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of tissue culture. The efficient multiplication technology of blueberry （Vaccinium corymbosum） plantlets in STTBs was systematically studied through L9（34） orthogonal experiment with three factors （culture medium volume， swing angle， and inoculation density） and completely randomized experiment with two factors （swing frequency and immersion time）. The results showed that the optimal culture parameters were set as follows： the culture medium volume of 250 ml/bottle， the swing angle at 45°， and the inoculation density of 60 plantlets/bottle. The optimal swing frequency was 1 time/6 h， and the immersion time was set as 60 s.
Key words Vaccinium corymbosum； Temporary immerion； Plantlets
Blueberry （Vaccinium corymbosum） is a perennial deciduous or evergreen shrub in Vaccinium of Ericaceae family. It has blue fruit， which is coated with a layer of white fruit powder and has fine， sweet and sour flesh with unique flavor[1]. The fruit has rich nutrition with healthcare functions such as antioxidation[2]， memory and visionimproving[3-4]， anticancer[5]， antiaging[6]and antibacterial functions[7]， and is thus listed as one of the five healthy foods by the United Nations Food and Agriculture Organization[8].
Blueberry is introduced into China from the mid1980s. With the development of economic society and the development of peoples living standard， it attracts more and more attention， its planting area is enlarging increasingly， and its seedlings are underproduced. Conventional cutting propagation hardly could satisfy the demand for blueberry seedlings now. At present， the propagation of highquality blueberry seedlings mainly depends on industrialized production through tissue culture technology. The tissue culture of blueberry is mainly carried out by solid culture method， which consumes a mass of manual labor in the operation process with lower proliferation rate and is thus a kind of labor intensive technique[9]. Furthermore， the technique has the disadvantages of complicated technical operation， low automatic and semiautomatic degree， and large consumption of nutrient substances， as well as higher cost. Temporary immersion liquid culture technology is a new kind of tissue culture technology， and it gradually overcomes these limits[10]. The research of this technology was started as early as the 1980s at abroad， and it has been widely applied in agriculture， forestry and food industry[11-13]. Related research was started later in China， but in recent years， the plant species propagated using this technology are increasing gradually[14-20]， which promotes the largescale production of highquality seedlings of some economic crops in China. Temporary immersion bioreactors system （TIBs for short） is used for periodic immersion culture of culture materials in medium under aseptic condition， realizing the purpose of rapid propagation. Such culture method allows close contact of plant tissues with liquid medium， thereby stimulating and promoting the absorption of nutrients and plant hormones into explants. Furthermore， the temporary and continuous vibration of medium avoid the lack of oxygen in liquid medium， so the cultured tissue culture plantlets have higher multiplication rate[21]. Meanwhile， such culture method could reduce culture links and reduce labor cost and production cost.
Jiangsu Vocational College of Agriculture and Forestry introduced TIBs from 2010， and through continuous improvement， the swinging type temporary immersion bioreactors system （STIBs for short） （patent number： CN201220094482.8） was developed. This reactor requires the inclined swing of swinging frames， and realizes the axial inclined swing of rows of containers on the swinging frame. The liquid in the containers immerses the plant tissues temporarily and periodically， and the timer and inclined angle adjusting device installed on the reactor would adjust the immersion frequency and the inclined angle of the culture containers according to plant species and the volume of medium. The reactors use rectangular bigbottle incubators （length×width×height=25 cm×12 cm×13.5 cm）， which have the characteristics of simple structure， large volume， high propagation efficiency and high automatic degree， and could produce tissue culture plantlets continuously with high efficiency. In this study， with the patent technology as the culture system， the multiplication technology of blueberry tissue culture plantlets was systematically studied， and an STIBs multiplication system of blueberry tissue culture plantlets was constructed. This study will provide experimental data for efficient industrialized nursing of blueberry seedlings and promote the popularization and application of novel tissue culture plantlet raising system.
Materials and Methods
Materials
The third generation of healthy rootless tissue culture plantlets of the variety， V. corymbosum cv. Berkeley were obtained through solid tissue culture. The plantlets had a height of 6-8 cm， with 12-15 leaves.
