Key Technology of In-vitro Culture for a New Vaccinium uliginosum Cultivar Zishuijing

Hailong WENG,Xinhua TIAN,Deshan SHI

Heilongjiang Institute of Forestry Sciences,Harbin 150081,China

Key Technology of In-vitro Culture for a New Vaccinium uliginosum Cultivar Zishuijing

Hailong WENG,Xinhua TIAN,Deshan SHI*

Heilongjiang Institute of Forestry Sciences,Harbin 150081,China

[Objective]This study aimed to discuss the key technology ofin-vitroculture for a newVaccinium uliginosumcultivar Zishuijing.[Method]Through the screening and optimization of sterilization method for explants,sampling time,multiplication, nursing and rooting culture,a matching clone propagation system was established for the newVaccinium uliginosumcultivar Zishuijing.[Result]The explants were sterilized with 0.1%HgCl2for 3 min;the differentiation and multiplication medium of Zishuijing was composed of WPM(modified),6-BA(1.0 mg/L)and ZT(1.0 MG/L);the rootless tube seedlings were transplanted in organic matrix(sawdust∶bark∶peat=1∶1∶1)in September and cultured at air relative humidity of 80%-90%and temperature of 20-25℃,and after 50 d,the rooting rate reached 72.4%.[Conclusion]The key technology of in-vitro culture for the newVaccinium uliginosumcultivar Zishuijing was established,thereby providing technical support for large-scale industrialized seedling production of Zishuijing.

Vaccinium uliginosum;Zishuijing;In-vitroculture;Condition optimization

T he newVaccinium uliginosumcultivar Zishuijing was bred by crossing wildVaccinium uliginosum(♀)with North American blueberry(♂).It passed the variety identification in December,2014.The newlybred cultivar has tall plant height with plant height of adult trees ranging from 0.5-0.8 m.Its fruit setting amount is 1 200 kg/hm2.Zishuijing has racemes and blue-purple fruits that are suitable for processing.The 100-grain weight of Zishuijing is about 60 g.Compared with similar varieties at home and abroad,Zishuijing has advantages of drought resistance and cold resistance.The Zishuijing seedlings will grow into a garden one year after their transplanting,and from the second year(since the transplanting)on,a high yield can be obtained.The Zishuijing is suitable to be planted in Greater Khingan Mountains-Heihe-Lesser Khingan Mountains line.The active accumulated temperature of Zishuijing is below 2 300℃.

Currently,there have been scarce researches on application and new variety breeding ofVaccinium uliginosumin China.Thein-vitroculture system ofVaccinium uliginosumhas not been established.The survival rate of transplanted tissue culture seedlin-gs is still low[1-4],which is not conducive to large-scale production and promotion of new varieties. Therefore,the matching clone propagation system of a newVaccinium uliginosumcultivar Zishuijing was discussed in this study so as to provide technical support for large-scale industrialized seedling production.

Materials and Methods

Materials

The dormant branches of Zishuijing were used as test material.They were soaked in clean water for germination induction,and the water was changed every two days.The water temperature was controlled at 18-20℃.After about 7 d,the dormant

Methods

Sterilization of explantsAfter axillary buds sprouted,stem segments with buds,in length of about 1.0 cm, were cut off.They were washed repeatedly with water.The sterilization was performed on a super-clean bench.A total of 6 treatments were designed.The stem segments were soaked in 75%ethanol for 30 s and 0.1%HgCl2for 3 min,and then they were rinsed 3 times with sterile water (S1);the stem segments were soaked in 75%ethanol for 30 s and 0.1% Hg Cl2for 5 min,and then they were rinsed 3 times with sterile water(S2); the stem segments were soaked in 0.1%HgCl2for 3 min,and then rinsed 3 times with sterile water(S3);the stem segments were soaked in 0.1% HgCl2for 5 min,and then rinsed 3 times with sterile water(S4);the stem segments were soaked in 75%ethanol for 30 s and 5%sodium hypochlorite for 5 min,and then they were rinsed 3 times with sterile water(S5);the stem segments were soaked in 75%ethanol for 30 s and 5%sodium hypochlorite for 10 min,and then they were rinsed 3 times with sterile water(S6).After the sterilization,the excess water on the surface of stem segments was dried off with sterile filter paper.There were 10 stem segments in each treatment, and there were three replicates for each treatment.The sterilization effect was observed 5 d later.

