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.     

Effect of enzyme concentrations on protoplast isolation and protoplast culture of <Emphasis Type="Italic">Spathiphyllum</Emphasis> and <Emphasis Type="Italic">Anthurium</Emphasis>

Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 10⁶ protoplasts g⁻¹ and 1 × 10⁵ protoplasts g⁻¹ could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl₂·2H₂O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm⁻¹ alternating current for 60 s and subsequent generation of two pulses of 4500 V cm⁻¹ direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.     

Molecular mapping of leaf rust resistance gene <Emphasis Type="Italic">Rph14</Emphasis> in <Emphasis Type="Italic">Hordeum vulgare</Emphasis>

An incompletely dominant gene conferring resistance to Puccinia hordei, Rph14, identified previously in an accession of Hordeum vulgare, confers resistance to all known pathotypes of P. hordei in Australia. Knowledge of the chromosomal location of Rph14 and the identification of DNA markers closely linked to it will facilitate combining it with other important leaf rust resistance genes to achieve long lasting resistance. The inheritance of Rph14 was confirmed using 146 and 106 F₃ lines derived from the crosses ‘Baudin’/‘PI 584760’ (Rph14) and ‘Ricardo’/‘PI 584760’ (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks using DArT markers located Rph14 to the short arm of chromosome 2H. DArT marker bPb-1664 was identified as having the closest genetic association with Rph14. PCR based marker analysis identified a single SSR marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cm in the ‘Baudin’/‘PI 584760’and ‘Ricardo’/‘PI 584760’ populations, respectively.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Response of antioxidant activity to excess copper in two cultivars of <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis> Makino

Changes in ascorbic acid content and antioxidant enzyme activities were investigated in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) leaves of ‘Wutacai’ and ‘Erqing’ exposed to excess copper (Cu). Cu treatment reduced the fresh weight of shoot and root by 57% and 46% in ‘Wutacai’, and 60 and 54% in ‘Erqing’, respectively. The accumulation of copper in leaves was higher in ‘Wutacai’ than that in ‘Erqing’. Compared to the control, ascorbic acid (AsA) contents were significantly decreased after copper treatment in both cultivars, while they were higher in ‘Wutacai’ than in ‘Erqing’, which may explain the higher copper-tolerance of ‘Wutacai’ with higher copper accumulation. The higher AsA contents of ‘Wutacai’ resulted from their lower activities of degrading enzymes, such as ascorbate oxydase (AAO) and ascorbate peroxidase (APX), as well as the increasing activity of dehydroascorbate reductase (DHAR) after copper treatment compared with ‘Erqing’. Copper stimulated superoxide dismutase (SOD) activity in both cultivars, but for catalase (CAT), there was little difference between both cultivars. Peroxidases (POD) activity was decreased after copper treatment in ‘Erqing’, while in ‘Wutacai’, it was significantly increased at 14 days, and POD activity was higher in ‘Wutacai’ than that in ‘Erqing’ at 21 and 28 days. Therefore, the induced increasing activity of POD in ‘Wutacai’ also played an important role in its copper tolerance.     

An <Emphasis Type="Italic">in vitro</Emphasis> method to evaluate grapevine cultivars for <Emphasis Type="Italic">Erysiphe necator</Emphasis> susceptibility

Powdery mildew, caused by the obligate biotrophic ascomycete Erysiphe necator, is one of the most destructive grapevine diseases worldwide. Cultivars of Vitis vinifera L, for wine and table grape production, are all susceptible to E. necator, whose attacks result in severe epidemics under the warm and dry conditions of the Mediterranean basin. The aim of the present study was to compare the susceptibility of different grapevine cultivars to E. necator by an in vitro assay for assessing the potentiality of this method in breeding programs for resistance to the pathogen. Leaves of 12 grapevine cultivars were spot-inoculated in vitro with about 10 conidia from five different isolates of E. necator, using colony growth and conidiation 3 wk post-inoculation as indicators of susceptibility to the disease. A remarkable difference was observed between highly susceptible cultivars like ‘Baresana’, ‘Malvasia’, ‘Bianca’, and ‘Italia’, and the less susceptible ‘Alphonse Lavallée’ and ‘Ohanez’, in accordance with their behavior in the field. No statistically significant differences were found in the virulence of E. necator isolates.     

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.     

