2.2.2. Effect of Initial Explants
shows the SE initiation responses of different initial explants from sugi seed families carrying the male sterility gene
MS1. Similar to the experiment results with male-fertile-derived seed explants, the highest initiation frequency (30.57%) was achieved with megagametophytes, whereas the lowest rate (0.46%) was recorded when whole seeds were used as initial explants. The results of the statistical analysis indicated that the proportion of explants with SE initiation response significantly differed among initial explant types (χ
2 = 890.77, df = 4,
p < 0.001) and seed families (χ
2 = 693.45, df = 3,
p < 0.001). The induction of ECs from seeds stored at 5 °C for 1 and 4 weeks was achieved but with no improvement in SE initiation frequencies was observed compared to non-stored megagametophyte explants (). In contrast, cold storage of initial explants improved initiation frequencies in white spruce [
36] and radiata pine [
37]. The difference in these results could be attributed to the cold storage method used since, while the seeds of white spruce and radiata pine were stored before dissection, the seeds of sugi were stored after surface sterilization in our experiment. Similarly, Haggman et al. [
38] reported that, although the cold treatment of the cones had no significant effect on SE initiation in Scots pine, the cones can be collected and stored for at least 2 months without losing the ability to initiate SE. Park [
39] also reported that eastern white pine cones might be stored at 3 °C for at least 40 days without reducing embryogenic capacity. These results suggest that cold storage of explants could be a promising alternative for practical uses to improve induction rates and to extend the very narrow window of time when SE initiation is possible. Although in our experiments the cold storage treatment of the explant did not increase the SE initiation frequencies, more research is needed to clarify the potential of cold preconditioning of initial explants in sugi. Improving the cold preconditioning techniques of initial explants to increase induction frequencies (to capture as many genotypes as possible) and to extend SE initiation period is important to develop varietal lines and to manage genetic diversity [
11].
Table 4. Somatic embryogenesis (SE) initiation frequency of sugi seed families carrying the male sterility gene MS1. The data represent the explants with SE initiation response and the total number of explants tested, and the initiation frequency (%) for each of four seed families using different initial explants.
Seed Family $ |
SE Initiation Frequency (%) by Initial Explant Type |
Megagametophyte |
Megagametophyte from 5 °C (1 week) |
Megagametophyte from 5 °C (4 weeks) |
Whole Seed |
Seed with Coat Cut Lengthwise |
Total |
♀ “Shindai 3” ♂ “Suzu 2” |
284/707 (40.17) |
402/1416 (28.39) |
56/435 (12.87) |
0/48 (0.00) |
1/48 (2.08) |
743/2654 (28.00) *** |
♀ “Fukushima-funen 1” ♂ “S3-37(1)” |
81/1056 (7.67) |
66/932 (7.08) |
36/635 (5.67) |
0/252 (0.00) |
4/894 (0.45) |
187/3769 (4.96) *** |
♀ “Fukushima-funen 1” ♂ “Oi 7 |
355/936 (37.93) |
148/775 (19.10) |
19/192 (9.90) |
1/204 (0.49) |
33/477 (6.92) |
556/2584 (21.52) *** |
♀ “Fukushima-funen 1” ♂ “S3-118(2)” |
416/1017 (40.90) |
258/987 (26.14) |
42/293 (14.33) |
2/152 (1.32) |
71/884 (8.03) |
789/3333 (23.67) *** |
Total |
1136/3716 (30.57) *** |
874/4110 (21.27) *** |
153/1555 (9.84) *** |
3/656 (0.46) *** |
109/2303 (4.73) *** |
2275/12,340 (18.44) |
On the other hand, in contrast to our expectation, seeds with the coat cut lengthwise as initial explants showed low induction frequencies (4.73–5.00%) comparable with whole seed explants (0.46–4.17%). This result can be attributed to the improper cutting technique applied, which possibly did not allow the extrusion of ECs. More effort is needed to improve this technique to save energy and time in preparing the initial material.
