Embryo Development, Seed Germination, and the Kind of Dormancy of Ginkgo biloba L.

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1 Article Embryo Development, Seed Germination, the Kind Dormancy Ginkgo biloba L. Jing Feng 1,2, Yongbao Shen 2,3,4, *, Fenghou Shi 2,3,4 Chengzhong Li 5 1 College Lscape Architecture, Nanjing Forestry University, Nanjing , China; fengjing9520@yeah.net 2 Collaborative Innovation Center Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing , China; fhshi406@163.com 3 Southern Tree Seed Inspection Center, National Forestry Administration, Nanjing , China 4 College Forestry, Nanjing Forestry University, Nanjing , China 5 Department Horticulture, Jiangsu Agri-animal Husbry Vocational College, Taizhou , China; chengzhongli11@126.com * Correspondence: ybshen@njfu.edu.cn; Tel.: ; Fax: Received: 13 October 2018; Accepted: 9 November 2018; Published: 11 November 2018 Abstract: The embryos Ginkgo biloba L. are generally reported to undergo after-ripening be underdeveloped at the time dispersal, which suggests that the seeds have morphological dormancy (MD) or morphological physiological dormancy (MPD). The aim this work is to determine whether embryos a G. biloba population are well-developed at the time seed dispersal, whether the seeds are dormant or not. From 8 September, which was the 140th day after flowering (140 DAF), seeds were collected separately from trees (T) the ground (G) every 10 days until 7 December (230 DAF), resulting in a total 10 samples. The changes in vertical diameter, transverse diameter, fresh weight, water content, embryo length during seed were measured. Simultaneously, the effects different temperatures (15, 25, 30, 35 C) on seed germination, dormancy, germination characteristics G. biloba seeds were studied. Results showed that the embryos G. biloba seeds were well developed had no morphological dormancy. On 18 September (150 DAF), embryos were visible with a length 2.5 mm. On 7 December (230 DAF), at the time seed dispersal, their length was 17.1 mm. The germination percentage the isolated embryos seeds increased as the delay in seed collection increased, but there was no significant difference between T G (p > 0.05). On 7 December (230 DAF), the germination the isolated embryos reached 98%, indicating that the embryos were nondormant. Without pretreatment, seed germination was 82.57% within four weeks at 25 C. Furthermore, the germination test at different temperatures showed the highest germination percentage at 30 C (84.82%). Obviously, the G. biloba seeds were nondormant. The mean germination time (MGT) the seeds at C was significantly lower than that the seeds at C, the speed germination (SG) was significantly higher than that the seeds at C. Although there was no significant difference in the seed-germination percentage between C, a portion the seeds (9.5%) rotted at 35 C. Therefore, 30 C was the most favorable germination temperature for G. biloba seeds. This is the first study that reports G. biloba seeds with no dormancy. Keywords: after-ripening; morphological dormancy; embryo ; shedding; germination; temperature; gymnosperm Forests 2018, 9, 700; doi: /f

2 Forests 2018, 9, Introduction Both spore seed plants undergo growth reproduction stages throughout their lives. The whole process comprising the sequential, regular cycle these two stages is called the life history or life cycle. Seeds are the sexual reproduction organ or transmission unit in higher plants, a product the long-term evolution plants, a al stage an individual seed plant [1]. The life history seeds begins with flowering pollination, then proceeds through fertilization,, maturation, scattering, dormancy,, finally, germination. This series steps translate to a delicate, unique, inter-related life course [2]. Ginkgo pollen matures in late April begins to pollinate; however, fertilization does not occur immediately. The time between pollination fertilization is approximately 120 days. At the beginning September, the color the ginkgo s sarcotesta changes from green to yellow or orange white powder appears, signifying the maturity Ginkgo biloba seeds [3]. Usually, the harvest period starts in about late September or earlier, but the embryo is still underdeveloped at this time. After one month, the sarcotesta begins to shrink then a few seeds are shed, many researchers collect their seeds during this period. For example, Cao Cai [4] reported that the maturation harvest period Ginkgo biloba L. seeds was 20 September. Men s [5] research found that embryos grew slowly after the seeds were harvested in September. After removing the fleshy sarcotesta placing it in a warm environment, the embryos grow to their full size (10 13 cm) within 8 10 weeks begin to sprout [6,7]. Embryos in some seeds are differentiated at maturation dispersal, with a radicle, a hypocotyl, cotyledons, yet they are underdeveloped must grow before the radicle emerges. Most the space inside these relatively large seeds is occupied by the endosperm, with the embryos accounting for only 1% or less the seed volume. The underdeveloped state the embryos in these seeds means that they must grow to a critical size before germination. This growth causes delayed germination, which is called morphological dormancy [8]. It is generally believed that, when G. biloba seeds disperse, embryonic is incomplete, the seeds are small (but differentiated), that some time is required for the seeds to grow to a size sufficient for germination. Many seeds use dormancy to avoid adverse circumstances, germinate only when the living environment becomes suitable [9]. Dormancy classification is based on the embryonic state at the time seed dispersal, the physical properties the seeds, the physiological responses the seeds to environmental stimulation [10]. Nikolaeva [8] found that the type seed dormancy was determined by the seed s morphological physiological traits, based on this, a seed-dormancy classification system was proposed by Baskin Baskin [9], who divided seed-dormancy types into: physiological dormancy (PD), morphological dormancy (MD), morphophysiological dormancy (MPD), physical dormancy (PY), joint dormancy (PY + PD). MD is hypothesized to be the oldest type dormancy, occurring in ancient seeds (gymnosperms). The delay in seed germination may be due to a delay in fertilization, delayed embryo may be a response to specific light dark, moisture, temperature conditions, indicating that the