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Abstract [Objectives] This study was conducted to screen out a vernalization method of radish that is more suitable for breeding and hybrid production.
[Methods]Eight radish inbred lines with different bolting tolerance were used as the research objects for the experiment. The following three treatments were done： ① treating at 4 ℃ in the dark with heat preservation for 3， 4， and 5 weeks， respectively[1]， ② treating at 4 ℃ in the dark with heat preservation for 3 weeks after 24 h incubation in light at 20 ℃， and ③ treating under light at a low temperature of 6.5 ℃ for three weeks after cultivating under light at 20 ℃ to 4 true leaves. The effects of low temperature treatment on the bolting and flowering time of different varieties of radish treated at different developmental stages were analyzed.
[Results] When comparing these three methods， the low-temperature treatment of wet seeds had a better effect on early bolting of radish and is simpler and more feasible. The effect of treating four-leaf seedlings at 6.5 ℃ was the worst; most radish inbred lines could shorten bolting time when treated at 4 ℃ for one week; and within a certain range， the longer the 4 ℃ treatment time， the better the vernalization effect. Different radish inbred lines had different performances to low temperature treatment.
[Conclusions]This study provides a scientific basis for the theoretical research on vernalization of radish.
Key words Radish; Low temperature treatment; Bolting; Development period
Received： January 1 2021 Accepted： March 2 2021
Supported by Agricultural Improved Variety Project of Shandong Province （2017LZN045）.
Xiaowei YUAN （1983-）， female， P. R. China， senior agronomist， PhD， devoted to research about vegetable breeding and biotechnology.
*Corresponding author. E-mail： yuanxiaowei-yuan@163.com.
Radish （Raphanus sativus L.）， a two-year or annual cruciferous herb with rich edible and medicinal value， is an important mainland vegetable in China[1]. Radishes are native to China， with diverse varieties， and are currently grown all over China[2].
The traditional radish breeding method is to breed one generation in two years or one generation in a year. In the breeding of superior radish， it takes 6-8 years or longer to breed only a good inbred line， which seriously affects the progress of breeding[3]. In order to speed up the breeding process， we can make artificial treatment to make it bloom in advance， thereby shortening the breeding time. Now the commonly used treatment method is generally the method of low temperature treatment[4-5]. In this study， we planned to screen out a more suitable radish vernalization method for breeding and hybrid production by studying the differences in bolting and flowering time between different varieties of radish subjected to low temperature treatment at different developmental stages， so as to provide a scientific research basis for the theoretical research of radish vernalization. Materials and Methods
Experimental materials
Shandong Huasheng Agricultural Co.， Ltd. provided eight radish inbred lines with different bolting times， named No. No. No.3， No.4， No.5， No.6， No.7， and No.8. These eight inbred lines were fixed from Japanese radish Shiwu， Huasheng radish （Guandong， Yufeng， Tianshanxue， Shisanjibai）， Yangzhou Yuanbai， and Korean radish Taiqing.
Experimental methods
First， the radish seeds were soaked with distilled water， and put in prepared petri dishes （the petri dish was covered with a layer of absorbent cotton absorbed enough distilled water） after the surface moisture was absorbed， and then， the following three treatments were done： ① treating at 4 ℃ in the dark with heat preservation for 3， 4， and 5 weeks， respectively， ② after 24 h incubation in light at 20 ℃， treating at 4 ℃ in the dark with heat preservation for 3 weeks， and ③ after cultivating under light at 20 ℃ to 4 true leaves， treating under light at a low temperature of 6.5 ℃ for three weeks. The photoperiod during the light cultivation process was 16 h light/8 h dark， and the light intensity was 2 000 lx. A total of three biological replicates were set. From each treatment， 10 plants were taken and planted in the greenhouse per variety， and managed in a unified manner， and the number of days from sowing to bolting or flowering was counted.