Sampling time and seedling growth statusThe robust and healthy Zishuijing dormant branches were collected in April.The water culture was adopted for promoting sprouting.The young buds were inoculated in the modified WPM medium,in which the original CaCl2and K2SO4were replaced by Ca(NO3)2·4H2O and KNO3. The local-year stem segments with single buds were selected for inoculation in July and October,respectively. The contamination status was observed 7,14 and 21 d after the inoculation respectively so as to investigate the effect of different inoculation time on seedling growth status.In the medium,sucrose(30 g/L)and agar (Japan,6 g/L)were added.The pH was adjusted to 5.8.In the daytime, the culture temperature was(25±1)℃, and in the night,the culture temperature was(20±1)℃.The light intensity was 2 000 lx,and the lighting duration was 12 h/d.

Growth induction of lateral buds

The modified WPM medium(replacing CaCl2and K2SO4with Ca(NO3)2·4H2O and KNO3)was used as the basic medium.Under sterile conditions,the stem segments with single buds were inoculated into modified WPM medium that was added with ZT(0.5 mg/L),sucrose(30 g/L)and agar(6 g/L).The specific culture conditions were as described above.

Differentiation and multiplication culture of tube seedlingsAfter the axillary buds on stem segments elongated,the stem segments were inoculated into the modified WPM medium that was added with 6-benzyladenine (6-BA)and zeatin(ZT)respectively. The 6-BA and ZT were treated as two test factors,and total three levels were designed for each of the two factors. The L9(34)orthogonal test was designed for screening of multiplication culture medium for buds.There were two replicates for each treatment,and there were 10 culture bottles for each replicate.The seedling height and seedling number were investigated 35 d later.

Culture of robustVaccinium uliginosumseedlingsTheVaccinium uliginosumtube seedlings are relatively soft and thin,so they need for a period of seedling culture to induce rooting.The tube seedling segments,in length of about 1.0 cm,were cut off and inoculated in modified WMP medium added with ZT(1.0 mg/L)and sucrose(30 g/L).They were cultured at light intensity of 2 000 lx(I1)and 3 000 lx(I2)respectively.The culture all lasted for 50 h.There were two replicates for each treatment,and there were 10 culture bottles for each replicate.After a 50-d culture,the seedling height, stem diameter,multiplication multiple and growth status were investigated.

Rooting culture of tube seedlings Effect of different matrix on rooting ofVaccinium uliginosumseedlingsThe fermented organic matrixes were sterilized with saturated KMnO4solution for 30 min and then flushed with a large amount of water.After covered with plastic film,the matrixes were exposed to sunlight for more than 24 h. Subsequently,the organic matrixes were ventilated for use.A total of 6 kinds of matrixes were designed,including peat+husk+bark(1∶1∶1, Ma1),coniferous+bark+broadleaf (1∶1∶1,Ma2),coniferous+broadleaf+ husk(1∶1∶1,Ma3),bark+broadleaf(1∶1, Ma4),sawdust+bark+peat(1∶1∶1, Ma5)and straw rotting fungus fermented bran+bark(1∶1,Ma6).

After the robust seedling culture, theVaccinium uliginosumseedlings were planted in the six kinds of matrixes respectively.There were 20 plants for each treatment,and there were 3 replicates for each treatment.After 30 d,the survival rates ofVaccinium uliginosumtube seedlings in the six kinds of matrixes were investigated,and then the optimum matrix suitable forVaccinium uliginosumtube seedlings was screened out.

