Fingerprinting trifoliate orange germ plasm accessions with isozymes, RFLPs, and inter-simple sequence repeat markers

 Trifoliate orange [Poncirus trifoliata (L.) Raf.] is frequently used as a parent in citrus rootstock breeding, but the origin and amount of genetic diversity in germ plasm collections are poorly understood. Most accessions are self-compatible, but produce a mixture of sexual and apomictic seedlings. Variation among 48 vegetatively propagated trifoliate orange accessions was assessed at seven isozyme loci, together with the restriction fragment length polymorphisms (RFLPs) detected by 38 probe-enzyme combinations and the inter-simple sequence repeat (ISSR) markers generated by 11 primers. Isozymes and RFLPs detected few polymorphisms among accessions, although genetic analysis has shown that the common phenotype is heterozygous for four isozyme and at least four RFLP loci. ISSR amplification generated multiple banding profiles with an average of 58 fragments/primer/accession. These fragments were repeatable across DNA samples extracted from different trees of the same accession or extracted at different times, and across separate PCR runs. Seventeen unique marker phenotypes were identified. The 48 trifoliate orange accessions were classified into four major groups based on polymorphic ISSR markers. All large-flowered accessions are in group 4, while small-flowered accessions are in group 3. Many ISSR markers segregated in progeny derived by open-pollination (probably mostly selfing) of a common accession, indicating that these ISSR markers are also heterozygous. Accessions having identical genotypes for a large number of heterozygous markers are unlikely to have diverged by recombination. Thus the limited divergence we detected among most accessions most likely originated by mutation. ‘Monoembryonic’ and ‘Simmons’ differed from other accessions only in the loss of specific markers, indicating that they originated as zygotic seedlings of individuals similar to the common genotype. Three accessions recently introduced from China have relatively different fingerprints with 3–14 unique ISSR markers, and probably represent a much more divergent germ plasm that may be a valuable breeding resource. Received: 8 August 1996 / Accepted: 21 March 1997     

Restoration of vigor and rooting competence in stem tissues of mature citrus by repeated grafting of their shoot apices onto freshly germinated seedlings in vitro

Summary Repeated grafting of 0.2-cm shoot tips from fruiting-age trees ofCitrus reticulata Blanco ‘Ponkan’ mandarin andC. sinensis Osbeck ‘Liu Tseng’ sweet orange onto freshly germinated ‘Troyer’ citrange [Poncirus trifoliata (L.) Raf. X.C. sinensis Osbeck] seedlings in vitro resulted in progressive restoration of rooting competence and vigor of regenerated roots and shoots. The restored traits were retained through the course of the investigation and suggested a phase reversal phenomenon.     

Genetic transformation of three sweet orange cultivars from explants of adult plants

The difficulty in adult tissue genetic transformation in woody species is still an obstacle to be overcome, including in most sweet orange cultivars of the Brazilian citrus industry. This work reports that, after in vitro culture adjustments, transgenic adventitious buds of ‘Hamlin’, ‘Pêra’, and ‘Valencia’ sweet oranges (Citrus sinensis L. Osbeck) were recovered using adult material as explant source, in genetic transformation experiments via Agrobacterium tumefaciens. The transgenic buds were identified by the GUS histochemical analysis and confirmed by PCR analysis, which indicated the presence of an amplified fragment of 817 bp corresponding to the uidA gene sequence. The efficiencies of genetic transformation for ‘Hamlin’, ‘Pêra’, and ‘Valencia’ sweet orange cultivars were 2.5, 1.4, and 3.7%, respectively. Media supplemented with auxins and cytokinins during co-culture, and media with high concentrations of cytokinins (3 mg L⁻¹) during transgenic selection led to the transformation and, consequently, the regeneration of adequate number of adventitious buds for the three cultivars. The use of sonication during the explant disinfection was not effective to reduce endophytic contamination and reduced transformation efficiency.     

Obtaining autotetraploids in vitro at a high frequency in Citrus sinensis

Tetraploid plants are essential for interploid hybridization to create triploid seedless citrus. Here we report a simple and efficient in vitro method for generating autotetraploids for sweet orange (Citrus sinensis). Cell-division activity in ‘Anliucheng’ sweet orange callus was analyzed using flow cytometry to determine the peak frequency of cell division at which time callus in a liquid media and solid media was treated with 1000 mg l⁻¹ colchicine. The percentage of the DNA-content-varied cells in the callus increased markedly from 11.0% to 44.4% and to 59.0% for liquid and solid media respectively. A total of 20 tetraploid plantlets were recovered via embryogenesis from 47 plantlets regenerated from the treated callus. All the autotetraploids were derived from different embryoids. Autotetraploids will be useful parents for interploid hybridization to generate commercially valuable seedless triploid citrus cultivars.     

Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy <Emphasis Type="Italic">Citrus</Emphasis> cultivars

Cytoplasmic male sterility (CMS) is known to be controlled by mitochondrial genome in higher plants including Satsuma mandarin (Citrus unshiu Marc.). Citrus symmetric fusion experiments often produce diploid cybrids possessing nuclear DNA from the mesophyll parent and mitochondrial DNA (mtDNA) from the embryogenic callus parent. Therefore, it is possible to transfer CMS from Satsuma mandarin as callus parent to seedy citrus cultivars as leaf one by somatic cybridization. Herein, symmetric fusion technique was adopted to create cybrids for potential seedlessness by transferring CMS from Citrus unshiu Marc. cv. Guoqing No. 1 (G1) to two traditional Chinese seedy citrus cultivars, ‘Shatian’ pummelo (C. grandis (L) Osbeck) and ‘Bingtang’ orange (C. sinensis (L) Osbeck). Flow cytometry analysis showed that 19 plants recovered from G1 + ‘Bingtang’ orange and 17 of 35 plants regenerated from G1 + ‘Shatian’ pummelo were diploid. The remaining plants from G1 + ‘Shatian’ pummelo were tetraploid. The diploid plants from the two combinations were confirmed as true cybrids by simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) analysis, with nuclear DNA from their corresponding leaf parent and mtDNA from their common suspension parent, G1 Satsuma mandarin. The remaining plants from G1 + ‘Shatian’ pummelo were identified as somatic hybrids with mtDNA from G1. The chloroplast simple sequence repeat (cp-SSR) analysis revealed somatic hybrid/cybrid plants from the two combinations in most cases possessed either of their parental chloroplast type, and two plants from G1 +‘Shatian’ pummelo and all embryoids analyzed from G1 + ‘Bingtang’ orange possessed chloroplast DNA (cpDNA) from both parents. These results demonstrated that we succeeded in introducing mtDNA from G1 Satsuma mandarin into the two target seedy citrus cultivars for potential seedlessness through symmetric fusion.     

High-efficiency <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of citrus via sonication and vacuum infiltration

An improved method for the Agrobacterium infiltration of epicotyl segments of ‘Pineapple’ sweet orange [Citrus sinensis (L.) Osbeck] and ‘Swingle’ citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional Agrobacterium-mediated inoculation method (‘dipping’ method). It was observed that the morphogenic potential of the epicotyl segments decreased as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments, but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4% for ‘Pineapple’ sweet orange and 11.2% for ‘Swingle’ citrumelo) are the highest ones reported for both cultivars.     

An alternative method for the genetic transformation of sweet organce

Summary An alternative method for transforming sweet organe [Citrus sinensis (L.) Osbeck] has been developed. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein gene was introduced using polyethylene glycol into protoplasts of ‘Itaborai’ sweet organe isolated from an embryogenic nucellar-derived suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of the green fluorescent protein, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots. To expedite transgenic plant recovery, regenerated shoots were also micrografted onto sour orange seedling rootstocks. Presence of the transgene in calluses and regenerated sweet organe plants was verified by gene amplification and Southern analyses. Potential advantages of this transformation system over the commonly used Agrobacterium methods for citrus are discussed.     

Assessment of fire blight tolerance in apple based on plant inoculations with Erwinia amylovora and DNA markers

