Key Techniques for Somatic Embryogenesis and Plant Regeneration of Pinus Koraiensis

Article Key Techniques for Somatic Embryogenesis and Plant Regeneration of Pinus koraiensis

Fang Gao 1, Chunxue Peng 1, Hao Wang 1, Iraida Nikolaevna Tretyakova 2, Alexander Mikhaylovich Nosov 3,4, Hailong Shen 5,* and Ling Yang 1,* 1 State Key Laboratory of Tree Genetics and Breeding, School of Forestry, Northeast Forestry University, Harbin 150040, China; [email protected] (F.G.); [email protected] (C.P.); [email protected] (H.W.) 2 Laboratory of Forest Genetics and Breeding, Institution of the Russian Academy of Sciences V.N., Sukachev Institute of Forest Siberian Branch of RAS, 660036 Krasnoyarsk, Russia; [email protected] 3 Department of Cell Biology and Institute of Plant Physiology K.A., Timiryazev Russian Academy of Sciences, 127276 Moscow, Russia; [email protected] 4 Department of Plant Physiology, Biological Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia 5 State Forestry and Grassland Administration Engineering Technology Research Center of Korean Pine, Harbin 150040, China * Correspondence: [email protected] (H.S.); [email protected] (L.Y.)

Received: 23 June 2020; Accepted: 17 August 2020; Published: 20 August 2020

Abstract: Korean pine is the dominant species of Korean pine forests. It is an economically valuable species that yields oil, high-quality timber and nuts, and it offers great prospects for further development. Complete regenerated plants of Korean pine were obtained via somatic embryogenesis using megagametophytes as the explant. The seeds of 27 families of Korean pine were collected to induce embryogenic lines. We compared the effects of explant collection time, family and medium components (concentrations of sucrose, plant growth regulators and acid-hydrolyzed casein) on embryogenic lines induction. The effects of plant growth regulators and L-glutamine contents on the proliferation and maturation of embryogenic cell lines were studied, and the germinating ability of different cell lines was evaluated. The embryogenic lines induction percentage of Korean pine reached 1 1 33.33%. When 4.52 µmol L− 2,4-D and 2.2 µmol L− 6-BA were added to the medium of embryogenic · · 1 lines proliferation, the ability of embryo maturation was the best (cell line 001#-100 was 135.71 g− 1 · fresh weight). Adding 1–1.5g L− L-glutamine to the proliferation medium can improve the ability of embryo maturation (cell line 001#-100 was 165.63 g 1 fresh weight). The germination percentage of the · − three cell lines tested was significant, and the highest was 66%. We report on successful regeneration and cryopreservation methods for somatic embryos of Korean pine. This technology could be used to propagate the excellent germplasm resources of Korean pine and to establish multi-varietal forestry.

Keywords: Korean pine; embryogenic lines; somatic embryos; regenerated plant; megagametophytes; cryopreservation

1. Introduction Somatic embryogenesis is the formation of embryo-like structures from somatic cells without gametic fusion [1,2]. Somatic embryos (SEs) are not only the best receptor system for genetic transformation [3,4], but are also invaluable for the large-scale propagation of excellent germplasm resources, and have many potential applications [5–7]. To date, somatic embryogenesis has been achieved for nearly 30 pine species. This method is used in production for some species, and has had remarkable economic beneﬁts [8].

Forests 2020, 11, 912; doi:10.3390/f11090912 www.mdpi.com/journal/forests Forests 2020, 11, 912 2 of 15

Korean pine (Pinus koraiensis Sieb. Et Zucc.) is a species in the Pinaceae that is mainly distributed in northeast China, the far east of Russia, the Korean Peninsula and Japan [9]. Korean pine is an economically valuable species that yields oil, high-quality timber and nuts, and it has great prospects for further development [10]. In recent decades, the populations of natural broad-leaved Korean pine mixed forests have been sharply reduced [11,12], and the species has been listed as nationally endangered in China. Thus, the protection of P. koraiensis has become urgent [12]. The development of powerful clonal propagation methods, such as somatic embryogenesis, has potentially numerous application advantages over conventional rooted cuttings [13]. Somatic embryogenesis is considered the main way to achieve true rejuvenation in vegetative propagules, because SEs develop both embryonic apical and root meristems [14]. Somatic embryogenesis comprises the coordinated execution of four steps: embryogenic lines (EC) induction, proliferation, maturation and plant regeneration [15]. One of the main factors affecting SEs induction in pines is the developmental stage of the zygotic embryo [16]. Immature embryos are the most successful explant for the somatic embryogenesis of pine species, and the induction percentage of EC from immature embryos is higher than that from mature embryos [17–19]. For most pine species, the induction percentage of EC is higher when seeds are at the cleavage polyembryony or cotyledon embryo stages of development. However, the response of different tree species is different, which needs to be determined by experiments. Most Pinus species are recalcitrant to micropropagation and regeneration by SE [20], and Korean pine is not an exception. Bozhkov described two alternative pathways of SEs origin of P. koraiensis [21]. Gao induced EC and proliferated, but the induction percentage of EC was low, and SEs could not be obtained [19]. Once SEs are initiated and EC can be identified, the next challenge is to ensure the rapid proliferation of EM (embryonal mass, EM) on subculture onto fresh medium, so as to generate the amounts that are needed for various steps [22]. The quality of EC during proliferation impacts not only the yield but also the quality of the cotyledonary embryos, leading to lower germination rates [23]. At the same time, it is necessary to optimize the culture conditions of the EC proliferation stage. In this study, the seeds from 27 families were collected. Using immature megagametophytes as the explant, we established an efficient EC induction system. The key techniques of EC proliferation and maturation were optimized. The germinating abilities of SEs of different cell lines were evaluated. These methods can be used to propagate the excellent germplasm resources of P. koraiensis, and to establish multi-varietal forestry.

2. Materials and Methods

2.1. EC Induction

2.1.1. Selection of Explant Source From June to July 2015, open-pollinated cones of three families (numbered 057#, 108# and 135#) were collected from the Qingshan Forest Seed Orchard (Weihe Forestry Bureau, Heilongjiang Province, China). The age of each family was 28 years, and the dates of seed collection were 23 June, 30 June, 6 July and 13 July (representing seeds in the E1, E2, E3 and E4 phases, respectively). The cones were cut from the branches and stored at 4 ◦C. The physiological state of explants from seeds collected at the four times was determined by observation under a microscope, and they were photographed using a Moticam 3000C camera. At the same time, 10 seeds were randomly selected, and the fresh weights of the megagametophytes were measured by peeling oﬀ the seed coat, then putting them in the oven at 108 ◦C at constant weights and measuring the dry weight. On 1 July 2017, open-pollinated cones in the E1 phase were collected from 24 families, and their megagametophytes were used as explants. Forests 2020, 11, 912 3 of 15

2.1.2. Explant Sterilization Method The cones of P. koraiensis were washed with detergent for about 30 min and then washed with running water for 8 h. After peeling off the seed scales, the seeds were treated with 75% (v/v) alcohol for 1 to 2 min on a clean bench and then washed three to five times with sterile water. The washed seeds were treated with 10% (m/v) sodium hypochlorite solution for 15 min, and then rinsed three to five times with sterile water. The inner and outer testae were removed, and the whole megagametophyte was sterilized by soaking in 3% (m/v) hydrogen peroxide for 8 min and washing three to five times with sterile water.