Effects of carbon source and concentration on development of lingonberry (<Emphasis Type="Italic">Vaccinium vitis-idaea</Emphasis> L.) shoots cultivated <Emphasis Type="Italic">in vitro</Emphasis> from nodal explants

Summary Carbohydrate type and concentration and their interactive effects on in vitro shoot proliferation of three lingonberry (Vaccinium vitis-idaea ssp. vitis-idaea L.) cultivars (‘Regal’, ‘Splendor’, and ‘Erntedank’) and two V. vitis-idaea ssp. minus (Lodd) clones (‘NL1’ and ‘NL2’) were studied. Nodal explants were grown in vitro on medium with 2 μM zeatin and either glucose, sorbitol, or sucrose at a concentration of 0, 10, 20, or 30 gl⁻¹. The interactive effects of carbohydrate type and concentration and genotype were important for shoot proliferation. The best response was afforded by sucrose at 20 gl⁻¹ both in terms of explant response and shoot developing potential, although glucose supported shoot growth equally well, and in ‘NL1’ at 10 gl⁻¹ it resulted in better in vitro growth than sucrose. Carbohydrate concentration had little effect on shoot vigor. The genotypes differed in terms of shoots per explant, length, and vigor, leaves per shoot, and callus formation at the base of explants; this was manifested with various types and concentrations of carbohydrate. Changing the positioning of explants on the medium from vertically upright to horizontal increased the shoot and callus size, but decreased shoot height and leaves per shoot. Proliferated shoots were rooted on a peat:perlite (1∶1, v/v) medium and the plantlets were acclimatized and eventually established in the greenhouse.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Chrysanthemum</Emphasis> plantlets using light-emitting diodes

Effects of illumination spectrum on the morphogenesis of chrysanthemum plantlets (Chrysanthemum morifolium Ramat. ‘Ellen’) grown in vitro were studied using an illumination system consisting of four groups of light-emitting diodes (LEDs) in the following spectral regions: blue (450nm), red (640nm), red (660nm), and far-red (735nm). Taking into account all differences in shoot height, root length, and fresh and dry weight (FW and DW, respectively), observed while changing the total photon flux density (PFD), the optimal total PFD for growth of chrysanthemum plantlets in vitro was estimated. For 16 h photoperiod and typical fractions of the spectral components (14%, 50%, 28%, and 8%, respectively), the optimal total PFD was found to be 40 μmol m⁻² s⁻¹. Our study shows that the blue component in the illumination spectrum inhibits the plantlet extension and formation of roots and simultaneously increases the DW to FW ratio and content of photosynthetic pigments. We demonstrate photomorphogenetic effects in the blue region and its interaction with the fractional PFD of the far-red spectral component. Under constant fractional PFD of the blue component, the root number, length of roots and stems, and fresh weight of the plantlets have a correlated nonmonotonous dependence on the fractional PFD of the far-red component.     

Intertribal hexaploid somatic hybrid plants regeneration from electrofusion between diploids of Citrus sinensis and its sexually incompatible relative, Clausena lansium

 Chinese wampee [Clausena lansium (Lour.) Skeels], a sexually incompatible relative of citrus, is commercially cultivated in South China. In this study, embryogenic protoplasts of ‘Bonanza’ navel orange [Citrus sinensis (L.) Osbeck] were electrically fused with leaf protoplasts isolated from ‘Chicken Heart’ Chinese wampee. After 8 months of culture, fusion products regenerated into shoots. More than 70% of the shoots unexpectedly rooted well. Chromosome counting of several shoot- and root-tips revealed that their chromosome numbers were not 2n=4x=36 as expected, but 2n=6x=54, suggesting that chromosome doubling occurred rather than chromosome elimination in this intertribal fusion combination. RAPD analysis of embryoids and the leaves of unrooted and rooted shoots verified their hybridity. This is the first report of hexaploid somatic hybrid plant regeneration from fusion between diploids in Aurantioideae. Received: 20 June 1998 / Accepted: 18 August 1998     

Patterns of cytotype variation of <Emphasis Type="Italic">Turnera sidoides</Emphasis> subsp. <Emphasis Type="Italic">pinnatifida</Emphasis> (Turneraceae) in mountain ranges of central Argentina

Cytogeographical variability among 564 plants from 26 populations of Turnera sidoides subsp. pinnatifida in mountain ranges of central Argentina was analysed with meiotic chromosome counts and flow cytometry and is described at regional and local scales. Populations were primarily tetraploids (2n = 4x = 28), although diploid (2n = 2x = 14), hexaploid (2n = 2x = 42), and mixed populations of diploids and triploids (2n = 3x = 21) were also found. Diploids, triploids, and hexaploids were fewer in number and restricted to narrow areas, while tetraploids were the most common and geographically widespread cytotype. Diploids grew at higher altitudes and in colder and wet locations; tetraploids had the broadest ecological spectrum, while hexaploids occurred at the lowest altitudes and in drier conditions. The cytotypes were also spatially segregated at a microgeographical scale. Diploids grew in the piedmont, tetraploids were in the adjacent valley, and in the contact zone of both cytotypes, patches of diploids and triploids were found. At a regional scale, the distribution of the cytotypes may be governed by a combination of ecological and historical variables, while segregation in the contact zone may be independent of the selective environment because the cytotypes are unable to coexist as a result of reproductive exclusion. The role of triploids is also discussed.     

