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.

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

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.

Somatic Embryogenesis and Plant Regeneration from Sugi (Japanese Cedar, Cryptomeria japonica D. Don, Cupressaceae) Seed Families by Marker Assisted Selection for the Male Sterility Allele ms1.

One of the possible countermeasures for pollinosis caused by sugi (Cryptomeria japonica), a serious public health problem in Japan, is the use of male sterile plants (MSPs; pollen-free plants). However, the production efficiencies of MSPs raised by conventional methods are extremely poor, time consuming, and resulting in a high seedling cost. Here, we report the development of a novel technique for efficient production of MSPs, which combines marker-assisted selection (MAS) and somatic embryogenesis (SE). SE from four full sib seed families of sugi, carrying the male sterility gene MS1, was initiated using megagametophyte explants that originated from four seed collections taken at one-week intervals during the month of July 2017. Embryogenic cell lines (ECLs) were achieved in all families, with initiation rates varying from 0.6% to 59%. Somatic embryos were produced from genetic marker-selected male sterile ECLs on medium containing maltose, abscisic acid (ABA), polyethylene glycol (PEG), and activated charcoal (AC). Subsequently, high frequencies of germination and plant conversion (≥76%) were obtained on plant growth regulator-free medium. Regenerated plantlets were acclimatized successfully, and the initial growth of male sterile somatic plants was monitored in the field.