Radiation dose effects on the morphological development of M 1 generation pea(Pisum sativum)

Da-Peng Xu ?Hu-Yuan Feng?Jian-Bin Pan?Ze-En Yao ?Jun-Run Wang

Abstract We irradiated pea seeds with neutrons from a 252Cf source and studied the radiation dose effects on various morphological development parameters during the growth of M 1 generation peas.We found that in the dose range of 0.51–9.27 Gy,with the increase in neutron-absorbed dose,the morphological development parameters of M1 generation peas at the initial seedling stage showed an obvious trend with three fluctuations.With the development of pea,this trend gradually weakened.Further analysis and verification showed that the main trend in the M1 generation of peaseedswasan inhibitory effect induced by neutron irradiation and there was a good linear correlation between the inhibitory effect and neutron absorption dose.We successfully demonstrated the background removal of mutant plants and defined morphological development parameters for peas that match the overall development of plants.Our results will positively impact neutron mutation breeding and automatic agriculture.

Keywords Neutron irradiation.Pea(Pisum sativum).252Cf source.Radiation does effects.Absorbed dose.Morphological development

1 Introduction

Radiation is widely used in life science research.X-rays[1,2],γ-ray[3],proton beam[4],HZE ion,and252Cf fission neutron sources are used extensively[5].In 1928,the American botanist Stadler proved that X-rays can cause mutagenic effects based on research in barley irradiated by X-ray[6].Since then,scientists have carried out research on the biological effects and applications produced by the interaction between radiation and plantsat all levels.In this research process,the most commonly used radioactive sourcesare X-ray andγ-ray sources,which havebeen used to obtain excellent mutants,such as rust-resistant wheat mutants[7],high-palmitic acid mutant soybean[8],and Chinese Cabbage mutants[9].There have also been studies of the genetic characteristics[10]and molecular mechanisms[11]of plants using mutants obtained byγ-ray irradiation.Additionally,heavy ion[12],γ-ray[13,14],or UV ray[15]havebeen used to study theeffectsof radiation by dose.

Research on radiation dose effects is an important component of research into the biological effects of radiation,primarily because the biological effects of radiation often vary significantly with radiation dose.Studies have shown that the biological effects of radiation on plants at low doses are often stimulative.For example,rice and mung exposed to low doses ofγ-rays show significant biological effects that promote growth[16].Low doses of60Co-γray irradiation of Terminalia arjuna Roxb.seeds can induce such biological effects as improved seed germination rate,vigor index,relative growth rate of seedlings,and fresh weight[17].Other studies have shown that the biological effects of plants exposed to high doses of radiation mainly involve inhibitive effects.For example,after the dry seeds of Zoysia japonica were irradiated with high doses of60Co-γradiation above 300 Gy,such biological effects as plant height shortening,decreased seedling emergence rate,and decreased seedling fresh weight were observed[18].At present,the research in this field has mainly focused on the radiation dose effect of X-ray andγ-ray,whereas there is comparatively less research on theneutron radiation doseeffect[19].Neutrons,discovered by Chadwich et al.[20],are high linear energy transfer rays.The interaction between neutrons and matter is obviously different from other rays and neutron radiation has the characteristics of strong penetrability,a wide variation spectrum,a high variation rate,and stable characteristics of the offspring of variation.Therefore,neutrons can produce more obvious and greater biological effects[21].One study by Zhang et al.showed that the biological effects of irradiation of onion dry seeds by252Cf fission neutrons were 124 times that of60Co-γrays[22].Thus,neutronshave mostly been used to obtain mutants[23–25].However,the dose effects require further study.

Pea(Pisum sativum L.)is an important food crop[26],and it is also one of the top 10 vegetables preferred by consumers globally.Fresh pods and pea seedlings are very popular ingredients[27],and research on pea has always been of great scientific interest[28–30].Plant morphological parametersareclosely related to biological research and breeding.Existing studies have suggested that the development trendsof maizebreeding areclosely related to the morphological parameter indices [31].Therefore,selecting and implementing appropriate morphological parameters are conducive to plant biological research.Currently,the traditional plant morphological parameters fail to meet the requirements in the process of radiation plant research,which is one of the important factors hindering the progress of this research direction.

In this study,the morphological development of M1generation peas was studied in the field using dried pea seeds irradiated by thE252Cf neuton,and the relationship between the neutron absorption dose and morphological development of pea plants across a wide gradient dose range was first explored.This study provides a rich scientific basis for understanding biological effectsof neutron radiation and neutron mutation breeding.

