Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.]

Somatic embryogenesis was induced and plant regeneration was obtained in 11 different genotypes of sweet orange navel group [Citrus sinensis (L.) Osb.] from cultures of stigma/style explants and undeveloped ovules. Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg l-1 malt extract; 500 mg l-1 malt extract and 4.6 μM kinetin; and 500 mg l-1 malt extract and 13.3 μM 6-benzylaminopurine. Sucrose (146 mM) was used as carbon source. Somatic embryogenesis occurred 1–3 months after culture initiation from undeveloped ovule and stigma/style cultures of all the genotypes tested. Somatic embryos developed into plantlets with a high frequency (74%) after transfer to Murashige and Skoog medium supplemented with 146 mM sucrose and 500 mg l-1 malt extract. Plants were successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fertile fruit trees obtained by somatic hybridization: navel orange (Citrus sinensis) and Troyer citrange (C. sinensis x Poncirus trifoliata)

Summary Nucellar cell suspension protoplasts of navel orange (Citrus sinsensis Osb.) were chemically fused with mesophyll protoplasts of Troyer citrange (C. sinensis x Poncirus trifoliata) and cultured in hormone-free Murashige and Tucker medium containing 0.6 M sucrose. Two types of plant were regenerated through embryogenesis. One type showed intermediate mono-and difoliate leaves and the other types was identical to Troyer citrange. The regenerated plants with intermediate morphology were demonstrated by chromosome counts and rDNA analysis to be amphidiploid somatic hybrids. Five clones of these somatic hybrids were grafted in the field. After 4 years, they set flowers having a morphology intermediate between those of the two parents. The pollen grains showed high stainability and sufficient germinability, and were larger than those of Troyer citrange. The fruits of the somatic hybrids were large and spherical with thick rinds. Most of them contained seeds with normal germinability. These results indicate that somatic hybridization is a useful tool for Citrus breeding.     

DEMOGRAPHIC ASPECTS OF FLOWER AND FRUIT PRODUCTION IN BEAN PLANTS,PHASEOLUS VULGARIS L.

In this paper we assess the relative contributions to total pod yield of cohorts of flowers of known age. Bean plant (Phaseolus vulgaris L.) growth was monitored and 'births' of individual flowers were recorded and their fates followed. For every cohort of flowers an array of yield measures was calculated, including the mean and total cohort dry weight of fruits, and the numbers of viable and aborted seeds and completely undeveloped ovules. When mature, pods were removed from half of the plants. Plants whose mature pods were removed produced significantly greater numbers of flowers, ovules and viable seeds and a greater total weight of pods than did untreated plants. However, the proportion of ovules giving rise to viable seeds and to aborted seeds and undeveloped ovules was the same in plants of both groups. Approximately 50% of ovules of marketable pods, in all plants, yielded viable seeds. The 12 cohorts of flowers contributed markedly different amounts to plant yield. Early and later cohorts contributed much less than did middle cohorts in terms of marketable pod dry weight and numbers of fully developed seeds, aborted seeds, and undeveloped ovules. Peak productivity was therefore obtained from the middle phase of flowering in these annual bean plants. The lower pod yield of later cohorts is interpreted as a result of competition for limited resources between maturing pods and new flowers. We consider possible causes for the various fates of flowers and ovules and discuss flowers as plant modules suitable for demographic examination.     

Cultura in Vitro di Ovuli di Pinus Pinea L.

Abstract

Pinus Pinea ovules cultured in vitro. — The degree of growth and autonomical differentiation of Pinus pinea L. proembryo has been studied by means of controlled cultures in vitro of excised ovules.

Proembryos in vitro undergo involution and initials of their growth points change back into parenchimatoides cells.

Completely differentiated embryos cultivated in vitro behave as if they were not physiologicaly ripe in all their parts. Embryos cultured in august, september and october develop into rocotless seedlings. Only embryos cultured in november have roots able to elongate where germinating, but in a still scarce degree in comparison with hypocotile and cotiledons. Hypocotil root ratio is inverted as regards what happens in nature.

The primary endosperm of Pinus pinea L. cultivated in vitro undergoes surface diffuse proliferation.

A case of polyembriony has been observed.     

Contributo preliminare alla sistematica di Chamaecytisus hirsutus e Chamaecytisus supinus

Abstract

Chamaecytisus hirsutus and C. supinus: a preliminary report. – Chamaecytisus hirsutus (L.) Link and C. supinus (L.) Link have been described as twostrictly related species, which differ mainly in the form of inflorescence: C. hirsutus has lateral flowers, C. supinus has terminal flowers. Besides, most Floras provide a set of additional differential characters that should permit to di-C. hirsutus and C. supinus.

The author analyses such differential characters and demonstrates that all of them are inconsistent: therefore the inflorescence seems to be the only difference between C. hirsutus and C. supinus.

