Gynogenetic haploids of Citrus after in vitro pollination with triploid pollen grains

This study reports haploid plantlet regeneration through gynogenesis in Citrus clementina Hort. ex Tan., cv. Nules, induced by in vitro pollination with pollen grains of Oroblanco, a triploid cultivar of grapefruit. It indicates that parthenogenesis induced in vitro by triploid pollen can be an alternative method to obtain haploids in monoembryonic cultivars of Citrus. Actually, despite considerable efforts, androgenesis has not been yet successful in many genotypes of Citrus. Pollination and mature stage of pistils was necessary for gynogenic embryo regeneration. Fourteen haploid gynogenic embryos of Nules clementine were obtained. Embryo conversion was high (85.7%) and embryos vigorously germinated producing twelve plantlets. Chromosome counting, performed in the roots of obtained embryos, showed the haploid level (n=x=9). Isozyme analyses confirmed the expected homozygous state of embryos and plantlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improvement of<Emphasis Type="Italic"> Citrus clementina</Emphasis> Hort. ex Tan. microspore-derived embryoid induction and regeneration

An improvement of the protocol for haploid induction through anther culture of Citrus clementina Hort. ex Tan. cv. Nules was achieved following the evaluation of a number of the factors affecting androgenesis. The influence of thidiazuron (TDZ) and three temperature pre-treatments (4°C, 25°C, 32°C) on the floral buds with respect to anther culture of C. clementina Hort. ex Tan., cv. Nules was investigated. An increased embryoid production was induced in the medium supplemented with TDZ. Pre-treatment temperatures of 4°C and 25°C were more favorable for embryo production than 32°C. Regeneration of androgenic haploid plantlets from cv. SRA 63 of C. clementina is reported here for the first time.Abbreviations 6-BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - KI Kinetin - NAA -Naphthaleneacetic acid - TDZ Thidiazuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea) - ZEA Zeatin Communicated by L. PeñaBoth authors have contributed equally to this article.     

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.     

Gibberellic acid impairs fertilization in Clementine mandarin under cross-pollination conditions

We investigated the effect of gibberellic acid (GA₃) in the fertilization of Clementine mandarin cv. ‘Clemenules’ (Citrus clementina Hort. ex Tan.), a parthenocarpic variety that produces seedless fruit due to its self-incompatible nature, but yields seedy fruits when grown under cross-pollination conditions.Experiments were conducted with on-tree ‘Clemenules’ flowers and ‘Fortune’ mandarin pollen (C. clementina Hort. ex Tan. × C. reticulata Blanco), which is sexually compatible with the former. Preanthesis treatment at −2 days after anthesis (−2 DAA) enhanced ovule abortion in both unpollinated and cross-pollinated (at +2 DAA) flowers. In the latter, the number of pollen tubes reaching the ovules was significantly reduced although pollen grains were not treated; thus, fertilization was partially avoided and seed set was reduced. When GA₃ was applied at anthesis (0 DAA) at the time of pollination, ovule abortion was again enhanced, and pollen tube growth was completely arrested; thus, fertilization was prevented and seed set was impeded. When GA₃ was applied 24 h after pollination (+1 DAA in flowers pollinated at anthesis), pollen tube growth was impaired but not arrested and ovule abortion was enhanced; therefore, fertilization was not prevented but impaired.We conclude that, when applied the days around anthesis, GA₃ (10 mg l⁻¹) impairs fertilization by either enhancing ovule abortion or reducing pollen tube growth, in ‘Clemenules’ flowers under cross-pollination conditions. The intensity of the response depends on the physiological flower state at the moment of treatment.     

Restoration of vigor and rooting competence in stem tissues of mature citrus by repeated grafting of their shoot apices onto freshly germinated seedlings in vitro

Summary Repeated grafting of 0.2-cm shoot tips from fruiting-age trees ofCitrus reticulata Blanco ‘Ponkan’ mandarin andC. sinensis Osbeck ‘Liu Tseng’ sweet orange onto freshly germinated ‘Troyer’ citrange [Poncirus trifoliata (L.) Raf. X.C. sinensis Osbeck] seedlings in vitro resulted in progressive restoration of rooting competence and vigor of regenerated roots and shoots. The restored traits were retained through the course of the investigation and suggested a phase reversal phenomenon.     

