research article large-scale plantlet conversion...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 829067 8 pageshttpdxdoiorg1011552013829067

Research ArticleLarge-Scale Plantlet Conversion and Ex Vitro TransplantationEfficiency of Siberian Ginseng by Bioreactor Culture

Jingli Yang1 Shicheng Zhao2 Changyeon Yu3 and Chenghao Li1

1 State Key Laboratory of Tree Genetics and Breeding Northeast Forestry University Harbin 150040 China2Department of Crop Science College of Agriculture amp Life Sciences Chungnam National UniversityDaejeon 305-764 Republic of Korea

3 Division of Bioresource Technology College of Agriculture and Life Sciences Kangwon National UniversityChuncheon 200-701 Republic of Korea

Correspondence should be addressed to Chenghao Li chli0daumnet

Received 31 July 2013 Accepted 18 September 2013

Academic Editors S Del Gaudio P Fickers S P Govindwar S Menoret and S Rodtong

Copyright copy 2013 Jingli Yang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To achieve large-scale low-cost ex vitro acclimatization of Siberian ginseng plants heart- and torpedo-shaped secondary somaticembryos (SEs) induced from germinated SEs on agar mediumwere collected and then inoculated to 10-l bubble column bioreactorrespectively For plantlet conversion inoculation of torpedo-shaped secondary SEs was more effective than heart-shaped SEs TS2(culture of torpedo-shaped SEs in a bioreactor with a 2-week subculture interval) plantlets had a higher root number and leafnumber and larger leaf area than did HS3 (culture of heart-shaped SEs in a bioreactor with a 3-week subculture interval) and HS2(culture of heart-shaped SEs in a bioreactor with a 2-week subculture interval) plantlets Of these converted plants TS2 plantlets hadhigher survival rate (837) and growth characteristics after transplantation in a simple shed covered with a 50 sunshade net onlyfor 6 months TS2 plantlets also showed significantly lower H

2O2content and significantly increased superoxide dismutase (SOD)

glutathione peroxidase (GPX) and glutathione transferase (GST) expression levels as compared to HS2 plants when exposure toex vitro conditions

1 Introduction

Over the past decade there has been a significant applicationof somatic embryogenesis in rapid multiplication of plantswhich provided a possible from-laboratory-to-large-scaleindustrial production However the extensive use of somaticembryo (SE) is still restricted by its relative high productioncosts and low survival rate of in vitro plantlets during exvitro transplanting [1] During in vitro cultivation plantletsgrow in a unique aseptic microenvironment with specialclimatic conditions of high relative humidity and low lightintensity to allow heterotrophic growth These conditionsmay lead to changes in plant with abnormal morphologyand physiology which are often characterized by retardationin the deposition of epicuticular waxes and developmentof functional stomata associated with the low ability of theplantlets to regulate water loss which can lead to losses dur-ing acclimatization [2] Most of the management of environ-

mental growth conditions has been focused on the ex vitroacclimatization stage little attention has been placed in themanagement of environmental growth conditions of in vitroculture Nevertheless some studies demonstrated that partialdesiccation treatment for early stage SEs could facilitate exvitro establishment of SE-derived plantlets [1 3]

Siberian ginseng (Eleutherococcus senticosus) is a medic-inal woody plant species that is endangered because ofoverharvesting in its natural habitat The cortical tissues ofthe roots shoots and leaves are used for various medicinalpurposes and in subsidiary food production [4] Conven-tional propagation by seeds and stem cuttings is very difficultbecause of the long-term stratification needed to inducegermination of the zygotic embryos as well as the low rootingfrequency of cuttings [5] Therefore production of Siberianginseng plantlets by somatic embryogenesis as an alternativemethod has been reported [6 7]

2 The Scientific World Journal

The use of bioreactors is a technology suitable for large-scale plant production of Siberian ginseng [8] We previouslyused 10-l bubble column bioreactors to culture Siberianginseng secondary SEs which resulted in a higher pro-ductivity than in suspension flask cultures [9] Suspension-culture-propagated heart-shaped secondary SEs that wereinoculated in bioreactors could germinate uniformly in plantgrowth regulators-free medium Mass-produced germinatedSEs attained 647 conversion frequency About 90 of invitro plants survived after acclimatization but only obtainedunder controlled temperature of 21∘C requireing very highproduction costs [9] Thus a key problem of productionof high-quality plants to decrease the cost at ex vitroacclimatization stage needs to be resolved before widespreadcommercial application Moreover most work are restrictedto increase the induction germination rate and conversioninto plantlets with little focus on the underlying physiologicaland molecular processes during acclimatization [6 7 10]