2 Materials and methods

2.1 Radioactive sources and biological samples

The radioactive source used in this study was a252Cf isotope neutron source.The neutron source is a spontaneous fission neutron source,in which the neutron emissivity is 2.31×1012n/s.g and the half-life is 2.647 years.The neutron spectrum of spontaneous fission is close to a pure fission spectrum and the average neutron energy is 2.158 MeV[32].

Needle-leafed pea MZ-1(P.sativum var.MZ-1)is a leafless pea variety introduced from the USA by the Institute of Soil Fertilizer and Water-saving Agriculture,Gansu Academy of Agricultural Sciences[33].Dry pea seed pods that were full of seeds and that exhibited a germination rate higher than 96%were selected and divided into ten groups for different doses of neutron radiation.

2.2 Neutron radiation dose

We have fully considered the space that can accommodate samples in thE252Cf source irradiation device and placed ten groups of peas in the channel of the device(the device is detailed in reference[34]).To ensure that the irradiation dose can cover as wide a range as possible,we first conducted analysis and calculation and then set the irradiation time as 14,153.7 h.In this way,the neutronabsorbed doses of ten groups of peas were 0.51 Gy,0.64 Gy,0.81 Gy,1.03 Gy,1.32 Gy,1.80 Gy,2.40 Gy,3.55 Gy,5.47 Gy,and 9.27 Gy.

2.3 Field trials and recording of morphological characters

Ten irradiation groupsof pea seedstreated with different neutron absorption doses and the control group without radiation were seeded in the field.Five replications were set up with 16 plants in each replication to carry out the field planting experiment of the M1generation.The experimental field was located in Yuzhong Campus of Lanzhou University.From the 2nd day of pea sowing,the seedling emergence and the development of the stipules of each pea were recorded in detail daily at 9 a.m.,and these recordsweremade until the 52nd day(that is,theday when the fifth pair of stipules of the last pea was opened).The appearance of the flower buds and the flowering of peas were immediately recorded,and all peas were recorded for 25 days.

2.4 Confirmatory experiment

Needle pea seeds matching those in the field trial were selected.Five groups were irradiated in the scope of the middle-high doses.Neutron absorption doses in each irradiated pea group had an equal gradient interval of 2 Gy,4 Gy,6 Gy,8 Gy,and 10 Gy.Five treatments and one control group without irradiation were placed in germinating boxesfor seed germination at 20°C.Fivedayslater,when seeds basically completed germination,seven germinated peas chosen from each group at random were planted in flowerpots for the greenhouse experiment.Three repetitions were established in each group.The day after the peaswere planted,the stipule situation of each pea was observed in detail every morning until the32nd day(on the day when the fifth pair stipule of the last pea unfolded).A dividing ruler wasused to measure the height of all peasat three different growth periods for 15 days.The measurements were taken once every 5 days for a total of three measurements.

2.5 Definition of morphological development parameters

Based on years of experimental experience on pea and referring to the germination index and vitality index commonly used in seed germination experiments,we defined seven parameters that closely reflected the overall development of the plants.

2.6 Data analysis

Before the data analysis,it was necessary to calculate the background of spurious(potentially mutant)plants and remove these data.Following this calculation,the data for the slowest-growing pea were removed from each replication of each experiment group.One-way ANOVA and least significant difference(LSD)multiple comparison tests(SPSS 21.0,IBM Corp.,Armonk,NY,USA)were used to analyze theexperimental data of the M1generation pea in the control group and irradiation group.The standard deviation of each experimental group was calculated and plotted using Excel(Microsoft Corp.,Albuquerque,NM,USA)software.The flowchart of our research scheme is shown in Fig.1.

Fig.1 Flowchart of our study protocol

3 Results

3.1 Relationship between neutron absorption dose and main plant morphological development of M 1 generation pea

The coniferous pea we selected is a type of leafless pea.In the seedling stage,the main form of morphological development is the increase in the number of stipules.Therefore,as described in ‘Sect.2.5,’we defined the SEI and the n th pair of StEI.The data processing results are shown in Fig.2.

It can be observed from Fig.2a that the SEI had three regular fluctuations with the increase in neutron absorption dose;that is,the SEI initially exhibited three fluctuations with the decrease in neutron absorption dose,followed by an increase in the neutron absorption dose.Significance analysis showed that there was a significant difference between the highest point of SEI(dose points:0.81 Gy,1.80 Gy,9.27 Gy)and the lowest point of SEI(dose points:0.64 Gy,1.32 Gy,3.55 Gy)in each of the three fluctuations.Three regular fluctuations similar to those described above can also be found in Fig.2b–f.After analysis of the first,second,third,fourth,and fifth pair of StEI,it was also found that in any one of the fluctuations,there were significant differencesbetween the highest point and the lowest point.