2.3. Somatic Embryogenesis Initiation from Polycross-Pollinated-Derived Seed Explants Effect of Polycross Family and Seed Collection Times
Seeds derived from polycross pollination (artificial mating using mixed pollen of three and 10 parents) were used as the initial explant for SE initiation in sugi. As shown in Table 5, although initiation frequencies varied according to the polycross family and seed collection time, high induction rates ranging from 38.86–71.18% (with an overall average of 54.50%) were achieved. The results of the statistical analysis indicated that
the proportion of explants with SE initiation response significantly differed between the polycross family (χ2 = 28.997, df = 1, p < 0.001) and among seed collection times (χ2 = 178.63, df = 2, p < 0.001). Similar to the results obtained in the experiments with male-fertile and male-sterile-derived seed explants, the best responses from polycross families were recorded with plant materials collected in mid-July (Table 5). These results confirmed that, during mid-July, the zygotic embryos are highly responsive to induce ECs. On the other hand, the results regarding the effect of the polycross family indicated that the SE initiation frequency of seeds derived from mixed pollen of three parents was better than 10 parents. To clarify this response, studies are currently being carried out with molecular markers. Preliminary results indicate that the highest induction frequency achieved with three mix-pollinated-derived seeds is subordinate to the dominance of a specific parent. In contrast, the dominance of specific pollen parents was suppressed by using 10 mixpollinated-derived seeds [40]. Even though more studies are needed to better understand
the mechanism of polycross fertilization and its effect on SE process, these results suggest that polycross pollination using many individuals could be a practical alternative for the production of seedlings with high genetic diversity.
Table 5. Somatic embryogenesis (SE) initiation frequency from polycross-pollinated-derived seeds of sugi. The data represent the explants with SE initiation response and the total number of explants tested, and the number in the parentheses represent the initiation frequency (%) for each polycross family at different seed collection times.
Polycross Family 1
|
SE Initiation Frequency (%) by Seed Collection Time
|
Early-July
|
Mid-July
|
Late-July
|
Total
|
“S 11” x “3 Mix”
|
157/404 (38.86)
|
583/819 (71.18)
|
471/841 (56.00)
|
1,211/2,064
(58.67) ***
|
“S 11” x “10 Mix”
|
215/514 (41.83)
|
451/726 (62.12)
|
345/773 (44.63)
|
1,011/2,013
(50.22) ***
|
Total
|
372/918 (40.52) ***
|
1,034/1,545 (66.93) ***
|
816/1,614 (50.56) ***
|
2,222/4,077
(54.50)
|
1: Mother tree and pollen parents used for polycross seed families are shown in Supplementary Table S3; *** significantly different at p < 0.001 by post hoc analysis of Pearson’s Chi-squared test.
2.4. Summarized Results on SE Initiation in Sugi from Male-Fertile, Male-Sterile, and Polycross-Pollinated-Derived Seeds
The results of several years of experiments on SE initiation in sugi from male-fertile, male-sterile, and polycross-pollinated-derived seeds are summarized in Table 6. These initiation frequencies were consistent with the reports on SE in artificially pollinated seed families of sugi [41]. The best average induction frequency was achieved with polycross-pollinated-derived seeds (45.20%). However, despite the results suggesting that the statistical analysis indicated that the proportion of explants with SE initiation response significantly differed among the origen of families (χ2 = 618.55, df = 2, p < 0.001), it is important to note that the best average rate obtained with polycross-pollinated-derived seeds were the results of only two years of collection. In addition, SE initiation frequencies from male-fertile, male-sterile, and polycross-pollinated-derived seeds showed great variation ranging from 1.35–67.46% (Supplementary Table S1), 2.78–40.90% (Supplementary Table S2), and 8.37–58.67% (Supplementary Table S4), respectively. Therefore, in our opinion, these differences are attributable to the genotypic ability to induce a large number of ECs achieved in “S 11” X “3 Mix” (57.06%) and “S 11” X “10 Mix” (49.85%) families (Table 6). This result suggests that the observed differences in the efficiency of SE initiation among male-fertile, male-sterile, and polycross-derived families can be attributed to the genotype of the plant material regardless of its origin. The genotype of initial explant has been reported as the most influential factor in SE initiation in a number of conifer species [42–45].