2.1.3. Screening of Medium Components Four factors and four levels of orthogonal experiments were used. The orthogonal design is shown 1 1 in Table1. The basal culture medium was DCR [ 23], supplemented with 6.5 g L− agar and 500 mg L− 1 L-glutamine, and the four factors were sucrose (25, 30, 35 and 40 g L− ), 1-naphthalacetic acid (NAA) 1 (8.06, 10.74, 13.43 and 16.11 µmol L− ), 2.4-dichlorophenoxyacetic acid (2.4-D) (18.10, 27.14, 36.20 and 1 · 1 45.24 µmol L− ), N6-benzyladenine (6-BA) (2.22, 4.44, 6.66 and 8.88 µmol L− ) and acid-hydrolyzed · 1 · casein (CH) (0.3, 0.5, 0.8 and 1.0 g L− ). The pH of the medium was then adjusted to 5.8 before autoclaving (105 Pa at 120 ◦C, 20 min). L-glutamine was ﬁlter-sterilized and added to the medium after autoclaving. The sterilized fertilized megagametophytes were placed into Petri dishes (Jiafeng Horticultural Products Co., Ltd., Shanghai, China), which were 90 mm diameter 20 mm depth, × and ﬁve explants per dish. The number of replicates per treatment varied in the initiation experiments. This was due to the availability of cones on sampled trees in the seed orchards. After inoculation, the cultures were kept in a culture chamber at 23 2 C in the dark. The explants were observed ± ◦ regularly and the EC induction percentage was calculated after 12 weeks of culture.

Table 1. Inﬂuencing factors of source, acid-hydrolyzed casein (CH), 1-naphthalacetic acid (NAA) and N6-benzyladenine (6-BA) on embryogenic lines (EC) induction of Pinus koraiensis based on orthogonal experiments design.

Sucrose NAA 6-BA Total Induction Treatments Concentration Concentration Concentration CH (g L 1) − Explants Percentage (%) (g L 1) (µmol L 1) (µmol L 1) − · − · − 1 25 8.06 2.22 0.3 125 20.87 0.92 ± 2 25 10.74 4.44 0.5 141 12.27 1.26 ± 3 25 13.43 6.66 0.8 136 11.66 1.09 ± 4 25 16.11 8.88 1.0 96 0.00 0.00 ± 5 30 8.06 4.44 0.8 97 14.48 0.35 ± 6 30 10.74 2.22 1.0 123 18.73 1.22 ± 7 30 13.43 8.88 0.3 132 12.17 1.05 ± 8 30 16.11 6.66 0.5 136 12.43 1.05 ± 9 35 8.06 6.66 1.0 138 21.80 1.13 ± 10 35 10.74 8.88 0.8 132 33.33 0.69 ± 11 35 13.43 2.22 0.5 88 13.65 0.20 ± 12 35 16.11 4.44 0.3 115 3.52 1.34 ± 13 40 8.06 8.99 0.5 157 11.05 1.29 ± 14 40 10.74 6.66 0.3 142 12.02 1.13 ± 15 40 13.43 8.88 1.0 154 11.63 1.02 ± 16 40 16.11 2.22 0.8 70 0.00 0.00 ± Note: The values in the column Induction Percentage in the table represent the mean standard error. ±

In July 2017, the best scheme selected in 2015 was used for EC induction culture (DCR + 1 1 1 1 1 35 g L− sucrose + 10.74 µmol L− NAA + 6.66 µmol L− 6-BA + 0.8 mg L− CH + 6.5 g L− agar 1 · · and 500 mg L− L-glutamine). Other culture methods were the same as in 2015. The sterilized fertilized megagametophytes were placed into Petri dishes, ﬁve explants per dish, and each family had 50 explants. Forests 2020, 11, 912 4 of 15

The EC was cytologically observed. The fresh target EC was placed on a clean microslide, stained with 0.1% safranine staining solution, and then covered with a cover glass. The cover glass was tapped gently with the ﬂat end of a pencil to spread the plant tissue evenly. The cells were observed and photographed immediately under an optical microscope (Olympus BX51 equipped with a Moticam 3000C camera).

2.2. Proliferation and Maturation of the EC

2.2.1. Proliferation of the EC Proliferation experiment 1: Effect of plant growth regulator (PGRs) on the proliferation of Korean Pine EC. The EC of 001#-1, 001#-100 and 001#-34 were used as test materials. The proliferation medium 1 1 1 was mLV [24] basic medium supplemented with 0.5 g L− CH, 30 g L− sucrose, 0.5 g L− L-glutamine 1 and 4 g L− Gelrite (Phytagel™, Sigma-Aldrich, St. Louis, MO., USA). The hormone concentration was divided into three treatments: (1) 9.04 µmol L 1 2,4-D + 4.4 µmol L 1 6-BA; (2) 4.52 µmol L 1 · − · − · − 2,4-D + 2.2 µmol L 1 6-BA; (3) 2.26 µmol L 1 2,4-D + 0.44 µmol L 1 6-BA. Embryonal mass (0.2 g) was · − · − · − transferred to the same composition in each subculture, each cell line with five replicates, subcultured every 2 weeks. The fresh weight of the embryogenic cell masses was measured after four subculture cycles, and the maturation ability of the embryogenic cell lines was measured under three different PGRs conditions. Proliferation experiment 2: Effect of L-glutamine on the proliferation of Korean Pine EC. The EC of 001#-1, 001#-100 and 001#-34 were used as test materials. The proliferation medium was 1 1 1 1 mLV medium supplemented with 0.5 g L− CH, 30 g L− sucrose, 4.52 µmol L− 2,4-D, 2.2 µmol L− 1 · · 6-BA and 4 g L− Gelrite. L-glutamine concentration was divided into three treatments (0.5, 1.0 and 1 1.5 g L− ). Embryonal mass (0.2 g) was transferred to the same composition in each subculture, each cell line with four replicates, and subcultured every 2 weeks. The fresh weights of the embryogenic cell masses were measured after four subculture cycles, and the maturation ability of the embryogenic cell lines was measured under three different L-glutamine conditions.

2.2.2. Maturation of SEs The maturation ability of SEs was tested using the EC obtained from proliferation. The EC (100 mg) was transferred into a 50 mL centrifuge tube and the liquid proliferation medium without PGRs was added. Next, we shook the centrifuge tube violently to achieve full dispersal, and transferred the mixture to the filter paper with a pipette. A Brinell funnel was used for filtration, and then we put the filter paper with EC on the solid medium. Each cell line had between four and six replicates. The mLV medium contained 68 g L 1 sucrose, 75.66 µmol L 1 abscisic acid (ABA), 500 g L 1 CH, 500 g L 1 − · − − − L-glutamine and 10 g L 1 Gelrite. The number of SEs was recorded after being cultured at 23 1 C − ± ◦ for 3 months.