The culture device was a novel swinging type intermittent immersion bioreactor （STIBs） with independent intellectual property rights. Methods
Establishment of basic culture parameters
According to the culture characteristics of STIBs， at first， the three parameters， volume of nutrient solution， swing angle and inoculation density were studied. Threefactor threelevel orthogonal test L9（34） design was adopted， and the levels of the factors are shown in Table 1. The experiment had nine treatments， each including 15 bottles， with three replicates.
The inoculation adopted the simple method of cutting the tissue culture plantlets in the middle. Each explant had a length of 3-4 cm， with 4-5 internodes. The culture medium was the multiplication medium： 1/2 WPM+30.0 g/L sucrose +6BA 500 mg/L， with a pH of （5.2±0.1）. The plantlets were cultured under following conditions： the culture temperature at （25±1） ℃， the illumination intensity of 36-54 μmol/（m2·s）， and the photoperiod of 12 h illumination/12 h dark. The swing parameter was set as 60 s/6 h （i.e.， swinging once per 6 h， under immersion time of 60 s）. The growth condition of multiplied plantlets was observed 60 d after inoculation， the number of multiplied plantlets was investigated， and the multiplication coefficient and average fresh weight were calculated： Multiplication coefficient=Number of multiplied plantlets with a height over 1 cm/Number of inoculated plantlets； Average fresh weight=Fresh weight of multiplied plantlets in each bottle/Number of multiplied plantlets. Variance analysis was performed with SPSS 20.0， so as to determine the optimal culture parameters.
Screening of swing frequency and immersion time
On the basis of "Establishment of basic culture parameters"， the swing frequency was set with four levels （1 time/3 h， 1 time/6 h， 1 time/9 h， 1 time/12 h）， and the immersion time also had four levels （30， 60， 90， 120s）. The test adopted completely randomized arrangement. There were 16 treatments， each including 15 bottles， with three replicates. Sixty days later， the growth condition of the plantlets was observed and recorded， and the multiplication coefficient and average fresh weight were investigated. Variance analysis and multiple comparisons were performed， to screen the optimal swing frequency and immersion time.
Results and Analysis
Multiplication effect of blueberry tissue culture plantlets in STIBs under different culture parameters
According to the size and structural characteristics of culture containers， combining with the operating principle of STIBs， the multiplication condition of tissue culture plantlets was directly related to the volume of culture medium， swing angle and inoculation density. L9（34） orthogonal test design was performed with the three factors， and the results are shown in Table 2. It could be seen from the results in Table 2 that the multiplication coefficient and average fresh weight both appeared in treatment 4， which had the multiplication coefficient of 6.93 and the average fresh weight up to 73.47 mg/plant. Combining with the growth condition， it could be seen from the analysis that treatments 2， 4 and 5 achieved better results.
Range analysis
According to the results of the orthogonal test， the multiplication coefficient and average fresh weight of various treatments were subjected to range analysis， and the results are shown in Table 3.
Range （R） reflects the effects of various factors on the multiplication culture of blueberry tissue culture plantlets， and a larger R value indicates a more obvious effect. The illustrative diagram of the range analysis is shown as Fig. 1. It could be seen from Fig. 1 that the various culture parameters differed in the effect on the multiplication of blueberry tissue culture plantlets. As to the effects on both the multiplication coefficient and average fresh weight， the various culture parameters ranked as swing factor>inoculation density>culture medium factor. And the two indices were in positive correlation.
Variance analysis
The results of the orthogonal test were subjected to variance analysis with SPSS20.0 software. The results are shown in Table 4.
It could be seen from Table 4 that the effect of swing angle on multiplication coefficient reached the very significant level， and that on average fresh weight reached the significant level. However， the effects of culture medium volume and inoculation density on multiplication coefficient and average fresh weight were not significant. Therefore， multiple comparisons should be performed on the multiplication coefficient and average fresh weight at various swing angle levels.
Multiple comparisons
Multiple comparisons were performed on the multiplication coefficient and average fresh weight in different swing angle treatments by LSD test. The results are shown in Table 5.
