Evidence for host plant preference by Iphiseiodes quadripilis (Acari: Phytoseiidae) on Citrus

In this study, we present field and laboratory evidence on the preference of Iphiseiodes quadripilis (Banks) for grapefruit (Citrus paradisi Macfadyen) leaves compared with sweet orange (Citrus sinensis (L.) Osbeck) leaves. This preference was confirmed in four orchards whether leaf samples were taken from either border trees of contiguous grapefruit or sweet orange or interior row trees with both citrus species in adjacent rows. Iphiseiodes quadripilis was most abundant in grapefruit trees in spite of the greater abundance of the Texas citrus mite, Eutetranychus banksi (McGregor) (Acari: Tetranychidae) in sweet orange trees. Similar preference responses were observed in laboratory tests using a Y-tube olfactometer whether I. quadripilis were collected from sweet orange or grapefruit. Iphiseiodes quadripilis collected from grapefruit trees showed significant preference for grapefruit over sweet orange leaves in contact choice tests using an arena of alternating leaf strips (12 mm long × 2 mm wide) of sweet orange and grapefruit. However, I.␣quadripilis collected from sweet orange trees did not show preference for either grapefruit or sweet orange leaves. Based on these results, grapefruit leaves foster some unknown factor or factors that retain I. quadripilis in greater numbers compared with sweet orange leaves.     

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.     

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.     

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     

In vitro anther culture of sweet orange (Citrus sinensis L. Osbeck) genotypes and of a C. clementina × C. sinensis ‘Hamlin’ hybrid

Citrus, and particularly sweet oranges, are very recalcitrant to anther culture. In this paper it was evaluated for the first time the response of 27 genotypes of Citrus sinensis and of one hybrid C. clementina × C. sinensis, to in vitro anther culture. Ten genotypes of sweet oranges showed embryogenic callus induction, mostly blood sweet oranges genotypes, such as Tarocco, Moro and Sanguinelli. In vitro microspore developmental switches from the gamethophytic to the sporophytic pathway were shown by DAPI staining in microspores of these responsive genotypes, after 10 months in culture. However, microsatellite marker analyses showed that these calli were heterozygous. The flow-cytometric analysis of these embryogenic calli showed the presence of two peaks, corresponding to haploid (n) and diploid (2n) genotypes. Differently, anther cultures of the hybrid C. clementina × C. sinensis produced tri-haploid (3n) embryogenic calli and the embryos obtained were homozygous when analyzed by molecular markers (sample sequence repeats), confirming the more responsive characteristic of clementine to microspore embryogenesis through anther culture.     

ADVENTIVE EMBRYOGENESIS IN CITRUS (RUTACEAE) II. POSTFERTILIZATION DEVELOPMENT

Cytological and histological studies on postfertilization development of ovules were carried out in six facultatively apomictic Citrus cultivars. At the time of anthesis, adventive embryo initial cells (AEICs) were detected mainly in the cell layers of the nucellus around the chalazal half of the embryo sac. During the approximately 40 days rest period of the AEICs after fertilization, rapid cell division and enlargement in the endosperm and the chalazal half of the nucellus resulted in the split of AEICs into several separated areas forming the micropylar, lateral and chalazal islands surrounding the enlarging embryo sac. Both in diploid seeds with triploid endosperm and triploid seeds with pentaploid endosperm, the AEICs located in the micropylar half successfully developed into adventive embryos. In diploid seeds, almost all AEICs located in the chalazal half did not develop beyond the initial-celled stage, while in the triploid seeds, those located in the chalazal half occasionally developed into cotyledonary embryos. In seeds with aborted endosperm, the AEICs located in the chalazal half often developed into cotyledonary embryos. The chalazal expiants from normal seeds produced a large number of embryos in vitro. Four results can be summarized from these studies on adventive embryogenesis as follows: 1) All AEICs are initiated prior to anthesis. 2) Whether or not the AEICs successfully developed into adventive embryos is dependent upon their position in the seed. 3) The farther the AEICs are located from the micropylar end, the more adventive embryogenesis is suppressed by endosperm. 4) The degree of adventive embryogenesis in the chalazal half is affected by time and extent of malfunction of the endosperm. Under natural conditions, these regulatory systems of adventive embryogenesis contribute to high production of zygotic seedlings in apomictic Citrus species and cultivars.     

An efficient callus suspension culture system for triploid bermudagrass (Cynodon transvaalensis × C. dactylon) and somaclonal variations

An efficient callus suspension culture and regeneration system in a triploid bermudagrass (Cynodon dactylon × C. transvaalensis cv. Tifeagle) was studied in this report. Proline improved callus proliferation, but had no effect on regeneration. 0.6–1.2 mg l⁻¹ BA improved regeneration, but higher concentrations of BA (≤1.2 mg l⁻¹) resulted in the production of rootless plantlets. The embryogenic calli were able to proliferate continuously for at least 2 years with regeneration ability through the established suspension culture system. Observations with scanning electron microscope and light microscope showed somatic embryogenesis during the regeneration. Somaclonal variations were observed in regenerated plants. More than 2000 regenerants were screened for drought tolerance in the greenhouse, from which seven lines appeared to have increased drought tolerance relative to their parental variety. It is suggested that somaclonal variation in triploid bermudagrass offers an effective tool for its breeding.     

Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron

Summary A procedure for the regeneration of cacao (Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and 1.4 nM kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of 22.7 nM was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types. However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46. Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of a large number of plants from limited source materials.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

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.     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Citrus phylogeny and genetic origin of important species as investigated by molecular markers

Citrus phylogeny was investigated using RAPD, SCAR and cpDNA markers. The genotypes analyzed included 36 accessions belonging to Citrus together with 1 accession from each of the related genera Poncirus, Fortunella, Microcitrus and Eremocitrus. Phylogenetic analysis with 262 RAPDs and 14 SCARs indicated that Fortunella is phylogenetically close to Citrus while the other three related genera are distant from Citrus and from each other. Within Citrus, the separation into two subgenera, Citrus and Papeda, designated by Swingle, was clearly observed except for C. celebica and C. indica. Almost all the accessions belonging to subgenus Citrus fell into three clusters, each including 1 genotype that was considered to be a true species. Different phylogenetic relationships were revealed with cpDNA data. Citrus genotypes were separated into subgenera Archicitrus and Metacitrus, as proposed by Tanaka, while the division of subgenera Citrus and Papeda disappeared. C. medica and C. indica were quite distant from other citrus as well from related genera. C. ichangensis appeared to be the ancestor of the mandarin cluster, including C. tachibana. Lemon and Palestine sweet lime were clustered into the Pummelo cluster led by C. latipes. C. aurantifolia was located in the Micrantha cluster. Furthermore, genetic origin was studied on 17 cultivated citrus genotypes by the same molecular markers, and a hybrid origin was hypothesized for all the tested genotypes. The assumptions are discussed with respect to previous studies; similar results were obtained for the origin of orange and grapefruit. Hybrids of citron and sour orange were assumed for lemon, Palestine sweet lime, bergamot and Volkamer lemon, while a citron × mandarin hybrid was assumed for Rangpur lime and Rough lemon. For Mexican lime our molecular data indicated C. micrantha to be the female parent and C. medica as the male one. Received: 5 October 1999 / Accepted: 3 November 1999     

Susceptibility and Resistance of Citrus Genotypes to Alternaria alternata pv. citri

A survey of citrus cultivars in Israel in orchards where Alternaria brown spot was common on Minneola tangelos (mandarin × grapefruit), revealed the occurrence of the disease as typical foliar and fruit lesions on Dancy and Ellendale (mandarins), on Murcott tangor (mandarin × sweet orange), on Nova and Idith (mandarin hybrids), on Calamondin, and on Sunrise and Redblush (grapefruit). Isolates of Alternaria alternata from each of these hosts were proven to be pathogenic to Minneola tangelo.
The host range of A. alternata pv. citri from Israel was assayed by inoculating leaves of diverse citrus genotypes. Several mandarins and their hybrids (Dancy, Kara, King, Wilking, Satsuma, Minneola, Orlando, Mikhal, Idith, Nova, Page, Murcott), grapefruit (Marsh seedless), grapefruit × pummelo (Oroblanco), sweet orange (Shamouti, Valencia, Washington navel) Calamondin, and Volkamer citrus were susceptible. Several mandarins and their hybrids (Clementine, Avana, Yafit, Ortanique), Cleopatra, one sweet orange cultivar (Newhall), pummelo (Chandler), lemon (Eureka), Rough lemon, Rangpur lime, sweet lime, citron, limequat, sour orange, Troyer citrange and Alemow were resistant.     

The plastome of Citrus. Physical map,variation among Citrus cultivars and species and comparison with related genera

Summary A physical plastome map was constructed for Citrus aurantium, and the plastomes of species and cultivars of Citrus and of two Citrus relatives were analysed by Southern blot-hybridisation of labelled total tobacco cpDNA to digests of total Citrus DNA. A resemblance was found between the plastomes of cultivars of C. limon (lemon), C. sinensis (orange), C. aurantium (sour orange), C. paradisii (grapefruit) and C. grandis (pomello). The plastomes of other Citrus types such as mandarin (C. reticulata) and citron (C. medico) differed from each other as well as from the plastomes of the aforementioned group. The plastomes of Poncirus trifoliata and Microcitrus sp. are distinct from each other as well as from the Citrus types.     