2.3. SEs Germination and Plant Regeneration In total, 50 mature SEs were randomly selected from three cell lines for the germination test. The SEs needed to be desiccation treated before germination, and the desiccation treatment was completed with 6-hole cell plate (Corning-Costar 3516), in which 3 holes were filled with two layers of dry sterile filter paper. We then put the SEs on the filter paper, and the remaining three holes were filled with sterile water. Finally, the cell plates were sealed with preservative film and cultured in the dark at 4 ◦C for 7 days. The cotyledon embryos were placed onto the germination medium. The germination 1 1 1 medium was mLV medium supplemented with 2 g L− activated carbon, 20 g L− sucrose and 4 g L− Gelrite, cultured in the dark for 7 days, and then transferred to light culture (35 µmol cm 2 s 1 light, · − · − 16 h light/8 h dark photoperiod, 23 2 C). The regeneration percentage of plantlets was recorded after ± ◦ 8 weeks of culturing. Forests 2020, 11, 912 5 of 15

2.4. Transplanting and Acclimatization of Regenerated Plants After 16 weeks of culture, the rooted plantlets that developed from SEs were removed from the culture bottle with tweezers, the medium attached to the roots was washed oﬀ, and the plantlets were transplanted into a sterilized substrate (nutrient soil, vermiculite and perlite, at a volume ratio of 3:1:1). The plantlets were covered with plastic wrap to maintain high air humidity, and cultivated under the following conditions: 23 2 C, under a light intensity of about 35 µmol/(cm 2 s 1), and a 16 h ± ◦ − · − light/8 h dark photoperiod. The plastic wrap was removed after 2 weeks, and the plants were watered regularly. The survival percentage of the plantlets was determined at 6 weeks after transplanting into the soil substrate.

2.5. Microscopy Observation The paraffin section making method was performed as referred to by Li [25]. The cultures of different development stages were fixed in FAA fixative solution (formaldehyde: acetic acid:50% ethanol = 1:1:18). The fixed samples were dehydrated, soaked in wax and embedded in paraffin, stained with hematoxylin, then sectioned (the thickness of the section was 10 µm), and sealed with neutral balsam. The cells were observed under an optical microscope.

2.6. Data Statistics and Analysis Methods The experimental data were processed using Excel 2003, and the average value and proliferation eﬃciency were calculated from these data. Single-factor analysis of variance (ANOVA) to evaluate the eﬀects of PGRs, sucrose and CH was performed using SPSS 19 software (SPSS, Chicago, IL, USA). Graphs were drawn using Sigmaplot 12.0. The calculations used for the various indexes are:

Number of explants producing EC EC induction percentage(%) = 100 Number of living explants placed ×

(EC quality after culture EC quality before culture) EC proliferation eﬃciency(%) = − 100 Fresh quality of EC before culture ×

Number of SEs (per petri dish) SEs per gram of EC(g 1 FW) = 1000 − Fresh quality of EC before culture (mg) ×

Germination percentage (%) = Number of SEs with new shoots 100 Number of SEs placed ×

3. Results

3.1. Induction of EC

3.1.1. Development of Explants in Different Periods The development state of P. koraiensis seeds differed among the four collection times (Figure1), ranging from the early embryo stage (Figure1a, Phase E1; Figure1b, Phase E2) to the cleavage polyembryonic stage (Figure1c, Phase E3) and prophase at the columnar embryo stage (Figure1d, Phase E4). With the maturity of the megagametophyte, the fresh weight of the megagametophyte in three families increased first and then decreased (Figure2a), and the dry weight increased gradually (Figure2b). Forests 2020, 11, x FOR PEER REVIEW 6 of 15 Forests 2020, 11, 912 6 of 15 Forests 2020, 11, x FOR PEER REVIEW 6 of 15

Figure 1. Development status of immature seeds of Pinus Koraiensis at different collection times (2015). (a) Phase E1, collected on 23 June, bar = 0.2 cm; (b) Phase E2, collected on 30 June, bar = 0.2 cm; (c) Figure 1. Development status of immature seeds of Pinus koraiensis at diﬀerent collection times (2015). FigurePhase 1.E3, Development collected on status6 July, of bar immature = 0.2 cm; seeds (d) ofPhase Pinus E4, Koraiensis collected at on different 13 July, collection bar = 0.2 times cm. The (2015). red (a) Phase E1, collected on 23 June, bar = 0.2 cm; (b) Phase E2, collected on 30 June, bar = 0.2 cm; (c) Phase (arrowa) Phase points E1, collected to the embryo on 23 headJune, andbar =is 0.2indicated cm; (b )by Phase the lettersE2, collected ‘eh’, the on yellow 30 June, arrow bar =points 0.2 cm; to (thec) E3, collected on 6 July, bar = 0.2 cm; (d) Phase E4, collected on 13 July, bar = 0.2 cm. The red arrow Phasesuspensor E3, collected and is indicated on 6 July, by bar the = letter0.2 cm; s, (thed) Phaseblue arrow E4, collected points toon the 13 femaleJuly, bar gametophyte = 0.2 cm. The and red is points to the embryo head and is indicated by the letters ‘eh’, the yellow arrow points to the suspensor arrowindicated points by tothe the letters embryo ‘fg’, headthe yellow and is oval indicated is the byearly the embryogeny letters ‘eh’, theand yellow is indicated arrow by points the letter to the e, and is indicated by the letter s, the blue arrow points to the female gametophyte and is indicated by the suspensorlettersand the ‘fg’, red and the oval yellowis isindicated the oval corrosion is by the the earlycavity letter embryogeny and s, the is indicatedblue andarrow is by indicated pointsthe letter to by the‘c’. the female letter e,gametophyte and the red ovaland is indicatedthe corrosion by the cavity letters and ‘fg’, is indicated the yellow by oval the letteris the ‘c’. early embryogeny and is indicated by the letter e, and the red oval is the corrosion cavity and is indicated by the letter ‘c’.