It could be seen from Table 5 that the average multiplication coefficient was the highest in the 45° treatment， reaching 6.63， which was significantly different from the 30° treatment， and very significantly different from the 60° treatment. The difference between the 30° treatment and the 60° treatment also reached the very significant level， and specifically， the multiplication coefficient of the 60° treatment was the lowest of 4.32. The 45° treatment exhibited the largest average fresh weight， reaching 69.59 mg， which was not significantly different from the 30° treatment， but significantly different from the 60° treatment. Therefore， comprehensively from the multiplication coefficient and average fresh weight， the optimal swing angle was set as 45°. Consequently， it could be seen from the results of the orthogonal test and variance analysis， the optimal culture parameters were set as follows： the culture medium volume of 250 ml/bottle， the swing angle at 45°， and the inoculation density of 60 plantlets/bottle.
Multiplication effects of swing frequency and immersion time on blueberry tissue culture plantlets in STIBs
A completely randomized test was carried out on swing frequency and immersion time. The multiplication coefficient and average fresh weight values in different treatments are shown in Table 6 and table 7， respectively.
The results of the twofactor random test were subjected to variance analysis， as shown in Table 8.
It could be seen from the results of the variance analysis that swing frequency and immersion both had a very significant effect on multiplication coefficient； and swing frequency had a very significant effect on the average fresh weight， and immersion time also had a significant effect on the average fresh weight. Therefore， multiple comparisons should be further performed on the results of various treatments.
The results of the twofactor random test should be further subjected to multiple comparison by LSD test. The results are shown in Table 9.
It could be seen from Table 9 that among the various swing frequency treatments， the 1 time/6 h treatment had the highest multiplication coefficient， while the 1 time/12 h treatment showed the lowest multiplication coefficient， and the differences between any two of the various levels all reached the very significant level. As to the average fresh weight， the 1 time/6 h treatment had the highest average fresh weight， which was very significantly different from other treatments； there was no significant difference between the 1 time/3 h and 1 time/9 h treatments； and the 1 time/12 h treatment exhibited the lowest average fresh weight， which was different from other treatments at the very significant level.
Among the various immersion time treatments， the 30 s treatment exhibited the lowest multiplication coefficient and average fresh weight， and compared with other treatments， the differences in multiplication coefficient reached the very significant level， and the differences in average fresh weight reached the significant level. There were no significant differences in the average multiplication coefficient and average fresh weight between any two of the 60， 90 and 120 s treatments. However， the 90 and 120 s treatments showed partial vitrified plantlets to different degrees， while the 30 and 60 s treatments showed no vitrification phenomenon. Comprehensively form the multiplication effect and vitrification， the optimal immersion time was selected as 60 s. Conclusions and Discussion
The novel swinging type temporary immersion reactor is a kind of developed and improved novel temporary immersion liquid culture device， which has the advantages of simple structure， convenient control， large single bottle volume and high use ratio compared with the dualbottle and RITA temporary immersion liquid culture system commonly used at present. It is more beneficial to automatic control. STIBs tissue culture propagation technique is a kind of new industrialized plant tissue culture propagation technique with very high popularization and application value.
In this study， during the STIBs multiplication culture of blueberry tissue culture plantlets， the effects of culture medium volume， swing angle and inoculation density on the multiplication ranked as swing angle>inoculation density>culture medium volume. Among them， the effect of the swing angle factor reached the very significant level. This experimental result was mainly determined by the structure and operating principle of STIBs， and this result is also true for the multiplication culture of other plants in STIBs. Also， the optimal parameters set for the STIBs multiplication culture of blueberry tissue culture plantlets， the culture medium volume of 250 ml/bottle， swing angle at 45 ° and the inoculation density of 60 plantlets/bottle also have reference value for the STIBs multiplication culture of other plants.
It could be known comprehensively from the multiplication coefficient and average fresh weight， the optimal swing frequency for the STIBs multiplication culture of blueberry tissue culture plantlets was 1 time/6 h. Within the immersion time of 60-120 s， there were no significant differences in the multiplication effects， but taking the vitrification phenomenon factor into consideration， the optimal immersion time was selected to be 60 s， which also has reference value for the STIBs multiplication culture of other plants.