Effect of different hormone concentration on transplanting ofVaccinium uliginosumtissue culture seedlingsThe M5 and M6 were selected as culture matrixes.The rootless culture seedlings were dipped in 500 and 1 000 mg/L of ABT respectively for 30 s,and then planted in matrixes.After 50 d,the effect of different hormone treatment on rooting ofVaccinium uliginosumtube seedlings was analyzed.

Effect of different humidity on rooting ofVaccinium uliginosumseedlingsThe culture temperature ofVaccinium uliginosumseedlings was controlled at 20-25℃in the day and at 10-15℃in the night.The effect of different air humidity on rooting ofVaccinium uliginosumseedlings was investigated so as to screen out the optimum air humidity.

Effect of different transplanting time on rooting ofVaccinium uliginosumtube seedlingsThe M5 and M6 were selected as culture matrixes. The tissue culture seedlings were dipped in 500 and 1 000 mg/L of ABT respectively for 30 s,and then planted in matrixes in May,July and September.After 30 d,the survival rates were investigated so as to determine the most suitable transplanting time forVaccinium uliginosumtube seedlings.

Data processing and statistics

The calculation and graphingwere performed using Microsoft Excel 2003.The variance analysis and difference significance tests(SSR)were completed using DPS 7.05[5].

Results and Analysis

Sterilization effect of different sterilization method

As shown in Table 1,the contamination rates of S5 and S6 treatments were all higher than 60%,indicating that their sterilization was inadequate. However,the sterilization of S1,S2 and S4 treatments was excessive.The incisions of most explants browned.Although the contamination rates were low,the browning was relatively serious in seedlings,affecting the normal growth of seedlings.The contamination rate of S3 treatment was 16.7%, and its browning rate was 13.3%.So S3 was a more appropriate sterilization method.With the S3 sterilization method,the sterilization purpose can be achieved,and the explants will not be damaged.

Effect of different sampling time on growth ofVaccinium uliginosumseedlings

The inoculation ofVaccinium uliginosumexplants was carried out in March,July and October next year, and the results were observed 7,14 and 21 d after the inoculation respectively.The differentiation rate and shoot elongation all differed significantly among different sampling times. The contamination rate of stem segments inoculated in March was relatively low.Moreover,due to winter dormancy and nutrient accumulation, the self-regulation had been in the bud stage.The stem segments inoculated in spring had stronger growth,faster growth and earlier differentiation,so they began to differentiate as early as on the 8thd.The contamination was relatively serious in stem segments inoculated in July,which had faster growth upward,larger node spacing, larger,thinner and juicier leaves,easier vitrification,smaller differentiation and thinner seedlings.The growth of stem segments inoculated in October was relatively slow and even stagnated.They began to differentiate on the 16th d.

Growth status ofVaccinium uliginosumlateral buds

TheVaccinium uliginosumaxillary buds began to sprout in the modified WMP medium on the 10thd.Since the 15thd,the internodes began to be elongated,and the leaves began to be unfolded.After 25 d,the propagation coefficient became higher(average newly-sprouted bud number of 4.7); the leaves became more(average leaf number of 6.0);the growth of buds was relatively fast(average newlysprouted bud height of 0.67 cm);the average bud height was 1.54 cm.