Figure 2. (a) Fresh weight of megagametophyte (b) dry weight of megagametophyte in diﬀerent developmentalFigure 2. (a) Fresh stages weight of Pinus of koraiensismegagametophyte. Each data ( pointb) dry was weight repeated of megagametophyte for 10 samples. The in error different bars representdevelopmental the standard stages of error. Pinus koraiensis. Each data point was repeated for 10 samples. The error bars Figurerepresent 2. (thea) Fresh standard weight error. of megagametophyte (b) dry weight of megagametophyte in different 3.1.2.developmental EC Induction stages of Pinus koraiensis. Each data point was repeated for 10 samples. The error bars 3.1.2. EC induction representThe explants the standard at the error. E1 stage were placed onto the induction medium, and cell proliferation was initiatedThe explants at the at micropylar the E1 stage end were of the placed megagametophyte onto the induction within medium, 30 days and of culturingcell proliferation (Figure 3wasa). 3.1.2. EC induction Theinitiated cytological at the micropylar observation end indicated of the megagametoph that there wereyte vacuolatedwithin 30 days cells of (Figure culturing3b), (Figure embryogenic 3a). The cellcytological groupsThe explants (Figureobservation at3 c),the symmetrical E1 indicated stage were that embryogenic placed there ontowere cellsthe vacuolated induction (Figure3 d) cellsmedium, asymmetrical (Figure and 3b),cell embryogenic proliferationembryogenic cells was cell initiated at the micropylar end of the megagametophyte within 30 days of culturing (Figure 3a). The cytological observation indicated that there were vacuolated cells (Figure 3b), embryogenic cell

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Forests 2020, 11, x FOR PEER REVIEW 7 of 15 Forestsgroups2020 (Figure, 11, 912 3c), symmetrical embryogenic cells (Figure 3d) asymmetrical embryogenic 7cells of 15 groups(Figure (Figure3e) and early3c), symmetrical SEs (Figure 3f)embryogenic in the EC. Some cells of(Figure the embryogenic 3d) asymmetrical cells divided embryogenic symmetrically cells (Figure(Figure(Figure 3 3e)3d),e) andand which earlyearly made SEsSEs (Figure(Figurethe EC 3proliferate 3f)f) in in the the EC. EC. continuously. Some Some of of the the At embryogenic embryogenic the same time, cells cells some divided divided of the symmetrically symmetrically embryogenic (Figure(Figurecells divided 33d),d), whichwhich asymmetrically mademade thethe ECEC (Figure proliferateproliferate 3e) and continuously.continuously. gradually developed AtAt thethe samesame into time,time, early somesome SEs ofof(Figure thethe embryogenicembryogenic 3f). cellscells divided divided asymmetrically (Figure 33e)e) andand graduallygradually developeddeveloped intointo earlyearly SEsSEs (Figure(Figure3 f).3f).

Figure 3. Cytological observation of the EC of Pinus koraiensis after safranine staining: (a) EC, bar = Figure0.5 cm; 3.(b) VacuolatedCytological cells observation (vc), bar = of 20 the μm; EC (c)of Embryogenic Pinus koraiensis cell group, after bar safranine = 20 μm; staining: (d) Symmetrical (a) EC, Figure 3. Cytological observation of the EC of Pinus koraiensis after safranine staining: (a) EC, bar = barembryogenic= 0.5 cm;( cellsb) Vacuolated (sec), bar = 20 cells μm; (vc), (e) Asymmetrical bar = 20 µm; embryogenic (c) Embryogenic cells (aec), cell group, bar = 20 bar μm;= (f20) Earlyµm; 0.5 cm; (b) Vacuolated cells (vc), bar = 20 μm; (c) Embryogenic cell group, bar = 20 μm; (d) Symmetrical (SEs,d) Symmetrical bar = 60 μm; embryogenicThe letters ‘eh’ cells denote (sec), the bar embryo= 20 headµm; (eh), (e) Asymmetrical and the letter ‘s’ embryogenic denotes the cellssuspensor (aec), embryogenic cells (sec), bar = 20 μm; (e) Asymmetrical embryogenic cells (aec), bar = 20 μm; (f) Early bar(s). = 20 µm;(f) Early SEs, bar = 60 µm; The letters ‘eh’ denote the embryo head (eh), and the letter ‘s’ SEs, bar = 60 μm; The letters ‘eh’ denote the embryo head (eh), and the letter ‘s’ denotes the suspensor denotes the suspensor (s). (s). The induction percentage of EC differed significantly among different families (p < 0.05). In 2015, The induction percentage of EC differed significantly among different families (p < 0.05). In 2015, the highest induction percentage was in family 135#, followed by family 108# and then 057# (Figure the highestThe induction induction percentage percentage of wasEC differed in family significantly 135#, followed among by familydifferent 108# families and then (p < 057#0.05). (Figure In 2015,4). the4). Thehighest average induction induction percentage percentage was ofin ECfamily varied 135#, greatly followed among by familythe different 108# and cone then collection 057# (Figure times. TheThe averageEC induction induction percentage percentage of E1 was of EC the varied highest, greatly followed among by E2, the E3 di ffanderent then cone E4. collection times. 4).The The EC average induction induction percentage percentage of E1 was of theEC highest,varied greatly followed among by E2, the E3 different and then cone E4. collection times. The EC induction percentage of E1 was the highest, followed by E2, E3 and then E4.

Figure 4. Comparison of EC induction percentage of megagametophytes in three Pinus Koraiensis

familiesFigure 4. at Comparison different development of EC induction stages. percentage Each data point of megagametophytes was repeated for 16 in replicates. three Pinus The Koraiensis error bars representfamilies at the different standard development error. The distages.fferent Each small data letters point in thewas figure repeated show for a significant16 replicates. diff Theerence error at Figure 4. Comparison of EC induction percentage of megagametophytes in three Pinus Koraiensis pbars< 0.05 represent on the the medium standard of NAA error.+ The6-BA different combination, small letters and di inff theerent figure capital show letters a significant show a significant difference families at different development stages. Each data point was repeated for 16 replicates. The error diatff perence ˂ 0.05 atonp the< 0.05 medium on the of medium NAA + 6-BA of 2,4-D combination,+ 6-BA combination. and different capital letters show a significant bars represent the standard error. The different small letters in the figure show a significant difference difference at p ˂ 0.05 on the medium of 2,4-D + 6-BA combination. at p ˂ 0.05 on the medium of NAA + 6-BA combination, and different capital letters show a significant difference at p ˂ 0.05 on the medium of 2,4-D + 6-BA combination.