STIBs is a new way for raising tissue culture plantlets， while the research on its application is still very limited， and the setting of various culture parameters still needs further detailed study. Furthermore， the STIBs propagation research of more plants should be conducted， especially on the differences between different plants， which is of important significance to the popularization and application of the this new tissue culture plantlet raising method.
References
[1]HE Q， WU LR. Discussions on the biological function of nutrients in blueberry fruit[J]. Northern Horticulture， 2010 （24）： 222-224. （in Chinese） [2]BORNSEK SM， ZIBERNA L， POLAK T， et al. Bilberry and blueberry anthocyanins act as powerful intracellular antioxidants in mammalian cells[J]. Food Chem， 2012， 134（4）： 1878-1884. （in Chinese）
[3]ZHAO XL. Research progress of ingredient and healthy function of blueberry[J]. Chinese Wild Plant Resources， 2011， 30（6）：19-23
[4]WANG SS， SUN AD， LI SY. Advancement of health function of blueberry and its utilization[J]. ？Food and Nutrition in China， 2010 （6）：17-20. （in Chinese）
[5]WU X， RAHAL O， KANG J， et al. In utero and lactational exposure to blueberry via maternal diet promotes mammary epithelial differentiation in prepubescent female rats[J]. Nutr Res， 2009， 29 （11）： 802-811
[6]DEL BO C， MARTINI D， VENDRAME S， et al. Improvement of lymphocyte resistance against H2O2induced DNA damage in SpragueDawley rats after eight weeks of a wild blueberry （Vaccinium angustifolium）enriched diet[J]. Mutat ResGenet Toxicol Environ Mutagen， 2010， 703（2）： 158-162
[7]LACOMBE A， WU VCH， WHITE J， et al. The antimicrobial properties of the lowbush blueberry （Vaccinium angustifolium） fractional components against foodborne pathogens and the conservation of probiotic Lactobacillus rhamnosus[J]. Food Microbiol， 2012， 30 （1）： 124-131
[8]KADER F， ROVEL B， GIRARDIN M， METCHE M. Fractionation and identification of the phenolic compounds of Highbush blueberries （Vaccinium corgmbosum L.） [J]. Food Chem， 1996， 55（1）： 35-40
[9]GAO MP， LI ZC， ZHANG C， et al. Efficient propagation of Eleocharis dulcis plantlets in temporary immersion bioreactors（TIBs）[J]. Guangxi Agricultural Sciences， 2016， 47（10）： 1653-1657. （in Chinese）
[10]ZHAO WF， WANG LH. Study on temporary immersion bioreactor in tissue culture[J]. Journal of Anhui Agricultural Sciences， 2007， 35（2）： 317-320， 323. （in Chinese）
[11]MEYER GM， BANASIAK M， NTOYI TT， et al. Sugarcane plants from temporary immersion culture： acclimating for commercial production[J]. Acta Hortic， 2009， 812： 323-327
[12]PEЙARAMREZ YJ， JUREZGMEZ J， GMEZLPEZ L， et al. Multiple adventitious shoot formation in Spanish red cedar （Cedrela odorata L.） cultured in vitro using juvenile and mature tissues： an improved micropropagation protocol for a highly valuable tropical tree species[J]. In Vitro Cell DevPl， 2010， 46： 149-160
[13]SNYMAN SJ， MEYER GM， KOCH AC， et al. Applications of in vitro culture systems for commercial sugarcane production and improvement[J]. In Vitro Cell DevPl， 2011a， 47： 234-249 [14]YANG L， QIN G， SU J， et al. Micropropagation of banana （Musa AAA Cavendish Var. Williams B6） in Temporary immersion bioreactor system （TIBS） [J]. Journal of Fruit Science， 2010b， 27（6）： 1010-1013. （in Chinese）
[15]YANG L， QIN G， YANG LT， et al. Optimization of Saccharum sinensis Roxb rapid multiplication in temporary immersion bioreactors system[J]. Chinese Journal of Tropical Crops， 2010a ， 31 （4）： 614-620. （in Chinese）
[16]YANG L， QIN G， YANG LT， et al. Optimization of sugarcane rapid propagation in temporary immersion bioreactors system[J]. Journal of South China Agricultural University， 2011， 32（1）：37-41. （in Chinese）
