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.     

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.     

A somatic hybrid plant obtained by protoplast fusion between navel orange (Citrus sinensis) and satsuma mandarin (C. unshiu)

A somatic hybrid plant was obtained by protoplast fusion between navel orange and satsuma mandarin. Protoplasts isolated from nucellar calli of navel orange and from leaves of satsuma mandarin were fused by the PEG method. The fusion products were cultured in a Murashige & Tucker medium containing 0.6 M sucrose. In this medium, some colonies developed into whole plants through embryogenesis. One of the regenerated plants was shown to be a hybrid, which was proven by restriction endonuclease analysis of nuclear ribosomal DNA. The chromosome number of the hybrid was 36. Both parents have a chromosome number 2n=18.     

Somatic hybridization in citrus: An effective tool to facilitate variety improvement

Summary Citrus somatic hybridization and cybridization via protoplast fusion has become an integral part of citrus variety improvement programs worldwide. Citrus somatic hybrid plants have been regenerated from more than 200 parental combinations, and several cybrid combinations have also been produced. Applications of somatic hybridization to citrus scion improvement include the production of quality tetraploid breeding parents that can be used in interploid crosses to generate seedless triploids, and the direct production of triploids by haploid + diploid fusion. Applications of somatic hybridization to citrus rootstock improvement include the production of allotetraploid hybrids that combine complementary diploid rootstocks, and to combine citrus with sexually incompatible or difficult to hybridize genera that possess traits of interest for germplasm expansion. A few somatic hybrid tetraploid breeding parents have flowered, are fertile, and are being used as pollen parents to generate triploids. Several allotetraploid somatic hybrid rootstocks are performing well in commercial field trials, and show great promise for tree size control. Seed trees of most of these somatic hybrid rootstocks are producing adequate nucellar seed for standard propagation. Somatic hybridization is expected to have a positive impact on citrus cultivar improvement efforts.     

Are citrus species favorable hosts for the Mediterranean fruit fly? A demographic perspective

We studied, under laboratory conditions, demographic parameters of adult Mediterranean fruit flies, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (medfly), obtained from three sweet orange varieties, lemon, and bitter oranges. These data were combined with immature developmental rates and survival on the same hosts to estimate host‐specific population parameters. Pairs of newly emerged adults from each citrus variety were held individually in transparent plastic cages, and females were allowed to oviposit in either red domes (artificial, pre‐punctured plastic oviposition devices), or intact, whole citrus fruits. We found strong effects of larval host (citrus fruits) on adult longevity and fecundity. In all five citrus varieties, females did not manage to deposit eggs into fruit pulp. The proportion of eggs laid in either the flavedo or albedo area of the fruit peel differed depending on the citrus variety. In all cases except bitter oranges, females oviposited fewer eggs in citrus fruits than in the artificial oviposition substrates, suggesting that most citrus fruits cause a significant reduction in the reproductive potential of medflies. Negative correlations were found between fecundity and (a) the density of oil glands, and (b) the amount of essential oils in the flavedo area of citrus fruits. There was no correlation between fecundity and other fruit physical characteristics, such as resistance of fruit peel to pressure and thickness of the flavedo. Apparently, resistance of citrus fruits to medfly infestation is directly related to citrus essential oils. The intrinsic rate of increase (r) was higher in bitter oranges than in the three sweet orange varieties tested. A negative r was estimated for flies that developed and oviposited in lemons, indicating a tendency for population decrease in this host. The suitability of citrus fruits for medfly development and the practical implications of our findings for management of medflies in citrus orchards are discussed.     

