Development of an Efficient Protocol to Obtain Transgenic Coffee, Coffea arabica L., Expressing the Cry10Aa Toxin of Bacillus thuringiensis

This report presents an efficient protocol of the stable genetic transformation of coffee plants expressing the Cry10Aa protein of Bacillus thuringiensis. Embryogenic cell lines with a high potential of propagation, somatic embryo maturation, and germination were used. Gene expression analysis of cytokinin signaling, homedomains, auxin responsive factor, and the master regulators of somatic embryogenesis genes involved in somatic embryo maturation were evaluated. Plasmid pMDC85 containing the cry10Aa gene was introduced into a Typica cultivar of C. arabica L. by biobalistic transformation. Transformation efficiency of 16.7% was achieved, according to the number of embryogenic aggregates and transgenic lines developed. Stable transformation was proven by hygromycin-resistant embryogenic lines, green fluorescent protein (GFP) expression, quantitative analyses of Cry10Aa by mass spectrometry, Western blot, ELISA, and Southern blot analyses. Cry10Aa showed variable expression levels in somatic embryos and the leaf tissue of transgenic plants, ranging from 76% to 90% of coverage of the protein by mass spectrometry and from 3.25 to 13.88 μg/g fresh tissue, with ELISA. qPCR-based 2−ΔΔCt trials revealed high transcription levels of cry10Aa in somatic embryos and leaf tissue. This is the first report about the stable transformation and expression of the Cry10Aa protein in coffee plants with the potential for controlling the coffee berry borer.

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Soybean somatic embryo maturation: composition, respiration and water relations

Seed Science Research ◽

10.1017/s0960258500000970 ◽

1991 ◽

Vol 1 (4) ◽

pp. 251-262 ◽

Cited By ~ 12

Author(s):

J. Slawinska ◽

R. L. Obendorf

Keyword(s):

Somatic Embryo ◽

Somatic Embryos ◽

Soybean Seed ◽

Dry Weight ◽

Zygotic Embryos ◽

Naphthaleneacetic Acid ◽

Embryo Maturation ◽

Fresh Weight ◽

Somatic Embryo Maturation ◽

2,4 Dichlorophenoxyacetic Acid

AbstractEmbryogenesis was induced on cotyledons of immature zygotic embryos of soybean (Glycine max (L.) Merrill) placed on solid medium containing 62.5 mm glutamine, soybean seed growth medium salts and vitamins, and 40 mg I−1 2,4-dichlorophenoxyacetic acid (2,4-D) plus 175 mm maltose, or 8 mg I−1 α-naphthaleneacetic acid (NAA) plus 88 mM sucrose. Somatic embryo development was continued in liquid medium containing 0.16 mg I−1 indole-3-butyric acid and 2.64 mg I−1 abscisic acid, glutamine and salts as above, and 88–438 mM sucrose in progressively increasing steps. Germination was on solid half-strength Murashige-Skoog medium. During maturation, somatic embryos mimicked zygotic embryos in colour, protein concentration, water and solute potentials, and respiration. Protein and lipid accumulated to 329 and 86 g kg−1 dry weight in somatic embryos. Fatty acid composition was similar to that of axes of mature seeds. Before desiccation, the water and solute potentials of maturing somatic embryos declined to −1.13 and −1.99 MPa while turgor increased to 0.86 MPa. Concomitantly, a 60% reduction in activity of the cytochrome oxidase pathway of respiration occurred with somatic embryo maturation at 600 g water kg−1 fresh weight. Although small (about 8 mg per embryo), 60% of the somatic embryos formed roots and shoots during germination after maturation without drying and 30% germinated after drying to 60 g water kg−1 fresh weight. In the greenhouse, somatic plantlets grew to mature plants with seeds.

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Factors Influencing Somatic Embryo Maturation in Sugi (Japanese Cedar, Cryptomeria japonica (Thunb. ex L.f.) D. Don)

Plants ◽

10.3390/plants10050874 ◽

2021 ◽

Vol 10 (5) ◽

pp. 874

Author(s):

Tsuyoshi E. Maruyama ◽

Saneyoshi Ueno ◽

Hideki Mori ◽

Takumi Kaneeda ◽

Yoshinari Moriguchi

Keyword(s):

Somatic Embryo ◽

Cryptomeria Japonica ◽

Japanese Cedar ◽

Embryo Maturation ◽

Embryogenic Cell ◽

Factors Affecting ◽

Somatic Embryo Maturation ◽

Maturation Medium ◽

Embryogenic Cell Lines ◽

Basal Salts

This paper presents the results of several experiments identifying basal salts (BS) contained in maturation medium, polyethylene glycol (PEG) concentration, abscisic acid (ABA) concentration, additional supplementation with potassium chloride (KCl), amino acid (AA) concentration, and proliferation culture medium (PCM) as the main culture factors affecting somatic embryo maturation in sugi (Japanese cedar, Cryptomeria japonica, Cupressaceae). Highly efficient embryo maturation was achieved when embryogenic cell lines (ECLs) were cultured on media supplemented with a combination of PEG, ABA, and AAs. More than 1000 embryos per gram of fresh weight (FW) can be produced on EM maturation medium supplemented with 175 g L−1 PEG, 100 µM ABA, 2 g L−1 glutamine, 1 g L−1 asparagine, and 0.5 g L−1 arginine.

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