Construction of an intra-specific sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.) genetic linkage map and synteny analysis with the <Emphasis Type="Italic">Prunus</Emphasis> reference map

Linkage maps of the sweet cherry cultivar ‘Emperor Francis’ (EF) and the wild forest cherry ‘New York 54’ (NY) were constructed using primarily simple sequence repeat (SSR) markers and gene-derived markers with known positions on the Prunus reference map. The success rate for identifying SSR markers that could be placed on either the EF or NY maps was only 26% due to two factors: a reduced transferability of other Prunus-species-derived markers and a low level of polymorphism in the mapping parents. To increase marker density, we developed four cleaved amplified polymorphic sequence markers (CAPS), 19 derived CAPS markers, and four insertion–deletion markers for cherry based on 101 Prunus expressed sequence tags. In addition, four gene-derived markers representing orthologs of a tomato vacuolar invertase and fruit size gene and two sour cherry sorbitol transporters were developed. To complete the linkage analysis, 61 amplified fragment length polymorphism and seven sequence-related amplified polymorphism markers were also used for map construction. This analysis resulted in the expected eight linkage groups for both parents. The EF and NY maps were 711.1 cM and 565.8 cM, respectively, with the average distance between markers of 4.94 cM and 6.22 cM. A total of 82 shared markers between the EF and NY maps and the Prunus reference map showed that the majority of the marker orders were the same with the Prunus reference map suggesting that the cherry genome is colinear with that of the other diploid Prunus species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">MlAG12</Emphasis>: a <Emphasis Type="Italic">Triticum timopheevii</Emphasis>-derived powdery mildew resistance gene in common wheat on chromosome 7AL

Wheat powdery mildew is an economically important disease in cool and humid environments. Powdery mildew causes yield losses as high as 48% through a reduction in tiller survival, kernels per head, and kernel size. Race-specific host resistance is the most consistent, environmentally friendly and, economical method of control. The wheat (Triticum aestivum L.) germplasm line NC06BGTAG12 possesses genetic resistance to powdery mildew introgressed from the AAGG tetraploid genome Triticum timopheevii subsp. armeniacum. Phenotypic evaluation of F₃ families derived from the cross NC06BGTAG12/‘Jagger’ and phenotypic evaluation of an F₂ population from the cross NC06BGTAG12/‘Saluda’ indicated that resistance to the ‘Yuma’ isolate of powdery mildew was controlled by a single dominant gene in NC06BGTAG12. Bulk segregant analysis (BSA) revealed simple sequence repeat (SSR) markers specific for chromosome 7AL segregating with the resistance gene. The SSR markers Xwmc273 and Xwmc346 mapped 8.3 cM distal and 6.6 cM proximal, respectively, in NC06BGTAG12/Jagger. The multiallelic Pm1 locus maps to this region of chromosome 7AL. No susceptible phenotypes were observed in an evaluation of 967 F₂ individuals in the cross NC06BGTAG12/‘Axminster’ (Pm1a) which indicated that the NC06BGTAG12 resistance gene was allelic or in close linkage with the Pm1 locus. A detached leaf test with ten differential powdery mildew isolates indicated the resistance in NC06BGTAG12 was different from all designated alleles at the Pm1 locus. Further linkage and allelism tests with five other temporarily designated genes in this very complex region will be required before giving a permanent designation to this gene. At this time the gene is given the temporary gene designation MlAG12.     

Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Lilium</Emphasis>

A highly efficient Agrobacterium-mediated transformation system for Lilium × formolongi was established by modifying the medium used for inoculation and co-cultivation. Meristematic nodular calli of Lilium were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm harboring an intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase, and neomycin phosphotransferase II genes. The effects of ten different types of media and carbohydrates (sucrose, d-glucose, and l-arabinose) in both inoculation and co-cultivation media were evaluated. Interestingly, a dramatic increase in the frequency of transformation (25.4%) was observed when Murashige and Skoog (MS) medium containing sucrose and lacking KH₂PO₄, NH₄NO₃, KNO₃, and CaCl₂ was used. Hygromycin-resistant transgenic calli were obtained only in medium supplemented with sucrose. The effects of this modified medium were also investigated for Lilium cultivars ‘Acapulco’, ‘Casa Blanca’, and ‘Red Ruby’. The highest frequency of transformation (23.3%) was obtained for cv. Acapulco. Hygromycin-resistant calli were successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were confirmed by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot analyses.     

