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

Abstract

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.

Marker-Assisted Selection for Pollen-Free Somatic Plants of Sugi (Japanese Cedar, Cryptomeria japonica): A Simple and Effective Methodology for Selecting Male-Sterile Mutants With ms1-1 and ms1-2

“…SE and plant regeneration of C. japonica were described in our previous reports (Maruyama et al, 2020(Maruyama et al, , 2021a(Maruyama et al, , 2021b. The extrusion of ECs from the explants could begin about 2 weeks after the start of culture (Figure 1A) and the establishment of stable lines with evident EC proliferation was most frequently observed 4 weeks after it (Figure 1B).…”

“…However, for many coniferous species, in vitro clonal propagation is still difficult or inefficient (Bonga et al, 2010). Currently, the most well-established source of SE propagation in sugi is immature zygotic embryos, for which protocols have been established up to the stage of plant regeneration (Maruyama et al, 2020(Maruyama et al, , 2021a(Maruyama et al, , 2021b. Selecting a male-sterile strain as early as possible in this process will improve the efficiency of MSP production.…”

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

“…Embryogenic cells (ECs) were induced by Maruyama et al [19] from entire megagametophytes of immature seeds collected from a full sib seed family ('Shindai 3' × 'Suzu 2') carrying the male sterility allele ms1, in July 2016. Of induced ECLs, 19 male sterile lines from different megagametophytes were used for experiments.…”

“…Since plant regeneration through SE was first reported for sugi [10], several studies on SE initiation [11][12][13], somatic embryo maturation [14][15][16][17], and plantlet conversion [4,18] were reported, showing progress in enhancing SE protocols. In recent years, our group reported an improved methodology for SE initiation from the analysis of results on the influence of plant material, explant collection time, explant genotype, and culture conditions [19]. Subsequently, an optimized protocol for somatic embryo maturation from studies on the effect of basal salts contained in maturation medium, polyethylene glycol (PEG) concentration, abscisic acid (ABA) concentration, additional supplementation of potassium chloride, amino acid concentration, and proliferation culture medium, as the main culture-related factors affecting somatic embryo maturation in sugi, was reported [20].…”

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

“…Since SE was first reported for spruce species, numerous studies on conifer trees have been reported [ 10 , 11 , 12 ]. For sugi, after the first report on plant regeneration via SE [ 13 ], more results including studies on SE initiation [ 14 , 15 , 16 , 17 , 18 ], somatic embryo maturation [ 19 , 20 , 21 , 22 ], plant conversion [ 9 , 23 ], cryopreservation [ 24 , 25 ], and plant transformation [ 26 , 27 ] were published, showing progress in optimizing SE protocols. However, detailed reports on the factors influencing somatic embryo maturation in several ECLs from different seed families of sugi have not yet been published.…”