Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

Abstract Background Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley. Results We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

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Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

10.21203/rs.3.rs-42246/v1 ◽

2020 ◽

Author(s):

Monika Gajecka ◽

Marek Marzec ◽

Beata Chmielewska ◽

Janusz Jelonek ◽

Justyna Zbieszczyk ◽

...

Keyword(s):

Microspore Culture ◽

Doubled Haploids ◽

Microspore Embryogenesis ◽

Rrna Genes ◽

23S Rrna ◽

Gene Encoding ◽

Plastid Differentiation ◽

Barley Cultivars ◽

Albino Plants ◽

Genes Encoding

Abstract Background: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH), which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.Results: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. The impaired PEP activity caused a very low level of 16S and 23S rRNA transcripts, lack of plastid translation machinery and inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP, the highly increased expression of Sig2, plastid rRNA and tRNAGlu transcripts, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoint of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was the main cause of the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanism underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

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Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

10.21203/rs.3.rs-42246/v3 ◽

2020 ◽

Author(s):

Monika Gajecka ◽

Marek Marzec ◽

Beata Chmielewska ◽

Janusz Jelonek ◽

Justyna Zbieszczyk ◽

...

Keyword(s):

Microspore Culture ◽

Doubled Haploids ◽

Microspore Embryogenesis ◽

Rrna Genes ◽

23S Rrna ◽

Gene Encoding ◽

Plastid Differentiation ◽

Barley Cultivars ◽

Albino Plants ◽

Genes Encoding

Abstract Background: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.Results: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoint of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanism underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

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Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

10.21203/rs.3.rs-42246/v2 ◽

2020 ◽

Author(s):

Monika Gajecka ◽

Marek Marzec ◽

Beata Chmielewska ◽

Janusz Jelonek ◽

Justyna Zbieszczyk ◽

...

Keyword(s):

Microspore Culture ◽

Doubled Haploids ◽

Microspore Embryogenesis ◽

Rrna Genes ◽

23S Rrna ◽

Gene Encoding ◽

Plastid Differentiation ◽

Barley Cultivars ◽

Albino Plants ◽

Genes Encoding

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The genetics of 5S rRNA encoding multigene families in barley

Genome ◽

10.1139/g93-136 ◽

1993 ◽

Vol 36 (6) ◽

pp. 1023-1028 ◽

Cited By ~ 22

Author(s):

Vladimir Kanazin ◽

Evgeny Ananiev ◽

Tom Blake

Keyword(s):

Tandem Repeats ◽

Doubled Haploids ◽

Gene Clusters ◽

Variable Number ◽

5S Rrna ◽

Rrna Genes ◽

Multigene Families ◽

Obvious Effect ◽

Barley Cultivars ◽

5S Rrna Genes

Two loci containing genes encoding 5S rRNA were mapped on the second and third chromosomes of barley. The two gene clusters located on different chromosomes differed in the length of the nontranscribed spacer separating the 5S rRNA genes. All nontranscribed spacers contained a variable number of trinucleotide tandem repeats. The distribution of 5S genes between these two clusters and their copy number varied widely between cultivars and doubled haploids derived from a cross between two barley cultivars. However, this variation had no obvious effect on plant phenotype.Key words: 5S rRNA genes, multigene families, nontranscribed spacers, trinucleotide tandem repeats, barley, phenotype.

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Study of androgenesis and spontaneous chromosome doubling in barley ( Hordeum vulgare L.) genotypes using isolated microspore culture

Acta Agronomica Hungarica ◽

10.1556/aagr.57.2009.2.7 ◽

2009 ◽

Vol 57 (2) ◽

pp. 155-164 ◽

Cited By ~ 8

Author(s):

D. Kahrizi ◽

R. Mohammadi

Keyword(s):

