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P S Testillano - One of the best experts on this subject based on the ideXlab platform.
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autophagy is activated and involved in cell death with participation of cathepsins during stress induced Microspore embryogenesis in barley
Journal of Experimental Botany, 2018Co-Authors: Ivett Barany, Mariateresa Solis, Mariacarmen Risueno, Eduardo Berenguer, Yolanda Perezperez, Estrella M Santamaria, Jose L Crespo, Isabel Diaz, P S TestillanoAbstract:Microspores are reprogrammed towards embryogenesis by stress. Many Microspores die after this stress, limiting the efficiency of Microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced Microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during Microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance Microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.
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early embryo achievement through isolated Microspore culture in citrus clementina hort ex tan cvs monreal rosso and nules
Frontiers in Plant Science, 2015Co-Authors: Benedetta Chiancone, P S Testillano, Ivett Barany, Valeria Gianguzzi, Marines Marli Gniech Karasawa, A Abdelgalel, Daniela Torello Marinoni, Roberto Botta, Maria GermanaAbstract: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 eleven 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’.
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a new Microspore embryogenesis system under low temperature which mimics zygotic embryogenesis initials expresses auxin and efficiently regenerates doubled haploid plants in brassica napus
BMC Plant Biology, 2012Co-Authors: Deepak Prem, Mariateresa Solis, Mariacarmen Risueno, Ivett Barany, Hector Rodriguezsanz, P S TestillanoAbstract:Background Microspore embryogenesis represents a unique system of single cell reprogramming in plants wherein a highly specialized cell, the Microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway. In Brassica napus, a model species for this phenomenon, incubation of isolated Microspores at 32°C is considered to be a pre-requisite for embryogenesis induction.
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no ros and cell death associated with caspase like activity increase in stress induced Microspore embryogenesis of barley
Journal of Experimental Botany, 2012Co-Authors: Maria Rodriguezserrano, Mariacarmen Risueno, Ivett Barany, Deepak Prem, Maria Jose Coronado, P S TestillanoAbstract:Under specific stress treatments (cold, starvation), in vitro Microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic Microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley Microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in Microspore and suspension cultures, while ROS increased in non-induced Microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in Microspore cultures, NO production was detected after stress, and, in the case of 4-day Microspore cultures, in embryogenic Microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated Microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in Microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced Microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed Microspores.
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early markers of in vitro Microspore reprogramming to embryogenesis in olive olea europaea l
Plant Science, 2008Co-Authors: Mariateresa Solis, Beatriz Pintos, Mariajesus Prado, Mariaangeles Bueno, Ivan Raska, Mariacarmen Risueno, P S TestillanoAbstract:Microspore embryogenesis to form haploid and double-haploid embryos and regenerated plants is an efficient method of producing homozygous lines for crop breeding. In trees, the process is of special interest since classical methods are impractical in many cases, as in Olea europaea L. Recently, a convenient method has been developed for Microspore embryogenesis induction by stress in olive isolated Microspores in vitro cultures. In the present work, the switch of the Microspore developmental pathway and the formation of Microspore-derived multicellular proembryos have been achieved and a cytochemical and immunocytochemical analysis was performed in the early stages. The young Microspore proembryos displayed defined features different to both, the in vivo gametophytic, and the in vitro non-responsive Microspores. Reprogrammed Microspores showed an absence of starch, the occurrence of a first symmetrical division and cytokinesis, the presence of an abundant ribosomal population, and changes in cellulosic and pectic cell wall components which constituted early markers of the embryogenic Microspore process. They provided new insights on the molecular and cellular events associated with the Microspore reprogramming of woody plants, and specifically in olive, providing interesting knowledge which could guide future selection and regeneration strategies in this fruit tree of high economic interest.
János Pauk - One of the best experts on this subject based on the ideXlab platform.
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improvement of isolated Microspore culture of pepper capsicum annuum l via co culture with ovary tissues of pepper or wheat
Plant Cell Tissue and Organ Culture, 2009Co-Authors: Csaba Lantos, R Mihaly, Aniko Gemes Juhasz, Gyorgy Somogyi, Krisztina Otvos, Pal Vagi, Zoltan Kristof, Norbert Somogyi, János PaukAbstract:The influence of the developmental stage of Microspores on establishing isolated Microspore cultures of three Hungarian (‘Szegedi 80’, ‘Szegedi 178’, and ‘Remeny’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated. Donor anthers containing 80% uninucleated and 20% binucleated Microspores yielded the highest frequency of successful Microspore cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with pepper ovaries. Differences in efficiency of isolated pepper Microspore culture establishment were observed among different pepper genotypes. Green plantlets were regenerated from Microspore-derived embryoids, but some were exhibited abnormal growth habits, such as leaf rosetting. A total of seven fertile Microspore-derived plants were obtained, including three ‘Jariza’, three ‘Jaranda’, and a single ‘Szegedi 80’ plant.