The aim of this study was to establish an efficient plantpropagation and ex vitro acclimatizationmethod for Siberianginseng using bioreactor culture of agar-medium-developedsecondary SEs and determine the effect of such conditionson the rate of plant conversion and subsequent ex vitrogrowth in a shed covered with a 50 sunshade net andthe underlying physiological and molecular processes duringacclimatization

2 Materials and Methods

21 Induction andDevelopment of Secondary Somatic Embryo-genesis onAgarMedium Germinated SEs of Siberian ginseng(Eleutherococcus senticosus) developed in bioreactor culturefrom highly cyclic somatic embryogenic-competent cell lines(see [9]) were used as initial materials for the inductionof secondary SEs Bioreactor culture-germinated SEs weretransferred to 13 MS [11] agar medium containing 1 (wv)sugar and 25 (wv) gelrite (DUCHEFA The Netherlands)without plant growth regulators (PGRs) and were cultured at21∘C 25∘C or 29∘C Ten plantlets were cultured in each 500-mL plastic culture vessel containing 70mLmedium (adjustedto pH 58 autoclaved at 121∘C for 15min) and were culturedwith a 16-h photoperiod at 36120583molmminus2 sminus1 (cool whitefluorescent tubes) After 8 weeks the induction frequencyof secondary SEs cultured at the different temperatures wascalculated

22 Cultivation of Secondary SEs in Bioreactors Clustersof heart- and torpedo-shaped secondary SEs were syn-chronously developed from germinated SEs cultured on 13MS agar medium for 6ndash8 weeks as described (see [9]) Thesesecondary SEs were initially separated by gentle choppingwith tweezers and inoculated into 250-mL Erlenmeyer flaskscontaining 50mL 13 MS liquid medium and 1 (wv) sugarAbout 3000 heart-shaped or 1000 torpedo-shaped embryoswere cultured in each flask Cultures were agitated at 100 rpmon a gyrating shaker at 21∘C

After adapting to suspension culture for 1 week SEswere inoculated in 10-l bubble column bioreactors (30 cm

times 15 cm) containing 8-L 13 MS liquid medium (see [9])About 12000 heart-shaped or torpedo-shaped SEs were cul-tured in each bioreactor The cultures were agitated at onevolume of air per volume of medium per min at 21∘C Theeffect of medium subculture period (a 2- or 3-week intervalbetweenmedium changes) on SE developmentwas evaluatedAfter 4 weeks (for torpedo-shaped SEs) 6 weeks (for heart-shaped SEs with 2-week subculture intervals) or 9 weeks(for heart-shaped SEs with 3-week subculture intervals) ofbioreactor culture fresh weight hypocotyls diameter andlength and primary root number of germinated SEs weredetermined Each set of bioreactor treatment conditions wasreplicated in three bioreactors and each experiment wascarried out twice Germinated SEs were placed into 500-mLplastic culture vessels containing 70-mL 13MS agar medium(sim200 SEsvessel) and stored at 4∘C before being transferredto conversion medium

23 Comparison of Plantlet Conversion and Transplanta-tion Ability For plantlet conversion germinated SEs weretransferred to conversion medium which was 13 MS agarmedium containing 1 (wv) sugar and 25 (wv) gelriteTen germinated SEs were cultured in each vessel To improvethe conversion frequency germinated SEs stored in thevessel were exposed to light conditions (16-h photoperiod at36 120583mol mminus2 sminus1) for 10 days before transfer to conversionmedium After 6 weeks plantlet conversion frequencies weredetermined

Randomly selected in vitro plantlets were transferredto plastic boxes (35 times 55 times 15 cm) containing a mixtureof autoclaved sand and potting soil (1 3 vv) About 50plantlets were cultured in a box Boxes were covered withperforated polythene bags to maintain high humidity andremoved after 4 weeks Plants were grown in a shed coveredwith a 50 high-density polyethylene sunshade net Each ofabout 1500 1000 and 500 germinated TS2- HS2- and HS3-derived plantlets were used for transplantation experimentrespectively The survival rate area of leaf length and freshweight of whole plants and root systems were compared aftertransplantation for 6 months

24 Comparisons of H2O2Content of Transplanted Plants

Differences between TS2- and HS2-derived in vitro plantletsafter 0 1 3 and 5 days soil transplantation were investigatedat the physiological level H

2O2content was measured by

Zhang et al [12] with some modification Fresh leaves (05 g)were ground to powder in liquid nitrogen and extracted with5mL of 5 trichloroacetic acid (TCA) and 015 g activatedcharcoal The mixture was centrifuged at 10000 g for 20minat 4∘C The supernatant was adjusted to pH 84 with 17Mammonia solution and then filtered The filtrate was used fordetermination of H