environment plays a certain role in regulating germination time [10,11]. Even after many studies over a long time, the dormancy type ginkgo seeds is still not clearly defined. Li Chen [12] were the first authors to demonstrate that the underdeveloped state the embryos at the time dispersal was the cause germination delay. After the seed disperses, the embryo continues to develop until cold weather comes. West et al. [6] believed that the seeds Ginkgo biloba were characterized by MPD because the germination rate was only 10% after 12 weeks when they performed germination tests at room temperature (22 ± 2 C) on unstratified seeds with underdeveloped embryos, while the germination rate could be significantly increased by cold stratification or spraying exogenous GA 3 (90% 75%, respectively). Wang [13] believed that ginkgo seeds were unfertilized at the time dispersal that fertilization occurred when the sarcotesta rotted, the germ cells divided, the sperm was released. Cao et al. [4,14] reported that ginkgo seeds underwent after-ripening had embryonic dormancy. The embryo was still in the prophase stage after morphological maturity, germination could be achieved after a period

3 Forests 2018, 9, maturation. The research Qin et al. [15] suggested that low-temperature storage delayed embryo in G. biloba, mainly affecting the growth embryo length. However, none these authors reported any direct embryo observations or measurements to check whether they were fully developed when still on the tree. It is not known when G. biloba seeds naturally mature. In the existing literature, the seed dormancy Ginkgo biloba the the G. biloba embryo are still controversial. Moreover, to our knowledge, there are no detailed studies to confirm when seeds naturally disperse whether or not the embryos undergo physiological after-ripening. Therefore, the main purpose this study is to reveal (1) whether the seeds G. biloba are dormant by observing the whole process from seed to natural maturation shedding, (2) the relationship between embryo size germination, (3) the characteristics in vitro embryo seed germination. 2. Materials Methods Ginkgo seeds (Ginkgo biloba Jiafozhi ) were collected from three trees in Jiangsu Agri-Animal Husbry Vocational College, Taizhou City, Jiangsu Province ( N E). From 8 September 2017, just when the Ginkgo biloba seeds reached morphological maturity (the color the sarcotesta changed from green to orange), 50 seeds from each the three trees (T) were romly collected from the southeast northwest with a tree pruner, all fallen seeds were counted taken from the ground to avoid mixing with the next sample (number seeds (G) used for the germination test were the same as T). After putting them in an ice box, the seeds were immediately transported to the laboratory Nanjing Forestry University. Thus, the first sample was taken. A total 10 samples were taken every 10 days until the seeds were dispersed Morphological Characteristics Seeds Variation Embryo Length Three samples 30 seeds were romly chosen. The vertical horizontal diameter the seeds were measured by a vernier caliper, the seeds were weighed with an electronic balance (SECURA513-1CN, Sartorius, Gottingen, Germany). After removing the sarcotesta the seed coats with a pair pliers, the seeds were cut longitudinally with a razor blade, the lengths the embryos were measured by stereomicroscope (SZX16, OLYMPUS Co., Tokyo, Japan) Determination Moisture Content (MC) According to the International Seed Testing Association [16], an empty aluminum box with a lid (W1) was weighed, the seeds were sliced into small pieces by a single-edge razor blade. Then, the samples were placed into the aluminum box (together with the lid, W2) weighed. After drying at 130 ± 5 C for 4 h in an oven (101A-1E, Shanghai Laboratory Instrument Works Co., Ltd., Shanghai, China), the samples the aluminum box (together with the lid, W3) were weighed again. Three replications 5 seeds were used to determine the seed MC with the following equation: MC = W2 W3 100% (1) W2 W1 where W1 is the weight the container with the lid; W2 is the weight the container with the lid the sample before drying; W3 is the weight the container with the lid the sample after drying Seed Germination Characteristics In Vitro Embryo Culture Three samples 50 embryos were excised from the seeds incubated on absorbent cotton moistened with deionized water in plastic germination boxes; then, they were incubated in a growth chamber at a constant temperature 25 C. The embryos were observed in vitro every day

4 Forests 2018, 9, germination was recorded when the radicle had elongated to 2 mm over the 14 day incubation period. The absorbent cotton was kept moist during the whole process, the germination rate the in vitro embryos was determined after the end the culture period Germination Test Four samples 50 seeds were taken every 10 days from 7 November (200 days after flowering (DAF)) to 17 December (230 DAF). According to the International Seed Testing Association [16], the seeds were put on absorbent cotton moistened with deionized water in plastic germination boxes incubated in a growth chamber at a constant temperature 25 C with an 8 h photoperiod. Seed germination was recorded when the radicle had elongated to 2 mm (n). Seed germination was scored every 3 days over the 30 day incubation period. G. biloba seeds experience an inembryonate phenomenon, so to eliminate the effect embryoless seeds (NE) on the tests obtain an accurate germination percentage, both the germination percentage embryoless seeds were accounted for. The total germination (TGP) [17] was calculated by the following formula: TGP = n 100% (2) N NE where TGP is the seed-germination percentage; n is the number seeds that germinated normally; N is the sample size the tested seeds (50); NE is the number embryoless seeds. The mean germination time (MGT) [18] the speed germination (SG) [19] were calculated simultaneously. MGT = (D n) (3) n where MGT is the mean germination time; n is the number seeds which germinated on day D; D is the number days counted from the beginning germination. SG = ( n ) t where SG is the speed germination; n is the number seeds newly germinating at time t; t is the day from sowing Effect Temperature on Seed Germination On 7 December, four temperature gradients (15, 25, 30, 35 C) were set up for the germination seeds that matured naturally were shedding. Four samples 50 seeds were romly selected for each temperature. The other germination conditions were the same as in Section 2.3.2, the germination percentage was calculated at the end germination Statistical Analyses The data were analyzed with SPSS 23.0 (IBM, Armonk, NY, USA) Excel (Microst Office Home Student Edition 2016, Microst Corporation, Redmond, WA, USA) stware. Statistical analysis was performed using one-way or two-way analysis variance (ANOVA) followed by Duncan s Multiple Range Test (DMRT). Values are expressed as the mean ± SD (stard deviation) three replicates in each the independent experiments; p-values < 0.05 were considered statistically significant. 3. Results 3.1. Embryo Development Fertilization Ginkgo biloba occurred in middle late August, the zygote proceeded to rapidly undergo continuous mitosis, forming embryos after experiencing the free nuclear (4)