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Megagametophytes at the E1 stage from the 135# family were used as the explant. Among the media with combinations of NAA and 6-BA, the optimal medium for EC induction was DCR + 35 g ForestsL−1 sucrose2020, 11 ,+ 912 10.74 μmol·L−1 NAA + 6.66 μmol·L−1 6-BA + 0.8 mg L−1 CH, with the highest induction8 of 15 percentage of 33.33% (Table 1). Among the media with combinations of 2,4-D + 6-BA, the optimal medium was DCR + 35 g L−1 sucrose + 27.14 μmol·L−1 2,4-D + 6.66 μmol·L−1 6-BA + 0.8 g L−1 CH, and Megagametophytes at the E1 stage from the 135# family were used as the explant. Among the the maximum induction percentage was 31.90% (Table 2). Across all media (combinations of NAA 1+ media with combinations of NAA and 6-BA, the optimal medium for EC induction was DCR + 35 g L− 6-BA and 2,4-D + 6-BA),1 the factors could be1 ranked, from strongest1 influence on the induction sucrose + 10.74 µmol L− NAA + 6.66 µmol L− 6-BA + 0.8 mg L− CH, with the highest induction percentage to weakest,· as follows: NAA/2,4-D· > sucrose > 6-BA > CH. percentage of 33.33% (Table1). Among the media with combinations of 2,4-D + 6-BA, the optimal medium was DCR + 35 g L 1 sucrose + 27.14 µmol L 1 2,4-D + 6.66 µmol L 1 6-BA + 0.8 g L 1 CH, Table 2. Influencing factors− of source, acid-hydrolyzed· − casein (CH), 2,4-dichlorophenoxyacetic· − acid− and the maximum induction percentage was 31.90% (Table2). Across all media (combinations of NAA (2,4-D) and N6-benzyladenine (6-BA) on embryogenic lines (EC) induction of Pinus Koraiensis based + 6-BA and 2,4-D + 6-BA), the factors could be ranked, from strongest influence on the induction on orthogonal experiments design. percentage to weakest, as follows: NAA/2,4-D > sucrose > 6-BA > CH. Sucrose 2,4-D 6-BA CH Induction Total TreatmentsTable 2. Concentration Concentration Concentration (g Percentage Influencing factors of source, acid-hydrolyzed casein (CH), 2,4-dichlorophenoxyaceticExplants acid (2,4-D) and N6-benzyladenine(g L−1) (6-BA)(μmoL·L on embryogenic−1) (μ linesmoL·L (EC)−1) inductionL−1) of Pinus koraiensis based(%) on orthogonal1 experiments 25 design. 18.1 2.22 0.3 96 11.65 ± 0.90 2 25 27.14 4.44 0.5 127 10.32 ± 1.33 Sucrose 2,4-D 6-BA 3 25 36.2 6.66 0.8 Total 69 Induction 11.66 ± 1.85 Treatments Concentration Concentration Concentration CH (g L 1) 4 25 45.24 8.88 1.0− Explants 81 Percentage 0.00 ± 0.00 (%) (g L 1) (µmoL L 1) (µmoL L 1) 5 − 30 · 18.1− · − 4.44 0.8 72 12.57 ± 1.49 1 25 18.1 2.22 0.3 96 11.65 0.90 6 30 27.14 2.22 1.0 87 25.32± ± 1.51 2 25 27.14 4.44 0.5 127 10.32 1.33 ± 37 25 30 36.2 36.2 6.66 8.88 0.8 0.3 69 103 11.66 11.63 1.85± 1.23 ± 48 25 30 45.24 45.24 8.88 6.66 1.0 0.5 81 113 0.00 13.370.00 ± 1.00 ± 59 30 35 18.1 18.1 4.44 6.66 0.8 1.0 72 102 12.57 23.54 1.49± 1.37 ± 6 30 27.14 2.22 1.0 87 25.32 1.51 10 35 27.14 8.88 0.8 116 31.90±± 1.23 7 30 36.2 8.88 0.3 103 11.63 1.23 ± 118 30 35 45.24 36.2 6.66 2.22 0.5 0.5 113 112 13.37 12.42 1.00± 1.06 ± 129 35 35 18.1 45.24 6.66 4.44 1.0 0.3 102 117 23.54 4.35 ±1.37 1.28 ± 1013 35 40 27.14 18.1 8.88 8.99 0.8 0.5 116 130 31.90 11.60 1.23± 1.22 ± 11 35 36.2 2.22 0.5 112 12.42 1.06 14 40 27.14 6.66 0.3 104 8.64± ± 1.21 12 35 45.24 4.44 0.3 117 4.35 1.28 15 40 36.2 8.88 1.0 119 9.29± ± 1.28 13 40 18.1 8.99 0.5 130 11.60 1.22 ± 1416 40 40 27.14 45.24 6.66 2.22 0.3 0.8 104 153 8.64 0.65 1.21± 0.65 ± 15 40 36.2 8.88 1.0 119 9.29 1.28 ± Note:16 The values 40in the column Induction 45.24 Percentage 2.22 in the table represent 0.8 the mean 153 ± standard 0.65 error.0.65 ± Note: The values in the column Induction Percentage in the table represent the mean standard error. In 2017, EC was produced from explants from 6 of the 24 families;± the highest induction percentage was 12.00% and the lowest was 2.00% The induction percentage for other families was In 2017, EC was produced from explants from 6 of the 24 families; the highest induction percentage zero, and the induction percentage differed significantly among family sources (Figure 5). was 12.00% and the lowest was 2.00% The induction percentage for other families was zero, and the induction percentage differed significantly among family sources (Figure5).

Figure 5. Explant EC induction results from different sources families of Pinus koraiensis. Each data pointFigure was 5. repeatedExplant EC for induction 10 replicates. results The from error different bars represent sources the families standard of Pinus error. Koraiensis The different. Each small data letterspoint inwas the repeated figure show for 10 a significantreplicates. The difference error bars at p

