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

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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Plant Regeneration and In Vitro Growth Performance of Male-Sterile Somatic Plantlets of Sugi (Japanese Cedar, Cryptomeria japonica) Derived from Different Embryogenic Cell Lines

Forests ◽

10.3390/f12111592 ◽

2021 ◽

Vol 12 (11) ◽

pp. 1592

Author(s):

Momi Tsuruta ◽

Tsuyoshi E. Maruyama ◽

Saneyoshi Ueno ◽

Takumi Kaneeda ◽

Yoshinari Moriguchi

Keyword(s):

Growth Performance ◽

Somatic Embryo ◽

Japanese Cedar ◽

In Vitro Growth ◽

Embryo Germination ◽

Embryogenic Cell ◽

Somatic Embryo Maturation ◽

Embryogenic Cell Lines ◽

Somatic Embryo Germination

With the spread of pollinosis caused by sugi (Japanese cedar, Cryptomeria japonica) pollen, the use of pollen-free somatic seedlings of sugi is expected in Japan. However, there is a lack of knowledge on the relationship between the abilities during somatic embryogenesis, initial in vitro growth traits, and subsequent growth of somatic seedlings. In the present study, we provide the first basic information on somatic embryo maturation efficiency, somatic embryo germination, and plantlet conversion frequencies, as well as on in vitro growth performance of pollen-free somatic plantlets derived from different embryogenic cell lines (ECLs). Somatic embryo maturation efficiency varied from 34 to 514 cotyledonary embryos per plate and the average for the 19 ECLs tested was 244 embryos per plate. Subsequently, the overall average rates of somatic embryo germination and conversion among ECLs were 87.8% and 85.3%, respectively. The results of in vitro growth performance of pollen-free somatic plantlets showed significant differences in growth rate among ECLs.

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

International Journal of Molecular Sciences ◽

10.3390/ijms20215334 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5334 ◽

Cited By ~ 2

Author(s):

Eliana Valencia-Lozano ◽

José L. Cabrera-Ponce ◽

Miguel A. Gómez-Lim ◽

Jorge E. Ibarra

Keyword(s):

Mass Spectrometry ◽

Bacillus Thuringiensis ◽

Somatic Embryo ◽

Somatic Embryos ◽

Leaf Tissue ◽

Stable Transformation ◽

Embryo Maturation ◽

Embryogenic Cell ◽

Somatic Embryo Maturation ◽

Coffee Plants

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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Somatic Embryogenesis Initiation in Sugi (Japanese Cedar, Cryptomeria japonica D. Don): Responses from Male-Fertile, Male-Sterile, and Polycross-Pollinated-Derived Seed Explants

Plants ◽

10.3390/plants10020398 ◽

2021 ◽

Vol 10 (2) ◽

pp. 398

Author(s):

Tsuyoshi E. Maruyama ◽

Saneyoshi Ueno ◽

Yoshihisa Hosoi ◽

Shin-Ichi Miyazawa ◽

Hideki Mori ◽

...

Keyword(s):

Somatic Embryogenesis ◽

Cryptomeria Japonica ◽

Japanese Cedar ◽

Influential Factor ◽

Male Sterile ◽

Embryogenic Cell ◽

Factors Affecting ◽

Seed Collection ◽

Fertile Male ◽

Collection Time

This study aimed to obtain information from several embryogenic cell (EC) genotypes analyzing the factors that affect somatic embryogenesis (SE) initiation in sugi (Cryptomeria japonica, Cupressaceae) to apply them in the improvement of protocols for efficient induction of embryogenic cell lines (ECLs). The results of several years of experiments including studies on the influence of initial explant, seed collection time, and explant genotype as the main factors affecting SE initiation from male-fertile, male-sterile, and polycross-pollinated-derived seeds are described. Initiation frequencies depending on the plant genotype varied from 1.35 to 57.06%. The best induction efficiency was achieved when seeds were collected on mid-July using the entire megagametophyte as initial explants. The extrusion of ECs started approximately after 2 weeks of culture, and the establishment of ECLs was observed mostly 4 weeks after extrusion on media with or without plant growth regulators (PGRs). Subsequently, induced ECLs were maintained and proliferated on media with PGRs by 2–3-week-interval subculture routines. Although, the initial explant, collection time, and culture condition played important roles in ECL induction, the genotype of the plant material of sugi was the most influential factor in SE initiation.

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