High-resolution mapping of <Emphasis Type="Italic">Rsn1</Emphasis>, a locus controlling sensitivity of rice to a necrosis-inducing phytotoxin from <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG1-IA

Rhizoctonia solani is a necrotrophic fungal pathogen that causes disease on many crop-plant species. Anastomosis group 1-IA is the causal agent of sheath blight of rice (Oryza sativa L.), one of the most important rice diseases worldwide. R. solani AG1-IA produces a necrosis-inducing phytotoxin and rice cultivar’s sensitivity to the toxin correlates with disease susceptibility. Unlike genetic analyses of sheath blight resistance where resistance loci have been reported as quantitative trait loci, phytotoxin sensitivity is inherited as a Mendelian trait that permits high-resolution mapping of the sensitivity genes. An F₂ mapping population derived from parent cultivars ‘Cypress’ (toxin sensitive) and ‘Jasmine 85’ (toxin insensitive) was used to map Rsn1, the necrosis-inducing locus. Initial mapping based on 176 F₂ progeny and 69 simple sequence repeat (SSR) markers located Rsn1 on the long arm of chromosome 7, with tight linkage to SSR marker RM418. A high-resolution genetic map of the region was subsequently developed using a total of 1,043 F₂ progeny, and Rsn1 was mapped to a 0.7 cM interval flanked by markers NM590 and RM418. Analysis of the corresponding 29 Kb genomic sequences from reference cultivars ‘Nipponbare’ and ‘93-11’ revealed the presence of four putative genes within the interval. Two are expressed cytokinin-O-glucosyltransferases, which fit an apoptotic pathway model of toxin activity, and are individually being investigated further as potential candidates for Rsn1.     

<Emphasis Type="Italic">Rpv10</Emphasis>: a new locus from the Asian <Emphasis Type="Italic">Vitis</Emphasis> gene pool for pyramiding downy mildew resistance loci in grapevine

A population derived from a cross between grapevine breeding strain Gf.Ga-52-42 and cultivar ‘Solaris’ consisting of 265 F1-individuals was genetically mapped using SSR markers and screened for downy mildew resistance. Quantitative trait locus (QTL) analysis revealed two strong QTLs on linkage groups (LGs) 18 and 09. The locus on LG 18 was found to be identical with the previously described locus Rpv3 and is transmitted by Gf.Ga-52-42. ‘Solaris’ transmitted the resistance-related locus on LG 09 explaining up to 50% of the phenotypic variation in the population. This downy mildew resistance locus is named Rpv10 for resistance to Plasmopara viticola. Rpv10 was initially introgressed from Vitis amurensis, a wild species of the Asian Vitis gene pool. The one-LOD supported confidence interval of the QTL spans a section of 2.1 centi Morgan (cM) corresponding to 314 kb in the reference genome PN40024 (12x). Eight resistance gene analogues (RGAs) of the NBS–LRR type and additional resistance-linked genes are located in this region of PN40024. The F1 sub-population which contains the Rpv3 as well as the Rpv10 locus showed a significantly higher degree of resistance, indicating additive effects by pyramiding of resistance loci. Possibilities for using the resistance locus Rpv10 in a grapevine breeding programme are discussed. Furthermore, the marker data revealed ‘Severnyi’ × ‘Muscat Ottonel’ as the true parentage for the male parent of ‘Solaris’.     

Effect of ploidy increase on transgene expression: example from <Emphasis Type="Italic">Citrus</Emphasis> diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from ‘Murcott’ tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic ‘Valencia’ orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.     

