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     

Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata [L.] RAF.)

Halved shoot bases of Allium tuberosum Rottl. ex Spreng. proliferated both axillary and adventitious shoots on B5 medium (1968) supplemented with either 6-benzylaminopurine (0.5 mg/l) or 1-naphthalene acetic acid (0.1 mg/l) and 2-isopentenyladenine (0.5 mg/l). In vitro shoots proliferated further numerous shoots upon subculture to fresh medium, and these shoots rooted spontaneously. Plantlets were transplanted successfully to soil and retained the diploid condition of the parents.Abbreviations B5 Gamborg et al. (1968) medium - BAP 6-benzylaminopurine - 2ip 2isopentenyladenine - NAA 1-naphthalene acetic acid     

Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata [L.] RAF.)

Seeds of trifoliate orange (Poncirus trifoliata (L.) Raf.) are sensitive to desiccation, and could not withstand reduction in moisture level below 20%, whereas the excised embryonic axes could be easily desiccated to moisture levels as low as 14% without much loss in viability. Axes could be successfully cryopreserved in liquid nitrogen (–196°C) for eight months. The viable embryonic axes exhibited good growth on modified Murashige and Skoog medium supplemented wiith 1-Naphthalene acetic acid (NAA) and 6-Benzylaminopurine (BAP). Growth of cryopreserved axes was promoted in the presence of charcoal in the medium allowing for plant recovery.Abbreviations NAA Napthaleneacetic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) - LN Liquid nitrogen     

五种丛枝菌根真菌对枳实生苗耐锌污染的影响

通过盆栽实验研究丛枝菌根(AM)真菌Glomus versiforme(G.v)、G.mosseae(G.m)、G.intraradices(G.i)、G.aggregatum(G.a)和G.etunicatum(G.e)在锌污染条件下枳实生苗的菌根侵染、生长、叶片和根系锌、磷含量及部分生理指标的影响.结果表明:锌污染...     

New gene(s) involved in the resistance of Poncirus trifoliata (L.) Raf. to citrus tristeza virus

 Citrus tristeza virus (CTV) causes important economic losses in the citrus industry worldwide. Resistance to CTV is present in Poncirus trifoliata and is known to be controlled by a dominant gene at the Ctr locus. Short-distance movement of CTV around the inoculum, as well as passive movement through the phloem vessels, were studied in segregant plants derived by self-pollination from P. trifoliata var. “Flying Dragon” in order to genetically analyze the mechanism of CTV resistance. Accumulation of CTV in the vicinity of the inoculum and in new flushes was studied by means of a direct tissue-blot immunoassay (DTBIA). CTV is able to passively move with the phloematic flux from inoculated resistant genotypes Ctr-Rr and Ctr-RR up to a susceptible scion cultivar (Ctr-rr). Differences regarding CTV accumulation around the inoculum were found among Ctr-Rr individuals of the progeny. Bulked segregant analysis identified five RAPD markers linked to a locus (Ctm), or a genomic region, involved in short-distance accumulation of CTV but located in a different linkage group from Ctr. This result indicates that Ctr is not the only locus responsible for resistance to CTV in P. trifoliata, and that at least one other gene is involved. Given that citrus is a perennial crop, breeding for durable disease resistance should take into account selection at both the Ctr and Ctm loci. Received : 13 March 1996 / Accepted : 18 April 1997     

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as −26°C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.     

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.     

激素处理及非生物胁迫下枳PtPPF-1基因的表达特性

PtPPF-1是从枳叶片中分离出的与豌豆中的PPF-1具有很高同源性的基因。PPF-1基因能通过控制叶绿体发育而延缓叶片衰老,同时PPF-1能被外源GA3诱导,是与植物营养生长密切相关的基因。本研究以实生苗枳[Poncirus trifoliata(L.)Raf.]为材料,采用半定量RT-PCR对不同激素处理及非生物胁迫(低温、干旱、盐)下枳叶中PtPPF-1的表达情况进行了分析,以期明确PtPPF-1的功能。结果表明,GA3I、AA、ABA促进了PtPPF-1的表达,KT对PtPPF-1的表达有抑制作用;低温、干旱、盐胁迫能诱导PtPPF-1的表达。推测GA3、IAA、ABA信号通路可能与KT信号通路作用于PtPPF-1的表达存在不同的机制。     

Construction of a 1.2-Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of Poncirus trifoliata (L.) Raf.

