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

Tsuyoshi E Maruyama,Hideki Mori,Yukiko Itoh,Yukiko Bamba,Shin-Ichi Miyazawa,Yoshinari Moriguchi,Saneyoshi Ueno,Takumi Kaneeda,Satoko Hirayama,Kiyohisa Kawakami,Yoshihisa Hosoi

doi:10.3390/plants10020398

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.

Highlights

Somatic embryogenesis (SE) is currently the most efficient technique for large-scale propagation in clonal forestry and a powerful tool for plant regeneration in basic biological studies, genetic engineering, and implementation of multi-varietal forestry [1,2,3]
The first experimental approach was to determine the effect of plant growth regulators (PGRs) and seed collection times on SE initiation
The statistical analysis indicated that the proportion of explants with SE initiation response significantly differed among seed collection times (χ2 = 12.829, df = 3, p < 0.05 for mid-June, and p < 0.1 for late-July), but no significant differences were observed between the late-June and early-July collection times

Summary

Introduction

Somatic embryogenesis (SE) is currently the most efficient technique for large-scale propagation in clonal forestry and a powerful tool for plant regeneration in basic biological studies, genetic engineering, and implementation of multi-varietal forestry [1,2,3]. Precotyledonary and cotyledonary zygotic embryos are reported as the best initial explants for pine and spruce species, respectively [7]. These results indicate that the timing of the explant excision when zygotic embryos are highly responsive to inducing embryogenic cells (ECs) is the key to a successful. For many species, the time for a successful SE initiation is limited to a very short period of only a few weeks each year [9]. Optimal seed collection time as a critical factor in SE initiation ( in open-pollinated cones) may be influenced by weather, location, and cross variation [10,11,12,13]. Regardless of method, enhancing the success rate in the initiation stage is of vital importance for the improvement of SE protocols in breeding programs

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Conclusion