In addition,as observed from Fig.2a,the lower dose of neutron irradiation reduced the SEI of M1generation pea.The decrease in this index indicated that it could promote seedling emergence.The ANOVA indicated a significant difference between the SEIof thebest dose point(0.64 Gy)and that of the control group(0 Gy).Figure 2a also indicated that when the neutron irradiation dose was≥0.81 Gy,the SEIof all irradiation groupswas higher than that of the control group,indicating that it had an inhibitory effect on seedling emergence.We also analyzed the differences between the M1generation pea 0.64 Gy dose group and the control group in all the StEI and found no significant differences.This indicated that with the development of M1generation pea,the promoting effect of the low dose was gradually weakened.

Fig.2 Relationship between neutron absorption dose and main plant morphological development parameters of M 1 generation pea.a Relationship between seedling emergence index and neutron absorption dose.b Relationship between the expansion of the first pair of stipules index and the neutron absorption dose.c Relationship between the expansion of the second pair of stipules index and the neutron absorption dose.d Relationship between the expansion of the third pair of stipules index and the neutron absorption dose.e Relationship between the expansion of the fourth pair of stipules index and the neutron absorption dose.f Relationship between the expansion of the fifth pair of stipulesindex and theneutron absorption dose.The error bar represents the standard error within the group

3.2 Relationship between the neutron absorption dose and plant height development of M 1 generation pea

Plant height is affected by many biological and environmental factors and so the accuracy of the traditional random sampling methods is limited.Therefore,we used a comprehensive method whereby we measured each plant and defined the PHCI.In addition,in order to reflect the development of plant height morphology,we selected four equal interval time points(time interval of 5 days)to measure the plant height of all M1generation peas.The data processing results are shown in Fig.3.

It isevident from Fig.3a and b that the PHCIfluctuated three times with the increase in neutron absorption dose.After analyzing the significant difference of PHCI at the first time point and second time point,it was found that there were significant differences between the maximum PHCI(dose points:0.51 Gy,1.32 Gy,2.40 Gy)and the minimum of the corresponding fluctuation PHCI(dose points:0.81 Gy,1.80 Gy,9.27 Gy)in each fluctuation.However,the analysis of the PHCI of the third time point and fourth time point showed that there was no significant difference between the PHCIof the 0.51 Gy dose point of the first waveform and that of the 0.81 Gy dose point.It can also be seen from Fig.3c and d that thewaveform also weakened.The results showed that with the development of M1generation pea,the regularity of the three wave shapes of PHCI gradually weakened with neutron absorption dose.

It wasobserved in Fig.3 that the PHCIof M1generation pea could be increased by low-dose neutron irradiation.The significant difference analysis showed that there was no significant difference between the highest PHCI(0.51 Gy dose point)and the PHCI of the control group in the(a),(c),and(d).This indicated that the strength of promoting the plant height development of M1generation pea by low-dose neutron irradiation was limited.

Fig.3 Relationship between the plant height composite index of M 1 generation pea and the neutron absorption dose.a Relationship between the plant height composite index and the neutron absorption dose at the first time point.b Relationship between the plant height composite index and the neutron absorption dose at the second time point.c Relationship between the plant height composite index and the neutron absorption dose at the third time point.d Relationship between the plant height composite index and the neutron absorption dose at the fourth time point.The error bar represents the standard error within the group

3.3 Relationship between the neutron absorption dose and morphological development of M 1 generation pea floral organs

We defined four parameters that could accurately and reasonably reflect the morphological development of the reproductive organ flowers:FBI,FI,BSDI,and FSDI(see‘Sect.2.5’for details).The processing and analysisresults of the four parameters are shown in Fig.4.

It is evident from Fig.4a and b that,with the exception of the lowest dose of neutron irradiation(0.51 Gy),the other higher doses of neutron irradiation will cause a decline in the FBI and fiof M1generation peas.The decrease in FBI and fimeans that the reproductive phenology of pea will be delayed.After analyzing the difference in FBIand FI,it was found that there were significant differences between the FBI of the neutron irradiation group with an absorbed dose of 1.03 Gy or more and that of the control group and between the neutron irradiation group with an absorbed dose of≥0.81 Gy and that of the control group.Therefore,this suggested that when the neutron irradiation dose was higher than a certain value,the reproductive phenology of M1generation pea could be significantly delayed.