Chromosome Doubling ◽

Microspore Culture ◽

Doubled Haploids ◽

Negative Relationship ◽

Microspore Embryogenesis ◽

Culture Technique ◽

Hordeum Vulgare L ◽

Spontaneous Chromosome ◽

Barley Genotypes

This research aimed to study the androgenesis and spontaneous chromosome doubling of five barley genotypes using an isolated in vitro microspore culture technique, involving a completely randomized design (CRD) with three replications. Statistical analysis of embryogenesis and cytogenetic results showed that genotype had a significant effect on haploid embryogenesis (P<0.01) and on spontaneous chromosome doubling (P<0.05). The genotype Igri was found to have the highest potential to produce haploid embryos (1577 embryos from 100 anthers), followed by the genotypes Boyer/Rojo, Afzal/Turkman/Kavir, Ashar/Hebo and Agrigashar/Matico with 369, 304, 278 and 150 embryos from 100 anthers, respectively. The highest percentage of spontaneous chromosome doubling (76%) was observed for the genotype which had the lowest embryogenesis (Agrigashar/Matico) and the lowest (65%) for the genotype with the highest androgenic capacity (Igri). Microspore embryogenesis also showed comparatively higher genotypic (109.2) and phenotypic (109.5) coefficients of variation, heritability (99.62) and genetic advance (1206.77), indicating the pre-dominance of additive gene action in the control of this character in the material studied. Estimates of genetic parameters (PCV, GCV and heritability) for microspore embryogenesis were higher than for spontaneous doubled haploids. These results indicated that selection for androgenic capacity would be more effective than for spontaneous doubled haploids. The findings showed a negative relationship (r= −0.68) between embryogenesis and spontaneous chromosome doubling in the barley genotypes studied. All the large embryos used had high regenerability and good plantlet formation.

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Effects of Genotype and Culture Conditions on Microspore Embryogenesis and Plant Regeneration in Brassica Rapa ssp. Rapa L.

Plants ◽

10.3390/plants9020278 ◽

2020 ◽

Vol 9 (2) ◽

pp. 278 ◽

Cited By ~ 3

Author(s):

Daria Shumilina ◽

Dmitry Kornyukhin ◽

Elena Domblides ◽

Alexey Soldatenko ◽

Anna Artemyeva

Keyword(s):

Microspore Culture ◽

Doubled Haploids ◽

Genetic Disorder ◽

Cold Treatment ◽

Microspore Embryogenesis ◽

Culture Conditions ◽

Secondary Embryogenesis ◽

Isolated Microspore Culture ◽

Pure Lines

Turnip is a biennial crop and, consequently, the creation of pure lines for breeding is a time-consuming process. The production of pure turnip lines using doubled haploids produced in isolated microspore culture has not been sufficiently developed. The aim of the present work was to determine some key factors inducing embryogenesis in the isolated microspore culture of turnip, as well as investigating the manners of embryo development. It was shown that the acidity of the medium is an important factor in embryo production; different optimal pH levels ranging from 6.2 to 6.6 corresponded to individual genotypes. Such factors as the cold treatment of buds and the addition of activated charcoal to the nutrient medium increased the responsiveness of all genotypes studied. The turnip variety ‘Ronde witte roodkop herfst’ demonstrated a genetic disorder in the development of microspores; namely, non-separation of some microspores from tetrads. In the in vitro culture, each of the daughter microspores developed on its own. This indicates the dependence of the possibility of embryogenesis in the turnip microspore culture on the genotype. Results suggest that the initiation of secondary embryogenesis in primary embryos leads to an increase in the proportion of doubled haploid plants.

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The effect of copper on plant regeneration in barley microspore culture

Czech Journal of Genetics and Plant Breeding ◽

10.17221/82/2016-cjgpb ◽

2017 ◽

Vol 53 (No. 1) ◽

pp. 17-22 ◽

Cited By ~ 4

Author(s):

K. Makowska ◽

S. Oleszczuk ◽

J. Zimny

Keyword(s):

Plant Regeneration ◽

Copper Sulphate ◽

Microspore Culture ◽

Doubled Haploids ◽

Spring Barley ◽

Culture Method ◽

Induction Medium ◽

Isolated Microspore Culture ◽

Albino Plants ◽

Effect Of Copper

Isolated microspore culture is an excellent system for the production of doubled haploids in many crops, including barley. In a more traditional barley anther culture method copper sulphate is known to enhance plant regeneration. Here we report that one hundred times higher concentration of copper sulphate in the isolated microspore culture of two spring barley genotypes compared to the standard content in the induction medium resulted in a 34% increase of total plant regeneration. Detailed analysis of plant regeneration showed that additional supplementation of copper sulphate increased not only the regeneration of green plants but also proportionately that of albino plants. Hence, the results from two studied genotypes do not support an assumption that the addition of copper reduces albinism in barley microspore culture.

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INDUCTION OF SPOROPHYTIC DIVISION IN ORCHIDS MICROSPORES BY STRESS

KnE Life Sciences ◽

10.18502/kls.v2i1.181 ◽

2015 ◽

Vol 2 (1) ◽

pp. 390

Author(s):

Ari Indrianto ◽

Chairani Siregar ◽

Sutikno Linuhung ◽

Mekartinita _ ◽

Tri Sartikoningsih

Keyword(s):

