Mesophyll Parent Research Articles

Spatiotemporal profiles of gene activity in stamen delineate nucleo-cytoplasmic interaction in a male-sterile somatic cybrid citrus.

Cytoplasmic male sterility (CMS) has long been used to produce seedless fruits in perennial woody crops like citrus. A male-sterile somatic cybrid citrus (G1 + HBP) was generated by protoplast fusion between a CMS callus parent 'Guoqing No. 1' Satsuma mandarin (Citrus unshiu, G1) and a fertile mesophyll parent Hirado Buntan pummelo (Citrus grandis, HBP). To uncover the male-sterile mechanism of G1 + HBP, we compared the transcriptome profiles of stamen organ and cell types at five stages between G1 + HBP and HBP, including the initial stamen primordia, enlarged stamen primordia, pollen mother cells, tetrads, and microspores captured by laser microdissection. The stamen organ and cell types showed distinct gene expression profiles. A majority of genes involved in stamen development were differentially expressed, especially CgAP3.2, which was downregulated in enlarged stamen primordia and upregulated in tetrads of G1 + HBP compared with HBP. Jasmonic acid- and auxin-related biological processes were enriched among the differentially expressed genes of stamen primordia, and the content of jasmonic acid biosynthesis metabolites was higher in flower buds and anthers of G1 + HBP. In contrast, the content of auxin biosynthesis metabolites was lower in G1 + HBP. The mitochondrial tricarboxylic acid cycle and oxidative phosphorylation processes were enriched among the differentially expressed genes in stamen primordia, meiocytes, and microspores, indicating the dysfunction of mitochondria in stamen organ and cell types of G1 + HBP. Taken together, the results indicate that malfunction of mitochondria-nuclear interaction might cause disorder in stamen development, and thus lead to male sterility in the citrus cybrid.

Mitochondrial Genome of Callus Protoplast Has a Role in Mesophyll Protoplast Regeneration in Citrus: Evidence From Transgenic GFP Somatic Homo-Fusion

Protoplast fusion has great potential in citrus improvement. Although citrus mesophyll protoplasts usually cannot divide and regenerate, symmetric protoplast fusion of embryogenic callus protoplast + mesophyll protoplast sometimes results in the regeneration of mesophyll-parent-type cybrids. It suggested that mitochondrial DNA (mtDNA) from protoplasts of embryogenic callus parent plays an important role in stimulating division and regeneration of mesophyll protoplasts. Herein, somatic fusion was conducted via electrofusion between callus protoplasts isolated from Valencia orange [Citrus sinensis (L.) Osbeck] cell suspension cultures and transgenic GFP-tagged mesophyll protoplasts from the same genotype, i.e. transgenic Valencia orange plants containing the green fluorescent protein (GFP) gene, in an effort to elucidate whether mtDNA of callus line could stimulate the division and regeneration of mesophyll protoplasts from the same genotype. Two embryoids and one plantlet with GFP expression were successfully obtained and subsequent ploidy analysis by flow cytometry indicated that they were all diploids. The regenerated diploid embryoids and plantlet with GFP expression could be considered as ‘cybrids’ with mtDNA from the callus protoplasts of Valencia orange. The result indicated that citrus mesophyll-parent-type cybrid regeneration needed the stimulation of mtDNA from protoplasts of embryogenic callus parent regardless of their origin either from another genotype or the same genotype as the mesophyll parent.

Production of somatic hybrids between satsuma mandarin (Citrus unshiu) and navel orange (Citrus sinensis) by protoplast fusion

We have regenerated altotetraploid plants that are interspecific somatic hybrids between Citrus sinensis Osbeck cv. Yoshida navel orange and Citrus unshiu Marc cv. Okitsu satsuma mandarin. Protoplasts isolated from ‘Yoshida’ leaves were chemically fused with call us-derived protoplasts from ‘Okitsu’. After 6 months of culture, 102 plants were obtained. These hybrids were identified by differential leaf morphology, DNA fluorescence intensity, and DNA analysis. Ploidy analysis via the flow cytometry revealed that 15 of the 102 plants were tetraploids, with the rest being diploids that morphologically resembled their mesophyll parent. SRAP analysis confirmed that 9 of the tetraploid plants were allotetraploid somatic hybrids. These will be utilized as a possible pollen parents for improving seedy citrus cultivars, e.g., ponkan, mandarin, lemon and kumquat, in order to produce triploid seedless hybrids.

Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy Citrus 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.

GFP expression as an indicator of somatic hybrids between transgenic Satsuma mandarin and calamondin at embryoid stage

Somatic hybridization was performed via electrofusion between embryogenic suspension-derived protoplasts of transgenic green fluorescent protein (GFP) Satsuma mandarin (Citrus unshiu Marc. cv. Guoqing No. 1) (G1) callus and mesophyll protoplasts of calamondin (Citrus microcarpa Bunge), and three embryoids expressing GFP under UV light were obtained after 60 days of culture. The three embryoids were considered not as diploid cybrids but true allotetraploid somatic hybrids, as it was based on: (1) citrus heterokaryons are generally more vigorous and have higher capacity for embryogenesis as compared with unfused and homo-fused embryogenic callus protoplasts; (2) the callus line of G1 Satsuma mandarin has lost the embryogenesis capacity; and (3) citrus diploid cybrids produced by symmetric fusion always possess nuclear genome of mesophyll parent, and calamondin without GFP gene was used as leaf parent in this study. Subsequent flow cytometry, simple sequence repeat and cleaved amplified polymorphic sequence analysis of one regenerated callus mass and three resulting plants validated this supposition, i.e., the callus was derived from transgenic G1 callus protoplasts, and the three plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from callus parent. The potential of transgenic GFP citrus callus as suspension parent in citrus somatic fusion to study the mechanism of cybrid formation, create new citrus cybrids, and transfer organelle-encoded agronomic traits was also discussed.

Somatic hybrid vigor in Citrus: Direct evidence from protoplast fusion of an embryogenic callus line with a transgenic mesophyll parent expressing the GFP gene

Previous experience with citrus somatic fusion suggests that hybrid cells are generally more vigorous and have higher capacity for embryogenesis, compared to unfused cells from the embryogenic callus parent. However, this observation has never been validated due to the unavailability of an appropriate experimental design. Citrus mesophyll protoplasts never divide and regenerate into plants. Herein, mesophyll protoplasts of transgenic Valencia orange ( Citrus sinensis) which expressed the green fluorescent protein (GFP) gene were fused with protoplasts isolated from an embryogenic callus line of Shekwasha mandarin ( C. reticulata) to visually screen somatic hybrids. At the multi-cell cluster stage, the regenerated callus lines expressing GFP were morphologically identical to the callus parent regenerate. All hybrid callus lines expressing the GFP developed into embryoids, while most callus parent regenerates still remained in a callus stage. Embryoids with strong or weak GFP expression were verified to be diploid cybrid and tetraploid somatic hybrid embryoids, respectively, by flow cytometry, simple sequence repeat (SSR) and chloroplast SSR analyses. Diploid cybrid globular embryoids appeared light green in color and had a tendency to enter subsequent developmental stages earlier than the dark green tetraploids; however, GFP plants were regenerated from both sources. Thus, expression of the GFP marker gene was effectively used to visually screen somatic hybrids, and confirmed our previous observation that somatic hybrid tissue has a regeneration advantage. The intensity of GFP fluorescence and the color difference of regenerated embryoids was also an early indicator of ploidy level (based on visual GFP observation and confirmed by flow cytometry analysis), and could serve as a tool to study the regeneration mechanism of diploid cybrids via symmetric fusion in citrus and other higher plants.

GISH, AFLP and PCR-RFLP analysis of an intergeneric somatic hybrid combining Goutou sour orange and Poncirus trifoliata.

