Accepted 17 June, 2011

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1 Africn Journl of Biotechnology Vol. 1(59), pp , 3 Octoer, 11 Aville online t DOI: 1.597/AJB1.17 ISSN Acdemic Journls Full Length Reserch Pper Microspore derived emryo formtion nd douled hploid plnt production in roccoli (Brssic olerce L. vr itlic) ccording to nutritionl nd environmentl conditions Heyoung N 1, Guiyoung Hwng 1, Jung-Ho Kwk 1, Moo Koung Yoon 1 nd Chnghoo Chun,3 * 1 Ntionl Institute of Horticulturl nd Herl Science, Suwon -7, Kore. Deprtment of Horticulturl Science, Seoul Ntionl University, Seoul , Kore. 3 Reserch Institute of Agriculture nd Life Sciences, Seoul Ntionl University, Seoul , Kore. Accepted 17 June, 11 In cell culture, the mintennce of proper growing conditions is key pproch for improving the formtion of emryos, nd is useful in the production of douled hploid (DH) plnts. Optiml nutritionl nd environmentl conditions for the microspore culture of Brssic olerce L. vr itlic were determined in order to reduce time nd effort in reeding. The optiml conditions for microspore emryo formtion differed depending on genotype. Microspore-derived emryos (MDE) formtion ws influenced y the strength of the NLN medium, the microelement nd sugr concentrtion, nd the het shock temperture nd period. The.5XNLN liquid medium ws the most fvorle for MDE formtion. The most efficient formtion of MDE ws oserved in the.5x NLN liquid medium, without the ddition of microelements. When 13 or 15% sucrose ws dded to the.5x NLN liquid medium, the mount of norml MDE formtion incresed. The optimum het shock temperture nd period for MDE formtion ws 3.5 C nd h, respectively. A polyploidy test indicted tht 3% of the microspore derived plnts were diploid throughout the emryogenesis process. Key words: Emryogenesis, het shock, microelements, NLN medium, polyploidy test. INTRODUCTION Broccoli is considered s mjor vegetle, hving high nutritionl vlue with vrious functionl mterils such s selenium, sulforphne, indol-3-crinol nd folic cid. It is lso well-known ntioxidnt food. The microspore culture of Brssic plnts is very vlule tool for genetic mnipultion vi hploid reeding; however, the production of homozygous lines through ud pollintion is time consuming nd lor intensive. Microspore culture is n efficient technology for the production of homozygous lines when producing F 1 hyrids of modern cultivrs, leding to n increse in selection efficiency for desirle genetic recominnts (Dis, 1999). Microspore derived plnts provide rpid mens of otining homozygous nd homogeneous lines of griculturlly importnt plnts *Corresponding uthor. E-mil: chnghoo@snu.c.kr. (Dis, 1). Microspore culture hs een used to produce hploid nd douled hploid plnts in the genus Brssic (Keller nd Armstrong, 1979; Lichter, 199). These plnts cn e utilized in vrietl development, mutnt selection, nd iochemicl nd genetic engineering studies (Swnson nd Erickson, 199; Swnson et l., 19; Tylor et l., 1993). The douled hploid prentl lines cn enhnce nd ccelerte plnt reeding progrms y sving lor nd time. These lines hve lredy een developed using nther culture (Frnhm, 199), nd they hve een introduced into reeding schemes. Successful microspore culture in different roccoli genotypes hs een descried y Duijs et l. (199) nd Tkht nd Keller (1991). One prolem with the prcticl ppliction of microspore culture, reported y different uthors (Dis, 1999; Duijs et l., 199), is the very low emryo yield in mny

153 Afr. J. Biotechnol. length of the stigm were chosen. The uds of this stge contined nthers t the lte uninuclete stge of microspore development, nd their size ws to mm (Figure 1).