The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.     

Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.)

A map-based cloning strategy has been employed to isolate Ctv, a single dominant gene from Poncirus trifoliata that confers resistance to citrus tristeza virus (CTV), the most important viral pathogen of citrus. Cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. A high-resolution genetic linkage map of the Ctv locus region was developed using a backcross population of 678 individuals. Three DNA markers that were closely linked or co-segregated with Ctv were identified and used to screen BAC libraries derived from an intergeneric hybrid of Poncirus and Citrus. Through chromosome walking and landing, two BAC contigs were developed: one encompassing the Ctv region, and the other spanning the allelic susceptibility gene region. The resistance gene contig consists of 20 BAC clones and is approximately 550 kb in length; the susceptibility gene contig consists of 16 BAC clones and extends about 450 kb. The Ctv locus was localized within a genomic region of approximately 180 kb by genetic mapping of BAC insert ends. The BAC contigs were integrated with the genetic map; variation in the ratio of genetic to physical distance was observed in the vicinity of Ctv. Southern hybridization data indicated that a few copies of NBS-LRR class sequences are distributed at or around the Ctv locus. Efforts are being made to assign the Ctv locus to a smaller genomic fragment whose function can be confirmed through genetic complementation of a CTV susceptible phenotype. These results indicate that map-based gene cloning is feasible in a woody perennial.     

Inhibition of colon cancer cell growth and antioxidant activity of bioactive compounds from Poncirus trifoliata (L.) Raf

Recently several plant derived natural compounds have been screened for their anticancer activity in order to identify putative compounds with novel structures or mechanism of action. In the present study, fruits of Poncirus trifoliata were extracted with acetone and loaded onto silica gel column chromatography. The column was eluted with different solvents to obtain two bioactive compounds. The purity of compounds was analyzed by HPLC and their structures were identified by 1H and 13C NMR experiments as beta-sitosterol and 2-hydroxy-1,2,3-propanetricarboxylic acid 2-methyl ester (HPCME). beta-Sitosterol, HPCME, and trolox were tested for their antioxidant capacity by oxygen radical absorbance capacity (ORAC) measurement. Further, these compounds were tested for their inhibition of cancer cell proliferation and apoptosis using human colon cancer cell line (HT-29). These results were compared with the corresponding activity on non-cancerous (COS-1 fibroblast) cells. Cell proliferation and induction of apoptosis were determined by MTT assay and nuclear staining. The MTT assay indicated that both the compounds exhibited differential inhibition at various concentrations. Significant arrest of cell growth was observed with beta-sitosterol even at low concentration such as 0.63 microM in 48 h and none of the compounds exerted any apparent cytostatic effects on the non-cancerous COS-1 fibroblast cells. Growth inhibition assay suggested the potential use of bioactive compounds as cancer chemopreventive and therapeutic agents. This is the first report on HPCME isolation and identification from Rutaceae family and beta-sitosterol from P. trifoliata.     

Sequence analysis of a 282-kilobase region surrounding the citrus Tristeza virus resistance gene (Ctv) locus in Poncirus trifoliata L. Raf

Citrus tristeza virus (CTV) is the major virus pathogen causing significant economic damage to citrus worldwide, and a single dominant gene, Ctv, provides broad spectrum resistance to CTV in Poncirus trifoliata L. Raf. Ctv was physically mapped to a 282-kb region using a P. trifoliata bacterial artificial chromosome library. This region was completely sequenced to about 8x coverage using a shotgun sequencing strategy and primer walking for gap closure. Sequence analysis predicts 22 putative genes, two mutator-like transposons and eight retrotransposons. This sequence analysis also revealed some interesting features of this region of the P. trifoliata genome: a disease resistance gene cluster with seven members and eight retrotransposons clustered in a 125-kb gene-poor region. Comparative sequence analysis suggests that six genes in the Ctv region have significant sequence similarity with their orthologs in bacterial artificial chromosome clones F7H2 and F21T11 from Arabidopsis chromosome I. However, the analysis of gene colinearity between P. trifoliata and Arabidopsis indicates that Arabidopsis genome sequence information may be of limited use for positional gene cloning in P. trifoliata and citrus. Analysis of candidate genes for Ctv is also discussed.