Embryogenic mango cultures selected for resistance toColletotrichum gloeosporioides culture filtrate show variation in random amplified polymorphic DNA (RAPD) markers

Summary Genomic DNA isolated from embryogenic cultures of two mango cultivars, ‘Hindi’ and ‘Carabao,’ that had been selected for resistance to the culture filtrate ofColletotrichum gloeosporioides, was analyzed using Randomly Amplified Polymorphic DNA (RAPD).In vitro selection caused changes in RAPD markers in the selected embryogenic cultures with respect to the unchallenged control cultures and the stock plants. The differences involved both the absence and the presence of additional RAPD markers in the resistant lines, although the former was most commonly observed. The absence of differences between the unchallenged control of either cultivar and DNA from the leaves of parent trees confirmed that the changes were not due to prolonged maintenance in liquid cultures.     

Recovery and regeneration of embryogenic cultures from female flowers of False Horn Plantain

Somatic embryogenesis from immature male flowers in Musa is only suitable for genotypes with a male bud. Six friable embryogenic cultures were obtained from 28 cultured buds of female flowers of the AAB False Horn Plantains, ‘Curraré’ and ‘Curraré Enano’. Embryogenic suspensions were established from these embryogenic cultures. Somatic embryogenesis was demonstrated histologicaly. Regeneration of plants was obtained either from somatic embryos directly isolated from embryogenic cultures or from suspensions after plating on a semi-solid medium. This study demonstrates that somatic embryogenesis from immature flowers is suitable for genotypes of Musa with or without male buds. This revised version was published online in June 2006 with corrections to the Cover Date.     

QTL mapping of aroma compounds analysed by headspace solid-phase microextraction gas chromatography in the apple progeny ‘Discovery’ × ‘Prima’

Improving fruit quality of apple varieties is an important but complex breeding goal. Flavour is among the key factors of apple fruit quality but in spite of the analytical and biochemical knowledge about volatiles little is known about the genetic and molecular bases of apple aroma. The aim of this study was to use a saturated molecular linkage map of apple to identify QTLs for aroma compounds such as alcohols, esters and terpenes, but also for a number of unidentified volatile compounds (non-targeted analysis approach). Two parental genetic maps were constructed for the apple cultivars ‘Discovery’ and ‘Prima’ by using mainly AFLP and SSR markers. ‘Discovery’ and ‘Prima’ showed very different volatile patterns, and ‘Discovery’ mostly had the higher volatile concentrations in comparison with the Vf-scab resistant ‘Prima’ which has its origin in the small-fruited apple species Malus floribunda. About 50 putative QTLs for a total of 27 different apple fruit volatiles were detected through interval mapping by using genotypic data of 150 F₁ individuals of the mapping population ‘C3’ together with phenotypic data obtained by head-space solid phase microextraction gas chromatography. QTLs for volatile compounds putatively involved in apple aroma were found on 12 out of the 17 apple chromosomes, but they were not evenly dispersed. QTLs were mainly clustered on linkage groups LG 2, 3 and 9. In a first attempt, a LOX (lipoxygenase) candidate gene, putatively involved in volatile metabolism, was mapped on LG 9, genetically associated with a cluster of QTLs for ester-type volatiles. Implications for aroma breeding in apple are discussed.     

The effects of low levels of chloramphenicol and methotrexate on somatic embryogenesis inCitrus

Summary The effects of the antibiotics methotrexate and chloramphenicol on somatic embryogenesis inCitrus were evaluated. Relatively low levels (0.1 to 1.0 μg/ml) of these antibiotics did not inhibit embryo production from undeveloped ovules of ‘Key’ lime [C. aurantifolia) (Christm.) Swing.]. Surprisingly, both antibiotics induced the formation of embryogenic callus in these cultures. This is usually a rare event in cultures of undevelopedCitrus ovules, and ‘Key’ lime is especially recalcitrant. The effects of these antibiotics on embryogenic callus appeared to be limited to the induction stage, because there was no consistent effect, either stimulatory or inhibitory, on established, lines of embryogenic callus. Florida Agricultural Experiment Station Journal Series No. 8958. This research was supported in part by a grant to Moore and Cline from the Competitive Grants Office of the SEA, USDA (85-CRCR-1-1623).     