2O2

25 Quantitative Reverse Transcription PCR (RT-qPCR) Assayof Antioxidant Gene Expression Differences between TS2and HS2-derived in vitro plantlets after 0 1 and 3 dayssoil transplantation were investigated at the molecularlevel Total RNA was extracted from leaf tissues using

The Scientific World Journal 3

(a) (b)

(c) (d)

(e) (f) (g)

Figure 1 Bioreactor culture of agar medium induced heart-shaped and torpedo-shaped secondary somatic embryos of Siberian ginseng andplantlet conversion (a) Numerous secondary SEs induced from germinated primary SEs on 13 MS medium without PGRs Bar = 1 cm (b)Germinated HS2 (left) HS3 (middle) and TS2 (right) SEs production in a 10 l bubble column bioreactor Bar = 10 cm (c) Germinated TS2(left) HS2 (middle) and HS3 (right) SEs production in a 10 l bubble column bioreactor Bar = 2 cm (d) Plantlets derived from germinatedHS3 (left) HS2 (middle) and TS2 (right) SEs Bar = 4 cm Plantlet conversion of germinated HS3 (e) HS2 (f) and TS2 (g) SEs Bar = 2 cm

the CTAB method [13] First-strand cDNA synthesizedwith 05120583g purified RNA was reverse-transcribed with areverse transcriptase kit (MBI Fermentas) and PCR wasperformed in a volume of 20120583L containing 10 120583L of 2times SYBRpremix ExTaq (TaKaRa Biotech Dalian China) 05120583Mof forward and reverse primers and 2 120583L cDNA template(equivalent to 005 120583g of total RNA) The primer sequencesof Siberian ginseng 18S rRNA (GenBank AB0802451) 120573-tubulin (GenBank KC542394) and the previously definedstress inducible ROS-scavenging related genes (unpublished)SOD (GenBank JQ3656251) GPX (GenBank KC542392)

and GST (GenBank KC542393) were given in Table 1 andamplified under the following cycling conditions 10 sec at95∘C followed by 40 cycles of 5 sec at 95∘C 30 sec at 60∘Cand 1 s at 78∘C for plate reading RT-qPCRwas repeated threetimes for each sample on a DNA Engine Opticon 2 (MJResearch) following the manufacturerrsquos recommendations

26 Statistical Analysis Thedatawere analyzedwithANOVAusing SPSS 160 for Windows (SPSS Inc Chicago IL USA)Means that differed significantly were compared using Dun-canrsquos multiple range test at the 5 probability level


b

a

c

TS2Type of germinated SEs

0

20

40

60

80

100

HS2 HS3

Con

vers

ion

frequ

ency

()

Figure 2 Effects of inoculation stage and medium exchange periodon plantlet conversion of bioreactor cultured germinated SEs ofSiberian ginseng after culturing on 13 MS medium for 2 monthsValues with different letters in a column are significantly differentaccording to Duncanrsquos multiple range test at the 5 level

Table 1 Primers used for quantification of antioxidant enzymegenes by real-time PCR

Primers Sequence (51015840 rarr 31015840)

18S F 51015840-GGTCGTGCCTCCGGCGCTGTTAC-31015840

R 51015840-CCTCTGACTATGAAATACGAATG-31015840

120573-tubulin F 51015840-GGGAATTCGACTTCCATTCAAG-31015840

R 51015840-CAGGCTTCAACTTCCTCTTCTTC-31015840

SOD F 51015840-GGCCCAACTACAGTTACTGGAAG-31015840

R 51015840-GTGGCAGTACCATGTTCACCAACTG-31015840

GPX F 51015840-GCCTCACCAATTCAAACTACACAG-31015840

R 51015840-GGAGCGGCATTGCAACCATTCAC-31015840

GST F 51015840-CTCAAGCTAGGTTCTGGGCTGAC-31015840

R 51015840-GACAAGGATTACATCAACATACC-31015840

3 Results and Discussion

31 Induction and Development of Secondary SEs on AgarMedium After transformation of the germinated primarySEs from the bioreactors to solid medium the frequency ofsecondary SE induction was significantly different among thethree culture temperatures At 21∘C and 25∘C the inductionfrequency was 30 and 52 respectively whereas almost906 of SEs induced secondary SEs at 29∘C (Figure 1(a))demonstrating that the higher temperature was better forsecondary SE inductionMost secondary SEs gradually devel-oped to the heart- and torpedo-shaped stage about 6 and 8weeks after culture with no medium exchange respectivelyFurther development of torpedo-shaped SEs was arrestedand relative water content decreased to about 54 after 8weeks The number of secondary SEs per explants variedsignificantly About 20 of germinated SEs produced 100 to1000 SEs per explants at 29∘C

32 Bioreactor Culture of Secondary SEs To allow the SEsto adapt to the bioreactor culture environment heart-and torpedo-shaped SEs were first inoculated into 250-mL