Screening of multiplication medium forVaccinium uliginosumtube seedlings

After the primary culture,the tube seedlings were cut into stem segments in length of about 1.0 cm.The stem segments were inoculated in different multiplication media with different hormone combinations.After a 35-d culture,the growth of adventitious buds was observed.Under conditions of different hormones and different hormone concentrations,the growth of adventitious buds was significantly different.The multiplication rate ofVaccinium uliginosumstem segments and shoot tips was regulated by cytokinin in the medium.The range analysis was performed for the statistic results of each trait in tube seedlings.The analysis results showed that both the seedling height and multiplication multiple of MeZT treatments were higher than those of Me6-BA treatments.It suggested that the zeatin plays a decisive regulatory role in the longitudinal growth and differentiation and multiplication of tube seedlings.For the trait of seedling height,the effect of first-level ZT treatment was most obvious;while for the differentiation and multiplication,the effect of second-level ZT treatment was most obvious.When 6-BA was added,the explants were also proliferated,but the proliferation effect was much worse than that of ZT.The variance analysis showed that for both seedling height and multiplication multiple ofVaccinium uliginosumtube seedlings,there were all significant differences among the three treatment levels of 6-BA and ZT(P＜0.01).In terms ofFvalue,the larger theFvalue is,the greater the effect is,and the results were also consistent with the analysis results above.As shown in Table 2,Me1 showed the fasted upward growth,followed by Me8,and there were significant differences between Me1 and the other treatments (P＜0.05);Me8 showed the largestmultiplication multiple,followed by Me5,and there were significant differences between Me5 and the other treatments(P＜0.05).In summary,in the premise of ensuring the multiplication rate of tube seedlings,the treatment in which the seedlings showed higher plant height and stronger growth should be selected.Therefore, the Me8(WPM(modified)+6-BA 1.0 mg/L+ZT 1.0 mg/L)was selected as the differentiation and multiplication medium forVaccinium uliginosumseedlings.

Table 1Sterilization effect of different sterilization time

Table 2Comparison ofVaccinium uliginosumplant traits among different treatments

Culture of robustVaccinium uliginosumtube seedlings

The tube seedlings were cultured at different illumination conditions.After a 50-d culture,the seedling growth was observed.Table 3 showed that, the growth ofVaccinium uliginosumtube seedlings differed among different illumination treatments.In the I1 treatment,the growth of tube seedlings was relatively weak;the seedlings were thin;the leaves were small and light green;the stems were tender and of light color.In the I2 treatment,the stems were red and relatively thick and strong,and they had certain toughness;the leaves were completely unfolded,dark green and shiny. Although the plant height in I2 treatment was lower than that in I1 treatment,the performance of the other traits in I2 treatment was all better than that in I1 treatment.Therefore,robust seedling culture is necessary before the transplanting of tube seedlings. Thus the tube seedling stems will become thicker,the leaves will become red,the tissues in young stems will get enriched,and the lignification will be improved.After hardened,the tube seedlings will have strong transpiration resistance and adaptability in leaves, significantly improved photosynthetic performance and enhanced adaptability to external environment,promoting root germination and improving rooting rate.

Rooting technology ofVaccinium uliginosumtube seedlings

Effect of different matrix on survival rate ofVaccinium uliginosumseedlingsAfter the robust seedling culture,the tube seedlings were removed.The top parts ofVaccinium uliginosumseedlings were cut off,and the remaining seedlings were kept in length of 2.0 cm.The medium around the bases of seedlings was washed with water.Subsequently,the bases were rapidly dipped in 0.5%KMnO4for sterilization and then dipped in rootingpromoting hormone ABT for 30 s.Finally,the seedlings were planted in organic matrixes that had been sterilized with KMnO4.

The planted seedlings began to wilt in varying degrees in different matrixes,especially in the matrixes mixed with husk,7 d after the transplanting. However,12 d after the transplanting, the seedlings started turning green from their bases;and 18 d after the transplanting,the survived seedlings all became green entirely and grew in varying degrees.The stems became thick and strong;the plants became erect;the leaves became unfolded and dark green.However,the wilted seedlings got withered and yellow entirely,and the parts buried in matrixes began to be rotted.Ma5 and Ma6 showed better treatment effect,and their survival rates were 63.4%and 56.8%,respectively(Table 4).