Intergeneric somatic hybrids combining Goutou sour orange (Citrus aurantium L.) with trifoliate orange Poncirus trifoliata (L.) Raf] were produced by electrofusion and their genetic inheritance analyzed by amplified fragment length polymorphism (AFLP), genomic in situ hybridization (GISH), and PCR-restriction fragment length polymorphism (PCR-RFLP). Sixteen mini-calluses were obtained after 20 days of culture; they all developed into embryoids on EME500 medium. Following several subcultures on shoot induction medium for a total culture period of 6 months, shoots regenerated. The plants grew vigorously with a well-developed root system and exhibited the trifoliate leaf character of P. trifoliata. Ploidy analysis verified that all of the regenerates were tetraploids (2 n=4 x=36) as expected. GISH analysis confirmed that 18 chromosomes came from trifoliate orange and the remaining 18 from Goutou sour orange, as with most symmetric somatic hybrid plants; moreover, chromosome translocations were also observed in one plant. AFLP analysis of 16 regenerates and their fusion parents indicated that all of the somatic hybrids except one were genetically uniform. Analysis of the somatic hybrid cytoplasmic genomes with universal primers revealed that their chloroplast DNA (cpDNA) banding patterns were identical to those of the mesophyll parent trifoliate orange, while their mitochondria (mt) genomes were of the callus parent sour orange. The potential of GISH in Citrus somatic hybrid analysis is discussed.

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

Somatic 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.

Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.

Citrus cybrid leaf essential oil

The essential oil from the leaves of a Citrus cybrid plant, obtained by somatic hybridization between ‘Valencia’ sweet orange (Citrus sinensis L. Osbeck), embryogenic parent, and ‘Femminello’ lemon (C. limon L. Burm f.), leaf parent, has been studied by steam distillation and GC–MS analysis and compared with the correspondent oils from the parent plants. Although the overall composition of the cybrid essential oil appears much closer to that of ‘Femminello’, the relatively higher percentage of β-caryophyllene, as well as the qualitative and quantitative modifications of the esteric fraction, are characteristic of the new hybrid. Since cybrid cells possess the nuclear genome of the mesophyll parent, ‘Femminello’ lemon, and the mitochondrial genome of the nucellar parent, ‘Valencia’ sweet orange, it seems possible to conclude that the genetic information coding for the biosynthesis of the essential oils is mainly embedded in the nucleus, although interactions between nuclear genome and cytoplasmic genome can not be excluded. Copyright © 2000 John Wiley & Sons, Ltd.

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.

Cybrid Plants Produced by Electrofusion Between Satsuma Mandarin (Citrus unshiu) and Yuzu (C.junos) or Lemon (C. limon), and Recombination of Mitochondrial Genomes.

Electrofusion was conducted to use the embryogenic callus cells of satsuma mandarin (Citrus unshiu cv. ‘Juman’ unshiu) and mesophyll cells of yuzu (C. junos) and lemon (C.limon cv. ‘Eureka’ lemon). Two plants were regenerated from the combination of satsuma mandarin and yuzu, and one plant from that of satsuma mandarin and lemon. The plants had 18 chromosomes (2n=2×=18 in each parent), and showed the same nuclear rDNA fragment pattern as that of the mesophyll parent. The mitochondrial DNA analysis showed the presence of specific yuzu or lemon bands in addition to specific satsuma mandarin bands. From these results with morphological evidence, the regenerated plants were considered as cybrids with recombined mitochondrial DNA.

Analysis of mitochondrial genomes amongCitrus plants produced by the interspecific somatic fusion of ?Seminole? tangelo with rough lemon

Interspecific somatic fusion was performed between 'Seminole' tangelo (Citrus reticulata Blanco xC. paradisi Macf.) protoplasts isolated from embryogenic callus and rough lemon (C. jambhiri Lush.) mesophyll protoplasts. Eight plants out of ten randomly selected regenerants had 18 chromosomes and the same nuclear rDNA fragment patterns as that of the mesophyll parent. The remaining two plants showed rDNA fragment patterns from both parents and had 36 chromosomes. For the analysis of mitochondrial DNA (mtDNA),rrn26 derived from pea was used to probeBamHI digests of the regenerants. All plants showed mtDNA band patterns identical to that of the callus parent, suggesting that eight plants were cybrids and the remaining two plants were somatic hybrids. In addition to the callus parent band patterns, additional fragments from the mesophyll parent and/or a novel band fragment were revealed in some of the putative cybrids by peaatpA probe after digestion withDraI andPstI. These results suggest the occurrence of mtDNA recombination/rearrangement inCitrus cybrids produced by somatic fusion in this interspecific combination.