[17]XU YL， ZHANG JM. A efficient largescale tissue culture methodtemporary immersion culture method[J]. Plant Physiology Communications， 2013， 49（4）：392-399 . （in Chinese）
[18]ZHANG ZZ， SHI K， YU ZR， et al. Research on rapid propagation of strawberry based on plant micropropagation bioreactor[J]. Chinese Agricultural Science Bulletin， 2014， 30（16）： 216-220. （in Chinese）
[19]SHI K， ZHENG CX. Liquid culture system of rapid propagation of Cotinus coggygria ‘royal purple’ [J]. Plant Physiology Communications， 2015， 51（4）：553-558. （in Chinese）
[20]GAO MP， LI ZC， ZHANG C， et al. Efficient propagation of Eleocharis dulcis plantlets in temporary immersion bioreactors（TIBs）[J]. Guangxi Agricultural Sciences， 2016， 47（10）：1653-1657. （in Chinese）
[21]MEHROTRA S， GOEL MK， KUKREJA AK， et al. Efficiency of liquid culture system over conventional micropropagation： A progress towards commercialization[J]. African Journal of Biotechnology， 2007， 6（13）：1484-1492.
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
Key words Vaccinium corymbosum； Temporary immerion； Plantlets
Blueberry （Vaccinium corymbosum） is a perennial deciduous or evergreen shrub in Vaccinium of Ericaceae family. It has blue fruit， which is coated with a layer of white fruit powder and has fine， sweet and sour flesh with unique flavor[1]. The fruit has rich nutrition with healthcare functions such as antioxidation[2]， memory and visionimproving[3-4]， anticancer[5]， antiaging[6]and antibacterial functions[7]， and is thus listed as one of the five healthy foods by the United Nations Food and Agriculture Organization[8].
Blueberry is introduced into China from the mid1980s. With the development of economic society and the development of peoples living standard， it attracts more and more attention， its planting area is enlarging increasingly， and its seedlings are underproduced. Conventional cutting propagation hardly could satisfy the demand for blueberry seedlings now. At present， the propagation of highquality blueberry seedlings mainly depends on industrialized production through tissue culture technology. The tissue culture of blueberry is mainly carried out by solid culture method， which consumes a mass of manual labor in the operation process with lower proliferation rate and is thus a kind of labor intensive technique[9]. Furthermore， the technique has the disadvantages of complicated technical operation， low automatic and semiautomatic degree， and large consumption of nutrient substances， as well as higher cost. Temporary immersion liquid culture technology is a new kind of tissue culture technology， and it gradually overcomes these limits[10]. The research of this technology was started as early as the 1980s at abroad， and it has been widely applied in agriculture， forestry and food industry[11-13]. Related research was started later in China， but in recent years， the plant species propagated using this technology are increasing gradually[14-20]， which promotes the largescale production of highquality seedlings of some economic crops in China. Temporary immersion bioreactors system （TIBs for short） is used for periodic immersion culture of culture materials in medium under aseptic condition， realizing the purpose of rapid propagation. Such culture method allows close contact of plant tissues with liquid medium， thereby stimulating and promoting the absorption of nutrients and plant hormones into explants. Furthermore， the temporary and continuous vibration of medium avoid the lack of oxygen in liquid medium， so the cultured tissue culture plantlets have higher multiplication rate[21]. Meanwhile， such culture method could reduce culture links and reduce labor cost and production cost.
