Observations of anatomical structures of in vitro germination of stoneless and isolated olive embryos

Trabajo presentado en la 4th Conference Olivebioteq, celebrada en chania (Creta) del 31 de octubre al 4 de noviembre de 2011.Peer Reviewe

First embryogenic stages of Citrus microspore-derived embryos

6 p.-2 fig.This work is the first characterization at cellular and subcellular level of the main cellular events occurring in the first stages of microspore embryogenesis. Microspore embryogenesis was induced in two varieties of Citrus clementina (Nules and Monreal). The results showed that one of the most responsive stages for embryogenesis was the vacuolate microspore. Microscopic analysis revealed specific features of the young proembryos still surrounded by the exine:large nuclei, clear areas in the cytoplasm, starch accumulation, and an increase in the thickness of the wall under the exine. Immunogold labelling with JIM 5 antibody showed a high amount of non-esterified pectins in the surrounding cell wall. After exine rupture, different cell types were detected in late proembryos. As embryogenesis proceeded, the normal pattern of development was observed, including heart-shape, torpedo and cotyledonar embryos.The work was supported by the Spanish project DGESIC PB98-0678,CAM 07G/0054/2000 and the Spanish-Italian joint project CSIC/CNR 2001IT0017.Peer reviewe

Correlation of sequential floral and male gametophyte development and preliminary results on anther culture in Opuntia ficus-indica

8 pages.Before approaching anther culture as a tool to trigger an androgenic response in a new species, it is advisable to characterize and correlate flower and male gametophyte development to enable reproducible identification of the appropriate starting material. Buds and flowers of Opuntia ficus-indica cv. Gialla were classified in eight stages according to their total length at the earlier stages and the length of the corolla in flowers with emerging sepals. Due to the low condensation of chromatin in the microspore nucleus as well as in the vegetative nucleus of the bi- and tricellular pollen along with the high autofluorescence of the intricate exine, DAPI staining turned out not to be feasible in this species. Therefore an approach based on light-microscopy observation of semithin sections was used. These sections were stained with toluidine blue for general structure recognition and I2KI to study starch deposition. Correlations were made between the sequential floral and male gametophyte development. Using this approach we determined the timing of pollen formation and observed that pollen development is impaired in plants producing seedless fruits. Furthermore, anther culture was carried out with anthers collected from flower buds at stages 2 and 3. Most of the anthers produced callus, however no regeneration was obtained.Peer reviewe

Induction of pollen embryogenesis in seven cultivars of Prunus armeniaca l.

6 páginas -- PAGS nros. 273-278Haploids and doubled haploids (DHs), very important for their potential use in breeding and genetic analysis, can be induced mainly by two strategies, i.e. by regen¬eration from the female gamete or from the male gamete. Microspore embryogenesis through in vitro anther culture is a widely used method to obtain haploidization. In this research, the first stages of the gametic embryogenesis through the anther culture of seven apricot genotypes have been described and characterized through microscopical analysis, carried out at different times of culturePeer reviewe

Development of multicellular pollen of Eriobotrya japonica Lindl. through anther culture

8 pages, 5 figures.-- Printed version published Dec 2006.Eriobotrya japonica Lindl. is a worldwide known tree important for its use as horticultural and ornamental plant, especially in sub-tropical and Mediterranean countries. Microspore embryogenesis through in vitro anther culture is a widely used method to generate genetic variability by obtaining gametic or somatic embryos with many applications for plant breeding. In this work, a protocol has been set up for anther culture in loquat which resulted in the formation of multicellular pollen as a first step to further attempt haploid–plant production via pollen-derived structures. The response of nine of the most widely grown loquat cultivars to anther culture has been evaluated, and four cultivars being selected due to their higher response. The occurrence of anther swelling and the development of calli were analyzed as typical morphological features and potential markers that accompany pollen induction and reprogramming in many systems. Microscopical analysis in responsive anthers and the comparison with the normal gametophytic pollen development was carried out to characterize the cellular changes promoted by the treatment in the anther. The presence of multicellular pollen in the in vitro system developed here indicated the switch of developmental programme which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and plants. Monitoring structural analysis at different times of the culture revealed specific features of the early microspore embryogenic pathway as well as the cellular organization changes.This work is a collaboration between the CSIC (Spain) and the Università degli Studi di Palermo (Italy) in the frame of the joint bilateral Spanish-Italian programme of “Acciones Integradas” (HI2002-0099) and “Azione integrata” (IT 929). This work was partially supported by Spanish projects granted by MEC AGL2005-05104 and BFU2005-01094, and by the Italian PRIN 2004.Peer reviewe

Olive embryo in vitro germination potential: Role of explant configuration and embryo structure among cultivars

The in vitro germination of excised embryos can break dormancy rapidly and shorten the time required to produce seedlings, speeding up olive breeding programmes as well as rootstock production. In this study, the in vitro germination potential of four Sicilian olive cultivars was evaluated during two years of experiments, using explants with three different morphological configurations that represent three different degrees of embryo exposure: (1) intact stoneless seeds containing the embryo, the endosperm and the seed coat (Emb+En+SC), (2) seeds without the seed coat (Emb+En) and (3) naked, isolated embryos (seed coat and endosperm both removed: Emb). Differences were found in the germination percentages and timing due to both genotype and explant type. The root and shoot meristems, the radicle-hypocotyl axis, the provascular tissues and embryo storage reserves were identified as embryo anatomical structures which could influence germination capacity. Observation of these structures, however, indicated similar germination potential among cultivars, suggesting possible differences in other dormancy factors. In spite of variation in cultivar performance, after 60 days of in vitro culture all cultivars demonstrated the highest germination of naked embryos (explant type 3) and lowest for stoneless seeds (explant type 1); stoneless seeds without the seedcoat (explant type 2) showed intermediate germination percentages.This work was partially supported by the CORI (International Cooperation) 2006 program and by the project ‘‘Innovative Methods of propagation and Conservation of Mediterranean Trees’’, FFR 2012–2013, provided by the University of Palermo to Maria Antonietta Germana`.Peer Reviewe

First stages of microspore reprogramming to embryogenesis through anther culture in Prunus armeniaca L.