Erlenmeyer flasks with liquid medium After adapting tosuspension culture in flasks for one week secondary SEswere inoculated into bioreactors When cultured in 13 MSmedium the SEs inoculated in bioreactors grew quicklybut the growth rate decreased after sim2 weeks of cultureThe subculture period significantly affected SE growth HS2SEs grew normally and consistently with 2-week subcultureintervals but with a 3-week interval the HS3 SEs grew slowlyand then stopped until the medium was changed again Aftersubculture at 2-week intervals in bioreactors heart-shapedSEs (referred to as HS2) germinated and needed 6 weeks forgrowth whereas 9weekswere neededwith 3-week subcultureintervals (referred to as HS3) (Figure 1(b)) (data not shown)Similarly torpedo-shaped SEs germinated and grew fullyafter 4 weeks with a 2-week subculture interval (referredto as TS2 Figure 1(b)) The morphology of germinated SEsshowed a significant difference depending on the differentinoculation stages (Figure 1(c)) The hypocotyl length ofgerminated TS2 SEswas half of germinatedHS2 andHS3 SEsbut fresh weight hypocotyl diameter and root number in thegerminated TS2 SEs were all significantly higher than corre-sponding values for germinated HS2 and HS3 SEs (Table 2)

33 Plantlet Conversion Most bioreactor-cultured germi-nated SEs were pale yellow-green possibly because of theirhigh culture density which would result in less absorbedlight Exposing the storage vessels with the SEs to lightprior to culture on conversion medium resulted in quicklyturning greened SEs produced When exposing germinatedSEs to light for 10 days the conversion frequency extensivelyincreased and the conversion frequency of germinated TS2SEs was significantly higher than that of HS3 and HS2 SEs(Figure 2) TS2 plantlets had a higher root number leafnumber and larger leaf area than did HS3 and HS2 plantlets(Figures 1(d) to 1(g)) (data not shown) The leaf shape of TS2plantlets was also more similar to that of seedling plantlets(Figures 1(d) to 1(g))

34 Comparison of Transplantation Ability of HS2 HS3 andTS2 Plants In vitro plantlets were transferred to soil andgrown in a shed covered with a 50 sunshade net (Figures3(a) and 3(b)) After transplantation the leaves of HS2 andHS3 plants wilted or fell off within 2 weeks followed by newshoots and leaves forming at the shoot apex within 4 weeks(Figure 3(c)) On the contrary most leaves survived andmaintained vigorous growth for TS2 plantlets (Figure 3(d))Only for three days TS2 plantlets had formed newroots demonstrating that they can adapt quickly tothe ex vitro environment The results showed thatsurvival rate of TS2 plantlets was significantly higherthan that of HS2 and HS3 plantlets (Figure 4) TS2plantlets also have higher growth characteristicsthan HS2 plantlets (Figures 3(e) and 3(f) Table 3)Although the survival rate in this study was similar toit in our previous report [9] the cost fee of this shadingshed method was relative lower which would be of benefitto the commercial application of plantlet productionbecause the transplantation was completely performed in


(a) (b)

(c) (d)

(e) (f)

Figure 3 Transplantation of bioreactor cultured SE-derived plantlets of Siberian ginseng in simple sunshade shed (a) Simple shed coveredwith a 50 sunshade net Bar = 11m (b) In vitro plantlets were transferred to plastic boxes (35 times 55 times 15) containing a mixture of sand andsoil Bar = 20 cm (c) HS2 plantlets 2 weeks after transplantation Bar = 1 cm (d) TS2 plantlets after transplantation for 2 weeks Bar = 1 cm (e)HS2 (left) and TS2 (right) plantlets after transplantation for 6months Bar = 16 cm (f) TS2 (left) andHS2 (right) plantlets after transplantationfor 6 months Bar = 6 cm

Table 2 Effect of inoculation stage and medium exchange period on growth of SEs in 10-l bioreactor

Stage of inoculatedSEs

Medium exchangeperiod (weeks)

Bioreactor cultureperiod (weeks)

Fresh weight of germinatedSEs (mgSEs)z

Hypocotyl Primary rootnumberDiameter (mm) Length (cm)

Heart-shapex 2 6 66a 12a 50a 43a

Heart-shape 3 9 225b 12a 42b 17b

Torpedo-shape 2 4 137c 25b 24c 91cxAbout 12000 agar medium developed SEs were cultured in a bioreactorzValues with different letters in a column are significantly different according to Duncanrsquos multiple range test at the 5 level


Table 3 Comparison between plants obtained fromHS2 and TS2 SEs after transplantation for 6 months in a simple shed covered with a 50sunshade net

