X. X. Deng

Wide somatic hybrids of Citrus with its related genera and their potential in genetic improvement

Citrus wild relatives are an untapped germplasm reservoir, which possesses many elite resistance traits. Genetic introgression into Citrus by conventional methods has been greatly hindered by polyembryony, pollen / ovule sterility, sexual / graft incompatibility, long juvenility etc. Somatic hybridization via protoplast fusion may make it possible to transfer genes from wild relatives to Citrus. To date, more than sixty sexually compatible or incompatible intergeneric somatic hybrids between Citrus and its various related wild genera have been produced by PEG - orelectrically - induced fusion. These wide somatic hybrids were identified by morphology, cytology, isozyme, RAPD and RFLP analyses. Genetic variation or recombination was also revealed in some of them. Several sexually compatible combinations have flowered and set fruits. The agronomic performance of these wide somatic hybrids is diverse, and the current results are not conclusive. Somatic hybrids are being tested as rootstocks. The prospects of wide somatic hybridization of Citrus with its wild relatives are discussed.

Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein.

Regeneration and analysis of citrus interspecific mixoploid hybrid plants from asymmetric somatic hybridization

Embryogenic protoplasts of Dancy tangerine (Citrus reticulata Blanco) were X-ray irradiated at three doses and electrofused with iodoacetic acid-treated embryogenic protoplasts of Page tangelo (C. reticulata Blanco × C. paradisi Macf.). Shoots could regenerate only from the fusion combination with the lowest irradiation dose, but were recalcitrant to rooting. In vitro grafting was applied to obtain complete plants. Chromosome examination showed that the plants contained mainly diploid and aneuploid cells, together with few tetraploid cells, indicating that they were mixoploids. Random amplified polymorphic DNA analyses with 10-mer arbitrary primers confirmed the plants as true somatic hybrids. This is the first report on regeneration of mixoploid hybrid plants via protoplast asymmetric fusion in Citrus. Negative effects of ionizing irradiation on regeneration of embryoids and plantlets and possible agronomic interest of the mixoploid plants are also discussed.

Regeneration of citrus sinensis (+) clausena lansium intergeneric triploid and tetraploid somatic hybrids and their identification by molecular markers

SummarySomatic hybrid plants were regenerated via electrofusion between leaf-derived protoplasts of ‘Chicken heart’ sweet wampee (Clausena lansium) and embryogenic protoplasts of ‘Newhall’ navel orange (Citrus sinensis Osbeck). Most of the complete plantlets were formed via mini-grafting. Flow cytometry showed that most of the regenerants were tetraploids as expected, but unexpectedly three plantlets were triploids. Simple sequence repeat (SSR) analysis of seven randomly selected tetraploids and the three triploids showed that they had specific fragments from both fusion parents, thereby confirming their hybridity. Analysis of cytoplasmic genomes using universal primers revealed that their chloroplast DNA (cpDNA) band pattern was identical to the mesophyll parent, while their mitochondrial genomes were of the navel orange type. According to the SSR results, the triploids obtained in this study were most likely due to chromosome elimination of ‘Chicken heart’ sweet wampee prior to plant regeneration.

Somatic hybrids between navel orange (Citrus sinensis) and grapefruit (C. paradisi) for seedless triploid breeding.

Protoplasts from cell suspension cultures of ‘Bonnaza’ navel orange (Citrus sinensis (L.) Osbeck) were electrically fused with mesophyll protoplasts isolated from seedless ‘Red Blush’ grapefruit (Citrusparadisi). After 6 months of culture, a total of 20 plants were regenerated. Root tip chromosome counting revealed that 4 of them were tetraploids (2n = 4x = 36)and the rest were diploids (2n = 2x = 18) morphologically resembling the mesophyll parent. After 6 months of transplantation into the greenhouse, 4 of the diploidmesophyll regenerants unexpectedly flowered, but this phenomenon disappeared in the next year. This is the first report of precocious flowering in citrus via protoplast fusion. RAPD analysis further confirmed that the tetraploid regenerants were somatic hybrids while the diploid regenerants were mesophyll parent type. This somatic hybrid will be utilized as a possible pollen parent for improving the seedy pummelo cultivars in China by producing triploid seedless pummelo hybrid. The mechanism of early flowering was also discussed.

High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis

Transformation and high efficient regeneration of transgenic plants from embryogenic calluses of Bingtang sweet orange [Citrus sinensis (L.) Osbeck] was reported. Embryogenic calluses were inoculated with Agrobacterium tumefaciens strain EHA105, harboring the binary Ti plasmid pROK II and carrying a neomycin phosphotransferase II (NPTII) gene, an intron β-glucuronidase (GUS) gene and the Arabidopsis APETALA1 (AP1) gene. Transformation treatment was with inoculation time of 30 min, co-culture of 3 d at 23 °C and supplementation of the co-culture medium with 2 mg dm−3 acetosyringone (AS). Kanamycin (50 mg dm−3) was effective to inhibit the growth of non-transformed calluses while it did not affect the transformed ones. The total number of transformed callus lines was 7 with 100 % embryo induction. High efficient regeneration of the transgenic embryos (88 % with 4–5 shoots per embryoid) was realized within 3 months. Integration of the transgene into the citrus genome was confirmed by histochemical GUS staining, polymerase chain reaction (PCR) analysis with AP1-specific primer and Southern blot hybridization with a 712 bp PCR fragment of AP1 as the probe.