The inflorescence itself, however, is not a constant character: indeed, it is known that C. supinus may develop vernal latera flowers besides the normal aestival terminal ones.

The geographic distributions of the two inflorescence types are accurately examined (a report of the distributions is given, a list of herbarium specimens is presented in appendix, a point distribution map of the two types is given in figs. 1 and 2); the only differences are the following ones: plants with flowers in leafy racemes (usually identified as C. hirsutus) seem to be absent from Spain and from Southern Poland, and to be unfrequent in Central and Western France; instead, plants with flowers in heads (usually identified as C. supinus) are very unfrequent in the Southern Balcan Peninsula, and are absent from Southern Greece and the Italian Peninsula.

After a discussion of the biological significance of the capitate and lateral inflorescence, and on the basis of in vivo observations, the author argues that probably the same taxonomic unit is present in the whole area, showing some differences in its flowering behaviour; in the largest part of the areal – including the center of distribution of the species – most individuals flower twice, and therefore have been recorded as two different species; a trend toward the capitate inflorescence is remarkable in the North and in the West; instead, in the South and the East the trend is toward lateral flowers (fig. 3).

Further biometrical and biochemical studies on the species are now in progress; more observations in field in different parts of Europe are necessary in order to get conclusive evidence of the identity of these two so-called «species».     

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.     

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

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

Il sospensore embrionale nelle Cruciferae. I. Morfologia e struttura

Abstract

Embryo suspensor in Cruciferae. I. Morphology and structure. — Embryo suspensors in seven species of Cruciferae (Alyssum argenteum All., Brassica nigra (L.) Koch, Capsella bursa-pastoris (L.) Medicus, Diplotaxis erucoides (L.) DC., Eruca sativa Miller, Lepidium graminifolium L., Matthiola tricuspidata (L.) R. Br.) were morphologically investigated. These species have significant differences in their seeds, so that probable differences could be found in the structure of their ovules and therefore of their suspensors. Results indicate that it is impossible to generalize on the suspensor morphology and structure even in the same family. In Eruca sativa and Brassica nigra polyploidy and polyteny were found, suggesting a probably important metabolic function of their suspensors. In the other species polyteny was not present; a various degree of polyploidy could probably be assumed, suggesting a less active function of suspensors, maybe absorption and transport of nutritive material from the integuments to the developing embryo. The development of the endosperm is almost the same in all species, so that seems to be improbable a negative correlation with the suspensor and endosperm function in the early embryogenesis.     

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).     

Floral rewards and pollination in Cytiseae (Fabaceae)

 Cytiseae have been reported to be mostly nectar-lacking, yet some taxa secrete nectar from extrastaminal nectaries. We studied the pollination biology of four shrubby species of Cytiseae (Cytisophyllum sessilifolium (L.) Lang, Spartium junceum L., Genista radiata (L.) Scop., Genista cilentina Valsecchi) which differ for ecology, distribution and population size. All species resulted obliged xenogamous, insect visits being necessary for successful pollination. Bumblebees and solitary bees are the principal pollinators, but also many beetles, some hover-flies, and few bugs visit flowers. Pollinator specificity is low, and this may be the reason of the scarce seed set compared to the number of ovules. Pollen is the main reward, but traces of glucose were detected in all species, at the base of vexillum or on the reproductive column. Nectar production is irregular in time, and apparently unpredictable. We suppose that nectar may play a role in attracting pollinators determining their right position for a successful pollination. Received August 12, 2002; accepted November 25, 2002 Published online: June 2, 2003     

Biosynthesis and Contents of Gibberellins in Seeded and Seedless Sweet Orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> L. Osbeck) Cultivars

In this work, we study the capacity to biosynthesize gibberellins (GA) of ovules (either fertilised or unfertilised), developing seeds and pericarp from fruitlets and their relation with fruit set capacity. Experiments were performed in adult, 12-year-old trees of seeded (Pineapple) and seedless parthenocarpic (Washington navel) sweet orange [Citrus sinensis L. Osbeck] cultivars. The activity of GA20-, GA3- and GA2-oxidases and gibberellin levels were measured in the ovules and pericarp of fruitlets in different development states. The results indicate that ovules are the main sites of gibberellin synthesis in fruitlets during the post-anthesis period. The most intense GA₁ synthesis—coincident with the highest expression of GA20ox2, GA3ox1 and GA2ox1—was detected in the ovules of the seeded cultivar, probably induced by fecundation and associated with low early fruitlet abscission rates. By contrast, the low activity detected in the sterile cultivar appears to be rather developmentally or constitutively regulated. As a fruitlet develops, the GA₁ concentration is augmented in the pericarp in comparison to ovules or developing seeds, and levels therein did not exhibit noticeable differences between varieties. Furthermore, developing seeds from pineapple had higher GA₁ content than the unfertilised abortive ovules from Washington navel. Taken together, data suggest a main role for this hormone in the control of fruitlet abscission, and also demonstrate a function in seed development.     