Origin of plants Leaf area (cm2)z Plant height (cm) Plant fresh weight (g) Root system fresh weight (g)HS2 SEs 55a 173a 73a 39a

TS2 SEs 17b 125b 28b 12bzValues with different letters in a column are significantly different according to Duncanrsquos multiple range test at the 5 level

Surv

ival

freq

uenc

y (

)

Type of plantlets

b

a

c

TS20

20

40

60

80

100

HS2 HS3

Figure 4 Survival frequency of HS2 HS3 and TS2 plantlet after exvitro transplantation for 6 months Values with different letters ina column are significantly different according to Duncanrsquos multiplerange test at the 5 level

020406080

100120140160

0 1 3 5Time (days)

TS2HS2

H2O2

(mg g

minus1

FW)

Figure 5 Comparison of H2O2content between HS2 and TS2

plantlets after transplantation in a simple shed covered with a 50sunshade net

a simple sunshade shed The previous reported method needan accurate temperature (21∘C) environment for ex vitroacclimatization therefore it requires an instrument highdemand of

To achieve large-scale vegetative propagation by somaticembryogenesis the biggest obstacle is the mass productionof SEs with high plantlet conversion ability [14] Previousreports of somatic embryogenesis and plantlet production inSiberian ginseng showed that SEs germinate easily but havea relatively low conversion frequency and ex vitro acclima-tization difficulty [6 7 10] In the present study the main

cause for the high conversion and acclimatization abilityof SEs was probably due to their long partial desiccationprocess at earlier stages of development over 6 to 8 weeksA partial desiccation treatment is a common method forimproving the plantlet quality of fully developed SEs whichcan be otherwise difficult to germinate [15] In coniferousspecies SE desiccation treatment is the primary means forimproving conversion rates [14] Roberts et al [15] improvedthe mature postembryonic development of SEs of PiceaglaucatimesP engelmanniiby gradually lowering the high relativehumidity to achieve partial desiccation which decreased thegermination time synchronized stem and root developmentand resulted in gt90 conversion frequency In additionpartial desiccation treatment also successfully applied onlarge-scale SE production of coffee rubber and citrus trees byRITA bioreactor with a temporary immersion system [16ndash18]

35 Decreased H2O2Content and Enhanced Expression of

Antioxidant Genes in TS2 Plants Abiotic stress may increasethe level of reactive oxygen species (ROS) which is toxic toplant cells [18] During desiccation free radical scavengersmay provide additional protection because the developmentof desiccation tolerance coincides with an increase in antioxi-dant gene expression in seeds [19 20] To investigate whetherthe decreased sensitivity to environmental stress inTS2 plantsas compared to HS2 plants may be due to decreased ROSaccumulation theH

2O2content of TS2 andHS2 plants leaves

was compared at initiation period of ex vitro transplantationUnder these conditions H

2O2levels in TS2 plants were

less than half of those in HS2 plants (Figure 5) Thus TS2plantsmay havemore normal functional leaves and suffer lessenvironmental stress as compared to HS2 plants Our resultstrongly suggests that there is a correlation between the leafdamage and the imbalance of ROS production in early stageof transplantation

It has been accepted that antioxidant defense systemsmdashincluding nonenzymatic antioxidants such as ascorbate andreduced glutathione and enzymatic antioxidants such asSOD and GPXmdashplay a crucial role in plant defense mech-anisms against various stresses [21] The expression levelsof three stress inducible antioxidant enzyme genes SOD(GenBank JQ3656251) GPX (GenBank KC542392) andGST (GenBank KC542393) under ex vitro environment werecompared between TS2 and HS2 plants using RT-qPCR Theresults showed SOD GPX and GST expression levels in TS2plants were increased significantly as compared to that inHS2 plants when exposed to ex vitro conditions (Figure 6)We hypothesized that ex vitro transplanted plant cells caninduce expression of various antioxidant enzymes underenvironmental stresses and the increased expression of these


0

1

2

3

4

5

0 1 3 5Time (days)

SOD

relat

ive e

xpre

ssio

n

TS2HS2

(a)

0 1 3 5Time (days)

0

1

2

3

4

GPX

relat

ive e

xpre

ssio

n

TS2HS2

(b)

0 1 3 5Time (days)

005

115

225

335

GST

relat

ive e

xpre

ssio

in

TS2HS2

(c)

Figure 6 Comparisons of (a) SOD (b) GPX and (c) GST expression levels in HS2 and TS2 plantlet after transplantation in a simple shedcovered with a 50 sunshade net by RT-qPCR

antioxidant enzyme genes in TS2 plantsmay contribute to thescavenging of ROS Antioxidant enzyme genesrsquo expressionlevels of HS2 plants also increased when exposed to envi-ronmental conditions in vitro may be because the far moreproduced ROS could not protected leaves from oxidativedamage

