Doubled haploid callus lines of Valencia sweet orange recovered from anther culture

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial plants using conventional breeding techniques is currently a challenge because of a long juvenile period, high heterozygosity and the substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In this study, we report the regeneration of doubled haploid lines of Valencia sweet orange cv. Rohde Red (Citrus sinensis [L.] Osbeck) via anther culture. Anthers at the uninucleate stage were induced and two embryogenic calli were obtained that further regenerated to embryoids (2/400). Plantlets were obtained after transferring the embryoids to a shoot regeneration medium, but were short-lived. Ploidy analysis via both flow cytometry and chromosome counting verified that these two lines were diploids. Additionally, 43 simple sequence repeat (SSR) markers which showed to be heterozygous in the Valencia sweet orange donor line confirmed homozygosity and doubled haploids in the anther-derived lines. Furthermore, analysis of the doubled haploids via cleaved amplified polymorphic sequence (CAPS) markers and target region sequencing confirmed the allelic state of two genes (LCYE and LCYB) involved in the carotenoid biosynthesis of sweet oranges.     

Accelerated production and identification of fertile,homozygous transgenic wheat lines by anther culture

Anther culture has been developed in the winter wheat cultivar Florida to achieve accelerated production and identification of homozygous transgenic lines. With untransformed, seed-derived plants to develop the culture system, it was shown that cold pre-treatment of spikes excised from donor plants and addition of 2,4-dichlorophenoxyacetic acid together with either kinetin or 6-benzylaminopurine in the callus induction medium improves the anther culture response. The procedure developed allowed production of fertile homozygous lines within 8–9 months, which includes an 8-week vernalisation period. With transgenic wheat plants produced by particle bombardment as donors, we show that the system can be used to produce homozygous transgenics, requiring one generation cycle. Both T₀ tissue culture-derived plants and their T₁ seed-derived descendents serve as suitable donors. We show that an anther culture response comparable to that of untransformed, seed-derived plants can be achieved with T₀ tissue culture-derived plants. PCR and Southern molecular analyses of anther culture-derived transgenics show that the transgenes are stably inherited; there are no perturbations at the chromosomal level around the sites of transgene integration as a result of in vitro chromosome manipulation during anther culture.     

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.     

Variation of cytosine methylation in 57 sweet orange cultivars

Sweet orange is an important group of citrus cultivars, which includes a number of bud sport cultivars. Little is known about the CpG methylation status of the CCGG sequences in the orange genome. In this study, methylation-sensitive amplification polymorphism (MSAP), based on the application of isoschizomers (Hpa II and Msp I), was first used to analyze cytosine methylation patterns in 57 orange cultivars that were not fully differentiated by regular DNA molecular markers. Three types of bands were generated from ten primer pairs. Type I bands were present following restriction with Eco RI + Hpa II and Eco RI + Msp I; type II or type III were present only following restriction with either Eco RI + Hpa II or with Eco RI + Msp I. The total number of these three types of bands was 802, 72, and 157, respectively. Among these, the number of polymorphic bands were 244 (30.2%), 23 (31.9%), and 32 (20.4%), in type I, II and III, respectively. The methylation patterns of these 57 cultivars are discussed and assessed by dendrograms derived from the analysis of polymorphic MSAP bands. The distribution of polymorphic bands of the above three types demonstrate the methylation patterns and frequency at the cytosine loci. We suggest that methylation events could be more frequent than demethylation events, and that the methylation patterns maybe associated with phenotypic traits.     

Cytodifferentiation and transformation of embryogenic callus lines derived from anther culture of wheat

Three types of callus tissues established from anther culture of eleven doubled haploid (DH) lines of wheat (Triticum aestivum L.) were evaluated for their ability in enhancing friable embryogenic (Type II) culture differentiation and genetic transformation. Differences between types of callus inocula were highly significant (P < 0.001), suggesting that the quality of the initial callus explant is of profound importance in encouraging the proliferation of Type II cultures. Other factors found to be crucial included weekly subculture of friable embryogenic callus tissues on a maintenance medium containing 30 microM dicamba and a predominance of amino-acid nitrogen supplement. Transfer and integration of the beta-glucuronidase gene was also affected by the type of inoculum when suitable embryogenic cell cultures were transformed using silicon carbide whiskers and high velocity microprojectiles. Expression of the hygromycin phosphotransferase selectable marker gene sequence was confirmed in all the stably transformed cell lines maintained on selection media containing lethal levels of hygromycin. Comparatively, there were differences in the frequency of regenerable, transgenic clonal segments between whisker-treated and microprojectile bombarded tissues mainly as a result of the fact that cultures vortexed with whiskers were more capable of post-treatment cell proliferation and embryo differentiation than those bombarded with cDNA-coated microprojectiles. Conditions for obtaining these results are outlined and discussed in relation to the suitability of the two transformation strategies for producing transgenic cell aggregates of wheat.     