Results and Analysis
In order to screen out more convenient and feasible methods for radish vernalization in breeding and hybrid production， especially to select more suitable radish development stages for vernalization， we investigated the effects of treating wet radish seeds at 4 ℃， treating germinated radish seeds at 4 ℃ and treating radish seedlings at 6.5 ℃ on the bolting time of eight different bolting-tolerant radish inbred lines. In the experiment， all radish plants were managed uniformly， and the number of days from sowing to bolting was counted in detail.
Effects of 4 ℃ treatment of wet seeds on bolting time of different radish varieties
According to previous studies， low-temperature vernalization can make plants change from vegetative growth to reproductive growth in advance. In order to verify whether 4 ℃ treatment of wet seeds has an effect on the bolting time of different radish varieties， we selected 8 inbred lines with different bolting tolerance， and fully soaked the seeds and treated them in the dark at 4 ℃ for 0， and 3， 4， and 5 weeks， respectively. After the low temperature treatment was completed， the seeds were uniformly sown， and the bolting days were counted. The results of this study are shown in Fig. 1. The bolting tolerance of the eight inbred lines without low temperature treatment was roughly No.8>No.7>No.5>No.6> No.4>No. among which No.1 had no significant differences in bolting tolerance from No. No.3 and No.6. After 1 week of dark heat preservation at 4 ℃， the bolting time of other radish inbred lines except No.1 was significantly shortened， and the bolting days of No.2 to No.8 were reduced by 10， 7， 10， 10， 15， 4， and 6 d， respectively. After 2 weeks of dark heat preservation treatment at 4 ℃， the bolting time of 8 radish inbred lines was significantly shorter than that of the non-low temperature treatments. Compared with the low temperature treatments for 1 week， only the bolting time of No.2 and No.7 was significantly shortened by 6 and 4 d， respectively. After 3 weeks of dark heat preservation treatment at 4 ℃， the bolting time of No.5， No.6 and No.7 was significantly shorter than that of 2 weeks of low temperature treatment， reduced by 4， 3 and 4 d， respectively. After 4 weeks of dark heat preservation treatment at 4 ℃， the bolting time of No.3， No.4， No.5， No.6， No.7 and No.8 was significantly shortened compared with that of low temperature treatments for 3 weeks， reduced by 7， 1 9， 6， 7 and 9 d， respectively. After 5 weeks of dark heat preservation treatment at 4 ℃， the bolting time of No. No. No.3， No.5， and No.8 was significantly shorter than that of 4 weeks of low temperature treatment at 4 ℃.
It is not difficult to see from Fig. 1 that there were certain differences in the response of wet seeds to 4 ℃ treatment between different radish varieties. Most radish inbred lines exhibited the effect of early bolting after 1 week of low temperature treatment. Although some radish inbred lines had no significant differences in bolting time between 1 week to 3 weeks of low temperature treatments， such as No. No.3， No.4 and No.8， their bolting time could be significantly shortened when the 4 ℃ treatment time was extended， and within a certain range， the longer the low-temperature treatment time， the better the relative vernalization effect.
Effects of 4 ℃ treatment of germinated seeds on bolting time of different radish varieties
In order to verify the effects of low temperature treatment on the bolting time of germinated radish seeds， we treated the radish seeds after germination treatment at 4 ℃ for 3 weeks， and counted the bolting time. The results showed （Table 2） that for the bolting time of germinated radish seeds that were treated at 4 ℃， the bolting days of No.2-8 were 104， 98， 11 77， 90， 66， and 66 d， respectively， which were 20， 6， 10， 20， 12 and 7 d earlier than those of the control groups at room temperature， respectively. Specifically， the 4 ℃ treatment caused no significant differences in the bolting time of No.1 and No.4， while for other varieties， the bolting time was significantly shortened under low temperature treatment. The bolting time of No.2 and No.5 was shortened the most during the low temperature treatment， that is， advanced by 20 d. In summary， treating germinated radish seeds at 4 ℃ for 3 weeks advanced bolting for most radish varieties， but a few radish varieties （such as No.1 and No.4） could not bolt in advance.