4 Conclusion

An efficient micropropagation protocol for Siberian ginsenghas been established Development of secondary SEs totorpedo-shaped stage on agar medium prior inoculated tobioreactor culture was found to be beneficial for plant conver-sion and thus low the cost acclimatization of Siberian ginsengTS2 plants were found to have higher environmental stressadaption ability compared to the HS2 plants The protocol inthe present study can be successfully exploited to widespreadcommercial propagation of Siberian ginseng elite clones

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This research was supported by the Key Project of ChineseNational Programs for Fundamental Research and Devel-opment (973 Program 2009CB119100) the Fundamental

Research Funds for the Central Universities (DL11EA02)and the National Natural Science Foundation of China (no30671701)

References

[1] D Barry-Etienne B Bertrand A Schlonvoigt and H EtienneldquoThe morphological variability within a population of coffeesomatic embryos produced in a bioreactor affects the regener-ation and the development of plants in the nurseryrdquo Plant CellTissue and Organ Culture vol 68 no 2 pp 153ndash162 2002

[2] S Chandra R Bandopadhyay V Kumar and R ChandraldquoAcclimatization of tissue cultured plantlets from laboratory tolandrdquo Biotechnology Letters vol 32 no 9 pp 1199ndash1205 2010

[3] S Y Park H K Moon H N Murthy and Y W KimldquoImproved growth and acclimatization of somatic embryo-derived Oplopanax elatus plantlets by ventilated photoau-totrophic culturerdquo Biologia Plantarum vol 55 no 3 pp 559ndash562 2011

[4] I I Brekhman and I V Dardymov ldquoNew substances of plantorigin which increase nonspecific resistancerdquo Annual Review ofPharmacology vol 9 pp 419ndash430 1969

[5] S Isoda and J Shoji ldquoStudies on the cultivation of Eleuthero-coccus senticosusMaxim II On the germination and raising ofseedlingrdquo Nature Medicine vol 48 no 1 pp 75ndash81 1994

[6] Y E Choi J W Kim and E S Yoon ldquoHigh frequency ofplant production via somatic embryogenesis from callus or cellsuspension cultures in Eleutherococcus senticosusrdquo Annals ofBotany vol 83 no 3 pp 309ndash314 1999


[7] Y E Choi D C Yang and E S Yoon ldquoRapid propagationof Eleutherococcus senticosus via direct somatic embryogenesisfrom explants of seedlingsrdquo Plant Cell Tissue and OrganCulture vol 58 no 2 pp 93ndash97 1999

[8] K Y Paek E J Hahn and S H Son ldquoApplication of bioreactorsfor large scale micropropagation systems of plantsrdquo In VitroCellular amp Developmental Biology vol 37 no 2 pp 149ndash1572001

[9] J L Yang C G Zhou L H Liu et al ldquoHigh conversionfrequency of germinated somatic embryos of Siberian ginseng(Eleutherococcus senticosus Maxim) using a bubble columnbioreactorrdquo Plant Cell Tissue and Organ Culture vol 110 no2 pp 289ndash298 2012

[10] L Yang J N Wang L Bian Y H Li and H L ShenldquoCyclic secondary somatic embryogenesis and efficient plantregeneration in mountain ash (Sorbus pohuashanensis)rdquo PlantCell Tissue and Organ Culture vol 111 pp 173ndash182 2012

[11] T Murashige and F Skoog ldquoA revised medium for rapidgrowth and bioassays with tobacco tissue culturesrdquo PhysiologiaPlantarum vol 15 pp 495ndash497 1962

[12] Y M Zhang J L Tan Z F Guo et al ldquoIncreased abscisicacid levels in transgenic tobacco over-expressing 9 cis-epoxycarotenoid dioxygenase influence H

2O2and NO produc-

tion and antioxidant defencesrdquo Plant Cell amp Environment vol32 no 5 pp 509ndash519 2009

[13] L Jaakola AM PirttilaMHalonen andAHohtola ldquoIsolationof high quality RNA from bilberry (Vaccinium myrtillus L)fruitrdquoMolecular Biotechnology vol 19 no 2 pp 201ndash203 2001

[14] A C Stasolla and E C Yeung ldquoRecent advances in conifersomatic embryogenesis improving somatic embryo qualityrdquoPlant Cell Tissue and Organ Culture vol 74 no 1 pp 15ndash352003

[15] D R Roberts B C S Sutton and B S Flinn ldquoSynchronous andhigh frequency germination of interior spruce somatic embryosfollowing partial drying at high relative humidityrdquo AmericanJournal of Botany vol 68 pp 1086ndash1090 1990