Fig.4 Relationship between the morphological developmental parameters of M 1 generation pea floral organ and the neutron absorption dose.a Relationship between the flower bud index and the neutron absorption dose.b Relationship between the flowering index and theneutron absorption dose.c Relationship between the bud stage duration index and the neutron absorption dose.d Relationship between the flower stage duration index and the neutron absorption dose.The error bar represents the standard error within the group

Figure 4c and d indicates that the BSDIand FSDIof all irradiation groups were higher than those of the control group.There were significant differences between the three high-dose groups(3.55 Gy,5.47 Gy,and 9.27 Gy)and the control group.This indicated that neutron irradiation could prolong the flower bud and flowering period of M1generation pea and the effect was significant at higher doses(3.55 Gy and above).

3.4 Linear correlation analysis between morphological development parameters and absorbed neutron dose in the high-dose area

We analyzed the linear correlation between the morphological development parameters and the absorbed neutron dose at three dose points(3.55 Gy,5.47 Gy,and 9.27 Gy)in the high-dose region.The linear correlation analysis results of the various morphological development indices and neutron absorption dose are shown in Fig.5.

The six linear equations shown in Fig.5 are all positive correlations,which demonstrate a phenomenon whereby the plant morphological development time of M1generation pea was continuously delayed with the increase in neutron absorption dose,i.e.,inhibited.Among these values,the R2values of the linear equations shown in Fig.5a–d suggest that the goodnessof fit of the equation was good and the linear correlation was high.The P-values obtained by the F-test were 0.0962,0.1021,0.0783,and 0.1468 in Fig.5a–d,respectively.This shows that there was a linear correlation between the growth time of plant morphology and neutron absorption dose during and before the three pairs of stipules were expanded in the high-dose range.Of course,we also found that the linear correlation shown in Fig.5e and f became lower.This suggests that with the development of M1generation pea,the linear correlation between the growth time of plant morphology and the absorbed neutron dose decreased.

Fig.5 Linear relationship of various parameters of main plant morphological development indices and absorbed neutron dose in M1 generation pea.a Linear relationship between theseedling emergence index and the neutron-absorbed dose.b Linear relationship between the expansion of the first pair of stipules index and the neutronabsorbed dose.c Linear relationship between the expansion of the second pair of stipules index and neutron-absorbed dose.d Linear relationship between the expansion of the third pair of stipules index and the neutron-absorbed dose.e Linear relationship between the expansion of the fourth pair of stipules index and the neutronabsorbed dose.f Linear relationship between theexpansion of thefifth pair of stipules index and the neutron-absorbed dose

The four linear equations shown in Fig.6 are all negative correlations,which suggest that the height morphological development of M1generation pea plant decreased with the increase in neutron absorption dose;that is,it was inhibited.The R2value of the linear equation shown in Fig.6a is 0.9983,indicating that the equation had a high goodness of fit,and the P-value of the F-test was 0.0258,indicating that the linear correlation wassignificant.As the first measurement time point was before the initial seedling stage of pea development state(the initial seedling has three pairsof stipules),it can beconsidered that there wasa good linear correlation between plant height morphological development and neutron absorption dose in the high-dose range.In addition,although the R2values of the linear equations in Fig.6b–d were all greater than 0.99,the P-values of the F-tests were all greater than 0.05(0.0502,0.0509,and 0.0525,respectively).Thisshowsthat with the development of M1generation pea,the linear correlation gradually decreased.

Fig.6 Linear relationship between plant height composite index of M1 generation pea and neutron-absorbed dose.a Linear relationship between the plant height composite index and the neutron-absorbed dose at the first time point.b Linear relationship between the plant height composite index and the neutron-absorbed dose at the second time point.c Linear relationship between the plant height composite index and the neutron-absorbed dose at the third time point.d Linear relationship between the plant height composite index and the neutron-absorbed dose at the fourth time point

The linear equations shown in Fig.7a and b are all negative correlations,which suggested that the morphological development of pea flower buds and flowers was delayed with the increase in neutron absorption dose;that is,it was inhibited.The two linear equations have high goodness of fit,and the P-values of the F-test were 0.0493 and 0.0378,which indicated that the linear correlation was significant.The two linear equations shown in Fig.7c and d are all positive correlations,which suggest that the duration of pea flower bud period and flowering period increased with theincreasein neutron absorption dose.The two linear equations also have high goodness of fit.The P-values of the F-test were 0.085 2 and 0.1067,respectively,which indicated that the linear correlation was not significant.

