Protoplast Fusion Technology – Somatic Hybridization and Cybridization

Grosser, Jude W.; Ćalović, Milica; Louzada, Eliezer S.

doi:10.1002/9780470686522.ch10

Cited by 20 publications

(13 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The protoplast fusion between wmCENH3-RFP transgenic wheat line C3216 and ZmCENH3-YFP transgenic maize was performed by PEG-mediated somatic hybridization [25] [37] [38]. After re-suspending wheat and maize pellets in P5 medium, wheat and maize protoplasts were mixed together at a ratio of 1: min.…”

Section: Symmetric Hybridization Between Wheat and Maizementioning

confidence: 99%

Synthesis of a Wheat/Maize Hybrid CENH3 Gene, the Genetic Transformation of Wheat, Its Chromosomal Localization and Effects on Chromosome Behaviors in Wheat/Maize Somatic Hybrids

Yang¹,

Li²,

Yu³

2019

View full text Add to dashboard Cite

Centromere-specific histone H3 (CENH3) replaces the canonical histone H3 in nucleosomes of functional centromeres, and plays important roles in faithful chromosome segregation during cell division. CENH3 is also important in the recognition of alien centromeres and determines the accommodation or elimination of alien chromosomes in interspecific or intergenic hybridization. In this study, a maize full length CENH3 with a yellow fluorescent protein (YFP) tag at C-terminus (ZmCENH3-YFP) and a synthetic hybrid wmCENH3 with the N-terminus from wheat CENH3 and the histone fold domain (HFD) from maize tagged with a red fluorescent protein (RFP) at the C-terminus (wmCENH3-RFP) were transformed to wheat by biolistics transformation. Transgenic wheat plants with both ZmCNEH3-YFP and wmCENH3-RFP genes were identified by PCR. The expression of ZmCENH3-YFP was not observed, while the expression of wmCENH3-RFP could be detected by RT-PCR, direct fluorescence microscopy, and immunostaining with anti-RFP antibody. The expressed wmCENH3-RFP was localized to nuclei as dotted patterns, indicating its targeting to wheat centromeres. Somatic hybridization was performed between wmCENH3-RFP transgenic wheat and transgenic maize that expressed a ZmCENH3-YFP gene to investigate chromosome behaviors in somatic hybrids. Cytological and FISH analyses of somatic hybrid 986 Agricultural Sciences cells showed the formation of micronuclei and lagging chromatin in both somatic hybridizations with or without the wmCENH3-RFP transgene, indicating that ectopically expressed wmCENH3 could not overcome chromosome elimination in wheat/maize somatic hybrids. Immunostaining of wmCENH3-RFP and ZmCENH3-YFP in early stage somatic hybrid cells indicated that both wmCENH3-RFP and ZmCENH3-YFP proteins were expressed, but their binding patterns changed from the commonly observed dotted patterns to diffused ones, suggesting that the inactivation of CENH3 might be a factor for chromosome elimination in wheat/maize somatic hybridization.

show abstract

Section: Symmetric Hybridization Between Wheat and Maizementioning

confidence: 99%

Synthesis of a Wheat/Maize Hybrid CENH3 Gene, the Genetic Transformation of Wheat, Its Chromosomal Localization and Effects on Chromosome Behaviors in Wheat/Maize Somatic Hybrids

Yang¹,

Li²,

Yu³

2019

View full text Add to dashboard Cite

show abstract

“…The embryogenic suspension culture of 'Dancy' mandarin was started from unfertilized ovule-derived friable embryogenic callus cultures maintained in the citrus embryogenic callus collection of the University of Florida's Citrus Research and Education Center, Lake Alfred [UF-CREC (Grosser and Gmitter, 1990)]. Suspension-derived protoplasts were isolated from 1-to 2-yearold suspension cultures maintained in H+H medium (Grosser and Gmitter, 1990) on a 2-week subculture cycle, with protoplasts isolated during days 4-12 (Grosser and Gmitter, 1990;Grosser et al, 2010b). Prepollen haploid protoplasts from 'Ruby Red' grapefruit were isolated as follows (Grosser and Gmitter, 1990): large elongated unopened flower buds were collected and meiotic stages were estimated by squashing anthers in water and examining the squashes using an inverted microscope at •200 magnification.…”