Doubled Haploid ◽

Male Gametophyte ◽

Microspore Culture ◽

Doubled Haploids ◽

Microspore Embryogenesis ◽

Flower Bud ◽

Culture Methods ◽

Cold Temperatures ◽

Media Formulation ◽

Isolated Microspores

Orchid is one of the important ornamental plants in Indonesia this plant generally propagated by seed. Enhancing quality of this plant through breeding technology by various plant tissue culture methods and biotechnology, including doubled haploid technology are necessary. The most efficient method in creating doubled haploids plant is via microspore embryogenesis. We have develop new, innovative doubled haploid technology using the technique of isolated microspore culture. The goals are to obtain data on the male gametophyte development, viable embryogenic microspores, microspores derived embryos and double haploid plants of Orchid. Development of male gametophytes were analysed by isolation of microspores and pollen at various stages and staining with DAPI. Isolated orchid buds of Dendrobium hybrid 1, Vanda tricolor and Spathoglotis plicata were subjected to cold temperatures (4oC) for 7 days, microspores were then isolated by crushing the pollinia using glass rod and cultured them in embryogenesis A2, NP, MS and VW medium, viability of the microspores were determine by using Flourescein diacetate (FDA). Isolated Orchid pollinia were cultured in starvation medium B at various temperatures and duration of time to evaluate embryogenic response, isolated microspores then were cultured further in the basic embryogenesis medium and incubated at 25 oC in the darkness. The result showed that floral characteristics for the late-uninucleate stage of the microspores were different for every orchid spesies. Ovulum lenght was used for Vanda, while in Dendrobium, Phalaenopsis, Arachnis, Spathoglottis plicata and Cattleya, varied length of flower bud was used. Isolated microspores of Dendrobium hybrid 1, Vanda tricolor and Spathoglotis plicata at 7th days of culture in different media formulation showing different respond of viability. Medium A2 keeping viability of Dendrobium hybrid 1 microspores better than any other medium, while in Vanda tricolor and Spathoglotis plicata embryogenesis NP medium was superior. Incubation of orchid pollinia at 4 and 25 oC were successfully maintain viability of the microspores during starvation periods but not able to block gametophytic development. In contrast starved pollinia at 33oC were succesfully block gametophytic development, percentage of embryogenic microspores after starvation of isolated pollinia at 33°C for 4 days was superior compare to any other treatments. Symmetrical divisions and some multicellular structures were observed, which were clear indication for the sporophytic development of microspore-derived embryos, they had developed and after a few weeks they degenerated and died. Keywords: flower bud-pollinia-microspore-stress-embriogenic-embryo-Orchid

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Microspore embryogenesis in vitro: the role of stresses

Vavilov Journal of Genetics and Breeding ◽

10.18699/vj19.466 ◽

2019 ◽

Vol 23 (1) ◽

pp. 86-94 ◽

Cited By ~ 1

Author(s):

T. I. Djatchouk ◽

O. V. Khomyakova ◽

V. N. Akinina ◽

I. A. Kibkalo ◽

A. V. Pominov

Keyword(s):

Microspore Culture ◽

Doubled Haploids ◽

Embryo Sac ◽

Microspore Embryogenesis ◽

Gametic Embryogenesis ◽

Chemical Pretreatments ◽

Dh Lines

Gametic embryogenesis is one form of totipotency of plant cells, in which either male or female gametes are induced to form embryoids (sporophytes). Regeneration of haploid plants from embryoids and subsequent chromosome duplication result in doubled haploids and DH-lines. The production of haploids and doubled haploids (DHs) through gametic embryogenesis allows a single-stage development of complete homozygous lines from heterozygous plants. The development of effective haploid protocols to produce homozygous plants has a significant impact on plant breeding, shorting the time and costs required to establish new cultivars. There are several available methods to obtain haploids and DHs-lines, of which anther or isolated microspore culture in vitro are the most effective. Microspore embryogenesis is more commonly applied. This is in part because more male gametophytes are contained in a single anther compared to the single female gametophyte per embryo sac. Microspore embryogenesis is regarded as one of the most striking examples of plant cell totipotency. The switch of cultured microspores from gametophytic to sporophytic mode of development has been induced by stress treatments of various kinds applied to donor plants, inflorescences, buds, anthers or isolated microspores both in vivo and in vitro. Physical or chemical pretreatments (cold and heat shock, sugar starvation, colchicine, n-butanol, gametocydes) act as a trigger for inducing the sporophytic pathway, preventing the gametophytic pathway development of microspore. The recent investigations have revealed that cold pretreatment during microspore reprogramming acts rather as an anti-stress factor alleviating the real stress caused by nutrient starvation of anthers or microspores isolated from donor plants. Under stress pretreatment a vacuolated and polarized microspore transformed into a depolarized and dedifferentiated cell, which is an obligatory condition for reprogramming their development. We summarize data concerning the role of various stresses in the induction of microspore embryogenesis and possible mechanisms of their action at cellular and molecular levels. Identification of new stresses allows creating efficient protocols of doubled haploid production for end-user application in the breeding of many important crops.

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