Fire blight (Erwinia amylovora) causes serious damage to pome fruit orchards, and identification of germplasm with heritable disease resistance is therefore crucial. Two dominant SCAR (sequence characterised amplified region) marker alleles (AE10-375 and GE-8019), flanking a previously identified QTL (quantitative trait locus) for resistance to fire blight on ‘Fiesta’ linkage group 7 in apple cultivars related to ‘Cox’s Orange Pippin’, were screened on 205 apple cultivars. Both marker alleles were present in 22% of the cultivars, indicating presence of the QTL allele for tolerance, and both were lacking in 25%, indicating homozygosity for absence of the QTL tolerance allele. However, 33% had only the marker allele AE10-375, while 20% had only GE-8019, suggesting that some cultivars with the dominant alleles for both of the flanking markers can carry these on separate chromosomes and may lack the QTL allele for tolerance. In 2009 and 2010, terminal shoots of greenhouse-grown grafted trees of 21 cultivars (only 20 in 2010) were inoculated with Erwinia amylovora. ‘Idared’ (susceptible) and ‘Enterprise’ (tolerant) were included as controls. Disease severity for each cultivar was expressed as percentage of necrosis in relation to entire length of shoot, and the ranking of cultivars in 2009 and 2010 was compared with a Spearman rank correlation test, P < 0.01. A relationship between presence of both flanking marker alleles for tolerance and level of fire blight tolerance was confirmed with a Mann–Whitney U-test, P < 0.01 in 2009, and P < 0.05 in 2010. A PCO (principal coordinate) analysis based on band profiles obtained with 12 SSR (simple sequence repeat) loci produced three loose clusters, two of which contained known offspring of ‘Cox’s Orange Pippin’, and one with cultivars that were either unrelated or had an unknown origin. Cases where DNA markers did not predict level of fire blight damage as expected, were, however, as common among descendants of ‘Cox’s Orange Pippin’ as among apparently unrelated cultivars. Obviously the ‘Fiesta’ LG 7 QTL has some predictive value, both for known ‘Cox’ relatives and others, but more efficient markers would be desirable for marker-assisted selection.     

AFLP analysis of nephthytis (Syngonium podophyllum Schott) selected from somaclonal variants

This study analyzed genetic differences of 19 cultivars selected from somaclonal variants of Syngonium podophyllum Schott along with their parents as well as seven additional Syngonium species and six other aroids using amplified fragment length polymorphism (AFLP) markers generated by 12 primer sets. Among the 19 somaclonal cultivars, ‘Pink Allusion’ was selected from ‘White Butterfly’. Tissue culture of ‘Pink Allusion’ through organogenesis resulted in the development of 13 additional cultivars. Self-pollination of ‘Pink Allusion’ obtained a cultivar, ‘Regina Red Allusion’, and tissue culture propagation of ‘Regina Red Allusion’ led to the release of five other cultivars. The 12 primer sets generated a total of 1,583 scorable fragments from all accessions, of which 1,284 were polymorphic (81.9%). The percentages of polymorphic fragments within ‘White Butterfly’ and ‘Regina Red Allusion’ groups, however, were only 1.2% and 0.4%, respectively. Jaccard's similarity coefficients among somaclonal cultivars derived from ‘White Butterfly’ and ‘Regina Red Allusion’, on average, were 0.98 and 0.99, respectively. Seven out of the 15 cultivars from the ‘White Butterfly’ group and three out of six from the ‘Regina Red Allusion’ group were clearly distinguished by AFLP analysis as unique fragments were associated with respective cultivars. The unsuccessful attempt to distinguish the remaining eight cultivars from the ‘White Butterfly’ group and three from the ‘Regina Red Allusion’ group was not attributed to experimental errors or the number of primer sets used; rather it is hypothesized to be caused by DNA methylation and/or some rare mutations. This study also calls for increased genetic diversity of cultivated Syngonium as they are largely derived from somaclonal variants.     

Genetic Linkage Maps of <Emphasis Type="Italic">Betula platyphylla</Emphasis> Suk Based on ISSR and AFLP Markers

Two separate genetic linkage maps for Chinese silver birch based on inter-simple sequence repeat (ISSR) and amplified fragment-length polymorphism (AFLP) were constructed by a pseudo-testcross mapping strategy. Eighty F₁ progenies were obtained from the cross between two parental trees with desirable traits (the paternal one selected from ‘Qinghai’ and the maternal one from ‘Wangqing’). A total of 46 ISSR primers and 31 AFLP primers were employed to generate 102 ISSR and 355 AFLP polymorphic markers in the F₁ progenies. About 5.7% of all the markers displayed high segregation distortion with a P value below 0.01 and such markers were not used for map constructions. The paternal map consisted of 137 loci, spread over 13 groups and spanned 694.2 cM at an average distance of 5.1 cM between the markers, while in the maternal map, 147 loci were distributed in 14 groups covering a map distance about 949.62 cM at an average distance of 6.5 cM. These initial maps can serve as the basis for developing a more detailed genetic map.