Effect of different hormone concentration on rooting ofVaccinium uliginosumtube seedlingsFrom the 2ndweek on since the transplanting ofVaccinium uliginosumtube seedlings,the same-concentration rooting-promoting hormone was sprayed once a week,and the air relative humidity was controlled within 80% and 90%.The test results showed that the under the conditions of same matrix,the tissue culture seedlings dipped in 1 000 mg/L of ABT rooted earlier and more rapidly.The seedlings planted in the Ma5 treated by 1 000 mg/L of ABT rooted earliest,and they began to root on the 13thd since the transplant-ing,and the root(with branches) length reached 1.2 cm on the 18th d. However,the seedlings cultured in the Ma5 treated by 500 mg/L of ABT began to root on the 21st d since the transplanting,and their rooting speed was relatively low.The tube seedlings cultured in the Ma5 showed faster rooting and developed roots with branches.Furthermore,the average rooting rate of tube seedlings planted in the Ma5 dipped with 1 000 mg/L of ABT was highest(72.4%)(Table 5).

Table 3Growth status ofVaccinium uliginosumtube seedlings in different illumination treatments

Table 4Survival rates ofVaccinium uliginosumseedlings in different matrixes %

Table 5Rooting conditions ofVaccinium uliginosumtube seedlings in different hormone treatments

Effect of different air humidity on survival rate ofVaccinium uliginosumseedlings

The seedlings were transplanted in Ma5,and the survival status was observed 20 d later.Table 6 showed that within a certain range of air humidity,the survival rate of tissue culture seedlings was positively related to air relative humidity.The higher the air relative humidity is,the higher the survival rate of tissue culture seedlings is. The A3 treatment showed the highest survival rate.It suggests that after the transplanting,installing a shed above the seedling bed and covering a layer of transparent plastic film are conducive to the maintenance of air humidity,improving survival rate of tissue culture seedlings.

Effect of different transplanting time on survival rate ofVaccinium uliginosumtube seedlingsThe tissue culture seedlings along with callus were transplanted outdoor.The results showed that the survival rate ofVaccinium uliginosumtube seedlings differed greatly among different transplanting months(Table 7).In May,the temperature inside the shed could reach 30℃at noon,so the survival rate ofVaccinium uliginosumtissue culture seedlings was relatively low; In July,the temperature reached the peak across the year,and the survival rate ofVaccinium uliginosumtissue culture seedlings was lowest; In September,the temperature began to drop,and it was remained at 20-26℃in the day and at 5-10℃in the night.The low temperature was more suitable for the growth of sciophilousVaccinium uliginosumseedlings,so the survival rate was increased dramatically.

Table 6Effect of air humidity on transplanting survival rate ofVaccinium uliginosumtissue culture seedlings

Table 7Survival rates ofVaccinium uliginosumseedlings in different transplanting time treatments%

Discussion

(1)Targeting at difficult and poor rooting,poor absorption ability and low survival rate ofVaccinium uliginosumseedlings in tubes,the rooting and acclimation have been tested to be combined.The ex vitro rooting technology is adopted,so the breeding cycle is effectively shortened,the production costs are reduced and the needed space is saved.At the same time,the death phenomenon of seedlings caused by hardening transplanting is reduced,thereby improving survival rate of seedlings.

(2)Tube seedlings are generally cultured in conditions of high humidity, low light intensity and constant temperature.So when the seedlings are removed outside culture bottles,they are prone to dying of dehydration.So ex vitro rooting often adopts covering film or spraying to ensure air relative humidity(80%-90%).The temperature is more appropriate to be controlled at about 25℃,and the light intensity should be increased timely to ensure the normal breathing of seedling bases.At the same time,the wilting of leaves caused by dehydration should be avoided.In the late period ofex vitrorooting,the ventilation should be enhanced to reduce humidity and temperature,thereby enhancing the self-support ability of young seedlings and promoting the rapid improvement of leaf protection function. The pores will become smaller,and the resistance and adaptability of seedlings to external environment will be enhanced,and the rooting rate will be improved.The balance among temperature,light intensity,matrix moisture content and air humidity is a guarantee for high rooting rate[6].

