurine (BAP 1-50 μM) and N-phenyl-N'-1,2,3-thidiazol-5-ylurea (thidiazuron, TDZ) (0.1–10 μM). The optimal concentrations for bud induction were 25 μM BAP and 1 μM TDZ leading to 87 and 72 % of responsive tTCLs and 24 and 15 buds per tTCL, respectively. A higher percentage of responsive tTCLs and a higher frequency of bud regeneration were obtained with BAP and TDZ combined. With a combination of 10 μM BAP and 1 μM TDZ, 90 % of responsive tTCLs forming 37 buds per tTCL were obtained. Shoot elongation occurred after a transfer onto a medium containing 1 μM GA₃. Rooting of individual shoot was induced using 5 μM NAA. One hundred per cent of rooted shoots developed normally after transfer to the greenhouse; no phenotype variation was observed. High numbers of regenerated viable plants can be produced directly without callus formation from tTCL after 9 weeks of culture.