5 Forests 2018, 9, cellular stages [20 22]. The process embryo can be divided into three stages: proembryo, embryonic differentiation, late embryonic. At the beginning proembryo formation, when the original embryo was formed, there was no apparent Forests 2018, 9, x FOR PEER REVIEW 5 15 polar differentiation in morphology, but with the division the cells, polar differentiation began to occuras in shown the tissue. in Figure The differentiation 1A, from 18 September Ginkgo biloba (150 embryos DAF), when took about the embryo a month. was visible, with a length As shown 2.5 in mm, Figure the 1A, shape from 18the September proembryo (150 DAF), was stemlike, when the embryo the cotyledon was visible, primordia with a length were differentiated, 2.5 mm, the shape the top the proembryo the was formed stemlike, an arc the depression. cotyledonon primordia 28 September were (160 differentiated, DAF), the chalazal the top end cells the were embryo divided formed into antwo arc depression. cotyledons, the On micropylar 28 September end (160 cells DAF), formed theundeveloped chalazal end cells suspensor were tissue, divided into the two embryo cotyledons, length was the micropylar 4.5 mm. On end 8 October cells formed (170 DAF), undeveloped the cotyledon, suspensor germ, tissue, hypocotyl, the embryo radicle were length gradually was 4.5 mm. differentiating, On 8 October (170 the embryo DAF), the length cotyledon, was 8.6 germ, mm. Thereafter, hypocotyl, at the radicle late embryonic were gradually differentiating, stage, the embryo the embryo continued lengthto was grow. 8.6 mm. On 18 Thereafter, October (180 at the DAF), late embryonic the chalazal end endosperm stage, cells thedisintegrated embryo continued to form tothe grow. embryo On 18 cavity, October (180 the DAF), cotyledons the chalazal turned end green. endosperm On 17 November cells disintegrated (210 DAF), to the form length the embryo the embryo cavity, had increased the cotyledons to 16.5 mm turned (Figure green. 2); On after 17that, November the embryo (210 slowly DAF), the grew length to its size theat embryo the time had increased seed dispersal, to mm mm (Figure (7 December, 2); after that, 230 the DAF), embryo at which slowly point grew it basically to its size filled at the whole time embryo seed dispersal, cavity. Figure 17.1 mm 1B shows (7 December, the morphological 230 DAF), at changes which point the it sarcotesta, basically filled which the whole the embryo color was cavity. initially Figurepale 1B shows yellowish-green the morphological (140 DAF) changes but gradually the sarcotesta, changed to which orange the (160 color DAF), wasyellowish-brown initially pale yellowish-green (180 DAF), (140 grayish-brown DAF) but gradually covered changed with a thick to orange layer (160 white DAF), powder yellowish-brown (230 DAF). The (180sarcotesta DAF), began grayish-brown to shrink at covered 140 DAF with a thick was layer easily peeled whitef. powder The volume (230 DAF). the The G. sarcotesta biloba seeds began had to decreased shrink atto 140 its DAF minimum was at 230 easily DAF, peeled that f. is, The 3/5 volume its size the at 140 G. biloba DAF a seeds decrease had decreased 39%. tothe its minimum measurement at 230 DAF, the vertical that is, 3/5 transverse its size at 140 diameter DAF a G. decrease biloba seeds 39%. (Table The1) measurement showed that the thevertical diameter transverse the seeds diameter remained G. biloba fairly seeds constant (Table from 1) 150 showed to 230 that DAF, the while vertical the transverse diameter diameter the seeds remained the seeds decreased fairly constant significantly from 150(p to< ) DAF, while the water the transverse content diameter the seeds decreased the seedssignificantly decreased significantly (p < 0.05) during (p < 0.05) the time the period water content DAF. the seeds This decreased was caused significantly by the loss (p < 0.05) water during the time sarcotesta period during the DAF. late This stage was caused by the (Figure loss water 1B, Table in the1). sarcotesta According during to the the correlation late stageanalysis each (Figure index, as 1B, shown Table 1). in According Table 2, there to the were correlation significant analysis negative each correlations index, as shown between in Table embryo 2, there length were significant seed transverse negative correlations diameter, water between content, embryo length fresh weight seed during transverse the diameter, water G. content, biloba seeds fresh (transverse weight during diameter thecorrelation = 0.942, G. biloba p < 0.01; seeds water (transverse content diameter correlation correlation = 0.908, = p 0.942, < 0.01; p < fresh 0.01; weight water content correlation correlation = 0.865, = 0.908, p < 0.01), p < 0.01; indicating fresh weight that there correlation was a = close 0.865, relationship p < 0.01), indicating between that embryo there was a close relationship seed water between content. embryo The time onset seed embryo water content. The was time late in onset the G. embryo biloba seeds, the embryos was late were in thewell-developed G. biloba seeds, at the thetime embryos dispersal. were well-developed Therefore, there at the was time no morphological dispersal. Therefore, dormancy there (after-ripening) was no morphological in these embryos. dormancy (after-ripening) in these embryos. (A) (B) Figure 1. Morphological changes during embryo in Ginkgo biloba L. Jiafozhi Bar = 1 cm. (A) days after flowering (DAF): embryo Ginkgo Ginkgo biloba biloba L. Jiafozhi L. Jiafozhi seeds; seeds; (B) (B) DAF: DAF: the the morphological changes changes the the sarcotesta.