6 páginas, 3 figuras, 2 tablas -- PAGS nros. 152-157Prunus armeniaca L. is a worldwide known species, very important particularly in the Mediterranean basin. Microspore embryogenesis through in vitro anther culture is a widely used method to obtain haploid and doubled haploid (DHs) plants which are being routinely used in breeding programmes for new superior cultivar development in many crops. Haploid–diploidization through gametic embryogenesis allows single-step development of complete homozygous lines from heterozygous parents. In the case of fruit crops, with long reproductive cycle, a high degree of heterozygosity, large size, and, often, self-incompatibility, there is no way to obtain haploidization through conventional methods. Induction of microspore embryogenesis in vitro is switched by a stress treatment. In many species, heat or cold stress has been reported to trigger pollen embryogenesis, the response being genotype dependent. In the present work we analyzed whether microspore reprogramming could be induced in apricot cultivars by cold stress through anther culture. We report the development of an in vitro anther culture protocol in P. armeniaca L. and analyse the response of several cultivars to stress treatments and culture media for inducing pollen embryogenesis. Results showed the formation of multicellular pollen and proembryos. The effect of two culture media in the embryogenic response was also analyzed, being the responses genotype-dependent. Monitoring of the cellular changes on the microspores was performed by structural and confocal microscopy analyses. Results indicated that the reprogramming of the microspore and the first steps of the embryogenic pathway have been achieved in different varieties of P. armeniaca, which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and DH plants, for future potential applications in breeding programmes of this economically important fruit treeThis work is a collaboration between the CSIC (Spain) and the Universita’ degli Studi di Palermo (Italy) in the frame of the Italian CORI 2006 and it is also a collaboration between the CNR (Italy) and the CSIC in the frame of the Spanish-Italian Joint Project CSIC-CNR 2008IT0046. This work was partially supported by Spanish MICINN projects BFU2008-00203 and AGL2008-04255Peer reviewe

Early in vitro development of isolated microspores of four sicilian olive cultivars

Nowadays there is a great interest in the advancement of the studies on gametic embryogenesis in olive, as a method for obtaining homozygous material, due to the enormous effort and relevance in its genome sequencing and gene mapping. In this study, in vitro isolated microspore culture of four Sicilian olive cultivars (‘Biancolilla napoletana’, ‘Nocellara del Belice’, ‘Tonda Iblea’ and ‘Verdello’) has been carried out, investigating the influence of two polyamines (putrescine and spermidine) and of two medium compositions on pollen embryogenesis induction. During in vitro development, different structural features have been observed: uninucleated microspores, with no development, binucleated with two asymmetrical nuclei (normal gametophytic pathway: one vegetative and one generative nucleus), binucleated with two equal-size vegetative-type nuclei that had just started their sporophytic pathway, trinucleated, tetranucleated and multinucleated, indicating steps towards the sporophytic development. Moreover, the presence of several calli has been detected. A strong genotype-medium interaction has been observed: each cultivar showed a different response depending on the medium tested. This research represents an advancement in the knowledge about gametic embryogenesis in olive, towards the regeneration of homozygous olive plants

Early embryo achievement through isolated microspore culture in Citrus clementina Hort. ex Tan., cvs. ‘Monreal Rosso’ and ‘Nules’

10 p.-5 fig.-3 tab.Microspore embryogenesis is a method of achieving complete homozygosity from plants. It is particularly useful for woody species, like Citrus, characterized by long juvenility, a high degree of heterozygosity and often self-incompatibility. Anther culture is currently the method of choice for microspore embryogenesis in many crops. However, isolated microspore culture is a better way to investigate the processes at the cellular, physiological, biochemical, and molecular levels as it avoids the influence of somatic anther tissue. To exploit the potential of this technique, it is important to separate the key factors affecting the process and, among them, culture medium composition and particularly the plant growth regulators and their concentration, as they can greatly enhance regeneration efficiency. To our knowledge, the ability of meta-Topolin, a naturally occurring aromatic cytokinin, to induce gametic embryogenesis in isolated microspores of Citrus has never been investigated. In this study, the effect of two concentrations of meta-Topolin instead of benzyladenine or zeatin in the culture medium was investigated in isolated microspore culture of two genotypes of Citrus. After 11 months of isolated microspore culture, for both genotypes and for all the four tested media, the microspore reprogramming and their sporophytic development was observed by the presence of multinucleated calli and microspore-derived embryos at different stages. Microsatellite analysis of parental and embryo samples was performed to determine the embryo alleles constitution of early embryos produced in all tested media, confirming their origin from microspores. To our knowledge, this is the first successful report of Citrus microspore embryogenesis with isolated microspore culture in Citrus, and in particular in Citrus clementina Hort. ex Tan, cvs. ‘Monreal Rosso’ and ‘Nules.’This work was partially supported by “Functional genomics, genetic improvement, and innovation for the valorization of Citrus industry” IT-Citrus Genomics project (PON01_01623) funded by the Italian MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca) PON Research and Competitiveness 2007–2013 and UE, projects BFU2008-00203 and AGL2014-52028-R funded by the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund(ERDF/FEDER).Peer reviewe