[16] C Cabasson D Alvard D Dambier P Ollitrault and CTeisson ldquoImprovement of Citrus somatic embryo developmentby temporary immersionrdquo Plant Cell Tissue and Organ Culturevol 50 no 1 pp 33ndash37 1997

[17] H Etienne and Y M Berthouly ldquoTemporary immersion sys-tems in plant micropropagationrdquo Plant Cell Tissue and OrganCulture vol 69 no 3 pp 215ndash231 2002

[18] D Etienne-Barry B Bertrand N Vasquez and H EtienneldquoDirect sowing of Coffea arabica somatic embryos mass-produced in a bioreactor and regeneration of plantsrdquo Plant CellReports vol 19 no 2 pp 111ndash117 1999

[19] C Haslekas R A P Stacy V Nygaard F A Culianez-Maciaand R B Aalen ldquoThe expression of a peroxiredoxin antioxidantgene AtPer1 in Arabiodopsis thaliana is seed-specific andrelated to dormancyrdquo Plant Molecular Biology vol 36 no 6 pp833ndash845 1998

[20] P Suprasanna C Rupali N S Desai and V A Bapat ldquoPar-tial desiccation augments plant regeneration from irradiatedembryogenic cultures of sugarcanerdquo Plant Cell Tissue andOrgan Culture vol 92 no 1 pp 101ndash105 2008

[21] H C Jin Y Sun Q C Yang et al ldquoScreening of genes inducedby salt stress from Alfalfardquo Molecular Biology Reports vol 37no 2 pp 745ndash753 2010

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Microbiology


The use of bioreactors is a technology suitable for large-scale plant production of Siberian ginseng [8] We previouslyused 10-l bubble column bioreactors to culture Siberianginseng secondary SEs which resulted in a higher pro-ductivity than in suspension flask cultures [9] Suspension-culture-propagated heart-shaped secondary SEs that wereinoculated in bioreactors could germinate uniformly in plantgrowth regulators-free medium Mass-produced germinatedSEs attained 647 conversion frequency About 90 of invitro plants survived after acclimatization but only obtainedunder controlled temperature of 21∘C requireing very highproduction costs [9] Thus a key problem of productionof high-quality plants to decrease the cost at ex vitroacclimatization stage needs to be resolved before widespreadcommercial application Moreover most work are restrictedto increase the induction germination rate and conversioninto plantlets with little focus on the underlying physiologicaland molecular processes during acclimatization [6 7 10]

The aim of this study was to establish an efficient plantpropagation and ex vitro acclimatizationmethod for Siberianginseng using bioreactor culture of agar-medium-developedsecondary SEs and determine the effect of such conditionson the rate of plant conversion and subsequent ex vitrogrowth in a shed covered with a 50 sunshade net andthe underlying physiological and molecular processes duringacclimatization

2 Materials and Methods

21 Induction andDevelopment of Secondary Somatic Embryo-genesis onAgarMedium Germinated SEs of Siberian ginseng(Eleutherococcus senticosus) developed in bioreactor culturefrom highly cyclic somatic embryogenic-competent cell lines(see [9]) were used as initial materials for the inductionof secondary SEs Bioreactor culture-germinated SEs weretransferred to 13 MS [11] agar medium containing 1 (wv)sugar and 25 (wv) gelrite (DUCHEFA The Netherlands)without plant growth regulators (PGRs) and were cultured at21∘C 25∘C or 29∘C Ten plantlets were cultured in each 500-mL plastic culture vessel containing 70mLmedium (adjustedto pH 58 autoclaved at 121∘C for 15min) and were culturedwith a 16-h photoperiod at 36120583molmminus2 sminus1 (cool whitefluorescent tubes) After 8 weeks the induction frequencyof secondary SEs cultured at the different temperatures wascalculated

22 Cultivation of Secondary SEs in Bioreactors Clustersof heart- and torpedo-shaped secondary SEs were syn-chronously developed from germinated SEs cultured on 13MS agar medium for 6ndash8 weeks as described (see [9]) Thesesecondary SEs were initially separated by gentle choppingwith tweezers and inoculated into 250-mL Erlenmeyer flaskscontaining 50mL 13 MS liquid medium and 1 (wv) sugarAbout 3000 heart-shaped or 1000 torpedo-shaped embryoswere cultured in each flask Cultures were agitated at 100 rpmon a gyrating shaker at 21∘C

