Articles
Coffee microspore cultivation to attain doubled-haploid plantlets
Article number
1359_14
Pages
123 – 130
Language
English
Abstract
Introgression of disease resistance from related Coffea species to C. arabica has been done by inter-specific hybridization. C. arabica (tetraploid) × C. racemosa (diploid) hybrids were obtained at the IDR-Paraná, Brazil.
Some of the hybrids display impaired self-pollination, despite backcrossed to C. arabica. As an attempt to regenerate stable doubled-haploid plants, anthers and isolated microspores have been cultivated in vitro.
It was considered that chromosomes captured in a microspore following meiosis could acquire homo and/or homeologous stability faster in vitro than in vivo.
A C. arabica progeny was taken as control.
Flowers were collected at the IDR campus in Londrina, when young microspores were uninucleate, and treated with 8% active chlorine.
Microspores were extracted in 90 mM mannitol using a food mixer set to function for a few seconds, washed and centrifuged at 100× g twice, and cultivated in modified N6 liquid medium (105 cells mL‑1 in 35 mm diameter plates). Anthers were excised using scalpels and cultivated in solid medium.
Explants were kept for six months of continuous cultivation on that induction medium containing 6.5 mg L‑1 auxins, 1.0 mg L‑1 cytokinins and 0.5 mg L‑1 gibberellin (GA3), at 27°C under dark.
Microspores produced embryo-like structures or microcalli in very low frequencies (0.3 per plate). Anthers, on the other hand, produced embryogenic calli.
Colonization by an invariant morphotype of fungus took away 70% of Arabica coffee control anthers and 5% of the asseptic anthers produced friable calli, some embryogenic but arrested when compared with calli produced by the inter-specific hybrid anthers.
Colonization by an invariant morphotype of bacteria took away 25% of hybrid anthers and 8% of produced embryogenic tissue, with globular embryos simultaneously multiplying and maturing upon transfer to N6 medium.
Embryo conversion and photomorphogenesis under light on auxin/cytokinin ratio = 2 plus GA3 are going on, 1.2 year after inoculation.
Some of the hybrids display impaired self-pollination, despite backcrossed to C. arabica. As an attempt to regenerate stable doubled-haploid plants, anthers and isolated microspores have been cultivated in vitro.
It was considered that chromosomes captured in a microspore following meiosis could acquire homo and/or homeologous stability faster in vitro than in vivo.
A C. arabica progeny was taken as control.
Flowers were collected at the IDR campus in Londrina, when young microspores were uninucleate, and treated with 8% active chlorine.
Microspores were extracted in 90 mM mannitol using a food mixer set to function for a few seconds, washed and centrifuged at 100× g twice, and cultivated in modified N6 liquid medium (105 cells mL‑1 in 35 mm diameter plates). Anthers were excised using scalpels and cultivated in solid medium.
Explants were kept for six months of continuous cultivation on that induction medium containing 6.5 mg L‑1 auxins, 1.0 mg L‑1 cytokinins and 0.5 mg L‑1 gibberellin (GA3), at 27°C under dark.
Microspores produced embryo-like structures or microcalli in very low frequencies (0.3 per plate). Anthers, on the other hand, produced embryogenic calli.
Colonization by an invariant morphotype of fungus took away 70% of Arabica coffee control anthers and 5% of the asseptic anthers produced friable calli, some embryogenic but arrested when compared with calli produced by the inter-specific hybrid anthers.
Colonization by an invariant morphotype of bacteria took away 25% of hybrid anthers and 8% of produced embryogenic tissue, with globular embryos simultaneously multiplying and maturing upon transfer to N6 medium.
Embryo conversion and photomorphogenesis under light on auxin/cytokinin ratio = 2 plus GA3 are going on, 1.2 year after inoculation.
Authors
P.C.S. Angelo, L.F.P. Pereira, G.H. Sera, T. Sera
Keywords
Rubiaceae, embryogenesis, pollen
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