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protocol of triticale x triticosecale wittmack Microspore culture
2003Co-Authors: János Pauk, R Mihaly, T Monostori, M PuolimatkaAbstract:Triticale (x Triticosecale Wittmack) is a spontaneous synthetic amphiploid cereal that has been considerably improved through breeding. Triticale is currently grown on about 2 million hectares worldwide. After the initial period, intensive breeding research on triticale was started in the early 1950s by Arpad Kiss in Hungary. Nowadays, the centre of European triticale breeding is in Poland. Polish breeders released excellent varieties that have had a good performance, especially in Europe. Since the induction of the first triticale anther culture derived haploid plantlets by Wang et al. (1973), the method of anther culture has been essentially modified and improved. Theoretically, two in vitro cell and tissue culture methods are used to induce androgenesis: anther and Microspore culture. The anther culture method seems to be more laborious. The blender isolation protocol provides a sufficient number of Microspores to avoid the necessity for anther isolation. In cereals, microblending isolation was earlier established for barley (Olsen, 1991) and wheat (Ziauddin et al.,1992), confirming its potential. The present protocol describes an in vitro process for blender-isolated triticale Microspores leading to doubled haploid lines from Microspore derived embryo-like structures (ELS). Embryogenesis was observed in isolated triticale Microspores. This phenomenon also occurs in Microspore culture of other cereals. From the ELS green plants can be regenerated, but albinism is still a typical problem of triticale Microspore culture. Depending on the genotype, more than half of the regenerants may be albino.
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protocol of triticale x triticosecale wittmack Microspore culture
2003Co-Authors: János Pauk, R Mihaly, T Monostori, M PuolimatkaAbstract:Triticale (x Triticosecale Wittmack) is a spontaneous synthetic amphiploid cereal that has been considerably improved through breeding. Triticale is currently grown on about 2 million hectares worldwide. After the initial period, intensive breeding research on triticale was started in the early 1950s by Arpad Kiss in Hungary. Nowadays, the centre of European triticale breeding is in Poland. Polish breeders released excellent varieties that have had a good performance, especially in Europe. Since the induction of the first triticale anther culture derived haploid plantlets by Wang et al. (1973), the method of anther culture has been essentially modified and improved. Theoretically, two in vitro cell and tissue culture methods are used to induce androgenesis: anther and Microspore culture. The anther culture method seems to be more laborious. The blender isolation protocol provides a sufficient number of Microspores to avoid the necessity for anther isolation. In cereals, microblending isolation was earlier established for barley (Olsen, 1991) and wheat (Ziauddin et al.,1992), confirming its potential. The present protocol describes an in vitro process for blender-isolated triticale Microspores leading to doubled haploid lines from Microspore derived embryo-like structures (ELS). Embryogenesis was observed in isolated triticale Microspores. This phenomenon also occurs in Microspore culture of other cereals. From the ELS green plants can be regenerated, but albinism is still a typical problem of triticale Microspore culture. Depending on the genotype, more than half of the regenerants may be albino.
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In vitro androgenesis of triticale in isolated Microspore culture
Plant Cell Tissue and Organ Culture, 2000Co-Authors: János Pauk, M Puolimatka, K. Lökös Tóth, T MonostoriAbstract:Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using Microspore culture. Sporophytic development of isolated triticale Microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of Microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale Microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid.
T Monostori - One of the best experts on this subject based on the ideXlab platform.
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protocol of triticale x triticosecale wittmack Microspore culture
2003Co-Authors: János Pauk, R Mihaly, T Monostori, M PuolimatkaAbstract:Triticale (x Triticosecale Wittmack) is a spontaneous synthetic amphiploid cereal that has been considerably improved through breeding. Triticale is currently grown on about 2 million hectares worldwide. After the initial period, intensive breeding research on triticale was started in the early 1950s by Arpad Kiss in Hungary. Nowadays, the centre of European triticale breeding is in Poland. Polish breeders released excellent varieties that have had a good performance, especially in Europe. Since the induction of the first triticale anther culture derived haploid plantlets by Wang et al. (1973), the method of anther culture has been essentially modified and improved. Theoretically, two in vitro cell and tissue culture methods are used to induce androgenesis: anther and Microspore culture. The anther culture method seems to be more laborious. The blender isolation protocol provides a sufficient number of Microspores to avoid the necessity for anther isolation. In cereals, microblending isolation was earlier established for barley (Olsen, 1991) and wheat (Ziauddin et al.,1992), confirming its potential. The present protocol describes an in vitro process for blender-isolated triticale Microspores leading to doubled haploid lines from Microspore derived embryo-like structures (ELS). Embryogenesis was observed in isolated triticale Microspores. This phenomenon also occurs in Microspore culture of other cereals. From the ELS green plants can be regenerated, but albinism is still a typical problem of triticale Microspore culture. Depending on the genotype, more than half of the regenerants may be albino.