Carotenoids synthesized in citrus callus of different genotypes

Eight carotenoids, such as phytoene, α-carotene, violaxanthin, etc., synthesized in citrus callus of 31 genotypes were identified and determined. Though varied with genotypes, the carotenoids composition of callus derived from a certain genotype was stable, while carotenoids contents altered between sub-cultures. Some specific carotenoids were produced in calluses of limited genotypes: β-citraurin was only synthesized in calluses of Nianju tangerine (Citrus reticulata Blanco) and Page tangelo (C. reticulata × C. paradisi); while 9-Z-violaxanthin was only detected in Nianju tangerine and Skaggs Bonanza navel orange (C. sinensis L. Osbeck). Notably, the only carotenoid detected in calluses of Natsudaidai (C. aurantium L.) and other two sweet oranges (C. sinensis L. Osbeck) was phytoene. It implied that citrus calluses could be employed to produce specific carotenoids in the future. To further elucidate the characters of callus carotenoids profile, comparisons of carotenoids profiles was made among calluses, fruit tissues and leaves of four selected citrus genotypes. Results showed that lycopene was not detected in leaves and calluses; nevertheless, both citrus fruits and calluses accumulated phytoene, whereas leaves did not except those of Cara Cara navel orange. It is postulated that citrus callus featured its carotenoids profile different from fruit tissues and leaves. In conclusion, the advantages of using citrus callus as an alternative model research system in understanding the regulation of carotenogenesis have been discussed.     

Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.)

Summary Protoplasts of navel orange, isolated from embryogenic nucellar cell suspension culture, were fused with protoplasts of grapefruit isolated from leaf tissue. The fusion products were cultured in the hormone-free medium containing 0.6 M sucrose. Under the culture conditions, somatic embryogenesis of navel orange protoplasts was suppressed, while cell division of grapefruit mesophyll protoplasts was not induced. Six embryoids were obtained and three lines regenerated to complete plants through embryogenesis. Two of the regenerated lines exhibited intermediate morphological characteristics of the parents in the leaf shape. Chromosome counts showed that these regenerated plants had expected 36 chromosomes (2n=2x=18 for each parent). The rDNA analysis using biotin-labeled rRNA probes confirmed the presence of genomes from both parents in these plants. This somatic hybridization system would be useful for the practical Citrus breeding.     

柑桔种间原生质体融合植株再生及染色体数目变异的研究

为了获得具有抗病、优质丰产等优良性状的柑桔体细胞杂种,本研究应用当前推广良种朋娜脐橙胚性细胞原生质体和抗裂皮病、耐盐碱的红桔叶肉细胞原生质体作为亲本进行体细胞杂交研究。通过对原生质体分离,融合和培养过程中培养基调控等环节的研究,建立起原生质体融合及其后的胚状体再生系统,并从融合处理后的原生质体培养中获得了大最的胚状体,进而获得个别体细胞杂种植株。同时对融合后再生的胚状体染色体数目和同工酶分析,还揭示了在柑桔原生质体融合再生中,胚状体水平上存在淹广泛的遗传变异。其中有14.1%的胚状体为四倍体,近20%为超倍体的非整倍体,并讨论了变异发生及由胚状体再生植株困难的原因。     

Performance of Cryptolaemus montrouzieri feeding on Pulvinaria aurantii ovisacs on citrus plants

We studied the performance of the coccinellid Cryptolaemus montrouzieri on citrus plant species infested with orange pulvinaria scale, Pulvinaria aurantii, under controlled conditions in the laboratory. The experimental host plants were blood orange, grapefruit, lemon, pummelo and kumquat. All life history parameters indicated best performance of C. montrouzieri, when reared on P. aurantii ovisacs on the leaves of grapefruit. Pummelo, blood orange, lemon or kumquat were less suitable.     

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.     

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.     

Survival and development of immature stages of the Mediterranean fruit fly (Diptera: Tephritidae) in citrus fruit

We studied, under laboratory conditions, the performance of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), immature stages in intact whole fruit of three sweet orange varieties, lemon, and bitter oranges. Both citrus variety and fruit part (flavedo, albedo, and pulp) had strong effects on larval performance, smaller effects on pupae, and no effects on eggs. Fruit peel was the most critical parameter for larval development and survival, drastically affecting larval survival (inducing very high mortality rates). Among fruit regions, survival of larvae placed in flavedo was zero for all varieties tested except for bitter orange (22.5% survival), whereas survival in albedo was very low (9.8-17.4%) for all varieties except for bitter orange (76%). Survival of pupae obtained from larvae placed in the above-mentioned fruit regions was high for all varieties tested (81.1-90.7%). Fruit pulp of all citrus fruit tested was favorable for larval development. The highest survival was observed on bitter oranges, but the shortest developmental times and heaviest pupae were obtained from orange cultivars. Pulp chemical properties, such as soluble solid contents, acidity, and pH had rather small effects on larval and pupal survival and developmental time (except for juice pH on larvae developmental duration), but they had significant effects on pupal weight.     