Fig.7 Linear relationship between the morphological developmental parameters of M1 generation pea floral organs and absorbed neutron dose.a Linear relationship between the flower bud index and the neutron-absorbed dose.b Linear relationship between the flowering index and the neutron-absorbed dose.c Linear relationship between the bud stage duration index and the neutron-absorbed dose.d Linear relationship between the flower stage duration index and the neutronabsorbed dose

4 Discussion and verification

4.1 Methodology

During plant growth and development,there is a certain probability that mutant progeny will be produced.The experimental data of these mutant progeny will have a negative impact on the overall data analysis and thus it is necessary to eliminate the experimental data obtained from mutant progeny.For this reason,we defined the background of plants believed to be mutant progeny.Through the study of the development time of pea in the control group and the overall data analysis,we calculated and determined the background value of the potential mutant plants as 5%,which was used in correcting all the data in the experimental group.

There are individual differences in plant growth and development.Under all circumstances,it is the best choice to fully record the characteristics of all plants.In addition,in the study of plant development,a continuous data record is better able to reflect the developmental process than a single data record at a selected time point.If there are data records of all plants,parameters can be set according to each repetition of each group.If there are continuous data records,time can be further integrated into the set parameters.Our research was based on full records and daily continuous records for all plants,and seven parameters closely associated with the overall development state of plants were defined.

4.2 Low-dose promotion and high-dose inhibition of neutron radiation

Through the above research and analysis,we can confirm that in the 0.51–9.27 Gy range of neutron radiation in the pea seeds,the development of main plant morphology and plant height morphology of M1generation pea exhibited three regular obvious fluctuations with the increase in neutron absorption dose,among which the biological effect of the first fluctuation was mainly manifested in a promoting effect and the biological effect of the second and third fluctuations was mainly reflected in an inhibitory effect,with the inhibitory effect of the third fluctuation being more obvious.Thisresult differsfrom the previously reported simple inhibitory effect of neutron irradiation on the seedling height and plant height of ornamental kale[35].The main reason is that there were many dosage groups and low-dose neutron irradiation in this study.The promoting effect of low-dose radiation and the inhibitory effect of high-dose radiation have been verified in research onγ-ray-irradiated plants[13,18].

We also found that in the neutron absorption dose range that we studied,with the development of M1generation pea,the waveform character of the three regular fluctuations gradually weakened and the amplitude gradually decreased.This shows that the pea plants were constantly repairing the effects of the neutron radiation on the plant morphology and height during the development of peas.

4.3 Neutron radiation hasa general inhibitory effect on the reproductive flower organs of M 1 generation pea

In the studied range of neutron absorption dose,neutron radiation generally caused a delay in the morphological development of M1generation pea flower organs and the prolonging of flower bud emergence and flowering duration;that is,it had a universal inhibitory effect.Although 0.51 Gy neutron irradiation could promote the growth of the pea flower organs,the effect was not obvious.Even so,we think that low-dose neutron irradiation could promote the growth of the M1generation pea flower organs.However,due to the relatively conservative and stable genetic characteristicsof theflower organs,thepromoting effect on them will be relatively weak.

4.4 Linear correlation between absorbed neutron dose and morphological development parameters in the full-dose range

According to the above analysisof the linear correlation between the absorbed neutron dose and the morphological development parameters in the high-dose range.We speculated that the linear correlation could be found in the whole-dose range.Therefore,the data of the control group(0 Gy)and three high-dose irradiation groups(3.55 Gy,5.47 Gy,and 9.27 Gy)were analyzed for linear correlations.The results are shown in Table 1.

It can be seen from Table 1 that although the determination coefficient R2values of the linear correlation analysisin the table were mostly lower than thoseof only three high-dose irradiation groups,most of the significant P-values in the table were smaller than those of the three high-dose irradiation groups.In addition,the significant P-values in the morphological development parameters of plant height and flower organs were less than 0.05,indicating that these linear correlations were significant.All of these results showed that thelinear correlation between the morphological parameters and the absorbed neutron dose increased following treatment of the control group and three high-dose irradiation groups.Within the range of 10 Gy after excluding low dose,there was a good linear correlation between the neutron absorption dose and the morphological parameters.