Obtaining autotetraploids in vitro at a high frequency in Citrus sinensis

Tetraploid plants are essential for interploid hybridization to create triploid seedless citrus. Here we report a simple and efficient in vitro method for generating autotetraploids for sweet orange (Citrus sinensis). Cell-division activity in ‘Anliucheng’ sweet orange callus was analyzed using flow cytometry to determine the peak frequency of cell division at which time callus in a liquid media and solid media was treated with 1000 mg l⁻¹ colchicine. The percentage of the DNA-content-varied cells in the callus increased markedly from 11.0% to 44.4% and to 59.0% for liquid and solid media respectively. A total of 20 tetraploid plantlets were recovered via embryogenesis from 47 plantlets regenerated from the treated callus. All the autotetraploids were derived from different embryoids. Autotetraploids will be useful parents for interploid hybridization to generate commercially valuable seedless triploid citrus cultivars.     

New universal mitochondrial PCR markers reveal new information on maternal citrus phylogeny

The aim of this work was to provide a set of mitochondrial markers to reveal polymorphism and to study the maternal phylogeny in citrus. We first used 44 universal markers previously described in the literature: nine of these markers produced amplification products but only one revealed polymorphism in citrus. We then designed six conserved pairs of primers using the complete mitochondrial DNA sequences of Arabidopsis thaliana and Beta vulgaris to amplify polymorphic intergenic and intronic regions. From these six pairs of primers, three from introns of genes coding for NADH dehydrogenase subunits 2, 5, and 7, revealed polymorphism in citrus. First, we confirmed that citrus have a maternal mitochondrial inheritance in two populations of 250 and 120 individuals. We then conducted a phylogenic study using four polymorphic primers on 77 genotypes representing the diversity of Citrus and two related genera. Seven mitotypes were identified. Six mitotypes (Poncirus, Fortunella, Citrus medica, Citrus micrantha, Citrus reticulata, and Citrus maxima) were congruent with previous taxonomic investigations. The seventh mitotype enabled us to distinguish an acidic mandarin group (‘Cleopatra’, ‘Sunki’ and ‘Shekwasha’) from other mandarins and revealed a maternal relationship with Citrus limonia (‘Rangpur’ lime, ‘Volkamer’ lemon) and Citrus jambhiri (‘Rough’ lemon). This mitotype contained only cultivated species used as rootstocks due to their good tolerances to abiotic stress. Our results also suggest that two species classified by Swingle and Reece, Citrus limon, and Citrus aurantifolia, have multiple maternal cytoplasmic origins.     

Sequence Analysis and Comparison of 16S rRNA, 23S rRNA and 16S/23S Intergenic Spacer Region of Greening Bacterium Associated with Yellowing Disease (Huanglongbing) of Kinnow Mandarin

High incidence (up to 40%) of symptoms of yellowing and yellow mottling was observed in 5–8 years old orchards of kinnow mandarin {Citrus reticulate Balanco (‘King’ × ‘Willow mandarin’)} in the Punjab state of India during a survey in January 2007. These symptoms are often confused with nutrient deficiency and other stress related disorders. However, a greening bacterium has been attributed to cause the disease. The disease was graft transmissible and sequencing of 16S rRNA, 16S/23S intergenic spacer region and 23S rRNA of the greening bacterium associated with yellowing disease in kinnow mandarin confirmed it to be Candidatus Liberibacter asiaticus (‘Ca L. asiaticus’) showing maximum identity of 95.9% with ‘Ca L. asiaticus’ from USA and Brazil in 16S rRNA. The study indicates definite association of ‘Ca L. asiaticus’ with yellowing/chlorotic mottling symptoms of greening disease of kinnow mandarin in Punjab state of India.     

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.     

Influence of endogenous and exogenous RNases on the variation of pollen cytosolic-free Ca2+ in Pyrus serotina Rehd

The objective of this study was to examine whether S-RNase plays a specific role in the pre-germinated Pyrus pollen. Effects of exogenous RNase and endogenous S-RNase on concentration of cytosolic-free calcium ([Ca²⁺]_i) variation of pre-germinated Pyrus pollen were studied. [Ca²⁺]_i variation caused by different RNases were complex. In 1 h after being cultured, exogenous RNase, RNase T₁ and RNase A, and endogenous incompatible ‘Hohsui’ RNase promoted the [Ca²⁺]_i of ‘Hohsui’ pollen. Acid proteins of ‘Hohsui’ had no remarkable influence on the [Ca²⁺]_i of self-pollen. Endogenous compatible ‘Kohsui’ RNase reduced the [Ca²⁺]_i of ‘Hohsui’ pollen, but compatible ‘Hohsui’ RNase can stimulate the [Ca²⁺]_i of ‘Kohsui’ pollen. RNase T₁, RNase A and incompatible ‘Kohsui’ S-RNase can also make ‘Kohsui’ pollen [Ca²⁺]_i increase. Different from ‘Hohsui’ pollen, acid proteins of ‘Hohsui’ pull down the ‘Kohsui’ pollen [Ca²⁺]_i remarkably. Conclusion can be made that during the prophase of pollen germination, endogenous S-RNase has no specific effect on pollen [Ca²⁺]_i changes.     