High frequency plant regeneration from mature embryo explants of highland barley (Hordeum vulgare L. var. nudum Hk. f.) under endosperm-supported culture

An in vitro protocol for efficient plant regeneration has been developed from mature embryo explants of highland barley (Hordeum vulgare L. var. nudum Hk. f.) under endosperm-supported culture. Embryos with (endosperm-supported culture, ES) or without endosperm (non-endosperm-supported culture, NES) were excised from mature seeds and cultured on MS medium supplemented with various concentrations of 2,4-D (1–5 mg l⁻¹) for callus induction. The percentage of callus induction from ES explants was significantly (P < 0.05) lower than that from NES. The highest frequency (97.6%) of callus induction was obtained from NES explants on MS medium containing 3 mg l⁻¹ 2,4-D. When the primary calli were maintained at a reduced concentration of 2,4-D (0.5 mg l⁻¹) for 3 weeks, embryogenic calli were formed. The embryogenic calli were then transferred to MS medium supplemented with different concentrations of BA (1–5 mg l⁻¹) and 500 mg l⁻¹ casein hydrolysate (CH) for shoot regeneration. However, the capacity of plant regeneration from ES explant-derived calli was significantly (P < 0.05) higher than that from NES. The best response (81.3%) was observed from ES explant-derived calli on MS medium containing 2 mg l⁻¹ BA. Regenerated plantlets with well-developed root systems were transferred to pots where they grew well, attained maturity and produced fertile seeds. This method could be employed for genetic manipulation studies.     

Nodular somatic embryogenesis and frond regeneration in duckweed,Lemna gibba G3

Duckweed(Lemna gibba) is a useful model system for elucidating plant development, but the techniques needed for regenerating fronds from calli are not yet well established. This study examined the effects of auxin, sucrose, and gelling agents on callus and frond formation inL. gibba G3. After three weeks of culturing on a solid medium, two types of calli were observed: watery, pale-green, and undifferentiated; or white, compact calli that were organized into nodules and which resembled somatic embryogenie calli. Homogeneous callus lines were produced through selective subculture. To induce nodular calli, auxin (2,4-D) was absolutely required, with an effective concentration of 5 to 20 μM; induction was found to be possible with up to a maximum concentration of 4.4%. The calli were then maintained on a medium with a reduced 2,4-D concentration (1 μM), and were transferred every three weeks. Optimal callus induction and growth were obtained by using 3% sucrose with a combination of 0.15% Gelrite and 0.4% agar. Fronds, however, could be regenerated only on distilled water solidified with a combination of 0.4% agar and 0.15% Gelrite. On this medium, 87% of the callus expiants regenerated into fronds after four weeks of culture. These new fronds were morphologically normal but small, approximately 15 to 20% of the size of stock fronds. Continued culture of these fronds in an SH medium produced normal duckweeds, and histological examination of the cultures revealed several distinct types of callus nodules. Nonetheless, because zygotic embryogenesis inL. gibba does not produce distinct bipolar structures, the developmental pathway of frond regeneration from these nodular cultures remains unknown.     

Asymmetric protoplast fusion between sweet potato and its relatives,and plant regeneration

Experiments were conducted to asymmetrically fuse protoplasts from sweet potato (Ipomoea batatas L. Lam.) and its wild relativesI. trifida Don. andI. lacunosa L. Protoplasts of sweet potato were treated with iodoacetamide, whereas those ofI. trifida Don. andI. lacunosa L. were irradiated with X-rays. The asymmetric protoplast fusion was carried out by the electrofusion method and by polyethylene glycol treatment. Electrically-fused protoplasts initiated cell division, and then formed calli earlier than the polyethylene glycol-fused protoplasts. Plant regeneration occurred only in electrofused calli, suggesting that polyethylene glycol had some toxic effect on plant regeneration ability. Analysis of peroxidase isozymes confirmed the interspecific hybrid characteristics of both the fusion-derived calli and regenerated plants.     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

Callus induction,organogenesis and somatic embryogenesis in three chromosomal races of Urginea indica and production of bufadienolides

Three chromosomal races of Indian squill, Urginea indica Kunth., were screened for their ability to produce bufadienolide in tissue cultures. The protocols for callus induction, organogenesis and somatic embryogenesis differed in the three races with respect to vitamin requirements and growth regulator additions. Bufadienolide contents were determined by HPLC. Undifferentiated calli and cell suspension cultures did not produce the bufadienolides. Shoot differentiating cultures contained only trace amounts of proscillaridin A while embryogenic cultures and developing embryoids did not contain bufadienolides. All regenerated bulbs (derived from diploid, triploid and tetraploid parents through organogenesis and/or somatic embryogenesis) were found to contain both proscillaridin A and scillaren A, the bufadienolide characteristic of the parent plant.     

High frequency somatic embryogenesis and plant regeneration from zygotic embryo-derived callus cultures of three Allium species

The plant regeneration ability of zygotic embryo-derived callus cultures was studied for 12 A. cepa varieties and accessions, two A. fistulosum varieties, one A. fistulosum x A. cepa interspecific hybrid and two A. porrum varieties. Compact embryogenic callus was induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid. The embryogenic calluses of all three Allium species were similar in appearance. For all accessions tested plants could be regenerated at a high frequency from this compact callus through somatic embryogenesis, when using kinetin supplemented MS medium (regeneration medium). Addition of abscisic acid to the regeneration medium stimulated the formation of both somatic embryos and shoots for a number of varieties. Concerning shoot regeneration from callus cultures, significant differences existed between genotypes of all accessions except one.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - VDH Van Der Have Seed company