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3.2. Proliferation and Maturation of EC Forests 2020, 11, 912 9 of 15 3.2.1. Proliferation Experiment 1: Effect of PGRs on the Proliferation and Maturation of Korean Pine EC3.2. Proliferation and Maturation of EC The proliferation efficiency of three cell lines was significant (p ˂ 0.05) under different PGRs 3.2.1. Proliferation Experiment 1: Effect of PGRs on the Proliferation and Maturation of Korean concentrations (Table 3), and it was increased with the increase of PGRs concentration. The Pine EC combination of 9.04 μmol·L−1 2,4-D and 4.4 μmol·L−1 6-BA had the strongest proliferation ability (cell line 001#-100The proliferation was 617.00%), efficiency followed of three by cell4.52 linesμmol·L was−1 2,4-D significant + 2.2 (μpmol·L< 0.05)−1 6-BA, under and diff erentfinally PGRs 2.26 μconcentrationsmol·L−1 2,4-D (Table+ 0.443 μ),mol·L and it−1 was 6-BA. increased with the increase of PGRs concentration. The combination of 9.04 µmol L 1 2,4-D and 4.4 µmol L 1 6-BA had the strongest proliferation ability (cell line 001#-100 · − · − was 617.00%), followedTable 3. Effects by 4.52 ofµ PGRsmol L concentration1 2,4-D + 2.2 onµ ECmol proliferationL 1 6-BA, andof Pinus finally Koraiensis. 2.26 µ mol L 1 2,4-D + · − · − · − 0.44 µmol L 1 6-BA. · − PGRs Concentration (μmol·L−1) Proliferation Efficiency (%) Treatments 2,4-D 6-BA 001#-100 001#-1 001#-34 Table 3. Effects of PGRs concentration on EC proliferation of Pinus koraiensis. 1 9.04 4.4 617.00 ± 31.33 a 524.00 ± 29.77 a 605.00 ± 37.52 a 2 4.52 2.2 1 483.00 ± 38.78 b 414.00 ± 25.47 b 496.00 ± 31.20 ab PGRs Concentration (µmol L− ) Proliferation Efficiency (%) Treatments3 2.26 0.44 · 414.00 ± 29.97 b 388.00 ± 23.64 b 461.00 ± 39.03 b 2,4-D 6-BA 001#-100 001#-1 001#-34 Note:1 Each cell line 9.04with five replicates. 4.4 The valu617.00es in column31.33 aProliferation 524.00 29.77 Efficiency a 605.00 in the 37.52table a ± ± ± 2 4.52 2.2 483.00 38.78 b 414.00 25.47 b 496.00 31.20 ab represent the mean ± standard error. Different letters ±in the same column± indicate a significant± 3 2.26 0.44 414.00 29.97 b 388.00 23.64 b 461.00 39.03 b difference at p ˂ 0.05. ± ± ± Note: Each cell line with five replicates. The values in column Proliferation Efficiency in the table represent the mean standard error. Different letters in the same column indicate a significant difference at p < 0.05. The microstructure± of the paraffin section is shown in Figure 6. The late stage of SEs gradually developedThe microstructure after 10 days ofof themature para fficulturingn section (stage is shown I, SEs in with Figure elongated6. The late suspensors, stage of SEs Figure gradually 6a,d), stagedeveloped II developed after 10 at days about of mature30 days culturing(precotyledon (stage embryo, I, SEs with Figure elongated 6b,e), and suspensors, stage III developed Figure6a,d), at aboutstage II45 developed days (cotyledonary at about 30 daysembryo, (precotyledon Figure 6c,f embryo,). There Figure were6 b,e),significant and stage differences III developed in the at developmentabout 45 days and (cotyledonary maturation embryo, abilities Figure of prolif6c,f).erative There SEs were under significant three PGRs di fferences concentrations in the development (p ˂ 0.05) (Tableand maturation 4), and the abilities ability ofin proliferativedifferent cell SEs lines under was threealso different. PGRs concentrations Under the combination (p < 0.05) (Table of 9.044), μmol·L−1 2,4-D and 4.4 μmol·L−1 6-BA, the proliferation efficiency of the embryogenic cell line was1 the and the ability in different cell lines was also different. Under the combination of 9.04 µmol L− 2,4-D best, but the development1 and maturity ability of SEs was the worst (cell line 001#-034 was· 17.86·g−1 and 4.4 µmol L− 6-BA, the proliferation efficiency of the embryogenic cell line was the best, but the FW). However,· under the combination of 4.25 μmol·L−1 2,4-D and 2.2 μmol·L−1 6-BA, the1 proliferation development and maturity ability of SEs was the worst (cell line 001#-034 was 17.86 g− FW). However, efficiency of the embryogenic cell line 1was less than with the combination1 of 9.04 μ· mol·L−1 2,4-D and under the combination of 4.25 µmol L− 2,4-D and 2.2 µmol L− 6-BA, the proliferation efficiency of the 4.4 μmol·L−1 6-BA, but the development· and maturation ability· of SEs was1 the best (cell line 001#-1001 embryogenic cell line was less than with the combination of 9.04 µmol L− 2,4-D and 4.4 µmol L− 6-BA, −1 · · wasbut the135.71·g development FW). and maturation ability of SEs was the best (cell line 001#-100 was 135.71 g 1 FW). · −

Figure 6. Morphological and paraffin section observation of the mature process of Pinus koraiensis SEs. Figure(a) Late 6. SEs Morphological stage I (stage I),and bar paraffin= 0.1 cm; section (b) Late observation SEs stage IIof (stagethe mature II), bar process= 0.2 cm; of ( cPinus) Late Koraiensis SEs stage SEs.III (stage (a) Late III), SEs bar stage= 0.2 Ⅰ cm; (stage (d) Ⅰ Para), barffi =n 0.1 section cm; ( ofb) lateLate SEs SEs stage stage I, Ⅱ bar (stage= 200 Ⅱ),µ barm; (=e )0.2 Para cm;ffi (nc) section Late SEs of stagelate SEs Ⅲ (stage stage IIⅢ bar), bar= =100 0.2 µcm;m; ( (df) Paraffin Paraffin section section of of late late SEs SEs stage stage Ⅰ, IIIbar bar = 200= 100μm;µ (em;) Paraffin The letters section ‘eh’ ofdenote late SEs the stage embryo Ⅱ bar head = 100 (eh), μm; and (f the) Paraffin letter‘s’ section denotes of late the suspensorSEs stage Ⅲ ‘s’. bar = 100 μm; The letters ‘eh’ denote the embryo head (eh), and the letter ‘s’ denotes the suspensor ‘s’.

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Forests 2020Table, 11 ,4. 912 Effects of PGRs concentration on the Pinus Koraiensis SEs’ development and maturation. 10 of 15

PGRs Concentration (μmol·L−1) Number of Cotyledon SEs of 3 Cell Lines (No./g−1FW) Treatments Table 4. Effects2,4-D of PGRs concentration6-BA on the Pinus001#-100 koraiensis SEs’ development001#-001 and maturation.001#-034 1 9.04 4.4 95.83 ± 7.68 b 97.50 ± 11.46 a 17.86 ± 5.36 b 1 1 PGRs Concentration (µmol L− ) Number of Cotyledon SEs of 3 Cell Lines (No./g− FW) Treatments2 4.52 2.2 · 135.71 ± 13.48 a 118.75 ± 13.21 a 37.50 ± 6.46 a 3 2.262,4-D 0.44 6-BA 100.00 ± 001#-100 13.31 ab 91.67 001#-001 ± 7.68 a 27.08 001#-034 ± 3.84 ab 1 9.04 4.4 95.83 7.68 b 97.50 11.46 a 17.86 5.36 b ± ± ± Note: Each2 cell line with 4.52 six replicates. The 2.2 values in 135.71column 13.48Number a of118.75 Cotyledon13.21 SEs a of37.50 3 Cell 6.46Lines a ± ± ± in the table3 represent the 2.26 mean ± standard 0.44 error. Data 100.00 represent13.31 abthe number 91.67 of7.68 SEs a formed 27.08 per3.84 gram ab ± ± ± ofNote: fresh Each weight, cell line and with different six replicates. letters The in valuesthe same in column column Number indicate of Cotyledona significant SEs ofdifference 3 Cell Lines at inp ˂ the 0.05. table represent the mean standard error. Data represent the number of SEs formed per gram of fresh weight, and ± 3.2.2.di Proliferationfferent letters in Experiment the same column 2: The indicate Effect a significant of L-Glutamine difference at onp < the0.05. Proliferation and Maturation of Korean Pine EC 3.2.2. Proliferation Experiment 2: The Effect of L-Glutamine on the Proliferation and Maturation of KoreanDifferent Pine EC L-glutamine concentrations had no significant effect on the proliferation efficiency of EC (TableDifferent 5) and L-glutamine cell structure concentrations (Figure 7a,b), had nobu significantt had a significant effect on theeffect proliferation on the development efficiency of and EC ˂ −1 maturation(Table5) and ability cell structure of SEs (Figure(p 0.05)7a,b), (Table but had 6). a When significant EC proliferated e ffect on the at development 0.5 g L L-glutamine, and maturation the number of late stage embryos in stage I was less than 0.5 g L−1 L-glutamine1 (Figure 7c,d), as was the ability of SEs (p < 0.05) (Table6). When EC proliferated at 0.5 g L − L-glutamine, the number of late number of mature embryos of stage Ⅲ (cell line1 001#-100 was the highest, which was 118.75·g−1 FW). stage embryos in stage I was less than 0.5 g L− L-glutamine (Figure7c,d), as was the number of mature In addition, when the concentration of L-glutamine was increased to 1 or1 1.5 g L−1, the development embryos of stage III (cell line 001#-100 was the highest, which was 118.75 g− FW). In addition, when the and maturation of the SEs of the three cell lines were improved,1 but the ·difference was not significant. concentration of L-glutamine was increased to 1 or 1.5 g L− , the development and maturation of the −1 WhenSEs of EC the proliferated three cell lines unde werer the improved, condition but of the1.5 g di Lfference L-glutamine, was not the significant. number Whenof late ECstage proliferated embryos of stage I was more than 0.5 g1 L−1 L-glutamine (Figure 7c,d, 001#-001 cell line had the best ability of under the condition of 1.5 g L− L-glutamine, the number of late stage embryos of stage I was more than embryo 1 development and maturation). Furthermore, when under the condition of 1 g L−1 L- 0.5 g L− L-glutamine (Figure7c,d, 001#-001 cell line had the best ability of embryo development and glutamine, 001#-100 and 001#-034 cell lines had the best capacities1 for SEs maturation (cell line 001#- maturation). Furthermore, when under the condition of 1 g L− L-glutamine, 001#-100 and 001#-034 100 was 165.63·g−1 FW, 001#-034 was 46.88·g−1 FW). 1 cell lines had the best capacities for SEs maturation (cell line 001#-100 was 165.63 g− FW, 001#-034 was 1 · 46.88 g− FW). · Table 5. Effects of L-glutamine concentration on the EC proliferation of Pinus Koraiensis.