Biochemical characterization of blood orange,sweet orange,lemon, bergamot and bitter orange

This paper reports on the composition of aroma compounds and fatty acids and some physico-chemical parameters (juice percentage, acidity and total sugars) in five varieties of citrus: blood orange, sweet orange, lemon, bergamot and bitter orange. Volatile compounds and methyl esters have been analyzed by gas chromatography. Limonene is the most abundant compound of monoterpene hydrocarbons for all of the examined juices. Eighteen fatty acids have been identified in the studied citrus juices, their quantification points out that unsaturated acids predominate over the saturated ones. Mean concentration of fatty acids varies from 311.8 mg/l in blood orange juice to 678 mg/l in bitter orange juice.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Recovery of whole plants of sweet orange from somatic embryos subjected to freezing thawing treatments

Freezing/thawing conditions for cryopreservation of somatic embryos of Washington Navel sweet orange (Citrus sinensis (L.) Osb.) were evaluated. No survival of fast-cooled embryos occurred regardless of the thawing method. Embryos subjected to slow cooling at an estimated rate of 0.5°C min^-1 down to –42°C followed by immersion in liquid nitrogen survived. Survival rate depended on the thawing method. An average survival of 30.5% was achieved when frozen embryos were thawed by immersion in a water bath at 37°C. Surviving embryos developed into whole plantlets and no phenotypic abnormalities have been observed during a growth period of four years. Total soluble proteins and peroxidase and esterase isoenzyme analysis did not show differences between treated plants and non-frozen controls.     

Production and molecular characterization of diploid and tetraploid somatic cybrid plants between male sterile Satsuma mandarin and seedy sweet orange cultivars

Seedlessness, an important economic trait for fresh fruit, is among the prior goal for all citrus breeding programs. Symmetric somatic hybridization provides a new strategy for citrus seedless breeding by creating cybrids transferring mitochondrial DNA (mtDNA) controlled cytoplasmic male sterility (CMS) from the callus parent Satsuma mandarin (C. unshiu Marc.) to seedy cultivars. In this study, protoplast fusion was adopted to transfer CMS from C. unshiu Marc. cv. Guoqing No. 1 (G1) to three seedy sweet oranges (C. sinensis L. Osb.), i.e. ‘Early gold’, ‘Taoye’ and ‘Hongjiang’. Flow cytometry analysis showed that 12 of 13 regenerated plants from G1 + ‘Early gold’, 9 of 12 from G1 + ‘Taoye’ and both two plants from G1 + ‘Hongjiang’ were diploids, while the remaining regenerated plants were tetraploids. Molecular analysis using 23 simple sequence repeat (SSR) markers previously proven to map to the citrus genome showed that the nuclear DNA from all recovered diploid and tetraploid plants derived from their corresponding leaf parent, while cleaved amplified polymorphic sequence analysis showed that the mtDNA of all regenerated plants derived from the callus parent, indicating that the regenerated 2X and 4X plants from all these three combinations are authentic cybrids. Furthermore, the Chloroplast SSR analysis revealed that somatic cybrid plants from the three combinations possessed either of their parental chloroplast type in most cases. These results demonstrated that mtDNA of G1 Satsuma mandarin was successfully introduced into the three seedy sweet orange cultivars for potential seedlessness via symmetric fusion.     

A new strategy employed for identification of sweet orange cultivars with RAPD markers

We optimized RAPD techniques by increasing the length of RAPD primers and performing a strict screening of PCR annealing temperature to distinguish 60 sweet orange cultivars from the Research Institute of Pomology at the Chinese Academy of Agricultural Sciences. A new approach called cultivar identification diagram (CID) was used to improve the efficiency of RAPD markers for cultivar identification. Thirteen effective primers were first screened from 54 RAPD arbitrary 11-mer primers based on their amplification products and amplified polymorphic bands; they were then used for PCR amplification of all 60 cultivars. All cultivars were manually and completely separated by the polymorphic bands appearing in DNA fingerprints from 13 primers; a CID of the 60 sweet orange cultivars was then constructed. This CID separated all the cultivars from each other, based on the polymorphic bands; the corresponding primers were marked in the correct positions on the sweet orange CID. The CID strategy facilitates the identification of fruit cultivars with DNA markers. This CID of sweet orange cultivars will be very useful for the protection of cultivar rights and for early identification of seedlings in the nursery industry.     