Table 6. Somatic embryogenesis (SE) initiation frequency of megagametophyte explants derived from seventeen male-fertile, eight male-sterile, and five polycross seed families of sugi. The data represent the explants with SE initiation response and the total number of explants tested, and the numbers in the parentheses represent the initiation frequency (%) for each seed family.
Seed Family1
|
SE Initiation Frequency (%) of Seed Explants
Derived From
|
Male-fertile
Family
|
Male-sterile
Family
|
Polycross
Family
|
Chiyoda 327 (OP)
|
12/100 (12.00)
|
|
|
Kuji 6 (OP)
|
9/60 (15.00)
|
|
|
Kuji 9 (OP)
|
1/74 (1.35)
|
|
|
Kuji 14 (OP)
|
545/1,224 (44.53)
|
|
|
Kuji 17 (OP)
|
17/264 (6.44)
|
|
|
Kuji 39 (OP)
|
5/94 (5.32)
|
|
|
Naka 3 (OP)
|
41/192 (21.35)
|
|
|
Naka 5 (OP)
|
10/122 (8.20)
|
|
|
Naka 6 (OP)
|
18/78 (23.08)
|
|
|
Nihari 2 (OP)
|
71/426 (16.67)
|
|
|
Taga 2 (OP)
|
191/502 (38.05)
|
|
|
Taga 4 (OP)
|
37/192 (19.27)
|
|
|
Taga 10 (OP)
|
2/24 (8.33)
|
|
|
Taga 14 (OP)
|
195/580 (33.62)
|
|
|
Tsukuba 2 (OP)
|
27/192 (14.06)
|
|
|
Yamazaki 5 (OP)
|
23/156 (14.74)
|
|
|
Yanase 104 (OP)
|
105/750 (14.00)
|
|
|
“Shindai 3” x “Suzu 2”
|
|
496/1,943 (25.53)
|
|
“Fukushima-funen 1” x “S3-37(1)”
|
|
81/1,056 (7.67)
|
|
“Fukushima-funen 1” x “Oi 7”
|
|
355/936 (37.93)
|
|
“Fukushima-funen 1” x “S3-118(2)”
|
|
416/1,017 (40.90)
|
|
“S1S1-35” x “Gosenshi 1”
|
|
10/74 (13.51)
|
|
“S1S1-23(1)” x “Gosenshi 1”
|
|
1/36 (2.78)
|
|
“S1S1-10(1)” x “Gosenshi 1”
|
|
54/186 (29.03)
|
|
“S1S1-51(1)” x “Gosenshi 1”
|
|
14/347 (4.03)
|
|
“S 1” x “3 Mix”
|
|
|
32/286 (11.19)
|
“S 1” x “9 Mix”
|
|
|
28/268 (10.45)
|
“S 11” x “3 Mix”
|
|
|
1,301/2,280 (57.06)
|
“S 11” x “10 Mix”
|
|
|
1,031/2,068 (49.85)
|
“G 1” x “10 Mix”
|
|
|
40/478 (8.37%)
|
Total
|
1,309/5,030 (26.02)***
|
1,427/5,595 (25.50)***
|
2,432/5,380 (45.20)***
|
1: Mother trees and pollen parents used for polycross seed families are shown in Supplementary Table S3; OP: open-pollinated; *** significantly different at p < 0.001 by post hoc analysis of Pearson’s Chi-squared test.
2.5. Maintenance and Proliferation of Embryogenic Cells
Cultures for the maintenance and proliferation of ECs were carried out at 2–3 week intervals. Despite differences among ECLs with regard to the proliferation rate and morphological structure observed (Figure 3), the culture medium was able to support the growth of almost all lines by subculture routines for several years without losing their proliferation potential and initial morphological characteristics, as described elsewhere [10]. Some lines have been maintained and proliferated for more than 10 years, although with differences among genotypes regarding plant conversion capacity [46]. The embryogenesis response of sugi largely differs among ECLs [47].
Figure 3. Proliferation of embryogenic cell lines (ECLs) with different morphological structures: (A) mucilaginous whitish embryogenic cells (ECs), (B) mucilaginous yellowish ECs, and (C) friable white translucent ECs. Bars: 5 mm.