Table 5. Effects ofL-glutamine L-glutamine concentration onProliferation the EC proliferation Efficiency of Pinus (%) koraiensis. Treatments Concentration (g L−1) 001#-100 001#-100 001#-034 L-glutamine Proliferation Efficiency (%) Treatments1 0.5 467.50 ± 44.84 a 410.00 ± 30.62 a 492.50 ± 27.73 a Concentration (g L 1) 2 1 − 481.25001#-100 ± 48.41 a 396.25 001#-100 ± 20.14 a 487.50 001#-034± 30.10 a 13 0.5 1.5 455.00 467.50 ± 52.0844.84 a a 408.75 410.00 ± 43.2230.62 a a 455.00 492.50 ± 56.2027.73 a a ± ± ± 2 1 481.25 48.41 a 396.25 20.14 a 487.50 30.10 a ± ± ± Note: Each3 cell line with four 1.5 replicates. The 455.00 values in52.08 column a408.75 Proliferation43.22 Efficiency a 455.00 in 56.20the table a represent the mean ± standard error. Different letters± in the same column± indicate a ±significant Note: Each cell line with four replicates. The values in column Proliferation Efficiency in the table represent the differencemean standard at p ˂ error. 0.05. Different letters in the same column indicate a significant difference at p < 0.05. ±

Figure 7. Cell viability of EC of Pinus koraiensis under diﬀerent L-glutamine conditions. (a) EC at Figure 7.1 Cell viability of EC of Pinus koraiensis under different1 L-glutamine conditions. (a) EC at 0.5 0.5 g L− L-glutamine, bar = 100 µm; (b) EC at 1.5 g L− L-glutamine, bar = 100 µm; (c) Late SEs with −1 −1 gpoor L L-glutamine, viability, bar =bar0.2 = cm;100 (μdm;) Late (b) EC SEs at with 1.5 g good L L-glutamine, viability, bar bar= 0.2 = 100 cm. μ Them; ( lettersc) Late ‘eh’SEs denotewith poor the embryo head ‘eh’, and the letter ‘s’ denotes the suspensor ‘s’.

Forests 2020, 11, x FOR PEER REVIEW 11 of 15

viability, bar = 0.2 cm; (d) Late SEs with good viability, bar = 0.2 cm. The letters ‘eh’ denote the embryo head ‘eh’, and the letter ‘s’ denotes the suspensor ‘s’. Forests 2020, 11, 912 11 of 15 Table 6. Effects of L-glutamine concentration on the Pinus Koraiensis SEs development and maturation.

Table 6. Effects of L-glutamineL-glutamine concentration onNumber the Pinus of koraiensis SEs of 3SEs Cell development Lines (No./g and−1FW) maturation. Treatments Concentration (g L−1) 001#-100 001#-001 001#-034 1 1 L-glutamine0.5 118.75Number ± 10.83 ofb SEs 103.13 of 3 Cell± 7.86 Lines b 28.13 (No. /±g −9.38FW) a Treatments 1 2 Concentration 1 (g L− ) 165.63001#-100 ± 5.98 a 146.88 001#-001 ± 5.98 a 46.88 001#-034± 7.86 a 3 1.5 162.50 ± 5.10 a 162.50 ± 5.10 a 43.75 ± 6.25 a 1 0.5 118.75 10.83 b 103.13 7.86 b 28.13 9.38 a ± ± ± 2 1 165.63 5.98 a 146.88 5.98 a 46.88 7.86 a Note: Each cell line with four replicates. The values ±in column Number± of Cotyledon SEs± of 3 Cell 3 1.5 162.50 5.10 a 162.50 5.10 a 43.75 6.25 a Lines in the table represent the mean ± standard error.± Data represent the± number of SEs formed± per gramNote: of Each fresh cell weight, line with and four different replicates. letters The values in the in same column column Number indicate of Cotyledon a significant SEs of 3difference Cell Lines at in p the ˂ table represent the mean standard error. Data represent the number of SEs formed per gram of fresh weight, 0.05.and di fferent letters in the± same column indicate a significant difference at p < 0.05.

3.3. SEs SEs Germination Germination and and Plant Plant Regeneration Regeneration The development andand maturationmaturation abilityability of of 001#-100 001#-100 SEs SEs was was the the best best (Table (Table6). 6). However, However, some some of ofthe the SEs SEs germinated germinated normally normally (Figure (Figure8a, 8a, letter letter n), n) and, and some some of themof them abnormally abnormally (Figure (Figure8a, letter8a, letter a). a).As As a whole, a whole, the the germination germination ability ability of theof the 001#-100 001#-100 SEs SEs was was the the worst worst (36.00%) (36.00%) (Table (Table7). Among 7). Among the thethree three cell cell lines lines tested, tested, 001#-034 001#-034 had had the the strongest strongest germinating germinating ability ability (66.00%), (66.00%), followed followed by 001#-001by 001#- 001(58.00%) (58.00%) and ﬁnallyand finally 001#-100. 001#-100. After 16After weeks 16 of weeks SEs germination of SEs germination and culture, and they culture, were transplanted they were transplantedinto the substrate into the (nutrient substrate soil: (nutrient vermiculite: soil: Perlitevermiculite:= 3:1:1) Perlite (Figure = 3:1:1)8b). (Figure 8b).