Carotenoids synthesized in citrus callus of different genotypes

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

Critical studies on the sexuality of southern Argentinian Ericaceae

Summary The family Ericaceae is considered to be mostly hermaphroditic and only in a few cases unisexual or dioecious. Five Argentinian species (Gaultheria antarctica Hook, f., G. caespitosa P. et E., G. phillyreaefolia (Pers.) Sleumer, G. tenuifolia (Phil.) Sleumer and Pernettya insana (Molina) Gunckel), previously described as hermaphroditic, were found by us to have two types of flowers: female and male. Female flowers have both a rudimentary androecium with stamens, in which the anthers are small, collapsed and without pollen grains or are reduced to filaments only, and a perfect gynoecium with well-developed ovules filling the carpel locules. The stigma is expanded and of the wet, papillate type, with copious to slight secretion. The stigma protrudes above the anthers. Male flowers have a well-developed androecium included within the corolla and a reduced gynoecium. Anthers are as long as the filaments, except in G. caespitosa. Pollen is shed as tetrads which are 100% viable. Ovule development ranges from total absence, passing through aborted ovules of various sizes to apparently normal ones. Stigmata are neither expanded nor papillate, except in Pernettya insana. Only in two species do the style and stigma extend beyond the level of the anthers. Heterostyly does not occur in any of the five species studied. Functional dioecy thus characterizes the five species considered and is reported for the first time in the genus Gaultheria.     

In vitro pollination of isolated ovules of Cichorium intybus L.

 A method for the in vitro pollination of chicory (Cichorium intybus L.) ovules was developed. The purpose was to avoid self-incompatibility after in vitro self-pollination of ovules isolated from flower buds before anthesis and from open flower heads. Seedlings were obtained at a low percentage (0.76%), and the results were explained in terms of pollen viability, pollen germination on the ovule, embryogenesis studies and ploidy analysis. Received: 9 April 1997 / Revision received: 23 July 1997 / Accepted: 6 September 1999     

Interspecific crosses in the genus Tulipa L.: identification of pre-fertilization barriers

 Pollen tube growth in the pistil and pollen tube penetration of ovules have both been studied in crosses between cultivars from Tulipa gesneriana L. and 12 tulip species from all eight sections of the genus Tulipa to identify pre-fertilization barriers. Depending on the cross, pollen tubes grew as far as the stigma or the style or continued growing down into the ovary. Pollen tubes penetrated none or only a few percent of the ovules of some crosses, despite the presence of many pollen tubes in the ovary. In other crosses, from which no or only a few hybrids have been obtained after seed maturation on the plant, pollen tube penetration was found in up to 79% of the ovules. Apparently, various kinds of barriers preventing fertilization or normal embryogenesis occur in interspecific tulip crosses. Received: 26 July 1996 / Revision accepted: 31 January 1997     

15N-enrichment of plant tissue to mark phytophagous insects, associated parasitoids, and flower-visiting entomophaga

New techniques are presented on the use of ¹⁵N to mark insects. ¹⁵N, a stable isotope of nitrogen, was enriched above natural abundance in plant and insect tissues. Two laboratory studies demonstrated that enriched ¹⁵N-concentrations could be tracked from plant to insect using mass spectrometry. In the first study, adult Cotesia plutellae (Kurdjimov) (Hymenoptera: Braconidae) and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) were allowed to feed at the flowers of rapid-cycling Chinese cabbage plants that had been fertilized with ¹⁵N-enriched potassium nitrate (KNO₃-¹⁵NO₃). Both insect groups were found to have significantly elevated ¹⁵N levels after visiting the flowers of the ¹⁵N-enriched plants for 48 hours. In the second study, ¹⁵N-enriched bean plant (Phaseolus vulgaris L.) tissue was incorporated into an insect diet and fed to navel orangeworms, Amyelois transitella (Walker) (Lepidoptera: Pyralidae). When the navel orangeworm larvae were 4th instars, they were removed from the diet and exposed to the parasitoid, Goniozus legneri Gordh (Hymenoptera: Bethylidae). Results indicated that the enriched ¹⁵N-concentration of the bean plants was transferred to the navel orangeworms and, subsequently, to the parasitoids. This work may provide useful techniques to help establish whether agriculturally important entomophaga visiting ¹⁵N-enriched flowers or parasitizing enriched sentinel larvae in the field can be effectively marked with ¹⁵N.     

