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.     

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.     

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...     

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     

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     

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。     

A high-resolution linkage map of the citrus tristeza virus resistance gene region in Poncirus trifoliata (L.) Raf.

Resistance to citrus tristeza virus (CTV) was evaluated in 554 progeny of 10 populations derived from Poncirus trifoliata. A dominant gene (Ctv) controlled CTV resistance in P. trifoliata. Twenty-one dominant PCR-based DNA markers were identified as linked to Ctv by bulked segregant analysis. Of the 11 closest markers to Ctv, only 2 segregated in all populations. Ten of these markers were cloned and sequenced, and codominant RFLP markers were developed. Seven RFLP markers were then evaluated in 10 populations. Marker orders were consistent in all linkage maps based on data of single populations or on combined data of populations with similar segregation patterns. In a consensus map, the six closest marker loci spanned 5.3 cM of the Ctv region. Z16 cosegregated with Ctv. C19 and AD08 flanked Ctv at distances of 0.5 and 0.8 cM, respectively. These 3 markers were present as single copies in the Poncirus genome, and could be used directly for bacterial artificial chromosome library screening to initiate a walk toward Ctv. BLAST searches of the GenBank database revealed high sequence similarities between 2 markers and known plant disease resistance genes, indicating that a resistance gene cluster exists in the Ctv region in P. trifoliata.     

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     

Expression of betaine aldehyde dehydrogenase gene and salinity tolerance in rice transgenic plants

Betaine as one of osmolytes plays an important role in osmoregulation of most high plants. Betaine aldehyde dehydrogenase C BADH) is the second enzyme involved in betaine biosynthesis. The BADH gene from a halophite, Atriplex hortensis, was transformed into rice cultivars by bombarment method. Totally 192 transgenic rice plants were obtained and most of them had higher salt tolerance than controls. Among transgenic plants transplanted in the saline pool containing 0.5% NaCl in a greenhouse, 22 survived, 13 of which set seeds, and the frequency of seed setting was very low, only 10% . But the controls could not grow under the same condition. The results of BADH ac-tivity assay and Northern blot showed that the BADH gene was integrated into chromosomes of transgenic plants and expressed.     

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.     

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。