Section: Plant Materialsmentioning

confidence: 99%

“…Fruit were twisted with each half in opposite directions until separated to allow for removal of intact seeds. Seeds were germinated and seedlings were maintained in Magenta vessels (GA-7; Magenta Corp., Chicago, IL) containing 50 mL RMA medium (Grosser and Gmitter, 1990;Grosser et al, 2010b) and wrapped with a sealing film (Nescofilm Ò ; Bando Chemical Industries, Kobe, Japan). Tender, expanded leaves from the cultured nucellar seedlings were used for protoplast isolation.…”

Section: Plant Materialsmentioning

confidence: 99%

“…PROTOPLAST ISOLATION AND FUSION. Protoplasts were isolated from the parental suspension cultures in a 2.5:1.5 (v:v) mixture of 0.7 M BH3 protoplast culture medium and enzyme solution as described previously (Grosser and Gmitter, 1990;Grosser et al, 2010b). Sterile leaves from in vitro cultured nucellar seedlings were feather-cut with a sharp scalpel, and macerated anthers were incubated overnight (including a 25-min vacuum infiltration) in an 8:3 (v:v) mixture of 0.6 M BH3 protoplast culture medium and modified enzyme solution.…”

Section: Plant Materialsmentioning

confidence: 99%

See 1 more Smart Citation

Cybridization of Grapefruit with ‘Dancy’ Mandarin Leads to Improved Fruit Characteristics

Satpute

Chen

Gmitter

et al. 2015

J. Amer. Soc. Hort. Sci.

Self Cite

View full text Add to dashboard Cite

In cybridization, new combinations of nuclear and cytoplasmic genes result in a unique genotype that may bring cellular, physical, physiological, and biochemical changes to the plant. This has been demonstrated in the unexpected cybrids generated from the fusion of citrus (Citrus sp.) protoplasts in two independent experiments. The first experiment was conducted to generate potentially seedless triploids by fusing diploid protoplasts of embryogenic ‘Dancy’ mandarin (Citrus reticulata) suspension culture cells with haploid ‘Ruby Red’ grapefruit (C. paradisi) protoplasts derived from tetrad-stage microspores. After multiple attempts, only one triploid was recovered, but several diploid plants with typical grapefruit morphology were also regenerated. In the second experiment, protoplasts derived from embryogenic ‘Dancy’ mandarin suspension culture were fused with nonembryogenic protoplasts from ‘Duncan’ grapefruit leaves in an effort to produce an allotetraploid somatic hybrid. The fruit from the resulting trees resembled grapefruit in morphology and type, and maintained excellent quality throughout the summer, when commercial grapefruit rapidly loses quality. Fruit on these trees remained firm with exceptional sweetness and good flavor into August, and without seed germination. The regenerants obtained in the protoplast fusion experiments were confirmed as cybrids by genetic marker analyses. The test grapefruit were identical to commercial ‘Ruby Red’ grapefruit at six nuclear simple sequence repeat (SSR) marker loci, but identical to ‘Dancy’ with respect to a mitochondrial intron marker. The plastid genomes of individual trees originated from either fusion partner. In the first experiment, haploid ‘Ruby Red’ protoplast preparations must have also contained contaminant diploid protoplasts. Apart from the value of altered fruit quality attributes in the marketplace, these plants provide an opportunity to understand the contributions of cytoplasmic organelle genetics to important citrus fruit-breeding objectives.

show abstract

“…Recently, Davey et al (2010) and Grosser et al (2010) published practical manuals with numerous protocols for isolation, culture, fragmentation and fusion.…”

Section: Introductionmentioning

confidence: 99%