Intergeneric somatic hybrid plants from sexually incompatible woody species: Citrus sinensis and Severinia disticha

Summary The fusion of Citrus sinensis cv. Hamlin (sweet orange) protoplasts isolated from an embryogenic suspension culture with Severinia disticha (Philippine box orange) protoplasts isolated from epicotyl-derived callus with organogenic potential, resulted in the regeneration of allotetraploid somatic hybrid plants. Plant regeneration was a function of complementation, combining the capacity for somatic embryogenesis of C. sinensis with the organogenic ability of S. disticha. Confirmation of somatic hybrid identity was based on leaf morphology, chromosome number, and analyses of phosphoglucose mutase (PGM) and malate dehydrogenase (MDH) zymograms. Hybrid plants were multiplied organogenically and exhibited morphology intermediate to that of the parents. This is the first example of somatic hybrid plants produced between sexually incompatible woody genera.Florida Agricultural Experiment Station Journal Series No. 8198     

Citrus somatic hybridization with potential for improved blight and CTV resistance

Summary The production of five new somatic hybrids with potential for improved disease resistance is reported herein. Protoplast isolation, fusion, and plant regeneration was achieved from Caipira sweet orange (Citrus sinensis L. Osbeck) as an embryogenic parental source and Volkamer lemon (C. volkameriana Pasquale), Cleopatra mandarin (C. reticulata Blanco), and Rough lemon (C. jambhiri Lushington) as non-embryogenic parental sources. Fusion involving Cleopatra mandarin and Rangpur lime (C. limonia L. Osbeck) as embryogenic parental sources with Sour orange (C. aurantium L.) also resulted in somatic hybrid plants. Somatic hybridization was confirmed by leaf morphology evaluation, chromosome counting, and randomly amplified polymorphic DNA (RAPD) analyses. Somatic hybrids may combine complementary characteristics from both parental sources and have potential for tolerance to blight and citrus tristeza virus (CTV).     

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.     

Transmission of organelle genomes in citrus somatic hybrids

Restriction fragment length polymorphisms (RFLPs), were used to analyze the organelle composition of two-year-old trees, recovered from two different experiments: protoplasts from embryogenic cell suspensions of `Succari' sweet orange (C. sinensis L. Osbeck) were fused with leaf protoplasts of Citropsis gilletiana Swingle & M. Kell or to leaf protoplasts of Atalantia ceylanica(Arn.) Oliv. The somatic hybrids of both fusion combinations had the mitochondrial genome from the embryogenic partner. In some somatic hybrids, non-parental fragments were observed among the mitochondrial patterns. Somatic hybrids between `Succari' + Atalantia had plastid DNA from the embryogenic parent, while the somatic hybrids of `Succari' + Citropsis all had both parental chloroplast genomes. The relative abundance of organelle DNAs in the donor embryogenic and leaf cells may explain the consistent transmission of the embryogenic parent mitochondrial DNA and the inheritance of the chloroplast genome from either parent. This revised version was published online in June 2006 with corrections to the Cover Date.     

Further evidence of a cybridization requirement for plant regeneration from citrus leaf protoplasts following somatic fusion

Summary Somatic hybridization experiments in Citrus that involve the fusion of protoplasts of one parent isolated from either nucellus-derived embryogenic callus or suspension cultures with leaf-derived protoplasts of a second parent, often result in the regeneration of diploid plants that phenotypically resemble the leaf parent. In this study, plants of this type regenerated following somatic fusions of the following three parental combinations were analyzed to determine their genetic origin (nuclear and organelle): (embryogenic parent listed first, leaf parent second) (1) calamondin (C. microcarpa Bunge) + Keen sour orange (C. aurantium L.), (2) Cleopatra mandarin (C. reticulata Blanco) + sour orange, and (3) Valencia sweet orange (C. sinensis (L.) Osbeck) + Femminello lemon (C. limon (L.) Burm. f.). Isozyme analyses of PGI, PGM, GOT, and IDH zymograms of putative cybrid plants, along with RFLP analyses using a nuclear genome-specific probe showed that these plants contained the nucleus of the leaf parent. RFLP analyses using mtDNA-specific probes showed that these plants contained the mitochondrial genome of the embryogenic callus donor, thereby confirming cybridization. RFLP analyses using cpDNA-specific probes revealed that the cybrid plants contained the chloroplast genome of either one or the other parent. These results support previous reports indicating that acquisition of the mitochondria of embryogenic protoplasts by leaf protoplasts is a prerequisite for recovering plants with the leaf parent phenotype via somatic embryogenesis following somatic fusion.Abbreviations cp chloroplast - GOT glutamateoxaloacetate transaminase - IDH isocitrate dehydrogenase - mt mitochondria - PEG polyethylene glycol - PGI phosphoglucose isomerase - PGM phosphoglucomutase - RFLP restriction fragment length polymorphism Florida Agricultural Experiment Station Journal Series No. R-04631.     