Morfologia fiorale embriologia ed embriog in Leontopodium alpinum Cass. var. α typicum fiori e Paoletti

Abstract

FLOWER MORPHOLOGY, EMBRYOLOGY AND EMBRYOGENESIS OF LEONTOPODIUM ALPINUM CASS. VAR. α TYPICUM FIORI E PAOLETTI. — The present work has been carried on with capitules of L. alpinum var. α typicum, included and dissected, differing thus from the previous research which is also summarised (BEAUVERD 1909 a e b, 1910, 1012, 1913 a e b, 1914).

The results are the following: all capitules of the same corymb of this species have the same structure, differing eventually in details. Generally they show outwards a few female flowers, inwards a relevant number of pseudohermaphrodite flowers (the sterile hermaphrodite of BEAUVERD). They are therefore subdioecious capitules with tendency to reduce the already scarce number of female flowers. Both female and pseudohermaphrodite flowers have been extensively described. Some rare hermaphrodite flowers with ovules and anthers, and very rare subhermaphrodite without ovules and empty or almost empty anthers have been also observed.

On the same material, embryological and embryogenetical research has been carried out: from it results that the microsporogenesis proceeds normally, without degeneration. The megasporogenesis and the development of the female gametophyte and the formation of an 8-nucleate have been observed and also several cases of poliantiopody. Many gamethophytes at this stage degenerate; the others develop particularly transversally; the antipodes completely degenerate and the secondary nucleus and the egg cell reach remarkable size. Synergids at this stage appear more or less completely fessurate, in their micropilar part.

Fertilisation occurs through porogamy, the zygote producing a bicellular proembryo which goes on dividing while the nucleus of the endosperm forms a cellular albumen.

The embryological and embryogenetical observations do not completely agree with those made by SOKOLOWSKA-KULCZYCKA (1959) on specimens of L. alpinum collected on the Tatra mountains (Carpathian Mts.), where the tetrad often degenerates and only exceptionally brings to the constitution of an aphid gamethophyte. More frequently apomixis has been observed, which implies a parthenogenetical development of the egg cell.     

柑桔细胞电融合再生两个种间体细胞杂种

朋娜脐橙(Citrussinensis Osbeck)胚性细胞悬浮系原生质体分别与粗柠檬(C.jambhiri Lush)、枸头橙(C.aurantium)叶肉原生质体经电场诱导而融合。经培养,两组合均获得再生植株。对朋娜脐橙+粗柠檬的再生胚状体进行染色体计数,随机取样的52个胚状体中,26个为四倍体,另外26个为二倍体;对74棵再生植株进行染色体计数,由此说明都为四倍体;表明体细胞杂种在植株再生过程中具有明显的竞争优势。朋娜脐橙+枸头橙再生的14棵植株都为四倍体。对朋娜脐橙+粗柠檬部分植株进行POX同工酶和RAPD分析,表明所有检测植株都为杂种。朋娜脐橙+枸头橙再生植株经RAPD分析,表明也为杂种。     

Growth characteristics of Glycyrrhiza glabra cell suspension cultures

Sweet potato (Ipomoea batatas (L.) Lam.) breeding has been hampered by self-and cross-incompatibilities that are frequently encountered among the plants in the section Batatas. Ovule culture techniques were developed to assist in overcoming some of these incompatibilities. Ovules that contain embryos at the late globular to heart shaped stage of development were cultured on MS medium containing full strength or one-half strength salts with 3%, 8% or 12% sucrose. Ovules were cultured either intact or after slicing. Ovules of I. triloba and I. trifida were successfully cultured as early as 3 and 4 days after pollination while sweet potato ovules were successfully cultured 5 and 6 days after pollination. The percentage of ovules with developing embryos on the media tested ranged from 27.8% to 50.2%. The highest percentage of embryos developed when the ovules were sliced and cultured on medium containing one-half MS salts and 8% sucrose. Three plants were recovered from cultured ovules of incompatible interspecific crosses.Abbreviations DAP days after pollination - MS medium Murashige and Skoog (1962) medium     

Megagametophyte abnormalities of near-isogenic female partial-sterile soybean mutants (Glycine max; Leguminosae)

Megagametogenesis of soybean, Glycine max (L.) Merr., cultivars Clark and Clark k2, and F1 hybrid of Clark (female parent) crossed with Clark k2 (male parent) were studied using stereo light microscopy and confocal scanning laser microscopy. Reproductive development in Clark and Clark k2 plants was compared to F1 hybrid plants. In mature pods, 6.4% of the ovules of Clark, 8.1% of the ovules of Clark k2, and 41.4% of the ovules of F1 hybrid plants were aborted. This female partial sterility was due to incomplete megagametophyte development: undeveloped polar nuclei—or developed but not in a position for fertilization; increased megagametophyte wall thickness; abnormal shape and/or premature degeneration of synergids and intact synergids throughout the life of the ovule; egg cell not well-developed or absent; and megagametophyte remaining uninucleate. Each of these abnormalities contributed to either lack of double fertilization or early megagametophyte abortion. Electronic Publication