Jiangsu Vocational College of Agriculture and Forestry introduced TIBs from 2010， and through continuous improvement， the swinging type temporary immersion bioreactors system （STIBs for short） （patent number： CN201220094482.8） was developed. This reactor requires the inclined swing of swinging frames， and realizes the axial inclined swing of rows of containers on the swinging frame. The liquid in the containers immerses the plant tissues temporarily and periodically， and the timer and inclined angle adjusting device installed on the reactor would adjust the immersion frequency and the inclined angle of the culture containers according to plant species and the volume of medium. The reactors use rectangular bigbottle incubators （length×width×height=25 cm×12 cm×13.5 cm）， which have the characteristics of simple structure， large volume， high propagation efficiency and high automatic degree， and could produce tissue culture plantlets continuously with high efficiency. In this study， with the patent technology as the culture system， the multiplication technology of blueberry tissue culture plantlets was systematically studied， and an STIBs multiplication system of blueberry tissue culture plantlets was constructed. This study will provide experimental data for efficient industrialized nursing of blueberry seedlings and promote the popularization and application of novel tissue culture plantlet raising system.
Materials and Methods
Materials
The third generation of healthy rootless tissue culture plantlets of the variety， V. corymbosum cv. Berkeley were obtained through solid tissue culture. The plantlets had a height of 6-8 cm， with 12-15 leaves.
The culture device was a novel swinging type intermittent immersion bioreactor （STIBs） with independent intellectual property rights. Methods
Establishment of basic culture parameters
According to the culture characteristics of STIBs， at first， the three parameters， volume of nutrient solution， swing angle and inoculation density were studied. Threefactor threelevel orthogonal test L9（34） design was adopted， and the levels of the factors are shown in Table 1. The experiment had nine treatments， each including 15 bottles， with three replicates.
The inoculation adopted the simple method of cutting the tissue culture plantlets in the middle. Each explant had a length of 3-4 cm， with 4-5 internodes. The culture medium was the multiplication medium： 1/2 WPM+30.0 g/L sucrose +6BA 500 mg/L， with a pH of （5.2±0.1）. The plantlets were cultured under following conditions： the culture temperature at （25±1） ℃， the illumination intensity of 36-54 μmol/（m2·s）， and the photoperiod of 12 h illumination/12 h dark. The swing parameter was set as 60 s/6 h （i.e.， swinging once per 6 h， under immersion time of 60 s）. The growth condition of multiplied plantlets was observed 60 d after inoculation， the number of multiplied plantlets was investigated， and the multiplication coefficient and average fresh weight were calculated： Multiplication coefficient=Number of multiplied plantlets with a height over 1 cm/Number of inoculated plantlets； Average fresh weight=Fresh weight of multiplied plantlets in each bottle/Number of multiplied plantlets. Variance analysis was performed with SPSS 20.0， so as to determine the optimal culture parameters.
Screening of swing frequency and immersion time
On the basis of "Establishment of basic culture parameters"， the swing frequency was set with four levels （1 time/3 h， 1 time/6 h， 1 time/9 h， 1 time/12 h）， and the immersion time also had four levels （30， 60， 90， 120s）. The test adopted completely randomized arrangement. There were 16 treatments， each including 15 bottles， with three replicates. Sixty days later， the growth condition of the plantlets was observed and recorded， and the multiplication coefficient and average fresh weight were investigated. Variance analysis and multiple comparisons were performed， to screen the optimal swing frequency and immersion time.
Results and Analysis
Multiplication effect of blueberry tissue culture plantlets in STIBs under different culture parameters
According to the size and structural characteristics of culture containers， combining with the operating principle of STIBs， the multiplication condition of tissue culture plantlets was directly related to the volume of culture medium， swing angle and inoculation density. L9（34） orthogonal test design was performed with the three factors， and the results are shown in Table 2. It could be seen from the results in Table 2 that the multiplication coefficient and average fresh weight both appeared in treatment 4， which had the multiplication coefficient of 6.93 and the average fresh weight up to 73.47 mg/plant. Combining with the growth condition， it could be seen from the analysis that treatments 2， 4 and 5 achieved better results.
Range analysis
According to the results of the orthogonal test， the multiplication coefficient and average fresh weight of various treatments were subjected to range analysis， and the results are shown in Table 3.