Flower Bud Culture of Shallot (Allium cepa L. Aggregatum group) with Cytogenetic Analysis of Resulting Gynogenic Plants and Somaclones

Unpollinated flower culture was applied for induction of gynogenesis and somatic organogenesis in three shallot strains, ‘Dili-white’, ‘Yogya’ and ‘Dili-red’, from Indonesia. Chromosome surveys were performed on the plants obtained. From a total of 6,812 flowers, 89 plantlets were obtained by gynogenesis, of which 10 could be acclimated. Most of the plantlets were induced from ‘Dili-white’. Of the gynogenetic plants examined, two were haploid (2n = 8), four were naturally doubled haploid (2n = 16) and the remaining four were mixoploid (2n = 8, 16 or 2n = 16, 32, 64). ‘Dili-red’ showed the highest frequency of somatic organogenesis. Fifty-nine directly regenerated plants and 293 callus-derived plants were obtained from somatic organogenesis from the cultured flowers. Based on the chromosome number, frequencies of somaclonal variation were high both in the directly regenerated plants and the callus-derived plants. The frequency of tetraploid plants (2n = 32) in the former (50%) was higher than in the latter (33%). From these results we conclude that unpollinated flower culture is an effective method for chromosome doubling simultaneously with haploid induction in shallot.     

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.     

High-efficiency <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of citrus via sonication and vacuum infiltration

An improved method for the Agrobacterium infiltration of epicotyl segments of ‘Pineapple’ sweet orange [Citrus sinensis (L.) Osbeck] and ‘Swingle’ citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional Agrobacterium-mediated inoculation method (‘dipping’ method). It was observed that the morphogenic potential of the epicotyl segments decreased as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments, but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4% for ‘Pineapple’ sweet orange and 11.2% for ‘Swingle’ citrumelo) are the highest ones reported for both cultivars.     

Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy <Emphasis Type="Italic">Citrus</Emphasis> 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.     

Repression of asexual embryogenesis in vitro by some plant growth regulators

Summary A factor that represses asexual embryogenesis has been observed in the Rutaceae, with particularly high concentrations in the naturally monoembryonic cultivars. This investigation was an initial step towards identifying the factor.Citrus reticulata Blanco Ponkan mandarin nucellus explants andDaucus carota L. ‘Queen Anne's Lace’ callus were employed to examine effects of known plant growth regulators and to determine possible identity of one or more of them with the repressive factor. The chalazal halves of ovules ofC. media L. ‘Citron of Commerce’ were used as control repressor source. Embryo initiation and growth of both test tissues were depressed markedly by 2,4-D, abscisic acid and ethephon. Slight inhibitions were obtained with IAA, kinetin and gibberellic acid. Recovery from the repressor did not occur readily inCitrus nucellus following recultures in citron-ovule-free medium; carrot callus resumed normal embryogenesis immediately upon transfer to suppressor-free medium. The repression by natural sources apparently involved the combined action of some or all natural hormones that are generically related to the above. This paper is part of B. Tisserat's Ph.D. dissertation in Botany at the University of California, Riverside. The research was supported in part by the Elvenia J. Slosson Fellowship in Ornamental Horticulture awarded to T. Murashige.     

A modifier locus affecting the expression of the S-RNase gene could be the cause of breakdown of self-incompatibility in almond

Self-compatibility has become the primary objective of most almond (Prunus amygdalus Batsch) breeding programmes in order to avoid the problems related to the gametophytic self-incompatibility system present in almond. The progeny of the cross ‘Vivot’ (S ₂₃ S _fa) × ‘Blanquerna’ (S ₈ S _fi) was studied because both cultivars share the same S _f allele but have a different phenotypic expression: active (S _fa) in ‘Vivot’ and inactive (S _fi) in ‘Blanquerna’. In addition, the microscopic observation of pollen tube growth after self-pollination over several years showed an unexpected self-incompatible behaviour in most seedlings of this cross. The genotypes of this progeny showed that the S _fi pollen from ‘Blanquerna’ was not able to grow down the pistils of ‘Vivot’ harbouring the S _fa allele, confirming the active function of this allele against the inactive form of the same allele, S _fi. As self-compatibility was observed in some S ₈ S ₂₃ and S ₈ S _fa individuals of this progeny, the S _f haplotype may not always be linked to the expression and transmission of self-compatibility in almond, suggesting that a modifier locus may be involved in the mechanism of self-incompatibility in plants.     