Genetic diversity in mandarin landraces and wild mandarins from China based on nuclear and chloroplast simple sequence repeat markers

Summary Landraces and wild germplasm are important citrus genetic resources. Knowledge of the genetic diversity of this germplasm provides an opportunity both to mandarin breeding programmes and to germplasm conservation strategies. Genetic diversity of 33 loose-skin mandarin landraces and 19 wild mandarin accessions were analysed with ten nuclear simple sequence repeat (nSSR) and eight chloroplast simple sequence repeat (cpSSR) markers. Our analysis of nuclear and chloroplast genome polymorphism showed that the genetic diversity in wild populations was slightly higher than that found in landraces, with polymorphic information content values of 0.6138 and 0.5091 at nSSR loci, and 0.4921 and 0.3769 at cpSSR loci, respectively. Overall, 60 nSSR alleles and 19 cpSSR haplotypes were identified, and all but seven accessions were unambiguously discriminated by nSSR markers. Five groups of landraces and two groups of wild mandarins were identified based on the nSSR data, and two groups each of landraces and wild mandarins were identified at the cpSSR loci. Different distribution patterns of landraces and wild mandarins were observed between dendrograms constructed using these two marker systems, and hybrid origins for several landraces were suggested by these results. Combined analysis also suggested that, among wild mandarin, ‘Chongyi’ showed the closest relationship to landraces, and that landrace ‘Chazhigan’ was the primary genotype among landraces.

Universal chloroplast primer pairs for Simple Sequence Repeat analysis in diverse genera of fruit crops

To provide an alternative method to investigate chloroplast genome (cpDNA) diversity in sub-tropical and tropical fruit crops, where cpDNA sequences are unknown, a universal set of chloroplast simple sequence repeat (cpSSR) primers were developed. Thirty-three cpSSR primers from rice (Oryza sativa), pine (Pinus thunbergii), tobacco (Nicotiana tabacum) and Arabidopsis thaliana were applied to 45 fruit species (or varieties) belonging to 27 genera in 20 families. After being screened, the utility of these primers was verified. Experiments revealed that nine out of 14 primer pairs from N. tabacum, and seven out of 11 from A. thaliana, specifically amplified bands in cpDNA from most species tested, and polymorphisms were observed. While four out of the five primers from O. sativa generated intensive bands in agarose gels, but only a smear in polyacrylamide gels, all cpSSR primers from P. thunbergii failed to generate any specific amplified products in polyacrylamide gels. The results illustrate the feasibility of exploring diversity among unknown cpDNA sequences in exotic minor fruit crops by PCR methods.

Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast-protoplast fusion.

Cytoplasm of Satsuma mandarin (Citrus unshiu Marc.) is known to influence seedlessness. Transfer of cytoplasm to a seedy cultivar could possibly lead to the production of seedless citrus fruits. In the present paper cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin via ultra-centrifugation in a discontinuous gradient. No nucleus could be detected in the cytoplasts by DAPI (4′, 6-diamidino-2-phenylindole) staining compared with normal protoplasts. The cytoplasts, with high viability and small size, did not divide during solid embedding culture. Cytoplasts of Satsuma mandarin were electrically fused with embryogenic protoplasts of Murcott tangor (C. reticulata × C. sinensis), which led to regeneration of several cell lines. Flow cytometry (FCM) indicated that the cell lines were diploids. Simple sequence repeats (SSR) and cleaved amplified polymorphism sequence (CAPS) showed that the cell lines got their nuclear DNA from the protoplast parent, whereas the cytoplast parent donated the mtDNA, confirming transfer of mtDNA from Satsuma mandarin into Murcott tangor via cytoplast–protoplast fusion though no polymorphism was detected in chloroplast DNA between the fusion partners. This is the first report on isolation and characterization of cytoplasts, together with cytoplast–protoplast fusion in Citrus, which has a potential for citrus cultivar improvement involving cytoplasm transfer via cytoplast–protoplast fusion.

Cell size as a morphological marker to calculate the mitotic index and ploidy level of citrus callus

Abstract. We have developed a new method for calculating the mitotic index (MI) and ploidy level of citrus callus. The first step was to symbolize the diameters of new daughter cells and metaphase cells with RD and RM, respectively. The formula RD=0.7937 RM was subsequently established. The validity of this hypothesis was verified by morphological, cytological and fluorescent observations as well as by a protoplast electrofusion test. The MI and the ploidy level of a cell population were then calculated according to this formula. As a final step, tetraploid and hexaploid cell lines were produced from a mixoploid cell population, and somatic embryos were regenerated from these cell lines. These results suggest that cell size can be used as a morphological marker to calculate MI and ploidy level of citrus callus.