High-resolution genetic map of the <Emphasis Type="Italic">Rvi15</Emphasis> (<Emphasis Type="Italic">Vr2</Emphasis>) apple scab resistance locus

The Rvi15 (Vr2) apple scab resistance locus found in the GMAL 2473 accession has been previously mapped to the top of the Linkage Group 2 (LG2) by analyzing 89 progeny plants of a cross between ‘Idared’ and GMAL 2473. A new population of 989 progeny plants, derived from a cross between ‘Golden Delicious’ and GMAL 2473, has been analyzed with the two SSR markers CH02c02a and CH02f06, previously found to be associated with Rvi15 (Vr2), and with two published markers derived from NBS sequences (ARGH17 and ARGH37) estimated to map close to the Rvi15 (Vr2) locus. ARGH17 and ARGH37, were found to be the closest markers to the resistance locus, bracketing it within an interval of 1.5 cM. The SSRs mapped one on each side of Rvi15 (Vr2). CH02f06 mapped at 2.9 cM from ARGH37 while CH02a02a mapped at 1.7 from ARGH17. The position of Rvi15 (Vr2) respect to CH02a02a indicates that Rvi15 (Vr2) and Rvi4 (Vh4), a second apple scab gene mapped on the top of LG2, are two different resistance genes. In order to develop even more tightly linked markers to Rvi15 (Vr2), ARGH17 was used as the starting point for chromosome walking through the Rvi15 (Vr2) homolog region of the cv. ‘Florina’. A single ‘Florina’ BAC clone, 36I17, was sufficient to span the homologous locus in the new population’s recombinant progeny. Sequencing of the 36I17 BAC clone allowed identifying seven putative ORFs, including two showing a TIR-NBS-LRR structure. Ten additional markers could be developed mapping within a 1.8 cM interval around the Rvi15 (Vr2) resistance gene. ARGH17 and GmTNL1 markers, the latter also derived from NBS-LRR resistance gene homolog sequence, are the closest markers to Rvi15 (Vr2) bracketing it within a 0.5 cM interval. The availability of 12 markers within the Rvi15 (Vr2) region, all within a small physical distance (kbp) in ‘Florina’, suggests that cloning of the Rvi15 (Vr2) apple scab resistance gene from GMAL 2473 will be possible.     

Genetic stability of cryopreserved shoot tips of <Emphasis Type="Italic">Rubus</Emphasis> germplasm

Questions often arise concerning the genetic stability of plant materials stored in liquid nitrogen for long time periods. This study examined the genetic stability of cryopreserved shoot tips of Rubus germplasm that were stored in liquid nitrogen for more than 12 yr, then rewarmed and regrown. We analyzed the genetic stability of Rubus grabowskii, two blackberry cultivars (“Hillemeyer” and ‘Silvan’), and one raspberry cultivar (“Mandarin”) as in vitro shoots and as field-grown plants. No morphological differences were observed in greenhouse-grown cryopreserved plants when compared to the control mother plants. In the field, cryopreserved plants appeared similar but were more vigorous than mother plants, with larger leaves, fruit, and seeds. Single sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) analyses were performed on shoots immediately after recovery from cryopreservation and on shoots subcultured for 7 mo before analysis. Ten SSR primers developed from “Marion” and “Meeker” microsatellite-enriched libraries amplified one to 15 alleles per locus, with an average of seven alleles and a total of 70 alleles in the four genotypes tested. No SSR polymorphisms were observed between cryopreserved shoots and the corresponding mother plants regardless of subculture. Although no polymorphisms were detected in shoots analyzed immediately after recovery from cryopreservation, AFLP polymorphisms were detected in three of the four Rubus genotypes after they were subcultured for 7 mo. Field-grown plants from the polymorphic shoot tips of R. grabowskii and ‘Silvan’ displayed the same AFLP fingerprints as their corresponding mother plants. Only long-cultured in vitro shoot tips displayed polymorphisms in vitro, and they were no longer detected when the plants were grown ex vitro. The transitory nature of these polymorphisms should be carefully considered when monitoring for genetic stability.     

In vitro induction, regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus

In the present paper attempts were made to induce chromosome doubling of ‘Meiwa’ kumquat (Fortunella crassifolia) protoplasts and ‘Frost’ navel orange (Citrus sinensis Osbeck) embryogenic callus via colchicine treatment. Colchicine decreased protoplast viability, delayed protoplast division and inhibited callus growth, indicating presence of toxicity to cells. Cell lines established from ‘Meiwa’ protoplasts treated with 0.01 and 0.1% colchicine for 8, 16 and 24 h at each concentration showed different responses when they were cultured on embryoid-induction medium. Flow cytometry (FCM) demonstrated that tetraploids were detected in cell lines and embryoids from all of the treatments, with the highest frequency being 19.23%. As for ‘Frost’, tetraploid cells were only detected when the callus was treated with 0.1% colchicine for either 4 or 8 days, from which plantlets were regenerated. FCM and chromosome counting confirmed them as true tetraploids. The diploid cells were more active in mitotic division during a 12-day culture and smaller in size than their tetraploid counterpart. Potential applications of the novel tetraploid germplasms obtained through in vitro chromosome doubling to citrus cultivar improvement are discussed.