Range （R） reflects the effects of various factors on the multiplication culture of blueberry tissue culture plantlets， and a larger R value indicates a more obvious effect. The illustrative diagram of the range analysis is shown as Fig. 1. It could be seen from Fig. 1 that the various culture parameters differed in the effect on the multiplication of blueberry tissue culture plantlets. As to the effects on both the multiplication coefficient and average fresh weight， the various culture parameters ranked as swing factor>inoculation density>culture medium factor. And the two indices were in positive correlation.
Variance analysis
The results of the orthogonal test were subjected to variance analysis with SPSS20.0 software. The results are shown in Table 4.
It could be seen from Table 4 that the effect of swing angle on multiplication coefficient reached the very significant level， and that on average fresh weight reached the significant level. However， the effects of culture medium volume and inoculation density on multiplication coefficient and average fresh weight were not significant. Therefore， multiple comparisons should be performed on the multiplication coefficient and average fresh weight at various swing angle levels.
Multiple comparisons
Multiple comparisons were performed on the multiplication coefficient and average fresh weight in different swing angle treatments by LSD test. The results are shown in Table 5.
It could be seen from Table 5 that the average multiplication coefficient was the highest in the 45° treatment， reaching 6.63， which was significantly different from the 30° treatment， and very significantly different from the 60° treatment. The difference between the 30° treatment and the 60° treatment also reached the very significant level， and specifically， the multiplication coefficient of the 60° treatment was the lowest of 4.32. The 45° treatment exhibited the largest average fresh weight， reaching 69.59 mg， which was not significantly different from the 30° treatment， but significantly different from the 60° treatment. Therefore， comprehensively from the multiplication coefficient and average fresh weight， the optimal swing angle was set as 45°. Consequently， it could be seen from the results of the orthogonal test and variance analysis， the optimal culture parameters were set as follows： the culture medium volume of 250 ml/bottle， the swing angle at 45°， and the inoculation density of 60 plantlets/bottle.
Multiplication effects of swing frequency and immersion time on blueberry tissue culture plantlets in STIBs
A completely randomized test was carried out on swing frequency and immersion time. The multiplication coefficient and average fresh weight values in different treatments are shown in Table 6 and table 7， respectively.
The results of the twofactor random test were subjected to variance analysis， as shown in Table 8.
It could be seen from the results of the variance analysis that swing frequency and immersion both had a very significant effect on multiplication coefficient； and swing frequency had a very significant effect on the average fresh weight， and immersion time also had a significant effect on the average fresh weight. Therefore， multiple comparisons should be further performed on the results of various treatments.
The results of the twofactor random test should be further subjected to multiple comparison by LSD test. The results are shown in Table 9.
It could be seen from Table 9 that among the various swing frequency treatments， the 1 time/6 h treatment had the highest multiplication coefficient， while the 1 time/12 h treatment showed the lowest multiplication coefficient， and the differences between any two of the various levels all reached the very significant level. As to the average fresh weight， the 1 time/6 h treatment had the highest average fresh weight， which was very significantly different from other treatments； there was no significant difference between the 1 time/3 h and 1 time/9 h treatments； and the 1 time/12 h treatment exhibited the lowest average fresh weight， which was different from other treatments at the very significant level.
Among the various immersion time treatments， the 30 s treatment exhibited the lowest multiplication coefficient and average fresh weight， and compared with other treatments， the differences in multiplication coefficient reached the very significant level， and the differences in average fresh weight reached the significant level. There were no significant differences in the average multiplication coefficient and average fresh weight between any two of the 60， 90 and 120 s treatments. However， the 90 and 120 s treatments showed partial vitrified plantlets to different degrees， while the 30 and 60 s treatments showed no vitrification phenomenon. Comprehensively form the multiplication effect and vitrification， the optimal immersion time was selected as 60 s. Conclusions and Discussion
The novel swinging type temporary immersion reactor is a kind of developed and improved novel temporary immersion liquid culture device， which has the advantages of simple structure， convenient control， large single bottle volume and high use ratio compared with the dualbottle and RITA temporary immersion liquid culture system commonly used at present. It is more beneficial to automatic control. STIBs tissue culture propagation technique is a kind of new industrialized plant tissue culture propagation technique with very high popularization and application value.