After adapting to suspension culture for 1 week SEswere inoculated in 10-l bubble column bioreactors (30 cm

times 15 cm) containing 8-L 13 MS liquid medium (see [9])About 12000 heart-shaped or torpedo-shaped SEs were cul-tured in each bioreactor The cultures were agitated at onevolume of air per volume of medium per min at 21∘C Theeffect of medium subculture period (a 2- or 3-week intervalbetweenmedium changes) on SE developmentwas evaluatedAfter 4 weeks (for torpedo-shaped SEs) 6 weeks (for heart-shaped SEs with 2-week subculture intervals) or 9 weeks(for heart-shaped SEs with 3-week subculture intervals) ofbioreactor culture fresh weight hypocotyls diameter andlength and primary root number of germinated SEs weredetermined Each set of bioreactor treatment conditions wasreplicated in three bioreactors and each experiment wascarried out twice Germinated SEs were placed into 500-mLplastic culture vessels containing 70-mL 13MS agar medium(sim200 SEsvessel) and stored at 4∘C before being transferredto conversion medium

23 Comparison of Plantlet Conversion and Transplanta-tion Ability For plantlet conversion germinated SEs weretransferred to conversion medium which was 13 MS agarmedium containing 1 (wv) sugar and 25 (wv) gelriteTen germinated SEs were cultured in each vessel To improvethe conversion frequency germinated SEs stored in thevessel were exposed to light conditions (16-h photoperiod at36 120583mol mminus2 sminus1) for 10 days before transfer to conversionmedium After 6 weeks plantlet conversion frequencies weredetermined

Randomly selected in vitro plantlets were transferredto plastic boxes (35 times 55 times 15 cm) containing a mixtureof autoclaved sand and potting soil (1 3 vv) About 50plantlets were cultured in a box Boxes were covered withperforated polythene bags to maintain high humidity andremoved after 4 weeks Plants were grown in a shed coveredwith a 50 high-density polyethylene sunshade net Each ofabout 1500 1000 and 500 germinated TS2- HS2- and HS3-derived plantlets were used for transplantation experimentrespectively The survival rate area of leaf length and freshweight of whole plants and root systems were compared aftertransplantation for 6 months

24 Comparisons of H2O2Content of Transplanted Plants

Differences between TS2- and HS2-derived in vitro plantletsafter 0 1 3 and 5 days soil transplantation were investigatedat the physiological level H

2O2content was measured by

Zhang et al [12] with some modification Fresh leaves (05 g)were ground to powder in liquid nitrogen and extracted with5mL of 5 trichloroacetic acid (TCA) and 015 g activatedcharcoal The mixture was centrifuged at 10000 g for 20minat 4∘C The supernatant was adjusted to pH 84 with 17Mammonia solution and then filtered The filtrate was used fordetermination of H

2O2

25 Quantitative Reverse Transcription PCR (RT-qPCR) Assayof Antioxidant Gene Expression Differences between TS2and HS2-derived in vitro plantlets after 0 1 and 3 dayssoil transplantation were investigated at the molecularlevel Total RNA was extracted from leaf tissues using


(a) (b)

(c) (d)

(e) (f) (g)






b

a

c


0

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Con

vers

ion

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ency

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(a) (b)

(c) (d)

(e) (f)















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ival

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y (

)

Type of plantlets

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a

c

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020406080

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SOD

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005

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4 Conclusion




Acknowledgments



References














2O2and NO produc-


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)

(e) (f) (g)






b

a

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0

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Con

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frequ

ency

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(a) (b)

(c) (d)

(e) (f)















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ival

freq

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)

Type of plantlets

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a

c

TS20

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020406080

100120140160

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1

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225

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ssio

in

TS2HS2

(c)



4 Conclusion




Acknowledgments



References














2O2and NO produc-


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


b

a

c


0

20

40

60

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Con

vers

ion

frequ

ency

()





















(a) (b)

(c) (d)

(e) (f)















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ival

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)

Type of plantlets

b

a

c

TS20

20

40

60

80

100

HS2 HS3


020406080

100120140160

0 1 3 5Time (days)

TS2HS2

H2O2

(mg g

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ssio

in

TS2HS2

(c)



4 Conclusion




Acknowledgments



References














2O2and NO produc-


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)

(e) (f)















Surv

ival

freq

uenc

y (

)

Type of plantlets

b

a

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TS20

20

40

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HS2 HS3


020406080

100120140160

0 1 3 5Time (days)

TS2HS2

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(mg g

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225

335

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relat

ive e

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ssio

in

TS2HS2

(c)



4 Conclusion




Acknowledgments



References














2O2and NO produc-


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Surv

ival

freq

uenc

y (

)

Type of plantlets

b

a

c

TS20

20

40

60

80

100

HS2 HS3


020406080

100120140160

0 1 3 5Time (days)

TS2HS2

H2O2

(mg g

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0

1

2

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115

225

335

GST

relat

ive e

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in

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(c)



4 Conclusion




Acknowledgments



References














2O2and NO produc-


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1

2

3

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5

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relat

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225

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4 Conclusion




Acknowledgments



References














2O2and NO produc-


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








2O2and NO produc-


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article large-scale plantlet conversion...

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