6 Forests 2018, 9, Figure 2. Changes in embryo length during the seed Ginkgo biloba Jiafozhi. Data are the mean (±SD) three replicates, bars labeled with Figure different 2. lowercase Changes in letters embryo are significant length during at p the < seed Ginkgo biloba Jiafozhi. Data are the mean (±SD) three replicates, bars labeled with different lowercase letters are significant at p < Table 1. Multiple comparisons the external morphological changes different stages during embryo Ginkgo biloba Jiafozhi. Table 1. Multiple comparisons the external morphological changes different stages during embryo Ginkgo biloba Jiafozhi. Vertical Diameter Transverse Embryo Length DAF Date Fresh Weight (g) Water Content (%) Features (mm) Diameter (mm) (mm) Fresh Weight Water Content Vertical Diameter Transverse Diameter Embryo Length DAF Date Features September (g) ± 1.00 a (%) ± 0.33 ab (mm) ± 0.17 a (mm) ± 0.30 a (mm) 2.5 ± 0.40 h Orange-yellow, slightly wrinkled September September ± 1.00 ± 0.33 a a ± 0.33 ab ± 0.55 a ± 0.17 a ± 0.24 a ± 0.30 ± 0.27 b 4.5 a 2.5 ± 0.40 ± 0.44 g idem h Orange-yellow, slightly wrinkled October ± 0.32 ab ± 0.96 b ± 0.92 ab ± 0.29 c 8.6 ± 0.40 f idem September 18 October ± 0.33 ± 0.14 a bcd ± a ± 0.72 c ± a ± 0.22 ab ± 0.27 ± b 0.30 d ± 0.44 ± 0.11 g e Yellowish-brown, idem wrinkled October ± 0.32 ab ± 0.96 b ± 0.92 ab ± 0.29 c 8.6 ± 0.40 f Brown, covered idem with white October ± 0.85 bc ± 0.90 c ± 0.27 bc ± 0.16 de 12.2 ± 0.64 d October ± 0.14 bcd ± 0.72 c ± 0.22 ab ± 0.30 d 9.5 ± 0.11 e Yellowish-brown, powder, wrinkled, stem wrinkled shrank November ± 0.30 cd ± 0.76 d ± 0.73 a ± 0.41 de 13.7 ± 0.50 c Brown, covered idemwith white October 17 November ± ± bc 0.40 d ± c ± 1.53 d30.20 ± bc ± 0.35 ab ± ± de 0.10 e ± 0.64 ± 0.61 d ab idem powder, wrinkled, stem shrank November ± 0.34 cd ± 1.03 d ± 0.20 c ± 0.30 f 16.0 ± 0.70 b idem November ± 0.30 cd ± 0.76 d ± 0.73 a ± 0.41 de 13.7 ± 0.50 c Grayish-brown, idem covered with November 7 December ± 0.40 ± 1.05 d cd ± d ± 1.03 d30.86 ± ab ± 0.35 bc ± 0.10 ± e 0.21 f ± 0.61 ± 0.66 ab a white powder, idem wrinkled, November ± 0.34 cd ± 1.03 d ± 0.20 c ± 0.30 f 16.0 ± 0.70 b fruit idem stalk shrank Note: the same lowercase letter does not differ significantly at the 0.05 probability level. Grayish-brown, covered with December ± 1.05 cd ± 1.03 d ± 0.35 bc ± 0.21 f 17.1 ± 0.66 a white powder, wrinkled, fruit stalk shrank Note: the same lowercase letter does not differ significantly at the 0.05 probability level.

7 Forests Forests 2018, 2018, 9, 700 9, x FOR PEER REVIEW Table 2. Correlation analysis between embryo length, vertical diameter, transverse diameter, water Table 2. Correlation analysis between embryo length, vertical diameter, transverse diameter, water content, fresh weight during seed Ginkgo biloba Jiafozhi. content, fresh weight during seed Ginkgo biloba Jiafozhi. Embryo Transverse Vertical Water Indicators Embryo Transverse Vertical Water Fresh Indicators Length Length Diameter Diameter Diameter Diameter Content Content Weight Weight Embryo Embryo Length Length ** ** 0.464* * 0.908*** ** Sig. (two-tailed) Sig. (two-tailed) Transverse Transverse Diameter Diameter ** ** ** ** ** ** Sig. (two-tailed) Sig. (two-tailed) Vertical Diameter * Vertical Diameter * Sig. (two-tailed) Water Sig. Content (two-tailed) ** Sig. (two-tailed) Water Content ** FreshSig. Weight (two-tailed) Fresh Weight 1 * Significant (Sig.) at 0.05 p level, ** significant at 0.01 p level; others are nonsignificant. * Significant (Sig.) at 0.05 p level, ** significant at 0.01 p level; others are nonsignificant Seed Seed Dispersal Dispersal As As shown shown in in Figure Figure 3, 3, as as time time elapsed, the the number shed shed G. G. biloba seeds increased. Between 8 8 September (140 DAF) October (190 (190 DAF), the the total total number dropped seeds seeds was was only only 388; 388; however, however, the the number number increased increased dramatically during during the the period period DAF: DAF: during during these these days, days, it reached it reached its its highest highest value value Analysis variance showed that the thenumber shed shedseeds seeds at at DAF DAFwas wassignificantly higher than that at 210 DAF (p (p < 0.05). Maximum shedding for for G. G. biloba biloba was was concentrated in in the the period period DAF, DAF, accounting for for 82.7% 82.7% the the total total number number shed shed seeds. seeds. As As indicated indicated in in Table Table 1, 1, embryo embryo length length was was mm, mm, only only 20% 20% the the seeds seeds were were embryoless. The The remaining remaining seed seed embryos embryos were were well-developed, which which indicates indicates that that the the embryos embryos G. G. biloba biloba were were well well developed developed at at the the time time seed seed dispersal. dispersal. Figure 3. Number seeds shed during the seed Ginkgo biloba Jiafozhi. Data are the mean Figure (±SD) 3. Number three replicates, seeds shed during bars labeled the seed with the different Ginkgo lowercase biloba letters Jiafozhi. are significant Data are the at mean p < (±SD) three replicates, bars labeled with the different lowercase letters are significant at p < In Vitro Embryo Culture 3.3. In Vitro Embryo Culture Figure 4 shows the germination percentages excised embryos that were collected at different stages. Figure On 8 October 4 shows (170 the germination DAF), the germination percentages percentages excised embryos after a 14 that day were incubation collected at excised different embryos stages. On G. 8 biloba October seeds (170 collected DAF), from the germination the T from percentages the G wereafter onlya 5.9% 14 day incubation 13.3%, respectively. excised However, embryos on 28G. October biloba (190 seeds DAF), collected the germination from the percentage T from Tthe G Gwere reached only 81.2% 5.9% 83.55%, 13.3%, respectively. However, on 28 October (190 DAF), the germination percentage T G reached

8 Forests 2018, 9, x FOR PEER REVIEW 8 15 Forests 2018, 9, % 83.55%, respectively. The variance analysis showed that the germination percentage excised respectively. embryos Theat variance 190 DAF analysis was significantly showed that different the germination from 170 DAF percentage (p < 0.05). With excised longer embryos delays at in 190seed-collection DAF was significantly time, the different germination frompercentage 170 DAF (p < 0.05). excised With embryos longer continued delays seed-collection to increase, time, at 220 the germination DAF, the germination percentagepercentages excised embryos T continued G had increased to increase, to the highest at 220 DAF, values the germination 97.8% 98.6%, percentages respectively. T Moreover, G had increased the germination to the highest process values excised 97.8% embryos 98.6%, from respectively. T G was Moreover, similar, the germination there was no process significant excised difference embryos in germination from T percentage G was similar, between T thereg was at any no significant the time points difference (p > in 0.05). germination percentage between T G at any the time points (p > 0.05). Figure 4. Germination isolated embryos from trees (T) the ground (G) Ginkgo biloba Jiafozhi. Figure Data are 4. Germination the mean (±SD) isolated three embryos replicates, from trees bars (T) labeled the ground with different (G) Ginkgo lowercase biloba letters Jiafozhi. are Data significant are the at mean p < (±SD) three replicates, bars labeled with different lowercase letters are significant at p < Seed Germination 3.4. Seed TheGermination effects different seed-collection times on the germination the seeds from T the G are shown The ineffects Table 3. On different 7 November seed-collection (200 DAF), times theon germination the germination percentages the seeds T from G were T 59.33% the G are shown 57.03%, in respectively, Table 3. On 7 November on 27 November (200 DAF), (220 the DAF), germination they were percentages 80.68% T 71.96%, G were respectively % In addition, 57.03%, respectively, the germination on percentage 27 November T(220 wasdaf), significantly they were higher 80.68% at 220 DAF 71.96%, than respectively. that at 200 In addition, 210 DAF the (p germination < 0.05), while percentage there was no T was significant significantly difference higher inat germination 220 DAF than between that at DAF. (p At< ), DAF, while the germination there was no rate significant G wasdifference significantly in higher germination compared between to the220 rates at , DAF. 210, At DAF, the, germination as shown inrate Table G 1 was Figure significantly 3, the seeds higher matured compared to dispersed the rates at at200, this 210, time. This 220 indicates DAF,, thatas seed-collection shown in Table time 1 hadfigure a significant 3, the seeds effect matured on the germination dispersed G. at biloba this time. seeds This (Table indicates 4), while that the seed-collection source the time seeds had (T or a significant G) had no effect significant on the effect germination the germination, G. biloba seeds indicating (Table that 4), the while germination the source percentage the seeds (T G. or biloba G) had seeds no was significant not related effect toon the the seed-collection germination, indicating method, but that increased the germination with the delay percentage in seed-collection G. biloba time. seeds was not related to the seed-collection method, but increased with the delay in seed-collection time. Table 3. Effects seed-collection time on the germination seeds from T G. Table 3. Effects seed-collection time on the Germination germination (%) seeds from T G. Time (Day) T Germination (%) G Time (day) 200 DAF T ± 8.14b ± 10.90b G 200 DAF 210 DAF ± 8.14b ± 5.00b ± 10.25b ± 10.90b 210 DAF 220 DAF ± 5.00b ± 9.03a ± 5.57b ± 10.25b 230 DAF ± 3.95a ± 5.32a 220 DAF ± 9.03a ± 5.57b The same lowercase letter does not differ significantly at the 0.05 probability level. T: Seeds from trees; G: Seeds from the ground. 230 DAF ± 3.95a ± 5.32a The same lowercase letter does not differ significantly at the 0.05 probability level. T: Seeds from trees; G: Seeds from the ground.

9 Forests 2018, 9, x FOR PEER REVIEW 9 15 Forests 2018, 9, Table 4. Effects different seed collection time methods on the germination Ginkgo biloba Jiafozhi. Table 4. Effects different seed collection time methods on the germination Ginkgo biloba Jiafozhi. Source Sum Squares df Mean Square F p Corrected Source Model Sum Squares a 7 df Mean Square F p Corrected Intercept Model a Methods Intercept 113, , Methods Time * Time * Methods Methods Time Time Error Total , Corrected Total * Significant at at p level; p level; others others are are nonsignificant. nonsignificant. df: Degree df: Degree freedom; freedom; F: F value; F: F value; a : R-Squared : R-Squared = (Adjusted R-Squared = 0.710). = (Adjusted R-Squared = 0.710). At 25 C, the water gradually saturated the endosperm G. biloba seeds the tissue reached an imbibition state. At 10 days after sowing, the seed suture line was cracked, a reddish-brown membranous endopleura could be seen (Figure 5A). The radicle started to sprout broke through the micropyle the oval protective film the endopleura. Then, embryo tissue began to emerge from the fissure through the the gap gap the the endopleura (Figure 5B). 5B). Figure 5. Process germination seedling Ginkgo Ginkgo biloba biloba Jiafozhi. Bar Bar = 1 cm. = 1 cm. (A) (A) Seed Seed dehiscent, dehiscent, showing showing reddish-brown membranous endopleura; (B) seed (B) seed after after germination; (C) (C) emerged emerged primary primary root; root; (D) (D) leaf leaf primordia-forming buds; buds; (E) bud (E) bud elongation; elongation; (F) erect (F) erect branch; branch; (G) (G) primary primary leaves; leaves; (H) (H) enlarged image image primary leaves from (G); (I) (I) true leaves; (J) enlarged image the true leaves from (I); (K) ginkgo seedling; (L) enlarged image fan-shaped leaves from (K); (M): enlarged image lateral roots from (K).

10 Forests 2018, 9, After germination, the division differentiation the embryo cells began to accelerate, the speed growth significantly increased. The primary root emerged after the micropylar end the seed was ruptured (Figure 5C). While the cotyledon grew thicker, the lower part the cotyledon became obviously elongated, the radicle hypocotyl continued to be pushed out the seed coat. The base the cotyledon was swollen extended in a sheath shape. With the formation the cotyledon sheath, the embryonic cells between the cotyledons divided formed a small dome shape. When the radicle germ developed to a certain extent, polar differentiation was obvious: the germ grew upward, the radicle grew down. The root primordium the radicle continuously differentiated so that it was obviously enlarged, the apex extended into the soil grew downward, forming the main root. The upper part the cotyledons almost stopped elongating, it was still contained in the endosperm continued to supply nutrients, while the lower part the cotyledons continued to grow elongate until it protruded from the seed coat appeared bent arched (Figure 5C). Then, the germ at the base began to differentiate to form the first vegetative leaf primordium. During the immediate period after, the main root continued to grow thicker, stretched into a cylindrical shape. The root tip slowly extended down into the soil, the root system was initially established. When the young root grew to cm in diameter cm in length, the first vegetative leaf primordium began to develop into a bud (Figure 5D,E). Then, the bud sprouted from the gap at the base the cotyledons stretched upward. The cotyledon was still wrapped in endosperm at the time germination, its lower surface was in close proximity to the endosperm; this allows the cotyledon to absorb nutrients from the endosperm, which supplies nutrition to various parts the embryo for growth. The epicotyl young stem were pale-green stout. The diameter was about 0.5 mm tapered so that it was finer toward the top. The young stem continued to grow upward, forming erect branches (Figure 5F). The first leaves were undeveloped primary leaves, which are always scale leaves, with 2 4 alternate leaves that were wide linear (Figure 5G,H), 3 5 cm long, 2 mm wide, flat or retuse at the apex. Leaves at the third or fifth position gradually changed to true leaves with the petiole elongated, fan-shaped leaves also slowly formed (Figure 5I,J). When the true leaves unfolded, they were 1 cm long 0.5 cm wide with a deep fissure at the apex. The edge the leaf was irregularly wavy (Figure 5K,L) the base the leaf was wedge-shaped; the veins were dichotomies, the surface the leaves was green, the abaxial surface was light-green covered with powder. The length the petiole was 0.4 cm. As the leaves matured, the texture changed from st to hard, the color changed from light-green to dark-green. The hypocotyl main root the seedlings were not hypertrophic. The hypocotyl was very short, white, smooth, with a length mm. The diameter the collar was about 3.5 mm, the taproot was thick strong, the lateral roots were short (Figure 5M) yellowish-brown, the tip the radicle was white Effect Temperature on Germination As shown in Figure 6, the seeds G. biloba grown at 15 C began to germinate on the 21st day, the germination process was slow. On the 30th day, seed germination was only 16.59%. The germination percentage generally increased with increasing temperatures: it was 82.57% at 25 C, the highest germination percentage was 84.82% at a temperature 30 C. However, it decreased to 73.73% at 35 C due to a large number the seeds rotting (9.5%) during the incubation. From Table 5, compared to 15 C, the other three temperatures had a significant effect on the germination percentage G. biloba seeds (p < 0.05), but there were no statistically significant differences between the three temperatures. The MGT G. biloba seeds at 30 C 35 C was significantly lower than that at 15 C 25 C, the SG at C was significantly higher than that at C, which indicates that higher temperatures were favorable for the germination G. biloba seeds. Furthermore, there was no significant difference in germination percentage, SG, MGT between C, but the rot rate G. biloba seeds at 35 C was significantly higher than that at 30 C. Therefore, 30 C was the most suitable germination temperature for G. biloba seeds.