[Methods]Eight radish inbred lines with different bolting tolerance were used as the research objects for the experiment. The following three treatments were done： ① treating at 4 ℃ in the dark with heat preservation for 3， 4， and 5 weeks， respectively[1]， ② treating at 4 ℃ in the dark with heat preservation for 3 weeks after 24 h incubation in light at 20 ℃， and ③ treating under light at a low temperature of 6.5 ℃ for three weeks after cultivating under light at 20 ℃ to 4 true leaves. The effects of low temperature treatment on the bolting and flowering time of different varieties of radish treated at different developmental stages were analyzed.
[Results] When comparing these three methods， the low-temperature treatment of wet seeds had a better effect on early bolting of radish and is simpler and more feasible. The effect of treating four-leaf seedlings at 6.5 ℃ was the worst; most radish inbred lines could shorten bolting time when treated at 4 ℃ for one week; and within a certain range， the longer the 4 ℃ treatment time， the better the vernalization effect. Different radish inbred lines had different performances to low temperature treatment.
[Conclusions]This study provides a scientific basis for the theoretical research on vernalization of radish.
Key words Radish; Low temperature treatment; Bolting; Development period
Received： January 1 2021 Accepted： March 2 2021
Supported by Agricultural Improved Variety Project of Shandong Province （2017LZN045）.
Xiaowei YUAN （1983-）， female， P. R. China， senior agronomist， PhD， devoted to research about vegetable breeding and biotechnology.
*Corresponding author. E-mail： yuanxiaowei-yuan@163.com.
Radish （Raphanus sativus L.）， a two-year or annual cruciferous herb with rich edible and medicinal value， is an important mainland vegetable in China[1]. Radishes are native to China， with diverse varieties， and are currently grown all over China[2].
The traditional radish breeding method is to breed one generation in two years or one generation in a year. In the breeding of superior radish， it takes 6-8 years or longer to breed only a good inbred line， which seriously affects the progress of breeding[3]. In order to speed up the breeding process， we can make artificial treatment to make it bloom in advance， thereby shortening the breeding time. Now the commonly used treatment method is generally the method of low temperature treatment[4-5]. In this study， we planned to screen out a more suitable radish vernalization method for breeding and hybrid production by studying the differences in bolting and flowering time between different varieties of radish subjected to low temperature treatment at different developmental stages， so as to provide a scientific research basis for the theoretical research of radish vernalization. Materials and Methods
Experimental materials
Shandong Huasheng Agricultural Co.， Ltd. provided eight radish inbred lines with different bolting times， named No. No. No.3， No.4， No.5， No.6， No.7， and No.8. These eight inbred lines were fixed from Japanese radish Shiwu， Huasheng radish （Guandong， Yufeng， Tianshanxue， Shisanjibai）， Yangzhou Yuanbai， and Korean radish Taiqing.
Experimental methods
First， the radish seeds were soaked with distilled water， and put in prepared petri dishes （the petri dish was covered with a layer of absorbent cotton absorbed enough distilled water） after the surface moisture was absorbed， and then， the following three treatments were done： ① treating at 4 ℃ in the dark with heat preservation for 3， 4， and 5 weeks， respectively， ② after 24 h incubation in light at 20 ℃， treating at 4 ℃ in the dark with heat preservation for 3 weeks， and ③ after cultivating under light at 20 ℃ to 4 true leaves， treating under light at a low temperature of 6.5 ℃ for three weeks. The photoperiod during the light cultivation process was 16 h light/8 h dark， and the light intensity was 2 000 lx. A total of three biological replicates were set. From each treatment， 10 plants were taken and planted in the greenhouse per variety， and managed in a unified manner， and the number of days from sowing to bolting or flowering was counted.