In this study， during the STIBs multiplication culture of blueberry tissue culture plantlets， the effects of culture medium volume， swing angle and inoculation density on the multiplication ranked as swing angle>inoculation density>culture medium volume. Among them， the effect of the swing angle factor reached the very significant level. This experimental result was mainly determined by the structure and operating principle of STIBs， and this result is also true for the multiplication culture of other plants in STIBs. Also， the optimal parameters set for the STIBs multiplication culture of blueberry tissue culture plantlets， the culture medium volume of 250 ml/bottle， swing angle at 45 ° and the inoculation density of 60 plantlets/bottle also have reference value for the STIBs multiplication culture of other plants.
It could be known comprehensively from the multiplication coefficient and average fresh weight， the optimal swing frequency for the STIBs multiplication culture of blueberry tissue culture plantlets was 1 time/6 h. Within the immersion time of 60-120 s， there were no significant differences in the multiplication effects， but taking the vitrification phenomenon factor into consideration， the optimal immersion time was selected to be 60 s， which also has reference value for the STIBs multiplication culture of other plants.
STIBs is a new way for raising tissue culture plantlets， while the research on its application is still very limited， and the setting of various culture parameters still needs further detailed study. Furthermore， the STIBs propagation research of more plants should be conducted， especially on the differences between different plants， which is of important significance to the popularization and application of the this new tissue culture plantlet raising method.
References
[1]HE Q， WU LR. Discussions on the biological function of nutrients in blueberry fruit[J]. Northern Horticulture， 2010 （24）： 222-224. （in Chinese） [2]BORNSEK SM， ZIBERNA L， POLAK T， et al. Bilberry and blueberry anthocyanins act as powerful intracellular antioxidants in mammalian cells[J]. Food Chem， 2012， 134（4）： 1878-1884. （in Chinese）
[3]ZHAO XL. Research progress of ingredient and healthy function of blueberry[J]. Chinese Wild Plant Resources， 2011， 30（6）：19-23
[4]WANG SS， SUN AD， LI SY. Advancement of health function of blueberry and its utilization[J]. ？Food and Nutrition in China， 2010 （6）：17-20. （in Chinese）
[5]WU X， RAHAL O， KANG J， et al. In utero and lactational exposure to blueberry via maternal diet promotes mammary epithelial differentiation in prepubescent female rats[J]. Nutr Res， 2009， 29 （11）： 802-811
[6]DEL BO C， MARTINI D， VENDRAME S， et al. Improvement of lymphocyte resistance against H2O2induced DNA damage in SpragueDawley rats after eight weeks of a wild blueberry （Vaccinium angustifolium）enriched diet[J]. Mutat ResGenet Toxicol Environ Mutagen， 2010， 703（2）： 158-162
[7]LACOMBE A， WU VCH， WHITE J， et al. The antimicrobial properties of the lowbush blueberry （Vaccinium angustifolium） fractional components against foodborne pathogens and the conservation of probiotic Lactobacillus rhamnosus[J]. Food Microbiol， 2012， 30 （1）： 124-131
[8]KADER F， ROVEL B， GIRARDIN M， METCHE M. Fractionation and identification of the phenolic compounds of Highbush blueberries （Vaccinium corgmbosum L.） [J]. Food Chem， 1996， 55（1）： 35-40
[9]GAO MP， LI ZC， ZHANG C， et al. Efficient propagation of Eleocharis dulcis plantlets in temporary immersion bioreactors（TIBs）[J]. Guangxi Agricultural Sciences， 2016， 47（10）： 1653-1657. （in Chinese）
[10]ZHAO WF， WANG LH. Study on temporary immersion bioreactor in tissue culture[J]. Journal of Anhui Agricultural Sciences， 2007， 35（2）： 317-320， 323. （in Chinese）
[11]MEYER GM， BANASIAK M， NTOYI TT， et al. Sugarcane plants from temporary immersion culture： acclimating for commercial production[J]. Acta Hortic， 2009， 812： 323-327
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Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU