Sen Cao ·Ruiyang Hu ·Xialei Wu ·Yuhan Sun ·Bo Wu ·Hongjing Duan ·Huazhong Lin·Mingjing Wu·Luming Fang·Xiaolong Yu·Wei Wu·Yun Li

Abstract Chinese fir (Cunninghamia lanceolata (Lamb.)Hook),a fast-growing and economically important timber tree species in China,is widely used in construction,furniture,and paper manufacture but has a long breeding cycle.Chemical mutagens,such as ethyl methane sulfonate (EMS)and sodium azide (SA),are widely used in crops such as rice,wheat,cotton,soybean and sugarcane but their utility for tree breeding is unknown.In this study we examined the effects of EMS and SA on Chinese fir seed germination and growth.Chinese fir seeds were treated with the two chemical mutagens;were planted in Jiangle County,Fujian Province,China;and their heights were measured from 2011 to 2017.The concentrations and durations of treatment with the two chemical mutagens were significantly associated with the Chinese fir seedling and mortality rates,as well as with the heights of trees from the seedling stage to 3 years old.We also generated 127 mutants with abnormal branches and reproductive growth.We report here the effects of two chemical mutagens on Chinese fir breeding;our data will contribute to knowledge of the utility of EMS and SA in forestry.

Keywords Chemical mutagens·Chinese fir·Seedling growth·Seedling rate·Mutants

Introduction

Forest ecosystems provide about 50% of terrestrial biodiversity (Petit and Hampe 2006;Neale and Antoine 2011) and the quality of wood from trees affects the economic value of forests.Genetic variability is important for tree species,especially for varieties with high economic value for conservation and breeding.Rich genetic variability can enhance adaptation to the environment and accelerate the breeding of trees.Mutation is key to the evolution of species diversity (Zhu et al.2001;Ahloowalia et al.2004) and can be induced by treatment with chemical,physical,or biological mutagens (Lagoda et al.2012).The most popular method of mutagenesis uses chemical mutagens,such as ethyl methane sulfonate (EMS),hydrogen fluoride,sodium azide (SA) and methylnitrosourea,which typically have higher mutation rates than physical mutagens (Kharkwal 1998).Of this,EMS and SA are widely used because they are easy to use and do not require special equipment.Chemical mutagens induce specific base substitutions at high frequency and have been used in breeding strategies (Caldwell et al.2004;Stephenson et al.2010;Okabe et al.2011)to increase morphological and genetic variability (Bhosle and Kothekar 2010;Goyal et al.2010;Prabha et al.2010a,2010b).EMS induces point mutations and chromosome damage at a rate far higher than spontaneous mutagenesis (Greene et al.2 003).SA is metabolized into mutagenic compounds by plant cells and readily penetrates the cell wall (Owais et al.1983);it inhibits DNA replication by replacing a base,thus inducing genetic mutations (More et al.2011;Singh and Singh 2004;Wani et al.2011).Chemical mutagens are widely used in crops,such as rice (Lee et al.2017),wheat(Mishra et al.2016),cotton(Witt et al.2018),soybean(Espina et al.2018) and sugarcane(Khalil et al.2018),but rarely in tree breeding because of their long growth cycle and large genome size.

Chinese fir (Cunninghamia lanceolata(Lamb.) Hook)is a major fast-growing and economically important timber tree species in southern China and has high yield,good wood quality,and multiple uses (e.g.,in construction,furniture,and paper manufacture) (Han 1980).Breeders have been working hard to breed suitable varieties with high yield and quality.Chinese fir has been the focus of provenance experiments and strategies to improve its growth and adaptability since 1957.The recent establishment of fourth-generation seed orchards has provided beneficial alleles for breeding and improving Chinese fir (Ming et al.2016).However,there is a limited amount of valuable genetic diversity in conventional varieties of Chinese fir.To overcome this limitation,mutagenesis has been used as an artificial method to increase genetic variation that can be used for breeding in practical forestry.The effects of EMS concentration and different treatment conditions on germination and seedling growth of the cucumber have been reported (Shah et al.2015),but there are almost no studies on the relationship between mutagenesis and early growth in forestry.

The main goal of this study was to investigate the relationships between mutagens and the growth of Chinese fir.In 2011 and 2013,Chinese fir seeds were collected from the seed orchard at Jiangle State Forestry Farm,Fujian Province,China.We examined the changes in seed germination and early growth of Chinese fir with the concentration and duration of mutagen exposure.We also monitored the generation of mutants for future breeding programs.We hope to generate positive mutations that might be used for breeding programs and ultimately improve the productivity of Chinese fir forests.

Materials and methods

Two experiments were conducted with Chinese fir seeds collected from the seed orchard in 2011 and 2013.The first examined the optimal concentration and duration of exposure for mutagenesis.The second experiment analyzed the early growth of Chinese fir grown from mutagenized seeds,and meanwhile,to obtain several mutants.

Plant material

Mixed seeds collecting of the Chinese f ri were obtained from the Jiangle State Forestry Farm in Jiangle County,Fujian Province,China (26° 71′ N,117° 47′ E,167 m above sea level).The area is suitable for growing Chinese fir due to its subtropical monsoon climate (average annual temperature 21 °C,average annual rainfall 1200 mm).The forestry farm has a clonal seed orchard of Chinese fir,aged between 18 and 21 years planted at a spacing of 5 m × 5 m.The 1000-seed weights of Chinese fir seeds were 6.0083 and 5.8328 g for seeds collected in 2011 and 2013.Experiments were performed from 2011 to 2017.

EMS concentration and duration

The seeds were presoaked in GA3for 24 h and treated with EMS at 0%,0.3%,0.6%,0.9%,or 1.2% v/v for 8,12,or 16 h;all treatments were performed at 25 ± 1 °C.Next,the seeds (60 g,～ 10,000 per treatment) were thoroughly washed in running tap water to remove residual mutagens.For the seeds collected in 2013,various concentrations of EMS were added (0,0.2,0.3,and 0.4% v/v) for 8 h to the seeds(300 g,～ 50,000 per treatment),while the other conditions were the same as in the first study.

SA concentration and duration

The seeds were presoaked in GA3for 24 h and treated with SA at 0,2,4,6,8,or 10 mM for 4,8,or 12 h;all treatments were performed at 25 ± 1 °C.Next,the seeds (60 g,～ 10,000 per treatment) were thoroughly washed in running tap water to remove residual mutagens.For the seeds collected in 2013,various concentrations of SA were added (0,2,4,and 6 mM) for 8 h to the seeds (300 g,～ 50,000 per treatment),while the other conditions were the same as in the f rist study.

Germination

The seeds from all experiments were sown in a field in Qiantan Town,Jiangle in January 2012 and January 2014.Approximately 1000 seeds per square meter were planted in rows 10 cm apart.In the spring of 2015,the seedlings were transplanted on a mountain in Taocun Town.The heights of 30 trees were measured for the next 3 years and the plants with the best growth characteristics in 2017 were identified.

The following data on germination parameters were recorded.In this study,the seedling rate is the survival rate of seedlings considering plants with heights of at least 10 cm.The mortality rate is the difference in the seedling rate between the control and the experimental groups accounted for the germination rate of the control group.A negative mortality rate means that the seedling rate of the experimental group exceeded that of the controls.treatments were evaluated by analysis of variance (ANOVA)with Duncan’s multiple-range tests for multiple comparisons.Apvalue≤ 0.05 for the ANOVAF-test was considered statistically significant.

Results

Effect of EMS on the height of Chinese Fir

Table 1 shows the effect of EMS on germination of Chinese fir seeds.As the concentration and duration of treatment with EMS increased,the seedling rate decreased,and the mortality

In October of the same year,the heights of ≥ 90 randomly selected seedlings per treatment,including heights less than 10 cm,were measured using a ruler.

Aff orestation and height survey

Seedlings were transplanted on a mountain in Taocun Town in the spring of 2013 and 2015,and planted at a spacing of 3 m × 3 m.The heights of 30 trees were measured for 1 year in the first study and the next 3 years in the second experiment using a box staff .

The maximum-to-minimum ratio is the ratio of maximum to minimum height for the same treatment.The maximumto-minimum ratio was calculated as:

Mutants

Our experiments cover a large area,so geographical differences have an effect.This is why we cannot clearly determine whether the Chinese firs had mutations affecting height.In Most of the variation that occurred could only be judged by observing the differences from the control group before the biochemical and molecular characteristics were investigated.

Design and analysis

The experiments used a completely randomized block design;experiments were replicated three times.Microsoft Excel 2016 and IBM SPSS Statistics 24.0 were used to assess height differences.Diff erences in height among rate increased.Few seeds survived treatment with 1.2% EMS irrespective of the treatment duration,and the seeds survived treatment with 0.9% EMS only for ≤ 8 h.Also,the seedling rate differed significantly according to EMS concentration,with the exception of treatments with 0.3% and 0.6% EMS for 16 h.However,treatment with EMS for 8 h resulted in a slightly higher seedling rate than did treatment for 12 h,and both were significantly higher than that after 16 h.

The mean height of Chinese fir decreased as the concentration and duration of treatment with EMS increased(Fig.1).The seeds treated with 0.9% EMS for 12 or 16 h were dead,the same situation occurred in 1-year-old trees.Treatment with 0% and 0.3% EMS for 16 h,but not for 8 h,resulted in a significant difference in seedling height.Also,treatment with 0% and 0.3% EMS decreased the mean height of seedlings as the treatment duration increased.The data showed that 8 h was the optimum treatment duration for EMS.

Fig.1 Effects of EMS concentration and treatment duration on the heights of Chinese fir seedlings and 1-year-old trees.Results are means of 90 independent biological replicates in October 2012 a,and of 30 independent biological replicates in 1-year-old trees in October 2013 b.Results are mean heights after treatment with 0 and 0.3%EMS.Diff erent lowercase letters above bars indicate significant differences (p <0.05)

In the second experiment,the height of Chinese fir trees had a significant negative association with EMS concentration (Table 2).From 2013 to 2017,treatment with 0%and 0.4%,but not 0.2%,EMS resulted in seedlings of significantly different heights.The height of 2-year-old trees was not significantly influenced by 0%,0.2%,or 0.3% EMS,but was significantly reduced by the 0.4% EMS treatment.The maximum-to-minimum ratio has a significant negative association with EMS concentration;treatment with 0.2% EMS yielded seedlings with a maximum-to-minimum ratio of 3.15,while 0.4% EMS resulted in seedlings withratios of 2.73,2.83,and 2.91 in the next 3 years.The coeff icient of variation (CV) also increased with increasing EMS concentration.

Table 1 Effect of EMS on the germination of Chinese fir seeds in 2011

Effect of SA on the height of Chinese fir

The germination rate of Chinese fir seeds decreased,and their mortality rate increased,as the concentration and duration SA treatment increased (Table 3).The seedling rate was significantly decreased by 2—8 mM SA and was 0% after treatment with 10 mM SA.However,treatment with 2 and 4 mM SA for 4 h resulted in a negative mortality rate,indicating promotion of seed germination.

Following treatment for 4,8,and 12 h,the mean height of Chinese fir decreased as the SA concentration increased(Fig.2).Treatment with 8 and 10 mM SA resulted in significant differences in height according to treatment duration.The height of plants treated with 8 and 10 mM SA was significantly different from that of plants treated with 0,2,4,and 6 mM SA,but that of plants treated with 4 and 6 mM SA was not.Treatment with 0—6 mM SA for 8 h resulted in the tallest seedlings and 1-year-old trees.Thus,8 h and 2—6 mM SA were the optimum treatment duration and concentration.

Fig.2 Effects of SA concentration and treatment duration on the heights of Chinese fir seedlings and 1-year-old trees.Results are means of 90 independent biological replicates in October 2012 (2A),and of 30 independent biological replicates in 1-year-old trees in October 2013 (2B).Results are mean heights after treatment with 0—6 mM SA.Diff erent lowercase letters above bars indicate significant differences (p <0.05)

The height of Chinese fir seedlings and 1-year-old trees decreased as the SA concentration increased (8-h treatment;Table 4).Treatment with 6 mM SA resulted in a significant difference in the height of Chinese fir.Treatment with 4 mM SA yielded the tallest 2-and 3-year-old trees,and 2 mM SA resulted in 3-year-old trees of increased height.However,none of these differences was significant compared to the control.The maximum-to-minimum ratio had a significant positive correlation with the SA concentration.Treatment with 0 mM SA resulted in seedlings and 1-year-old trees with the lowest maximum-to-minimum ratio,compared to 2 mM SA for 2-and 3-year-old trees.Also,4 mM SA resulted in the highest maximum-to-minimum ratio in seedlings in all 4 years.However,the CV did not differ according to the concentration or duration of treatment with SA.

Chinese fir mutants

In October 2017,127 trees with mutations were selected.The density of lateral branches,lengths of lateral branches,mutations of top branches,bud and needle color,and early reproductive growth of these trees are listed in Table 5.Intensive lateral branching occurred when the number of branches was at least twice that of the control plants.Sparse lateral branching was when there were few branches and secondary branches compared to the control plants.Abnormal branching refers to the formation of short branches and clusters.Abnormal color indicates light green compared with the dark green of the control plants.Early reproductive growth refers to the earlier appearance of female globules compared with the control plants.EMS and SA generated mutants with specific top branches and lateral branches;these accounted for～ 94.49% of the mutants and were induced more frequently by EMS than by SA (Fig.3).Treatment with 6 mM SA resulted in only one Chinese fir with male flowers,and only EMS produced trees with abnormal coloration of buds and needles.

Fig.3 Mutants of Chinese fir induced by EMS and SA.a,intensive lateral branch;b,sparse lateral branch;c,abnormal lateral branch;d,abnormal top branch;e,abnormal bud color;f,abnormal needle color;g,reproductive growth;h,control plant)

Discussion

EMS and SA exerted different effects on the growth of Chinese fir seeds.The traits we described here have also been reported in previous studies of the effects of chemical mutagens on plant growth (Koshiba 1993;Booker et al.2005;Souza et al.2017).Chemical mutagens are used in forestry to shorten the breeding cycle,which provides germplasm resources more rapidly.In a prior study,the effects of EMS,DEB,and SA changed linearly with the concentration and duration of treatment (Chopra 2005).In this study,we found that EMS and SA are potent mutagens in Chinese fir.

The seedling germination rate decreased,and the mortality rate increased,as the EMS and SA concentration and treatment duration increased.This may be attributable to differences in the genetic makeup of the seeds and/or the mechanism of action of the two mutagens.The germination of Chinese fir seeds was more sensitive to EMS than to SA (Tables 1,3).It was reported that EMS induced meiotic chromosomal abnormalities at a high frequency,which impaired the growth and development ofNigella sativaL.EMS induces fewer chromosomal abnormalities but more desired mutations than SA (Dixit et al.2012;VineetaDixit et al.2013).Such chromosomal abnormalities could affect the germination of Chinese fir seeds.The mortality rate was showed negative with 2 and 4 mM SA in 4 h.The seedling rates with 2 and 4 mM SA were higher than that of the control.This may be because SA induces mutations through an organic metabolite,which enters the nucleus and generates point mutations (Olsen et al.1993).

Table 2 Effects of EMS on the height of Chinese fir from 2014 to 2017

Table 3 Effects of SA on the germination of Chinese fir seeds in 2011

Table 4 Effects of SA on height of Chinese fir from 2014 to 2017

Table 5 Number of mutants with the indicated morphological characteristics in 2017

Plant height is an important indicator of the effect of EMS (Devi and Mullainathan 2011).Treatment with EMS for 24 h was reported to decrease the height of Chinese-long cucumber seedlings (9930) (Shah et al.2015).Mutagenesis induces biological damage (i.e.,decreased plant growth),the magnitude of which increases with increasing mutagen concentration and treatment duration (Thilagavathi and Mullainathan 2009).Our results are consistent with a previous report (Roychowdhury et al.2014) that the seedling rate and plant height decrease with increasing EMS concentration.In this study,the height of Chinese fir decreased as the SA concentration increased.The effect of SA on microbial growth was investigated in the 1940s (Cabrol et al.2017;Kempf and Nungester 1944;Lichstein and Soule 1944) reported that 50 mM SA inhibited the growth of free-living prokaryotes;however,under nutrient-rich conditions,SA promoted microbial growth.Treatment with 2 and 4 mM SA increased the height of 2-and 3-year-old Chinese fir trees;however,the underlying mechanism is unknown.

In 2013 the treatment of 300,000 seeds with EMS and SA resulted in a mutation rate of～ 0.04%,possibly because of the huge genome size of conifer species,which is 18—35 Gb and contains many repeat sequences (Matias et al.2003;Nathalie et al.2005;Mackay et al.2012).An unexpected finding was trees with male flowers;reproductive growth of Chinese fir typically begins 5—7 years after afforestation but can occur after only 3 years.Therefore,the underlying mechanisms causing early reproductive growth in individualtrees need to be determined.A further in-depth study of the mutant Chinese fir trees generated in this study is warranted.

This study has several strengths.First,we evaluated the effect of EMS and SA on the germination rate and height of Chinese fir.Second,many the treated trees were actively growing at the time of writing.Third,this is,to our knowledge,the first report of several traits in mutant lines of Chinese fir.However,this study also has the following limitations.First,we did not investigate the biochemical and molecular characteristics of the mutants with abnormal branching and coloration.Second,for unknown reasons,the effect of 2 and 4 mM SA was not in agreement with prior reports.Third,although the seeds and seedlings were planted in the same location,environmental factors (e.g.,light intensity and humidity) could have influenced their growth;this possibility should be evaluated in further works.

Conclusions

Chemical mutagens such as EMS and SA induce nucleotide—base substitutions at high frequency,and so are utilized in breeding strategies.The growth characteristics of Chinese fir decreased as the concentration and duration of treatment with EMS and SA increased.Also,127 mutants with different morphological characteristics were generated;these represent potential sources of variation for breeding of Chinese fir.

AcknowledgementsWe are thankful to Dr.Zheng Renhua and his research team of Chinese fir from Fujian Academy of Forestry for their contribution to the establishment of the Chinese fir seed orchard in the Jiangle State Forestry Farm in Jiangle County,Fujian Province,China,which provides us with experimental materials.