奉节脐橙果皮褐变差减文库的构建及初步分析

以奉节脐橙果实为材料,采用抑制差减杂交技术,分别以褐变与未褐变柑橘果皮作为检测方和驱动方,成功构建了果皮褐变的差减cDNA文库,对部分克隆进行了序列测定并与GenBank进行了同源性比较。选择其中的4个基因:钙结合蛋白同源基因、半胱氨酸蛋白酶同源基因、NAC蛋白质家族同源基因和膨胀素同源基因进行半定量RT-PCR分析,结果表明它们在褐变果皮中的表达量均高于未褐变果皮,说明这些基因的增强表达可能与脐橙果皮褐变有密切关系。     

Analysis of cytoplasmic genomes in somatic hybrids between navel orange (Citrus sinensis Osb.) and ‘Murcott’ tangor

Summary Somatic hybrid plants were produced by protoplast fusion of navel orange and Murcott tangor. Hybridity of the plants was confirmed by the restriction endonuclease analysis of nuclear ribosomal DNA. All of the plants (16 clones) were normal, uniform, and had the amphidiploid chromosome number of 36 (2n=2x=18 for each parent). The cpDNA analysis showed that each of the 16 somatic hybrids contained either one parental chloroplast genome or the other. In all cases, the mitochondrial genomes of the regenerated somatic hybrids were of the navel orange type.Contribution No. E-132 of the Fruit Tree Research Station     

Citrus somatic hybrid: an alternative system to study rapid structural and epigenetic reorganization in allotetraploid genomes

Citrus somatic hybrids produced in the past years provide a novel opportunity to study the immediate effects of allopolyploidization on genome structure and methylation. Here, we present a first attempt to investigate the alterations in genome structure and methylation in three sets of citrus somatic allotetraploids and their diploid parents using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. Our results indicate that all the allotetraploids mainly have the AFLP and MSAP banding patterns containing specific bands from both parents plus some alterations. The incidences of the AFLP polymorphic bands in allotetraploids show a range from 4.61 to 7.88 %, while from 12.50 to 15.67 % of the sites are methylated. In addition, the proportions of callus-parent-specific DNA structure and methylation alterations are much greater than those of leaf-parent-specific alterations in the somatic hybrids. Furthermore, we find that the somatic hybrids take on a greater divergence from the callus parent and a closer relationship to leaf parent in all groups of plants by dendrogram analysis based on AFLP or MSAP data. Taken together, our results suggest that somatic hybrids are very useful in elucidating the immediate changes that occur in newly synthesized allotetraploid.     

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.     

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.     

Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus

Protoplast fusion technology has been utilized in many crops to generate allotetraploid somatic hybrids, and sometimes autotetraploids as a byproduct of the process. A brief history of this technology development is provided, along with a simple protocol developed for citrus, which can be easily adapted to other plants. Protoplast fusion has become a significant tool in ploidy manipulation that can be applied in various cultivar improvement schemes. In rare cases, a new somatic hybrid may have direct utility as an improved cultivar; however, the most important application of somatic hybridization is the building of novel germplasm as a source of elite breeding parents for various types of conventional crosses for both scion and rootstock improvement. Somatic hybridization is generating superior allotetraploid breeding parents for use in interploid crosses to generate seedless triploids. Seedlessness is a primary breeding objective for new fresh fruit citrus varieties, and several thousand triploid hybrids have been produced using somatic hybrids as the tetraploid parent. Protoplast fusion is also being utilized to produce somatic hybrids that combine complementary diploid rootstocks, which have shown good potential for tree size control. Tree size control has gained importance as a means of reducing harvesting costs, maximizing the efficiency of modern cold protection methodology, and facilitating the adaptation of new fruit production systems. Successful somatic hybridization in citrus rootstock improvement has enabled rootstock breeding at the tetraploid level via sexual hybridization, which can yield maximum genetic diversity in zygotic progeny upon which to impose selection for the many traits required in improved rootstock cultivars, including disease and insect resistance, broad adaptation, tree size control, and the ability to consistently produce high yields of quality fruit. Recent progress and successful examples of these applications are discussed. Finally, a discussion of the genetic potential of somatic hybrids as breeding parents, including meiotic behavior and inheritance is provided.