Responses of embryogenic mango cultures and seedling bioassays to a partially purified phytotoxin produced by a mango leaf isolate of Colletotrichum gloeosporioides penz

Summary Colletotrichum gloeosporioides Penz., the causal agent of mango anthracnose, produces a phytotoxin in vitro. The partially purified phytotoxin, presumably colletotrichin, caused anthracnose-like symptoms on young mango leaves, was toxic to embryogenic suspension cultures of two mango cultivars, ‘Hindi’ and ‘Carabao,’ and inhibited in vitro seed germination of two nonhosts, lettuce and tobacco. There were linear relationships between concentration of the partially purified phytotoxin and mortality of mango embryogenic cultures. Embryogenic cultures grown in the presence of the partially purified phytotoxin showed significantly lower growth rates than the controls. Similarly, embryogenic cultures grown in the presence of 40% (vol/vol) fungal culture filtrate showed significantly lower growth rates than unchallenged controls. Medium containing 40% (vol/vol) Czapek-Dox fungal broth did not reduce growth of embryogenic cultures, indicating the production of phytotoxin in vitro. The results suggest that either fungal culture filtrate or purified phytotoxin can be used as in vitro selection agents to screen for resistance to this fungus.     

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.     

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan. × Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blanco × Citrus sinensis L. Osb) is reported here for the first time.     

Mutagenesis of Embryogenic Cultures of Soybean and Detecting Polymorphisms Using RAPD Markers

Embryogenic suspension cultures of soybean (Glycine max L. cv. Iroquois) were subjected to mutagenesis using varying concentrations (1, 3, 10, and 30 mM) of ethyl methanesulfonate (EMS). Depending on the concentration of EMS used, the mean survival rate of embryogenic cultures decreased from 74 % (1 mM EMS) to 43 % after 30 mM EMS treatment. Random amplified polymorphic DNA (RAPD) analysis was used to determine whether induction of genetic variability in embryogenic cultures in response to the different EMS treatments may result in identification of polymorphic markers. Two of 35 ‘core’ primers tested revealed polymorphisms. One of the primers, OPO-01/1150, revealed polymorphism in tissue treated with 10 mM EMS, while the other primer, OPO-05/1200, revealed polymorphism in tissue treated with either 1 or 30 mM EMS. These results suggest that RAPD markers are useful in detecting mutations in embryogenic cultures of soybean. This revised version was published online in July 2006 with corrections to the Cover Date.     

Use of lysozyme for treatment of bacterial contamination in <Emphasis Type="Italic">in vitro</Emphasis> shoot cultures of fruit plants

Summary Use of lysozyme was tested for treatment of bacterial contaminations in in vitro shoot cultures of quince (Cydonia oblonga) ‘BA 29’ and the hybrid (Prunus persica × P. amygdalus) rootstock ‘GF 677’. Shoots which had been contaminated for about 1 yr by Bacillus circulans and Sphingomonas paucimobilis were treated in liquid culture, at pH 4.5, with 9–36 mg ml⁻¹ egg white lysozyme (EWL), and compared to each other and to untreated cultures for their growth, proliferation, and number of bacterial colony-forming units in the tissues. EWL did not negatively affect shoot growth up to 18 mg ml⁻¹; furthermore, the proliferation rates of EWL-treated shoots were sometimes higher than those of controls. In contrast, the concentration of 36 mg ml⁻¹ had some deleterious effect on the regrowth capacity and shoot production of ‘GF 677’ at the first subculture to solid medium after EWL, treatments. EWL had a simple bacteriostatic effect against Sphingomonas paucimobilis; in contrast, it was effective at 18 mg ml⁻¹ in eliminating Bacillus circulans in both ‘BA 29’ and ‘GF 677’ cultures, after optimal treatment duration.     