(3)In this study,the fermented mixed organic matrixes are used to induce rooting.The results show that the rooting rate of tube seedlings differs greatly among different matrixes. Among all the 6 kinds of matrixes,the Ma5 is more appropriate.Moreover, the Ma5 dipped in 1 000 mg/L of ABT shows better rooting-promoting effect. Compared with inorganic matrix,the Ma5 is lighter,nutritious and easier to be carried,reducing labor intensity.At the same time,the Ma5 can significantly reduce the mortality of seedlings caused by changing soil in seedling beds.

Conclusions

(1)The explants of the newVaccinium uliginosumcultivar Zishuijing are more suitable to be sterilized with 0.1%HgCl2for 3 min.Thus the sterilization purpose can be achieved,and the explants will not be damaged.

(2)The contamination rate is relatively low in stem segments inoculated in March.They also show faster growth and earlier differentiation,and begin to differentiate on the 8th d since the inoculation.The stem segments inoculated in July show relatively high contamination rate,while the stem segments inoculated in October show relatively slow growth.

(3)The study results indicate that the medium composed of modified WMP,6-BA(1.0 mg/L)and ZT(1.0 mg/L)is the optimum medium for differentiation and multiplication of Zishuijing.After a 35-d culture,the average seedling number and seedling height reach 7.1 and 5.83cm,respectively.

(4)In the robust seedling culture, the light intensity can be increased (3 000 lx).Thus the stems of tube seedlings will become thicker,the leaves will become red,the tissues in young stems will become compacted, and the lignification will be improved, thereby enhancing the adaptability of seedlings to external environment.

(5)After the robust seedling culture,the tube seedlings can be dipped in 1 000 mg/L of ABT(30 s)and induced to root ex vitro in September. Under conditions of organic mixed matrix of sawdust+bark+peat(1∶1∶1), air relatively humidity of 80%-90%, culture temperature of 20-25℃,the rooting rate reaches 72.4%50 d after the transplanting.

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Responsible editor:Tingting XU

Responsible proofreader:Xiaoyan WU

笃斯越橘新品种"紫水晶"离体培养关键技术研究

翁海龙，田新华，石德山*
（黑龙江省林业科学研究所，黑龙江哈尔滨150081）

［目的］探讨笃斯越橘新品种"紫水晶"离体培养的关键技术。［方法］通过对外植体灭菌方法，取材时期，增殖、壮苗、生根培养的筛选与优化，建立适合笃斯越橘新品种"紫水晶"的无性配套繁殖体系。［结果］外植体灭菌采用0.1%HgCl2消毒3 min；WPM（改良）+ 6-BA 1.0 mg/L+ZT 1.0 mg/L为笃斯越橘新品种"紫水晶"分化增殖培养基；9月份将无根试管苗移栽到混配有机基质锯末∶树皮∶草炭= 1∶1∶1基质中，空气相对湿度80%~90%，培养温度为20~25℃，50 d后生根率达72.4%。［结论］建立了笃斯越橘新品种"紫水晶"离体培养关键技术，为大规模工厂化育苗提供技术支持。

笃斯越橘；紫水晶；离体培养；条件优化branches began to sprout.

国家“十二五”科技支撑计划项目“典型森林生态系统结构及功能优化技术集成与示范”（2011BAD08B01）子课题“特色浆果资源恢复及高效培育技术研究与示范”（2011BAD08B01-03）；黑龙江省林业厅资助项目（201004068-6）。

翁海龙（1981-），男，黑龙江铁力人，硕士，助理研究员，主要从事林木遗传育种研究。*通讯作者，副研究员，主要从事森林培育方面的研究，E-mail：tianjie1976@sina.com。

2015-05-04

修回日期 2015-06-05

Supported by Key Project of the National Twelfth-Five Year Research Program of China (2011BAD08B01-03);Funding Project of Department of Forestry of Heilongjiang Province(201004068-6).

*Corresponding author.E-mail:tianjie1976@sina.com

Received:May 4,2015 Accepted:June 5,2015