Figure 8. SEs germination and plant regeneration of Pinus koraiensis.(a) Germination of SEs, bar = 2 cm; Figure(b) Regenerated 8. SEs germination plant, bar =and2 cm;plant The regeneration letter ‘n’ indicates of Pinus normal koraiensis germination. (a) Germination of SEs, and of SEs, the letterbar = ‘a’2 cm;indicates (b) Regenerated abnormal germinationplant, bar = 2 of cm; SEs. The letter ‘n’ indicates normal germination of SEs, and the letter ‘a’ indicates abnormal germination of SEs. Table 7. Germination of mature SEs of Pinus koraiensis.

Cell LinesTable Number7. Germination of SEs (number)of mature SEs Normal of Pinus SEs koraiensis. Percentage (%) C001#-100ell Lines Number of SEs 50 (number) Normal SE 36.00s Percentage5.10 (%) ± 001#-001 50 58.00 5.83 001#-100 50 36.00 ±± 5.10 001#-034 50 66.00 8.72 001#-001 50 58.00 ±± 5.83 Note: The values in column Normal SEs Percentage in the table represent the mean standard error. 001#-034 50 66.00 ± 8.72 ±

4. DiscussionNote: The values in column Normal SEs Percentage in the table represent the mean ± standard error.

4.4.1. Discussion Initiation of EC

4.1. InitiationThe induction of EC percentage of coniferous EC is usually very low, and depends heavily on the physiological stage of the explant, the location of the explant, the genotype of the families, the nutrient compositionThe induction of the percentage medium, the of typesconiferous and concentrationsEC is usually very of growth low, and regulators, depends andheavily the cultureon the physiologicalconditions [23 ].stage Our of previous the explant, research the shows location that, of as the the explant, age and the physiological genotype statusof the offamilies, the mother the nutrienttree increases, composition the callus of inductionthe medium, percentage the types decreases and concentrations significantly of [19 growth]. In most regulators, previous and studies, the cultureEC has conditions been induced [23]. from Our previous young tissues research of coniferousshows that, species as the age [26]. and Many physiological studies have status reported of the motherthat the tree EC inductionincreases, percentagesthe callus induction of Pinus percenta and Piceage aredecreases higher atsignificantly the cleavage [19]. polyembryonic In most previous and prophase of cotyledon embryo stages than at other stages. In the present study, the best stage for the EC induction of P. koraiensis was the E1 stage (proembryo stage). The EC induction percentage Forests 2020, 11, 912 12 of 15 was lower at the E3 and E4 stages than at the E1 stage, different from most other conifer species [23]. Our previous research shows that the highest induction percentage of EC was 1.67% [19]. In this study, the highest induction percentage was 33.33%, the lowest was 0.14%, and the induction percentage of EC was significantly increased. We found that there were significant differences in EC induction percentage among different families, like in our previous research [19]. The induction percentages of 24 families in 2017 were lower than those in 2015 (the highest induction percentage is 12%), and this may be related to the genotype of the families, or to the unsuitable development of immature seeds. Further verification is required in the later test process. Phytohormones are the key substances controlling the process of somatic embryogenesis in culture in vitro. The induction and proliferation of the embryogenic cultures of many conifers proceed on standard nutrient medium supplemented with exogenous PGRs [27]. Auxin and cytokinin are used at the induction stage of Pinus [6]. In previous studies, the most commonly used auxin was 2,4-D, and the most commonly used cytokinin was 6-BA [28]. Auxin is considered to be the most critical factor in the induction stage [26]. In this study, combinations of NAA and 6-BA, or 2,4-D and 6-BA, at different concentrations were used to induce EC from explants of P. koraiensis. Both sets of combinations induced EC of P. koraiensis. Overall, the induction percentage was slightly higher with combinations of NAA and 6-BA than with combinations of 2,4-D and 6-BA, which was different from most other pine species [29–31].

4.2. Multiplication and Maturation of EC EC is the foundation of regenerating plants on a large scale, and serves as an important material in genetic transformation. It is also an ideal system for studying the entirety of single-cell differentiation and the expression of totipotency [19]. The quality of EC not only affects the proliferation efficiency, but also affects the quantity and quality of SEs [32,33]. The quality of SEs is also a key factor in evaluating the success of somatic embryogenesis. In our study, the PGRs concentration significantly affected the EC proliferation and maturation, while L-glutamine concentration had no significant effect on the proliferation efficiency, but had a significant effect on the SEs’ maturation. Therefore, during somatic embryogenesis, not only the proliferation efficiency but also the development and maturity ability of SEs should be considered.

4.3. Germination of Mature SEs Among these phases, germination/conversion is regarded as the most important step in obtaining plantlets; this determines the success of this technique. Morphologically, mature conifer somatic embryos cannot germinate or convert into viable plantlets unless the embryos undergo partial desiccation treatment. This treatment has been used effectively to improve the germination/conversion of somatic embryos [34,35]. When SE was partially desiccated, the highest germination percentage was 66%. In the process of SEs germination, some SEs germinate abnormally [32,36,37], which limits the process of large-scale breeding. In this study, we found that the germinating ability of SEs of different genotypes was different. In the next step, we need to further optimize the key technology of EC proliferation of Korean pine, prolong the retention time of EC, enhance the ability of embryo maturation and germination, and lay the foundation for large-scale propagation.

5. Conclusions In summary, this work demonstrates that efficient SE induction and establishment in continental and Mediterranean provenances of Korean pine depends not only on the mother tree but also on the developmental stage of the megagametophyte. Culture conditions during the EC proliferation stage significantly affect the maturation of SEs. Therefore, during somatic embryogenesis, not only the proliferation efficiency but also the development and maturity ability of SEs should be considered. We established systems for EC induction, somatic embryogenesis, plant regeneration Forests 2020, 11, 912 13 of 15 and cryopreservation. These systems can be used to propagate the excellent germplasm resources of P. koraiensis, and for the establishment of multi-varietal forestry.

Author Contributions: L.Y. and H.S. conceived and designed the study. F.G. and C.P. collected plant materials and prepared SE samples for analysis. F.G. analyzed the results for experiments. L.Y., F.G. and H.W. wrote the paper. I.N.T. and A.M.N. revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: The work was supported by the National Key R&D Program of China (2017YFD0600600), and the Innovation Project of State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University, 2016C01). Acknowledgments: We thank two anonymous reviewers and the editor for comments that improved an earlier draft of this article. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

2,4-D 2,4-dichlorophenoxyacetic Acid 6-BA N6-benzyladenine ABA Abscisic acid CH Acid-hydrolyzed casein DCR Basal culture medium (Gupta and Durzan 1985) DMSO Dimethyl sulfoxide minimum mLV Litvay medium (Litvay et al. 1985, modiﬁed by Hargreaves et al., 2009) NAA 1-Naphthalacetic Acid SEs Somatic embryos EC Embryogenic lines PGRs Plant growth regulator

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