Selection of Highly Informative Microsatellite Markers to Identify Pollen Donors in ‘Hass’ Avocado Orchards

‘Hass’ is the most popular avocado (Persea americana Mill.) cultivar in the world. It has been characterized as a crop requiring cross-pollination. However, the potential extent of self-pollination and the most effective pollen donors (best cross-pollinizing cultivars) have not been determined. In this study, 56 markers were screened against ‘Hass’ and nine commonly used pollinizing cultivars grown in southern California: ‘Bacon,’ ‘Ettinger,’ ‘Fuerte,’ ‘Harvest,’ ‘Lamb Hass,’ ‘Marvel,’ ‘Nobel,’ ‘Sir Prize,’ and ‘Zutano.’ Seventeen microsatellite, i.e., simple sequence repeat (SSR) markers, were found to be very promising for paternity analysis. Four highly informative SSR markers were selected to accurately and unequivocally identify pollen parents of ‘Hass’ fruit from an orchard interplanted with these pollinizing cultivars. From 2003 to 2006, 7,984 ‘Hass’ fruit were analyzed for their paternity. Overall, the pollen parents of 99.55% of the analyzed fruit could be unequivocally identified with a single multiplex polymerase chain reaction (PCR). Only 36 fruits (<0.45%) required a second PCR reaction to reach unequivocal identification of the pollen parents.     

Analysis of volatile compounds released from embryogenic cultures and somatic embryos of sweet oranges by head space SPME

Volatile compounds released from callus and nucellar embryo tissues of ‘Valencia late’ and ‘Washington Navel’ sweet oranges (Citrus sinensis, L. Osbeck) were collected/concentrated by head space solid phase micro extraction and analysed by gas chromatography-mass spectrometry. Friable, white embryogenic cultures released a number of volatile compounds, including some essential oils. Different samples of the same embryogenic culture showed variability, possibly related to the presence of tissues undergoing differentiation. Analyses of the somatic embryos permitted the identification of several components, including limonene and methyl anthranilate. Considering the simplicity and the very small sample required (0.3 g of fresh tissue) head space solid phase micro extraction is suitable for studies and comparisons of volatile metabolites released from in vitro Citrus tissue cultures suggesting its potential in Citrus biochemical, genetic and breeding research. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development of SCAR markers linked to self-incompatibility in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

‘SI1300’ is a self-incompatible Brassica napus line generated by introgressing an S haplotype from B. rapa ‘Xishuibai’ into a rapeseed cultivar ‘Huayou No. 1’. Five S-locus specific primer pairs were employed to develop cleaved amplified polymorphic sequences (CAPS) markers linked the S haplotype of ‘SI1300’. Two segregating populations (F₂ and BC₁) from the cross between ‘SI1300’ and self-compatible European spring cultivar ‘Defender’, were generated to verify the molecular markers. CAPS analysis revealed no desirable polymorphism between self-incompatible and self-compatible plants. Twenty primer pairs were designed based on the homology-based candidate gene method, and six dominant sequence characterized amplified region (SCAR) markers linked with the S-locus were developed. Of the six markers, three were derived from the SRK and SP11 alleles of class II B. rapa S haplotypes and linked with S haplotype of ‘SI1300’. The other three markers were designed from the SLG-A10 and co-segregated with S haplotype of ‘Defender’. We successfully combined two pairs of them and characterized two multiplex PCR markers which could discriminate the homozygous and heterozygous genotypes. These markers were further validated in 24 F₃ and 22 BC₁F₂ lines of ‘SI1300 × Defender’ and another two segregating populations from the cross ‘SI1300 × Yu No. 9’. Nucleotide sequences of fragments linked with S-locus of ‘SI1300’ showed 99% identity to B. rapa class II S-60 haplotype, and fragments from ‘Defender’ were 97% and 94% identical to SLG and SRK of B. rapa class I S-47 haplotype, respectively. ‘SI1300’ was considered to carry two class II S haplotypes and the S haplotype on the A-genome derived from B. rapa ‘Xishuibai’ determines the SI phenotype, while ‘Defender’ carry a class I S haplotype derived from B. rapa and a class II S haplotype from B. oleracea. SCAR markers developed in this study will be helpful for improving SI lines and accelerating marker-assisted selection process in rapeseed SI hybrid breeding program.