3. Materials and Methods
3.1. Plant Material
Sugi seeds collected from seed orchards were used as plant material for the SE initiation experiments. At each collection time, the samples of zygotic embryos were observed to determine their developmental stage according to the scale used to classify zygotic embryo development in loblolly pine [48]. The developmental stage of explants collected from mid-June to early-July was the pre-embryo stage equivalent to stages 1–3. Collections in mid-July were mostly represented by early embryo stages equivalent to stages 3–5, and seeds collected in late-July showed the pre-cotyledonary stages equivalent to stages 6–8. Except experiments with different explant types, in all the other experiments, the entire megagametophyte was used as the initial explant.
3.1.1. Somatic Embryogenesis Initiation from Male-Fertile-Derived Seed Explants
Male-fertile-derived seeds were collected from seventeen different OP mother trees in seed orchards at the Forestry and Forest Products Research Institute (Tsukuba, Ibaraki, Japan) and Ibaraki Prefectural Government Forestry Technology Center (Naka, Ibaraki, Japan) from 1997 to 2011 (Supplementary Table S1). To determine the effect of seed collection time and culture media on SE initiation, four seed collections from OP Yanase
104 mother tree were carried out at approximately two-week intervals from mid-June to mid-July (1997) and cultured on medium with or without PGRs (Table 1). The effect of different initial explants on SE initiation was tested with collected plant materials in early-July (2005) from six different OP mother trees (Table 2).
3.1.2. Somatic Embryogenesis Initiation from Male-Sterile-Derived Seed Explants
Male-sterile-derived seeds were collected from eight different full-sib seed families carrying the male sterility gene MS1 or MS2 [49] in seed orchards at Niigata Prefectural Forest Research Institute (Murakami, Niigata, Japan) from 2016 to 2018 (Supplementary Table S2). The effect of collection time on SE initiation was evaluated with seeds from four different seed families collected from early to late-July in 2016 and 2017 (Table 3). To determine the effect of initial explants on SE initiation, five different initial explants (including megagametophyte, megagametophyte isolated from seeds stored at 5 °C for 1 to 4 weeks, whole seeds, and seeds with coat cut lengthwise) collected in 2017 from four different seed families carrying the male sterility gene MS1 were tested (Table 4).
3.1.3. Somatic Embryogenesis Initiation from Polycross-Pollinated-Derived Seed Explants
Polycross-pollinated-derived seeds were collected from five different full-sib seed families using three parents of mixed pollen (3 mix) and nine or ten parents of mixed pollen (9 mix or 10 mix) in a seed orchard at the Niigata Prefectural Forest Research Institute (Murakami, Niigata, Japan) from 2019 to 2020 (Supplementary Table S3 and S4). The effect of collection time on SE initiation was evaluated using 3 mix and 10 mix
polycross-pollinated-derived seeds collected from early to late-July in 2019 (Table 5).
3.1.3. Somatic Embryogenesis Initiation from Polycross-Pollinated-Derived Seed Explants
Polycross-pollinated-derived seeds were collected from five different full-sib seed families using three parents of mixed pollen (3 mix) and nine or ten parents of mixed pollen (9 mix or 10 mix) in a seed orchard at the Niigata Prefectural Forest Research Institute (Murakami, Niigata, Japan) from 2019 to 2020 (Supplementary Table S3 and S4). The effect of collection time on SE initiation was evaluated using 3 mix and 10 mix
polycross-pollinated-derived seeds collected from early to late-July in 2019 (Table 5).
3.2. Surface Sterilization of Seeds
After isolation from collected cones, the seeds were surface sterilized with 1% (w/v available chlorine) sodium hypochlorite solution for 15 min and then rinsed three times with sterile distilled water for 5 min each time.
3.3. Media and Culture Conditions
For the induction of ECs, explants were placed horizontally onto initiation media contained in 90 x 15 mm quad-plates (three explants per well, 12 per plate) and cultured in darkness at 25 °C . The initiation medium containing basal salts reduced to half the concentration from the standard EM medium [10] was supplemented with 10 g L−1 sucrose, 10 μM 2,4-D, 5 μM BA, 0.5 g L−1 casein acid hydrolysate, and 0.5 g L−1 glutamine and was solidified with 3 g L−1 gellan gum (Gelrite®; Wako Pure Chemical, Osaka, Japan). The pH was adjusted to 5.8 prior to autoclaving the medium for 15 min at 121 °C . Media without PGRs but containing 2 g L−1 activated charcoal (Wako Pure Chemical, Osaka, Japan) were also tested to compare SE initiation frequencies with those media containing PGRs (Table 1). For all other experiments, media supplemented with 2,4-D and BA were used.