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.     

Somatic hybridization between Citrus reticulata and Citropsis gabunensis through electrofusion

Protoplasts from embryogenic calli of Citrus reticulata Blanco cv. Ponkan and Citropsis gabunensis (Engl.) Swing. & M. Kell (Cabon Cherry Orange), were isolated and fused using electric current. Maximum fusion frequency was obtained with AC at 75 kV/cm (1.0 MHz) for 15 s, followed by DC square-wave pulses at 1.25 kV/cm for 40 s. Fusion-treated protoplasts were cultured on MT medium containing no growth regulators, solidified with 0.6% Bacto Difco agar. Protoplast-derived calli were proliferated on MT medium containing 1 mg/l zeatin and 0.9% agar. A total of 31 lines of somatic hybrid calli were obtained by screening on the basis of chromosome count and isozyme analysis. The somatic hybrids were tetraploid (2n=36). Plants were regenerated from the calli via somatic embryogenesis. The somatic hybrid plants exhibited morphological characteristics intermediate to the parental plants.Abbreviations 2,4-D 2, 4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - MT Murashige and Tucker (1969) - PEG polyethylene glycol - AC alternating current - DC direct current     

Targeted cybridization in citrus: transfer of Satsuma cytoplasm to seedy cultivars for potential seedlessness

CMS (cytoplasmic male sterility) can be controlled by the mitochondrion genome in higher plants, including Satsuma mandarin. Somatic fusion experiments in citrus combining embryogenic callus protoplasts of one parent with leaf protoplasts of a second parent often produce cybrid plants of the leaf parent, a phenomenon occurring most often with interspecific fusion combinations. In an attempt to practically exploit this cybridization phenomenon, we conducted somatic fusion experiments combining embryogenic suspension-derived protoplasts of Satsuma mandarin, Citrus unshiu Marc. cv. Guoqing No. 1 (G1), a male-sterile cultivar, with leaf protoplasts of other seedy types—Hirado Buntan Pink pummelo (HBP) [Citrus grandis (L.) Osbeck], Sunburst mandarin (C. reticulata Blanco), Orie Lee hybrid (C. reticulata cv. Clementine × Murcott tangor), and Murcott tangor [C. reticulata × C. sinensis (L.) Osbeck], respectively—in an attempt to generate seedless cybrids by the targeted transfer of CMS. The genetic identities of regenerated plants from all four parental combinations were determined by flow cytometry, SSR, CAPS (or PCR-RFLP), RFLP, and chloroplast-SSR analyses. Regenerated plants from the first three parental combinations were diploids, and the cybrid nature of G1 + HBP with the mitochondrion genome from G1 and the chloroplast genome from HBP was confirmed, whereas the cybrid nature of the remaining two combinations was difficult to confirm because of the close phylogenetic relatedness of both fusion parents, as expected. Plants from G1 + Murcott were confirmed as tetraploid somatic hybrids. This is the first report of targeted citrus cybrid production by symmetric fusion with male-sterile Satsuma as the callus parent and other seedy cultivars as the leaf parents.Abbreviations CAPS: Cleaved amplified polymorphic sequence - CMS: Cytoplasmic male sterility - cp-SSR: Chloroplast simple sequence repeat - PEG: Polyethylene glycol - SSR: Simple sequence repeat - RFLP: Restriction fragment length polymorphism Communicated by G.C. Phillips     

Studies on Taxonomy and Evolution of Citrus and Its Related Genera by Isozyme Analysis