Citrus cybrid regeneration following cell fusion between nucellar cells and mesophyll cells

Through the cell fusion between nucellar callus cells and mesophyll cells in two Citrus combinations, we obtained the regenerated plants resembling mesophyll parent besides the somatic hybrids. In the fusion experiment between sudachi ( Citrus sudachi Hort.) and lime ( C. aurantifolia Swingle), 4 out of 12 regenerated clones were similar to mesophyll parent (lime) as we already reported. In another experiment of sudachi and lemon ( C. limon Burn) 4 out of 6 clones were similar to mesophyll parent (lemon). These regenerated plants had 18 chromosomes, which were equal to diploid parents and showed the same nuclear rDNA fragment patterns as those of mesophyll parents. The composition of the mitochondrial genomes of these regenerated plants was investigated. The mtDNA analysis revealed that all of the clones that resembled mesophyll parents had identical mtDNA fragment patterns to those of the nucellar callus parent, indicating that these regenerated clones resembling mesophyll parents were cybrids. These results suggest that the mitochondria of nucellar cells may play a significant role in Citrus embryogenesis.

Cell type determines plastid transmission in tomato intergeneric somatic hybrids

Mesophyll (M)- and suspension culture (S)-derived protoplasts of both Lycopersicon esculentum, tomato, and its wild relative Solanum lycopersicoides were fused as S + M, M + M and S + S combinations, respectively, to resolve the role of parental cell types in determining cpDNA transmission to intergeneric somatic hybrid plants. The mesophyll cpDNA was preferentially transmitted to 96% of the plants, each regenerated from a separate callus, in M + S and S + M fusion combinations. In contrast, for the M + M combination there was an equable distribution of either tomato cpDNA or that of S. lycopersicoides among the 34 hybrid plants. The number of plastids or proplastids in mesophyll or suspension protoplasts was not a factor regulating cpDNA transmission. Mesophyll or suspension protoplasts of both fusion partners had comparable frequencies of either plastid type with a mean of 23. The biased transmission of plastids from the mesophyll parent in somatic hybrid plants of S + M and M + S combinations appears to be due to differential multiplication of plastids, possibly conditioned by an unequal input of the nucleoids found in plastids versus proplastids. In the M + M fusion, plastid and nucleotide input and subsequent plastid multiplication are apparently equal, and when combined with random sorting out leads to an equal distribution of parental cpDNAs in the regenerated somatic hybrid plants. For the S+S combination, 22 somatic hybrid plants have exclusively tomato cpDNA, an outcome that is not readily explained by donor cell input.

Production and analysis of intraspecific somatic hybrids of potato ( Solanum tuberosum L.)

Photoplasts of two dihaploid lines of potato were fused to produce a large number of intraspecific somatic hybrid plants among which plants of the expected tetraploid level might be found. Fusion frequencies up to 12% (mean 7%) were observed using a revised polyethylene glycol fusion protocol. Fusion products were identified by the dual fluorescence emission from the chloroplasts in mesophyll protoplasts (red) and from the fluorescein diacetate stain in light and norflurazon bleached protoplasts (yellow-green). Hybrid cells were isolated 2–3 days after fusion and cultured at a cell density of 2000 cells/ml. From a total of 1363 isolated putative hybrid cells, 258 divided to form calli. Plants were regenerated from 166 of these. Isozyme analysis confirmed the hybrid nature in 57 of 58 analysed plants. Ploidy was determined in 51 plants; 12% were tetraploid, 41% hexaploid, 12% octoploid and 35% were mixoploid. Expression of isocitrate dehydrogenase isozymes indicated that the majority of the hexaploid hybrids contained 2 genomes of the bleached parent and one genome of the mesophyll parent. This study shows that tetraploid somatic hybrid potato plants can be obtained by the fusion and selection method presented.