3.4. Maintenance and Proliferation of Embryogenic Cells
Induced ECs were subcultured every 2–3 weeks on maintenance/proliferation medium containing basal salts reduced to half the concentration from the standard EM medium [10] supplemented with 3 μM 2,4-D, 1 μM BA, 30 g L−1 sucrose, 1.5 g L−1 glutamine, and 3 g L−1 gellan gum. Clumps of embryogenic cells (12 per plate) were cultured in darkness at 25 °C .
3.5. Statistical Analysis
The proportion differentiation of the explants with SE initiation response among seed families, seed collection times, and initial explant types were examined using Pearson’s Chi-squared test. To further elucidate which part of the data was causing the significant differentiation, the residuals of the Chi-squared test were used to conduct the post hoc analysis and the p-values were adjusted with a Bonferroni correction [50]. Pearson’s Chi-squared test was performed using R version 3.6.2 [51], and the post hoc analysis based on the residuals of the Chi-squared test was performed using the R package “chisq.posthoc.test” [52].
4. Conclusions
Our research in sugi proved that, although SE initiation was possible from mid-June to late-July, the best induction efficiency was achieved when seeds were collected in mid-July. The best collection time for SE initiation was confirmed in experiments with male-fertile, male-sterile, and polycross-pollinated-derived seed explants. Notwithstanding differences regarding SE initiation frequencies among families observed throughout our experiments, the optimal collection time for almost all seed families was determined around mid-July. Similarly, as reported for other conifers, the megagametophyte explant was also the best plant material for SE initiation in sugi. However, even though the initial explant, collection time, and culture condition played important role in ECL induction, the genotype of the plant material of sugi was the most influential factor in SE initiation. More effort is necessary to obtain experimental information about the SE initiation performance of sugi genotypes using control-pollinated families to select the most appropriate female and male parents. Emphasizing this point, we believe that the polycross pollination technique can be a practical tool for this purpose.
Supplementary Materials: The following are available online at https://www.mdpi.com/2223-7747/10/2/398/s1, Supplementary Table S1: Somatic embryogenesis (SE) initiation frequency from open-pollinated-derived seeds of male-fertile families of sugi. Supplementary Table S2: Somatic embryogenesis (SE) initiation frequency from sugi seed families carrying the male sterility gene MS1 or MS2. Supplementary Table S3: Mother trees and pollen parents used for polycross-pollinated derived seeds of sugi. Supplementary Table S4: Somatic embryogenesis (SE) initiation responses from polycross-pollinated-derived seed families of sugi.
Author Contributions: Conceptualization and methodology, T.E.M., S.U., H.M. and Y.M.; funding acquisition and project administration, T.E.M. and Y.M.; plant material preparation, T.E.M., S.H., Y.B. and Y.I.; data curation, T.E.M., H.M. and S.U.; experiments and data analysis, T.E.M., T.K., Y.B., K.K., S.-I.M., Y.H., S.U. and H.M.; writing—original draft, T.E.M.; writing—review and editing, T.E.M., S.U., H.M. and Y.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research was partly supported by research grants from the Forestry and Forest Products Research Institute; from the Ministry of Agriculture, Forestry, and Fisheries of Japan (MAFF); from NARO Bio-oriented Technology Research Advancement Institution (BRAIN) (the
Science and technology research promotion program for agriculture, forestry, fisheries, and food industry (No.28013B)); and from NARO Bio-oriented Technology Research Advancement Institution (BRAIN) (Research program on development of innovative technology (No.28013BC)).
Acknowledgments: We would like to thank the Ibaraki Prefectural Government Forestry Technology Center and Niigata Prefectural Forestry Research Institute for the logistic support in the plant material preparation.
Conflicts of Interest: The authors declare no conflict of interest.
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