The leaf or bark tissue extracts of 108 biotypes of Citrus and its five related genera were analyzed by polyacrylamide gel electrophoresis for isozymes of peroxidase, glutamate oxaloacetate transaminase, phosphoglucose isomerase, phosphoglucose mutase, superoxide dismutase, tetrazolium oxidase, NADP+- dependent malate dehydrogenase and esterase. The inter-or intra-generic differences of isozymograms were compared. Dissimilarities of the isozyme patterns among the samples were calculated by computer based on the UPGMA method to demonstrate the phylogenetic relationship of the biotypes. There are remarkable isozymogramatic differences among the six genera. Generally speaking, each genus possesses its own unique bands. Based on the cluster analysis, the total Citrus biotypes are classified into seven groups, i.e. Honghe papeda, Mauritius papeda, Pummelo, Ichang papeda, Citron, Lemon-lime and Mandarin-orange. Ichang papeda is suggested to be the third subgenus of the genus Citrus. The Fuming trifoliata orange is accepted as a new species of Poncirus-P. polyandra S. Q. Ding. It might be the evolutionary bridge between Poncirus and Citrus, and one of the “missing links” as suggested by W. T. Swingle. Microacrumen (small- fruited mandarin) is the primitive form of the mandarins. It seems better to consid er“ Horse-nose mandarin ”or “Xipigoushigan mandarin” as the typic mandarin originated in China. Euacrumen (large-fruited mandarin) might have arised through two ways. One is hybridization of Microacrumen and C. sinensis, and the other is just the variation of Microacrumen. Ancestors of some biotypes are inferred based on isozyme data. Fortunella obovata Tan. may be a hybrid of Fortunella and Citrus. C. unshiu Marc. arose from the nucellar mutation of“Huangyanbendiguangju mandarin”( C. nobilis Lour.) in Huangyan of Zhejiang Province. C. junos Sieb. came from hybridization of Ichang papeda and mandarin. C. grandis Osbeck and mandarin may have been involved in the origin of C. aurantium L.. C. limonia Osbeck may be a hybrid between C. medica L. and mandarin. The evolutionary trend of citrus fruit trees is proposed, anda tentative phylogenetic diagram is drawn.     

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.     

The response of citrus plants to the broad mite Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae)

1. Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) is a common polyphagous mite in tropical and subtropical areas and is considered as an important citrus pest.
2. To understand the response of citrus to P. latus infestation, we have characterized the volatile profile and the molecular defence mechanisms of two citrus genotypes, namely sour orange (Citrus aurantium) and Cleopatra mandarin (Citrus reshni), to P. latus infestation. These two species are important rootstocks for the citrus industry and display differential resistance to Tetranychus urticae Koch (Acari: Tetranychidae), with sour orange showing elevated levels of constitutive and induced resistance associated with the jasmonic acid (JA) pathway compared with Cleopatra mandarin.
3. P. latus infestation activated both the JA- and the salicylic acid-dependent pathways in sour orange but not in Cleopatra mandarin. However, this differential activation resulted in the production of similar volatile blends (a mixture of green leaf volatiles and aromatic compounds).
4. Contrary to T. urticae infestation, sour orange supported larger densities of P. latus than Cleopatra mandarin with similar injury levels.
5. Therefore, sour orange may be more tolerant to P. latus than Cleopatra mandarin.

     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

An alternative method for the genetic transformation of sweet organce

Summary An alternative method for transforming sweet organe [Citrus sinensis (L.) Osbeck] has been developed. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein gene was introduced using polyethylene glycol into protoplasts of ‘Itaborai’ sweet organe isolated from an embryogenic nucellar-derived suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of the green fluorescent protein, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots. To expedite transgenic plant recovery, regenerated shoots were also micrografted onto sour orange seedling rootstocks. Presence of the transgene in calluses and regenerated sweet organe plants was verified by gene amplification and Southern analyses. Potential advantages of this transformation system over the commonly used Agrobacterium methods for citrus are discussed.     

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.     

Effect of NaCl concentrations in irrigation water on growth and polyamine metabolism in two citrus rootstocks with different levels of salinity tolerance

Six-months-old, uniform sized seedlings of two citrus rootstocks; Cleopatra mandarin (Citrus reshni Hort. ex Tan) and Troyer citrange (Poncirus trifoliata × Citrus sinensis) were irrigated with half-strength Hoagland nutrient solution containing 0, 40 or 80 mM NaCl for 12 weeks. Shoot height, leaf number and fresh weights of the seedlings, and relative chlorophyll contents, chlorophyll fluorescence yields (Fv/Fm), net photosynthetic and respiration rates in the leaves decreased with the increase in salinity level in the irrigation water. The decrease was greater in Troyer citrange as compared to Cleopatra mandarin. The concentrations of sugars i.e. fructose, glucose and sucrose in the leaves of Cleopatra mandarin and both leaves and roots of Troyer citrange decreased with the increase in salinity level. However, the concentrations in the roots of Cleopatra mandarin increased with the increase in salinity level. Free proline content in the leaves of Troyer citrange and root tissue of Cleopatra mandarin also increased with the increased salinity level. Among the polyamines, spermine titer increased in the leaves of both rootstocks as a response to salinity treatments. Na⁺ concentrations were higher in leaf and root tissue of Cleopatra mandarin, while that of Cl⁻ were higher in Troyer citrange.     

An analysis of red cell enzymatic markers in the province of Bologna (Italy).