Results and Analysis
In order to screen out more convenient and feasible methods for radish vernalization in breeding and hybrid production， especially to select more suitable radish development stages for vernalization， we investigated the effects of treating wet radish seeds at 4 ℃， treating germinated radish seeds at 4 ℃ and treating radish seedlings at 6.5 ℃ on the bolting time of eight different bolting-tolerant radish inbred lines. In the experiment， all radish plants were managed uniformly， and the number of days from sowing to bolting was counted in detail.
Effects of 4 ℃ treatment of wet seeds on bolting time of different radish varieties
According to previous studies， low-temperature vernalization can make plants change from vegetative growth to reproductive growth in advance. In order to verify whether 4 ℃ treatment of wet seeds has an effect on the bolting time of different radish varieties， we selected 8 inbred lines with different bolting tolerance， and fully soaked the seeds and treated them in the dark at 4 ℃ for 0， and 3， 4， and 5 weeks， respectively. After the low temperature treatment was completed， the seeds were uniformly sown， and the bolting days were counted. The results of this study are shown in Fig. 1. The bolting tolerance of the eight inbred lines without low temperature treatment was roughly No.8>No.7>No.5>No.6> No.4>No. among which No.1 had no significant differences in bolting tolerance from No. No.3 and No.6. After 1 week of dark heat preservation at 4 ℃， the bolting time of other radish inbred lines except No.1 was significantly shortened， and the bolting days of No.2 to No.8 were reduced by 10， 7， 10， 10， 15， 4， and 6 d， respectively. After 2 weeks of dark heat preservation treatment at 4 ℃， the bolting time of 8 radish inbred lines was significantly shorter than that of the non-low temperature treatments. Compared with the low temperature treatments for 1 week， only the bolting time of No.2 and No.7 was significantly shortened by 6 and 4 d， respectively. After 3 weeks of dark heat preservation treatment at 4 ℃， the bolting time of No.5， No.6 and No.7 was significantly shorter than that of 2 weeks of low temperature treatment， reduced by 4， 3 and 4 d， respectively. After 4 weeks of dark heat preservation treatment at 4 ℃， the bolting time of No.3， No.4， No.5， No.6， No.7 and No.8 was significantly shortened compared with that of low temperature treatments for 3 weeks， reduced by 7， 1 9， 6， 7 and 9 d， respectively. After 5 weeks of dark heat preservation treatment at 4 ℃， the bolting time of No. No. No.3， No.5， and No.8 was significantly shorter than that of 4 weeks of low temperature treatment at 4 ℃.
It is not difficult to see from Fig. 1 that there were certain differences in the response of wet seeds to 4 ℃ treatment between different radish varieties. Most radish inbred lines exhibited the effect of early bolting after 1 week of low temperature treatment. Although some radish inbred lines had no significant differences in bolting time between 1 week to 3 weeks of low temperature treatments， such as No. No.3， No.4 and No.8， their bolting time could be significantly shortened when the 4 ℃ treatment time was extended， and within a certain range， the longer the low-temperature treatment time， the better the relative vernalization effect.
Effects of 4 ℃ treatment of germinated seeds on bolting time of different radish varieties
In order to verify the effects of low temperature treatment on the bolting time of germinated radish seeds， we treated the radish seeds after germination treatment at 4 ℃ for 3 weeks， and counted the bolting time. The results showed （Table 2） that for the bolting time of germinated radish seeds that were treated at 4 ℃， the bolting days of No.2-8 were 104， 98， 11 77， 90， 66， and 66 d， respectively， which were 20， 6， 10， 20， 12 and 7 d earlier than those of the control groups at room temperature， respectively. Specifically， the 4 ℃ treatment caused no significant differences in the bolting time of No.1 and No.4， while for other varieties， the bolting time was significantly shortened under low temperature treatment. The bolting time of No.2 and No.5 was shortened the most during the low temperature treatment， that is， advanced by 20 d. In summary， treating germinated radish seeds at 4 ℃ for 3 weeks advanced bolting for most radish varieties， but a few radish varieties （such as No.1 and No.4） could not bolt in advance.