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protocol of triticale x triticosecale wittmack Microspore culture
2003Co-Authors: János Pauk, R Mihaly, T Monostori, M PuolimatkaAbstract:Triticale (x Triticosecale Wittmack) is a spontaneous synthetic amphiploid cereal that has been considerably improved through breeding. Triticale is currently grown on about 2 million hectares worldwide. After the initial period, intensive breeding research on triticale was started in the early 1950s by Arpad Kiss in Hungary. Nowadays, the centre of European triticale breeding is in Poland. Polish breeders released excellent varieties that have had a good performance, especially in Europe. Since the induction of the first triticale anther culture derived haploid plantlets by Wang et al. (1973), the method of anther culture has been essentially modified and improved. Theoretically, two in vitro cell and tissue culture methods are used to induce androgenesis: anther and Microspore culture. The anther culture method seems to be more laborious. The blender isolation protocol provides a sufficient number of Microspores to avoid the necessity for anther isolation. In cereals, microblending isolation was earlier established for barley (Olsen, 1991) and wheat (Ziauddin et al.,1992), confirming its potential. The present protocol describes an in vitro process for blender-isolated triticale Microspores leading to doubled haploid lines from Microspore derived embryo-like structures (ELS). Embryogenesis was observed in isolated triticale Microspores. This phenomenon also occurs in Microspore culture of other cereals. From the ELS green plants can be regenerated, but albinism is still a typical problem of triticale Microspore culture. Depending on the genotype, more than half of the regenerants may be albino.
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In vitro androgenesis of triticale in isolated Microspore culture
Plant Cell Tissue and Organ Culture, 2000Co-Authors: János Pauk, M Puolimatka, K. Lökös Tóth, T MonostoriAbstract:Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using Microspore culture. Sporophytic development of isolated triticale Microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of Microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale Microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid.
Ivett Barany - One of the best experts on this subject based on the ideXlab platform.
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autophagy is activated and involved in cell death with participation of cathepsins during stress induced Microspore embryogenesis in barley
Journal of Experimental Botany, 2018Co-Authors: Ivett Barany, Mariateresa Solis, Mariacarmen Risueno, Eduardo Berenguer, Yolanda Perezperez, Estrella M Santamaria, Jose L Crespo, Isabel Diaz, P S TestillanoAbstract:Microspores are reprogrammed towards embryogenesis by stress. Many Microspores die after this stress, limiting the efficiency of Microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced Microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during Microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance Microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.
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early embryo achievement through isolated Microspore culture in citrus clementina hort ex tan cvs monreal rosso and nules
Frontiers in Plant Science, 2015Co-Authors: Benedetta Chiancone, P S Testillano, Ivett Barany, Valeria Gianguzzi, Marines Marli Gniech Karasawa, A Abdelgalel, Daniela Torello Marinoni, Roberto Botta, Maria GermanaAbstract: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 eleven 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’.
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a new Microspore embryogenesis system under low temperature which mimics zygotic embryogenesis initials expresses auxin and efficiently regenerates doubled haploid plants in brassica napus
BMC Plant Biology, 2012Co-Authors: Deepak Prem, Mariateresa Solis, Mariacarmen Risueno, Ivett Barany, Hector Rodriguezsanz, P S TestillanoAbstract:Background Microspore embryogenesis represents a unique system of single cell reprogramming in plants wherein a highly specialized cell, the Microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway. In Brassica napus, a model species for this phenomenon, incubation of isolated Microspores at 32°C is considered to be a pre-requisite for embryogenesis induction.