11 Forests 2018, 9, Forests 2018, 9, x FOR PEER REVIEW Figure 6. Germination Ginkgo biloba Jiafozhi seeds at at different temperatures. Data Data are are the the means means three three replicates ± SD. ± SD. Table 5. Effect temperature on germination Ginkgo biloba Jiafozhi seeds. Table 5. Effect temperature on germination Ginkgo biloba Jiafozhi seeds. Temperature ( Temperature Mean Germination Speed C) Germination (%) Speed Rot Rate (%) Germination Time Mean (MGT) Germination (day) Germination (SG) (%) Germination Rot Rate (%) 15 ( C ) ± (%) b Time ± (MGT) 0.53 a (day) 0.27 ± 0.21 c 0.00 ± 0.00 c ± 7.67 a ± 0.85 b 2.18 (SG) ± 0.22 (%) b 3.00 ± 2.58 bc ± ± a b ± 1.28 ± 0.53 c a ± ± a c ± ± c b ± 9.81 a ± 0.42 c 2.98 ± 0.43 a ± 7.39 a ± 7.67 a ± 0.85 b 2.18 ± 0.22 b 3.00 ± 2.58 bc The same lowercase letter does not significantly differ at the 0.05 probability level ± 1.69 a ± 1.28 c 3.10 ± 0.47 a 9.50 ± 4.12 b ± 9.81 a ± 0.42 c 2.98 ± 0.43 a ± 7.39 a The same lowercase letter does not significantly differ at the 0.05 probability level. 4. Discussion 4.1. Discussion Morphological Changes in Seed Development G. biloba 4.1. Morphological Studies on the Changes embryo in Seed Development G. G. biloba biloba have resulted in ambiguity. Some authors have reported observations the early embryo G. biloba. For example, Ball [23] confirmed Studies maturation on the embryo tissue was not complete G. biloba in have the roots resulted these in ambiguity. in vitro seedlings. Some authors Wang have Chen reported [24] observations suggested coconut the early milk, embryo both filtered autoclaved, G. biloba. which For example, was tested Ball for [23] its confirmed effect on the maturation growth tissue structure was not the complete young embryos. in the roots However, these due in vitro to the seedlings. long time Wang from pollination Chen [24] to fertilization suggested coconut ( milk, days), both in most filtered studies, autoclaved, seed collection which was was carried tested out for in the its middle effect on the September, growth since structure the volume the young G. biloba embryos. seeds was However, no longer due increasing to the long time the from sarcotesta pollination was orange-yellow, to fertilization st, ( days), slightly in shrunken, most studies, thus seed having collection a mature was appearance. carried out in However, the middle no research September, has reported since the the volume whole process G. biloba seeds G. biloba was no embryo longer increasing while the sarcotesta the seeds was were orange-yellow, on trees. In this st, study, most slightly shrunken, the embryos thus were having not a yet mature completely appearance. differentiated However, in no mid-september. research has reported According the whole to the anatomical process G. structure biloba embryo the seed, the embryos while differentiated the seeds were in early on trees. October, In this including study, most cotyledons, the germs, embryos hypocotyls, were not yet completely radicles. Thereafter, differentiated the embryos in mid-september. continued to According grow rapidly to the anatomical were fully developed structure in the early seed, December the embryos (reaching differentiated two-thirds in the early seed October, length). Seeds including were cotyledons, also naturally germs, shed, indicating hypocotyls, that there radicles. was no Thereafter, morphological the dormancy embryos in continued the embryo to grow G. biloba, rapidly the embryos were fully had fully developed developed in early on December the tree. The (reaching phenomenon two-thirds delayed the seed length). Seeds in the were embryos also naturally G. biloba shed, has caused indicating confusion that there in researchers. was no morphological The reason for dormancy delayed embryo in the embryo G. biloba, may be to prevent the embryos seeds from had fully germinating developed prematurely on the tree. in The autumn phenomenon so they can delayed survive the cold winter in the [25]. embryos G. biloba has caused confusion in researchers. The reason for delayed embryo may be to prevent seeds from germinating prematurely in autumn so they can survive the cold winter [25].

12 Forests 2018, 9, Seed Maturation Dispersal The seeds most plants shed rapidly at maturity, while other seeds take months to years to fall f, such as the seeds Pinus banksiana Lamb. Pinus albicaulis Engelm. [26]. Gymnosperms may control the time seed germination by regulating the time seed maturation shedding, or by delaying germination until environmental conditions become favorable [10]. G. biloba displays the seed-dropping phenomenon throughout its whole growth. Early seed dropping generally lasts from early May to early July, accounting for 10% the total, but it can reach 40% 50% in more extreme circumstances. The reasons for early seed dropping are related to the nutritional status the tree, poor pollination, hormones, climate. It was found that from 8 September (140 DAF) to 28 October (190 DAF), the number falling G. biloba seeds accounted for only 8% the total, while from 27 November (220 DAF) to 17 December (230 DAF), it was 82.7%. The results indicate that seeds matured shed during this period. To our surprise, our results showed the opposite the popular opinion that the embryos are small need after-ripening when seeds disperse. Thus, the collection G. biloba seeds for the purpose establishing seedlings should be delayed until the time seed dispersal Seed Germination Isolated Embryo Previous studies have examined the isolated embryos G. biloba. For example, Wang et al. [27] reported that the embryos G. biloba collected in Beijing on 24 September were mm long. The cotyledon had not differentiated, royal jelly could promote organ differentiation the young embryos G. biloba. Using an in vitro embryo culture G. biloba with 0.2% glucose solution or distilled water, Li [28] found that the former was beneficial to the growth young embryos, but, in the latter, the cotyledon hypocotyl were obviously grown only in large embryos. The radicle was slightly prolonged, yet no further growth occurred. When analyzing the studies these authors, we found that most the seeds used in their experiments were collected in September were in the early stage embryo, most the embryos were undifferentiated. In this study, the germination results embryos isolated from G. biloba seeds collected at different stages showed that the germination isolated embryos increased significantly during the 10 days between 18 October (180 DAF) 28 October (190 DAF). This was 30% higher than the sample collected in the previous period, which is probably because most the embryos differentiated during this period. Usually, in warm temperate regions, fertilization G. biloba occurs in August, embryos begin to develop in early October [29]. Wang et al. [30] observed the G. biloba in Beijing for three years found that fertilization occurred during August, by 26 October, the embryo had differentiated, including the cotyledon, germ, hypocotyl, radicle. The germination percentage the isolated embryos remained at a relatively low value, indicating that the embryos had not differentiated, most the embryos could not germinate. However, a few embryos still could germinate, which may be due to the influences external environmental conditions nutrient competition between seeds. There are great differences in fertilization, even in the same plant, because the phase from unformed sperm to the proembryo could exist simultaneously in the same plant [31]. As time delays in seed collection increased, the germination percentage isolated embryos increased. At 190 DAF, the germination percentage reached 80%, indicating that the embryos were nondormant. Willis et al. [10] reported that, when seeds with MD were mature shedding, the embryos had completely differentiated into the cotyledon, hypocotyl, radicle, but they were still small needed to grow to a critical size before germination. In related books articles on the germination G. biloba seeds, it is common to find ginkgo seeds described as dormant. Most researchers have suggested that the embryos in G. biloba seeds are incompletely developed have morphological or morphophysiological dormancy. However, in this study, we found that the embryos were well-developed (reaching two-thirds the seed length) at the time seed dispersal. Without pretreatment, seed germination reached >80% within four weeks at C, the

13 Forests 2018, 9, germination rate decoated seeds reached 80% within two weeks [32]. Therefore, the G. biloba seeds were nondormant Effect Temperature on Seed Germination A suitable temperature can promote the germination seeds the growth seedlings. The optimum germination temperature different seeds may vary, even among seeds the same species, temperatures have different effects depending on seed origin [33]. Temperature affects seed germination by affecting enzyme activity, respiration rate, water-absorption rate [34]. When the temperature is too low, the activation or catalysis ability the enzyme is inhibited, whereas a high temperature leads to the inactivation the enzyme, the destruction the cell-membrane system organelles, the inhibition the physiological metabolism the seeds. Therefore, an unfavorable temperature is not conducive to seed germination [35]. For example, the time at which the germination percentage Clinopodium salioticum (Lamiaceae) seeds reached 50% decreased with the increase in temperature from 10 C (18 days) to 20 C (6 days) [36]. Higher temperatures (27 35 C) were suitable for the seed germination Cycas revolute [37,38]. Yu et al. [39] reported that the seed germination G. biloba at 25 C was higher than that at 15 C, the respiration rate at 15 C was significantly lower than that at 25 C. In this experiment, when the temperature was 35 C, although the initial germination time G. biloba seeds was earlier than that at 30 C, the germination percentage was lower than that at 30 C. These results indicate that the higher temperature caused significant damage to the seeds, which succumbed to mildew rotted during germination. On the other h, at 15 C, the germination process was very slow. Although the germination percentage at 25 C was also high, the MGT SG had a better response at 30 C. Therefore, 30 C was the optimum temperature for the germination G. biloba seeds. This is inconsistent with the Xia et al. [40] study, where 25 C was the reported optimum temperature for germination. 5. Conclusions In conclusion, this study confirmed that: (i) The embryos G. biloba seeds were well-developed at the time seed dispersal the embryos were nondormant. Without any stratification treatment, seeds G. biloba achieved a high germination 82.57% at 25 C. It was clear that seeds G. biloba were nondormant. (ii) High temperature (30 C) was beneficial to the germination G. biloba seeds. In conclusion, different from the existing literature reporting that ginkgo seeds were dormant, we report the first, to our knowledge, embryos G. biloba seeds that were well-developed at the time seed dispersal embryos that were nondormant. This was mainly due to the seeds collected by researchers not being naturally matured. Moreover, without any stratification treatment, seeds achieved a high germination percentage; therefore, the seeds G. biloba were nondormant. A high temperature (30 C) was beneficial to the germination G. biloba seeds. Nevertheless, it should be noted that we did not investigate the relationship between the endosperm embryo during seed, the metabolism the endosperm, or the changes in endogenous hormone content, which are especially relevant during embryo ; this relationship still needs to be fully elucidated. Further experiments will be conducted to fully clarify these points. Author Contributions: J.F., Y.S., F.S. participated in the discussion experimental designs; C.L. prepared the experiment materials; J.F. undertook laboratory analyses drafted the manuscript. All authors read approved the final manuscript. Funding: This research was funded by the Priority Academic Program Development Jiangsu Higher Education Institutions (PAPD). Conflicts Interest: The authors declare no conflict interest.

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