Phenotypic characterization and bulk segregant analysis of anther culture response in two backcross families of diploid potato

Two diploid (2n=2x=24) backcross potato populations (PBCp, and CBC) were characterized for anther culture response (ACR). PBCp (Solanum phureja Juz. & Buk. genotype 1-3 × CP2) and CBC (CP2 × S. chacoense Bitt. genotype 80-1) resulted from a cross between CP2 (intermediate ACR) and its parents, S. chacoense 80-1(low ACR) and S. phureja 1-3 (high ACR). Three components of ACR were initially investigated: embryos per anther (EPA), embryo regeneration rate and percent monoploids (2n=1x=12) among regenerants. EPA was selected for further characterization because of its relative stability. In a series of studies of EPA on a total of 44 genotypes within CBC, nine high (mean EPA=2.5) and ten low (mean EPA=0.02) selections were made. In PBCp, ten high (mean EPA= 4.7) and ten low (mean EPA= 0.05) selections were made from 67 genotypes. High and low selections were used for bulk segregant analysis to screen 214 RAPD primers as candidate markers linked to EPA. Bands amplified by OPQ-10 and OPZ-4 were associated in coupling and repulsion, respectively, to ACR in PBCp. A band amplified by OPW-14 primer was associated in coupling to ACR in CBC. One-way ANOVAs using presence/absence of each candidate band to classify additional genotypes in each population verified association of the markers with EPA.     

Characteristics of Cd accumulation and distribution in two sweet potato cultivars

In this study, we compared the chemical forms and subcellular distribution of Cd in high-Cd (X16) and low-Cd (N88) sweet potato cultivars through hydroponic experiments and examined the Cd distribution in their roots by histochemical staining. The results showed that inorganic and pectate/protein-integrated Cd predominated in the leaves, and Cd concentrations were significantly higher in X16 than in N88. However, in the roots, Cd was mostly integrated with pectate and protein, and Cd concentration was higher in N88 than in X16. It was mainly stored through vacuolar sequestration and cell wall binding. In the leaves and stems, Cd concentrations in all subcellular fractions were higher in X16 than in N88; the opposite was observed in the roots. In X16, Cd was mostly accumulated in the root stele, and its Cd translocation factor was higher than that of N88. Overall, the subcellular fractions of X16 roots retained less Cd than N88 roots, and more Cd entered the root stele of X16 and subsequently moved to the shoots. The higher amounts of inorganic, water-soluble, and pectate/protein-integrated Cd with high mobility in the shoots of X16 than in N88 might facilitate Cd remobilization to other tissues, but this needs to be further studied.     

Breeding for nematode and virus resistance in potato via anther culture

Summary In Solanum tuberosum the production by parthenogenesis of 2x plants with 24 chromosomes, and the regeneration of microspores of such dihaploids to yield monohaploid (1x) plants is reproducibly possible, at least for some specific genotypes. Experiments are described using tissue culture techniques in an applied breeding program with the main aim of increasing the level of resistance to the potato cyst nematode Globodera pallida (Stone) and to the potato viruses X, Y and leaf roll. These resistances follow quantitative as well as qualitative modes of inheritance. Using anther culture it is demonstrated that doubled monohaploid clones can be produced which possess the resistance in the homozygous condition. In both ways of inheritance the ratio of resistant clones is rather high. The genotype of the anther donor plant has, however, a strong influence on the total number of androgenetic plants which can be regenerated. Therefore, experiments were initiated with the aim of integrating this capacity for regeneration (tissue culture ability) into valuable genotypes. The results show that the potentiality for regeneration is under genetic control and can be utilized by combination breeding. Its inheritance and physiological basis, as well as the behaviour of complete homozygous clones, is discussed.Dedicated to Prof. Dr. Joseph Straub on the occasion of his 70. birthday     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Recovery and characterization of transgenic plants from two spring wheat cultivars with low embryogenesis efficiencies by the bombardment of isolated scutella

Summary Two commercial wheat cultivars with low embryogenesis efficiencies, AC Karma and Hy417, were transformed by the bombardment of isolated scutella with two gene constructs. Three AC Karma plants (433, 436, and 437) carrying plasmid pRC62 containing a gus:npt fusion gene, and one Hy417 plant (438) carrying plasmid pBARGUS containing a bar gene and a gusA gene were recovered and characterized. Presence of transgenes in T0 and T1 plants was confirmed by both PCR and Southern hybridization. Copy number of transgenes varied from one to six in these four plants. The inheritance of transgenes in the progeny was characterized. The gusA gene and its activity in AC Karma plant 436 and bar gene and its activity in Hy417 plant 438 segregated in the selfed T1 progeny in a Mendelian 3:1 ratio, but gusA gene and its activity in AC Karma plants 433 and 437 segregated in selfed T1 progeny in a non-Mendelian 1:1 ratio. The gusA activity in all three AC Karma plants was stably transmitted to selfed T2 or T3 progenies. The levels of gusA and nptII activities in nine T1 plants from AC Karma plant 437 were also determined. A GusA fluorometric assay indicated that gusA activity in the nine T1 plants increased by 2.5–7.2-fold compared with the nontransformed control, while and NptII ELISA assay detected nptII activity only in two of the nine T1 plants, suggesting the nptII gene was silenced in the other seven T1 plants.     