Somatic embryogenesis from mesocotyl and leaf-base segments of <Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L., a minor millet

Summary Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N₆) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N₆ medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic calluses to hormone-free MS or N₆ regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N₆ medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’ explants such as immature embryos and unemerged inflorescences.     

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.     

Probable identity of substances in citrus that repress asexual embryogenesis

Summary The embryogenetic responseof culturedDaucus carota L. ‘Queen Anne's Lace’ callus was employed to attempt fractionation and identification of a repressive factor produced byCitrus medica L. ovules. The factor was evidently synthesized and released into the medium continuously, inasmuch as citron ovules that had been autoclaved with the medium were completely infeffective. The inhibition could be attributed to volatile and nonvolatile components. A substantial part of the inhibition was prevented by continuously refereshing the atmosphere within the cultures with filtered air. Monitoring of the gases produced by citron ovule sections under conditions simulating bioassays disclosed significant evolution of carbon dioxide, ethylene and ethanol. Repression of embryogenesis was not averted by trapping the liberated ethylene. On the other hand, ethanol in concentrations equivalent to those released by citron ovules suppressed asexual embryogenesis dramatically. The adverse effect of ethanol was reversed immeditaley upon transfer to ethanol-free medium. Another investigation had disclosed anti-embryogenetic effects of auxin, abscisic acid and gibberellin. Analysis ofCitrus ovules excised from young fuits disclosed those of monoembryonic citron to contain concentrations of IAA, ABA and GA₃ several times higher than those of polyembryonic Ponkan mandrain. The nonvolatile protion might be identified with these hormonal substances. This paper is part of B. Tisserat's PhD. 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.     

Long-term study of somatic embryogenesis from anthers and ovaries of 12 grapevine (<Emphasis Type="Italic">Vitis</Emphasis> sp.) genotypes

Summary Anthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V × Labruscana L. H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay’, ‘Merlot’, and ‘Pinot Noir’ occurred on medium C [Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose, 0.01% (w/v) inositol. 0.3% (w/v) Phytagel, 2.5 μM 2.4-dichlorophenoxyacetic acid, 2.5μM β-naphthoxyacetic acid, 5.0μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay’, ‘Merlot,’ and ‘Pinot Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture, with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet Franc,’ and ‘Pinot Noir’ grapevines.     

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.     

Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Summary Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.     

Characterization of embryogenic mango cultures selected for resistance to Colletotrichum gloeosporioides culture filtrate and phytotoxin

 Embryogenic nucellar cultures of two polyembryonic mango cultivars, ‘Hindi’ and ‘Carabao’, were selected for resistance to the culture filtrate and phytotoxin of a virulent strain of Colletotrichum gloeosporioides Penz. that was isolated from mango leaves. The cultures were recurrently selected either with progressively increasing concentrations of culture filtrate or by continuous challenge with the same concentration of either culture filtrate phytotoxin. Mycelium growth was inhibited when the pathogen was cocultured with the selected, resistant embryogenic cultures. Conditioned plant growth medium containing macerated resistant embryogenic cultures did not inhibit mycelium growth, confirming that extracellular antifungal compounds were involved in the defense response. Enhanced secretion of chitinase and glucanase was observed in the plant growth medium in which resistant embryogenic cultures and regenerated somatic embryos were grown in comparison with the controls. Received: 6 February 1997 / Accepted: 4 November 1997     

Induction of vesicle formation in a cell line derived from imaginal discs

Summary The ability of insect hemolymph to induce vesicles in a high passage insect cell line, IAL-TND1, is described. The factor responsible, designated VPA for ‘vesicle-promoting activity’, was determined to be heat sensitive, nondialyzable, and protease Type XIV sensitive but insensitive to trypsin digestion. In efforts to determine the source of VPA, hemolymph was collected from different developmental stages ofTrichoplusia ni, and certain tissues fromT. ni were cocultured with IAL-TND1 cells. Hemolymph from every developmental stage tested exhibited VPA although the effect was somewhat reduced in spinning-stage larvae. Additionally, several tissue, including fat body, tess, and imaginal discs, released VPA into the culture medium. Neural tissues and endocrine glands did not induce vesicle formation.     