4.5 The linear dependence verification of neutronabsorbed doses in the middle-high dosage range and each morphological development parameters

To verify the deduction in Sect.4.4,we used independently developed neutron-absorbed dose-distributed irradiation devices with high-dose evenness to irradiate five groups of pea seeds within the middle-high dose range(devices were shown in Ref.[3])and used a greenhouse experiment with a more uniform environment to carry out further verification.The relational relationsbetween stipule unfolding morphological development and neutron-absorbed doses are presented in Table 2,which depict that with the increase of neutron-absorbed doses,the StEI first increased,then decreased,and again increased.This law was consistent with the law that peas’main morphological development in middle-high dose areas changed with the doses as discussed in Sect.3.1.Through the linear dependence analysis in Table 3,it could be found that the slopes of five corresponding linear equations were all positive.The significance of each linear equation was high(P-value verified by F was less than 0.05),indicating the higher linear dependence between peas’main morphological development and neutron-absorbed doses.This result is highly consistent with the analysis result in Sect.4.4.

Table 1 Linear relationship between morphological development parameters and absorbed neutron dose

The result of relational analysis between the PHCI and the neutron-absorbed doses is described in Table 2,showing that with the increase of neutron absorption doses,the PHCI consistently declined.This was consistent with the law that PHCI showed a downward trend with the neutron absorption dose above 1.8 Gy as discussed in Sect.3.2.According to the linear dependence analysis in Table 3,we also found that the linear equations of PHCI belonged to negative correlation.The R2value of three equations was greater than 0.91.The P-value verified by F was less than 0.01,indicating that goodness of fit and significance of three equations were very high.This was highly consistent with the linear dependence results between the PHCI in the high-dose areas and the neutronabsorbed doses discussed in Sect.4.4.

Table 2 Morphological development parameters of M1 peas under different neutron absorption doses

Table 3 Linear relationship between the morphological development parameters and the absorbed neutron dose

The above-mentioned verification experiment further indicated that neutron radiation showed the inhibiting effect within 10 Gy range after excluding low doses.Neutron-absorbed doses showed the excellent linear dependence in this inhibiting effect.Further analysis showed that in the low-dose range(0.51–0.64 Gy),the protective system of pea was activated,which showed a certain promoting effect.The linear correlation between the inhibition effect and the neutron absorption dose was weakened.In the middle-dose range(0.81–2.40 Gy),the pea protection system reached its limit,resulting in an overall inhibitory effect that was not too significant.The linear correlation between the inhibitory effect and the neutron-absorbed dose will also be disrupted to some extent.In the high-dose range(3.55–9.27 Gy),the pea protection system was hardly activated.The inhibitory effect became increasingly significant with an increase in dosage.Thelinear correlation between theinhibitory effect and the neutron absorption dose was gradually restored.This is also the reason why SEI,StEI,and PHCIhad three regular fluctuations with the increase in neutron absorption dose.

5 Conclusion

In summary,the neutron-irradiated pea seeds obtained stress and showed a trend of gradual inhibition effect.The reproductive organs of plants have relatively conserved genetic characteristics that are highly stable and change little when affected by environmental factors.Therefore,the universal inhibitory effect of various morphological development parameters of M1generation pea flower organsin our study providessupport for thisinference.The verification experiment shows that there is a good linear correlation between the whole inhibition effect and the neutron absorption dose.Previous studies on peripheral blood in healthy adultsirradiated by X-rayshave found that there is a dose-dependent relationship between the expression of the Pig-a and Gadd45αgenes and the irradiation dosage[36].The dose-dependent relationship we found in our study of the morphological characters of neutron-irradiated plants offers a good reference for agriculture and for the study on the molecular biological mechanisms of neutron-irradiated plants.

We obtained the dose range of low-dose promotion effects,which could beuseful for the cultivation of pea and other plants.Moreover,the dose range for the inhibition effect is obtained,and the dose range given above is the reference doses for radiation mutation breeding.In addition,seven parameters closely associated with the overall development state of plants defined by us have been successfully applied.With the maturity of automatic agricultural data acquisition methods,these plant development parameters will be widely applicable.

Author contributionsDa-Peng Xu and Hu-Yuan Feng contributed to the study conception and design.Material preparation and data collection were performed by Da-Peng Xu,Jian-Bin Pan,and Jun-Run Wang.Data analysis were performed by Da-Peng Xu and Ze-En Yao.The first draft of the manuscript was written by Da-Peng Xu and all authors commented on previous versions of the manuscript.All authors read and approved the final manuscript.