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no ros and cell death associated with caspase like activity increase in stress induced Microspore embryogenesis of barley
Journal of Experimental Botany, 2012Co-Authors: Maria Rodriguezserrano, Mariacarmen Risueno, Ivett Barany, Deepak Prem, Maria Jose Coronado, P S TestillanoAbstract:Under specific stress treatments (cold, starvation), in vitro Microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic Microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley Microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in Microspore and suspension cultures, while ROS increased in non-induced Microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in Microspore cultures, NO production was detected after stress, and, in the case of 4-day Microspore cultures, in embryogenic Microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated Microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in Microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced Microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed Microspores.
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First Stages of Microspore Reprogramming to Embryogenesis through Isolated Microspore Culture in Loquat
Acta Horticulturae, 2011Co-Authors: Diego Padoan, Ivett Barany, B. Chiancone, Maria Germana, P. S. S. V. Khan, M.c. Risueño, Pilar S. TestillanoAbstract:The current experiments were undertaken to develop a method for regenerating doubled haploid (DH) plants of Eriobotrya japonica (Thunb.), Rosaceae, subfamily Maloideae through isolated Microspore culture of nine loquat cultivars. Protocols were developed for isolation and culture of loquat Microspores. Isolated Microspores in vitro cultured started dividing and developing multicellular, globular and irregular structures. After 4 weeks of culture, the responding Microspores produced yellowish/white callus. The embryogenic response of Microspores was highly dependent on the cultivar and on the medium composition.
Mariacarmen Risueno - One of the best experts on this subject based on the ideXlab platform.
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autophagy is activated and involved in cell death with participation of cathepsins during stress induced Microspore embryogenesis in barley
Journal of Experimental Botany, 2018Co-Authors: Ivett Barany, Mariateresa Solis, Mariacarmen Risueno, Eduardo Berenguer, Yolanda Perezperez, Estrella M Santamaria, Jose L Crespo, Isabel Diaz, P S TestillanoAbstract:Microspores are reprogrammed towards embryogenesis by stress. Many Microspores die after this stress, limiting the efficiency of Microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced Microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during Microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance Microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.
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a new Microspore embryogenesis system under low temperature which mimics zygotic embryogenesis initials expresses auxin and efficiently regenerates doubled haploid plants in brassica napus
BMC Plant Biology, 2012Co-Authors: Deepak Prem, Mariateresa Solis, Mariacarmen Risueno, Ivett Barany, Hector Rodriguezsanz, P S TestillanoAbstract:Background Microspore embryogenesis represents a unique system of single cell reprogramming in plants wherein a highly specialized cell, the Microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway. In Brassica napus, a model species for this phenomenon, incubation of isolated Microspores at 32°C is considered to be a pre-requisite for embryogenesis induction.
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no ros and cell death associated with caspase like activity increase in stress induced Microspore embryogenesis of barley
Journal of Experimental Botany, 2012Co-Authors: Maria Rodriguezserrano, Mariacarmen Risueno, Ivett Barany, Deepak Prem, Maria Jose Coronado, P S TestillanoAbstract:Under specific stress treatments (cold, starvation), in vitro Microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic Microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley Microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in Microspore and suspension cultures, while ROS increased in non-induced Microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in Microspore cultures, NO production was detected after stress, and, in the case of 4-day Microspore cultures, in embryogenic Microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated Microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in Microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced Microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed Microspores.
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early markers of in vitro Microspore reprogramming to embryogenesis in olive olea europaea l
Plant Science, 2008Co-Authors: Mariateresa Solis, Beatriz Pintos, Mariajesus Prado, Mariaangeles Bueno, Ivan Raska, Mariacarmen Risueno, P S TestillanoAbstract:Microspore embryogenesis to form haploid and double-haploid embryos and regenerated plants is an efficient method of producing homozygous lines for crop breeding. In trees, the process is of special interest since classical methods are impractical in many cases, as in Olea europaea L. Recently, a convenient method has been developed for Microspore embryogenesis induction by stress in olive isolated Microspores in vitro cultures. In the present work, the switch of the Microspore developmental pathway and the formation of Microspore-derived multicellular proembryos have been achieved and a cytochemical and immunocytochemical analysis was performed in the early stages. The young Microspore proembryos displayed defined features different to both, the in vivo gametophytic, and the in vitro non-responsive Microspores. Reprogrammed Microspores showed an absence of starch, the occurrence of a first symmetrical division and cytokinesis, the presence of an abundant ribosomal population, and changes in cellulosic and pectic cell wall components which constituted early markers of the embryogenic Microspore process. They provided new insights on the molecular and cellular events associated with the Microspore reprogramming of woody plants, and specifically in olive, providing interesting knowledge which could guide future selection and regeneration strategies in this fruit tree of high economic interest.