Triploid banana cell growth phases and the correlation of medium pH changes with somatic embryogenesis in embryogenic cell suspension culture

Embryogenic cell suspensions of Musa AAA and AAB genomic groups were cultured in a maintenance culture medium for 17 generations (lasting for 238 days). The cell growth phases and medium pH changes were also observed correspondingly. Three major growth phases of AAA genomic group have been focused, namely cell releasing, proliferation and globularization phases. During almost all the subculture generations the cell stocks of AAB ‘Raja’ were continuously characterized by proliferating cell aggregates while the globularization phase occurred only for short duration. The medium acidity levels of the cell stocks of AAA ‘Pei-Chiao’ and ‘Robusta’ were commonly scattered in a wider range of pH 3.3–5.3, while the AAB ‘Raja’ were deviated in a narrow range of pH 4.0–4.6. After subculture, culture medium showed biphasic pH changes, which were drastic pH falls followed by an auto-regulated steady-state level. The steady-state pH values in each of the three growth phases (i.e. cell releasing, proliferation and globularization phases) were of 3.3–4.0, 4.0–4.8 and 4.8–5.3 respectively. Morphological bipolarity and the efficiency in the formation of somatic embryos have been thoroughly discussed. Reported research indicates that the condition of pH below 4.6 may prevent the development of embryogenic cells towards polar growth.     

Effects of 1-aminocyclopropane-1-carboxylic acid,aminoethoxyvinylglycine, methylglyoxal bis-(guanylhydrazone) and dicyclohexylammonium sulfate on induction of embryogenic competence of mango nucellar explants

The effects of aminoethoxyvinylglycine (AVG),1-aminocyclopropane- 1 -carboxylic acid (ACC), dicycyclohexylammonium sulfate (DCHA) and methylglyoxal bis-(guanylhydrazone) (MGBG) on induction of embryogenic competence from the nucellus of two mango genotypes, ‘Tutehau’ (polyembryonic) and ‘Tommy Atkins’ (monoembryonic), were compared. Induction of embryogenic competence in the explanted nucellus of ‘Tommy Atkins’ was more sensitive to AVG and DCHA than ‘Tutehau’. MGBG had no effect on induction of embryogenic competence of either genotype, and ACC suppressed somatic embryogenesis with both cultivars. Ethylene biosynthesis was greater from explanted ‘Tommy Atkins’ cultures. Reducing ethylene biosynthesis with AVG marginally increased the number of embryogenic cultures of ‘Tutehau’, but completely inhibited somatic embryogenesis in ‘Tommy Atkins’ cultures. Ethylene biosynthesis was stimulated by ACC in nonembryogenic cultures of both genotypes and in ‘Tommy Atkins’ embryogenic cultures, in which ethylene biosynthesis was ca 7 X greater than in ‘Tutegau’ embryogenic cultures. The greater sensitivity of ‘Tommy Atkins’ to the ethylene antagonist, AVG, may be due to its greater sensitivity to disturbance of ethylene biosynthesis and spermidine synthesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis,plant regeneration and somaclonal variation in barley

In vitro culture of immature embryo and young leaf tissues was carried out with five cultivars of barley, Hordeum vulgare. Two cultivars (Albacete and Porthos) responded poorly from both types of explants, while the three others (Dissa, Golden Promise and Ingrid) produced a high frequency of embryogenic callus from these explants (25–60%). For Dissa and Ingrid, young leaf explants were slightly better than immature embryo explants for embryogenic callus induction, while immature embryo cultures of Golden Promise responded better than young leaf explants. Thus, there appears to be a significant genotype × explant interaction in the initiation of embryogenic callus in barley.Some phenotypic variants were detected among the regenerated plants of Golden Promise and Ingrid, most originating by epigenetic changes. Only in one case was the variant phenotype heritable, probably due to a mutation in the chloroplast DNA. Mitotic alteractions were not detected. Consequently, somaclonal variation did not appear to be a very frequent event in plants regenerated from 1- to 6- month-old cultures of barley.