Production of doubled-haploid plants from tritordeum anther culture

The effects of different media and cold pretreatment of spikes on the androgenic response and regeneration capacity from anther culture of tritordeum was studied. L5 medium gave the highest frequency of anther response. The frequency of cultures regenerating green or albino plantlets was not affected by the composition of the medium tested. Cold pretreatment of the spikes significantly increased the frequency of anther response and also the percentage of cultures giving albino plantlets. A mean of four green plants was obtained per 100 subcultured calli/embryos. The percentage of spontaneous chromosome doubling was only 1%. The addition of colchicine at 0.02% to the induction medium significantly increased the frequency of doubled haploids regenerated without any effect on regeneration capacity. This technique proved more efficient than a conventional chromosome-doubling method.     

Influence of anther pretreatment and culture medium composition on the production of barley doubled haploids from model and low responding cultivars

Different pretreatments were given to anthers of barley before culturing, and their effects assessed on the frequency of embryos and green doubled haploid plants produced. Mannitol pretreatment was better than cold pretreatment for some low responding cultivars. Optimal concentration of mannitol for pretreatment depended on cultivar. Low responding genotypes needed a higher concentration of mannitol than responsive ones. The addition of Ficoll to liquid medium increased the number of embryos and green plants. The influence of the growth regulators 2,4-D and TIBA was assayed using ten cultivars of barley grown in Spain. The anti-auxin TIBA gave good embryo production with some of the low responding cultivars. Two row-type cultivars always produced higher number of embryos and green plantlets than six row-type. The application of these modifications to 10 F₁ hybrids with potential agronomic value, allowed the production of almost 1000 doubled haploid plants from only 3500 anthers. Up to two doubled haploid plants per flower were produced from the cross Monlon × Sonja. This revised version was published online in June 2006 with corrections to the Cover Date.     

Temporal patterns of citrus greasy spot‐induced defoliation of sweet orange cultivars in Brazil

Recôncavo Baiano is an area favourable for the occurrence of citrus greasy spot (CGS) (Mycosphaerella citri), but there has been no study of this pathosystem in Brazil. This work aimed to characterise the temporal patterns of CGS‐induced defoliation in sweet orange cultivars ‘Bahia’ (Washington Navel) and ‘Pêra’. Temperature, rainfall and relative humidity were recorded, as well as weekly defoliation (fallen leaves/canopy m² or m³). Considering the mean of fallen leaves per canopy m², and mean canopy area, the total annual defoliation was estimated to be around 32 000 leaves per plant for ‘Bahia’ and 18 500 for ‘Pêra’ sweet orange. Spectral density analysis showed that defoliation has a 5‐week‐long main cycle for both cultivars. The proportion of symptomatic fallen leaves was never below 0.97. The monthly number of fallen leaves per canopy area was positively correlated with the mean CGS incidence on leaves. Defoliation was significant, resulting in a low leaf density throughout the year. Many defoliation cycles and the very high proportion of symptomatic fallen leaves assure a constant inoculum supply. Based on these results, CGS cannot be considered a minor disease, at least in Recôncavo Baiano.     

The improvement in regenerated doubled haploids from anther culture of wheat by anther transfer

This study was conducted to determine the most suitable method of regeneration by comparing two approaches: transfer of anthers (with and without embryo-like structures) to regeneration conditions after a period of two to four weeks on induction medium (= anther-transfer treatment) and transfer of embryo-like structures to regeneration conditions after five to eight weeks on induction medium. The early transfer of anthers brought about a significant reduction in the number of embryos formed, but nevertheless significantly improved the frequency of plant regeneration. Combining an optimal date of anther transfer with the early addition of colchicine to the induction medium (100 mg l⁻¹ for 1 and 3 days) led to an increase in the number of doubled haploid regenerants. The results indicate that transferring the anthers after 28 days and adding 100 mg l⁻¹ colchicine to the induction medium on one day only caused a significant improvement in the ability of green plants to regenerate (7.0 compared to 0.50) as well as in chromosome doubling (success index: 4.0 compared to 0.33). This revised version was published online in August 2006 with corrections to the Cover Date.