About 280 unrelated individuals living in the province of Bologna (Northern Italy) have been studied for the following red cell enzymatic markers: phosphoglucomutase (PGM), adenylate kinase (AK), adenosine deaminase (ADA) and phosphohexose isomerase (PHI). 116 subjects from the same sample have also been analysed for red cell acid phosphatase (ACP). The observed gene frequencies are PGM21 = 0.280; AK2 = 0.030; ADA2 = 0.091; ACPa = 0.297; ACPb = 0.647; ACPc = 0.056. In the PHI system two individuals with the variant PHI 3-1 phenotype have been found.     

Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus trifoliata cv. Flying Dragon

Intergeneric somatic hybrid plants between Hamlin sweet orange [Citrus sinensis (L.) Osbeck] and Flying Dragon trifoliate orange (Poncirus trifoliata Raf.) were regenerated following protoplast fusion. Hamlin protoplasts, isolated from an habituated embryogenic suspension culture, were fused chemically with Flying Dragon protoplasts isolated from juvenile leaf tissue. The hybrid selection scheme was based on complementation of the regenerative ability of the Hamlin protoplasts with the subsequent expression of the trifoliate leaf character of Flying Dragon. Hybrid plants were regenerated via somatic embryogenesis and multiplied organogenically. Hybrid morphology was intermediate to that of the parents. Chromosome counts indicated that the hybrids were allotetraploids (2n=4x=36). Malate dehydrogenase (MDH) isozyme patterns confirmed the hybrid nature of the regenerated plants. These genetically unique somatic hybrid plants will be evaluated for citrus rootstock potential. The cell fusion, selection, and regeneration scheme developed herein should provide a general means to expand the germplasm base of cultivated Citrus by intergeneric hybridization with related sexually incompatible genera.Abbreviations MDH malate dehydrogenase - CTV citrus tristeza virus - MT Murashige and Tucker basal medium - BH3 protoplast culture medium, Grosser and Chandler, 1987 - PEG polyethylene glycol - GA3 giberellic acid - BA N-(phenylmethyl)-1 H-purin-6-amine - HCl hydrochloric acid Florida Agricultural Experiment Station Journal Series No. 7972     

Asymmetric somatic hybridization between UV-irradiated <Emphasis Type="Italic">Citrus unshiu</Emphasis> and <Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">sinensis</Emphasis>: regeneration and characterization of hybrid shoots

In the present paper, attempts were made to explore the possibility of employing ultraviolet (UV) irradiation in citrus asymmetric fusion for transfer of limited amount of favorable traits from a desirable cultivar to a target one. Exposure of Satsuma mandarin (Citrus unshiu Marc.) embryogenic protoplasts to UV at an intensity of 300 μW cm⁻² led to reduced viability, especially under long irradiation duration. The protoplasts could not grow during culture when they were irradiated for over 30 s. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay revealed extensive DNA fragmentation in the UV-irradiated protoplasts compared with those without UV treatment. Electrofusion between UV-irradiated protoplasts of Satsuma mandarin (donor) with those of Jincheng (C. sinensis Osbeck, recipient), a local cultivar of superior quality, gave rise to regeneration of several lines of shoots, which failed to root despite enormous endeavors. Ploidy analysis via flow cytometry and chromosome counting showed that four selected shoots were either diploid, triploid or tetraploid. Random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) confirmed the shoots, irrespective of their ploidy level, as putative somatic hybrids. Cleaved amplified polymorphism sequences (CAPS) demonstrated that the shoots predominantly got their cytoplasmic components, in terms of chloroplast (cp) and mitochondrion DNA, from Jincheng, along with possible recombination of cpDNA in some shoot lines. The current data indicated that UV-based asymmetric fusion could also be employed in citrus somatic hybridization with the intention of creating novel germplasms, which may provide an alternative approach for cultivar improvement.     

Comparative electrophoretic study of phosphoglucose isomerase,phosphoglucose mutase and tetrazolium oxidase in some hydatid cysts of Echinococcus granulosus collected from different mammal hosts

Hydatid cyst scolexes of lung and liver from sheep and water buffalo were electrophoreticaily examined for phosphoglucose isomerase (PGI), phosphoglucose mutase (PGM) and tetrazolium oxidase (Te) activity. One each of phosphoglucose isomerase, phosphoglucose mutase and tetrazolium oxidase activities were found in all of the hydatid cysts studied. PGI and PM in ail hydatid cysts studied appeared te be controlled by two codominant alleles. PGI and PGM proved to be a good taxonomic criterion to differentiate the hydatid cysts of the water buffalo from those from sheep. On the other hand, TO failed to differentiate between the sheep and water buffalo hydatid cysts. The hydatid cysts that parasitise the sheep's organs can be considered as arising from a different strain from the cysts that parasitise the water buffalo organs, while lung and liver hydatid cysts of the sheep appeared to be a similar strain.     

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.