2 153 Afr. J. Biotechnol. length of the stigm were chosen. The uds of this stge contined nthers t the lte uninuclete stge of microspore development, nd their size ws to mm (Figure 1). The uds were wrpped in guze nd surfce sterilized in 1% sodium hypochlorite for 15 min on the shker t 7 rpm, nd then rinsed three times for 3 minutes ech, using sterile wter. The uds were gently mcerted with ml of B5-13 medium (Gmorg et l., 19), nd ground using mortr. They were filtered through 5 µm metl mesh screen, nd collected in 5 ml centrifuge tue. The microspore suspension ws wshed three times with 1 ml of B5-13 medium y centrifuging t 1, rpm for 3 min. Then, the superntnt ws removed nd pelleted microspores were re-suspended t density of, microspores to 1 ml of NLN liquid medium (Lichter, 19). The numer of microspores ws estimted using hemcytometer. The lst microspore suspension ws re-suspended in NLN liquid medium with 13% sucrose. We dispensed.5 ml of the microspore suspension into x 15 mm sterile Petri-dish tht ws susequently seled with prfilm. All culture medi ws djusted to ph 5. using NOH or HCl nd filter-sterilized using 5 µm low protein inding memrne filter (Corning, USA). After h het shock tretment nd 1dy incution in drkness, ll microspores were plced on shker t rpm nd 5 C under 1 h photoperiod with 5 µmol m - s -1 PPFD (cool, white fluorescent lmps) for weeks. Figure 1. Flower ud removed sepls of Brssic olerce L.vr itlic for microspore derived emryo culture. The stigm is longer thn the length of the florl lef. roccoli genotypes. There hs lwys een n ttempt to dpt nd improve the current microspore culture protocols to mke this technique ville for hploid reeding. There re few pulished reports on microspore emryogenesis in roccoli, ut improvement in the microspore culture protocols is required. Severl fctors influencing microspore emryogenesis re donor plnt conditions, genotype, developmentl stge, medi constituents nd culture conditions. The ojective of this pper ws to study nutritionl, chemicl nd physicl fctors ffecting microspore derived emryo (MDE) formtion in roccoli, nd to lso verify the polyploidy of microspore-derived plntlets. MATERIALS AND METHODS Gene source K5 provided y the Ntionl Agroiodiversity Center, locted t Suwon Kore ws used for donor plnts. The donor plnts were grown using plstic pots (5 x 9 cm) in greenhouse under 1 h photoperiod with µmol m - s -1 - photosynthetic photon flux density (PPFD). Lter, they were vernlized in cold room mintined t ±1 C under 1 h photoperiod with µmol m - s -1 PPFD for eight weeks. After florl differentition nd the strt of genertive development, plnts were trnsferred to greenhouse t 5 C under 1 h photoperiod with µmol m - s -1 PPFD. Microspore isoltion Flower uds hving shorter florl lef length s compred to the Microspore tretments Microspores were incuted in the drk t 3.5 C during the h het shock tretment, nd then trnsferred to 5 C in the drk. After 15 dys, the Petri-dishes were plced on shker nd gitted t rpm with 1 h photoperiod t 5 C. The emryo numer ws scored four weeks fter microspore isoltion (Figure A). Microspores were cultured with vrious NLN liquid medium strengths (.5X,.5X, 1.X,.X nd.x) to investigte the effect of NLN on MDE induction. To investigte the effects of sugr concentrtion on emryonic induction, microspores were cultured in.5x NLN liquid medium contining, 3, 5, 1, 13, 15 nd % sucrose. Microspores were cultured t four different het shock tempertures of 5, 3.5, 37 nd C for h in order to determine the optiml temperture for MDE formtion. The het shock period ws lso vried t,, nd 7 h t 3.5 C. After 3 dys of culture, the numer of emryos in ech Petri-dish ws counted. The experiment ws conducted with ten replictions. Emryo yields were clculted s the verge of ten Petri-dishes. Germintion of microspore derived emryos For the conversion of microspore emryos into plntlets, the fully developed dicotyledonous emryos nd torpedo emryos were picked up nd trnsferred directly to MS medium contining 3% sucrose nd % gr (Figure A nd B). All microspore emryos were incuted t 5 ± 1 C under 1 h photoperiod with 5 mol m - s -1 PPFD (cool, white fluorescent lmps) for weeks. These were trnsferred ex vitro (Figure C). Ploidy nlysis using flow cytometry The nucler DNA content of the leves of microspore-derived plntletswsmesured with flow cytometer (Cytoflow PA,Prtec GmH, Germny) using the protocol descried y Mishi et l. (). Seedling leves of K5 (n = x = 1) were used s stndrd. Young leves (.3-.5 cm) from microspore-derived plntlets nd seedlings were nlyzed for their nucler DNA content. Fresh tissues were individully chopped with shrp rzor lde to less thn 1 mm in cm glss petri-dish contining

N et l. 1537 A B C mm Figure. Microspore derived emryo of Brssic olerce L. vr itlic. A, Cotyledonry microspore emryo formtion fter weeks in culture on.

% gr); C, cclimtized microspore derived plnts in the greenhouse weeks fter trnsfer from in vitro culture. µl of extrcting uffer (Solution A inthe CyStin UV Precise P Kit, Prtec, Germny).

For ech smple,,5-5, nuclei were nlyzed using flow cytometer equipped with HBO-1 mercury lmp.

One wy ANOVA ws used to ssess differences of microspore-derived emryos formtion in NLN liquid medium strength, microelement strength of NLN medium, sucrose concentrtion, het shock temperture nd het

3 N et l A B C mm Figure. Microspore derived emryo of Brssic olerce L. vr itlic. A, Cotyledonry microspore emryo formtion fter weeks in culture on.5 X NLN medium contining 15 g L -1 sucrose; B, microspore derived plntlet formtion fter weeks on conversion medium (.5X MS medium contining 3 g L -1 sucrose,.% gr); C, cclimtized microspore derived plnts in the greenhouse weeks fter trnsfer from in vitro culture. µl of extrcting uffer (Solution A inthe CyStin UV Precise P Kit, Prtec, Germny). After chopping, 1,ml of the, - dimidino--phenylindol (DAPI) stining uffer (Solution B of the kit) wsdded. The suspension ws filtered through 3 µm nylon mesh (CellTricsTM, Prtec, Germny). For ech smple,,5-5, nuclei were nlyzed using flow cytometer equipped with HBO-1 mercury lmp. Sttisticl nlysis Sttisticl nlysis ws done to evlute significnt differences mong microspore-derived emryos formtion nd vrious nutritionl nd environmentl conditions. One wy ANOVA ws used to ssess differences of microspore-derived emryos formtion in NLN liquid medium strength, microelement strength of NLN medium, sucrose concentrtion, het shock temperture nd het shock temperture period. ANOVA were crried out using sttisticl nlysis systems softwre SAS 9. (SAS Institute., Cry, NC, USA). Mens were seprted using Duncn s multiple rnge tests t the.5 significnce level. RESULTS AND DISCUSSION The MDE formtion ws. nd. in the.5x nd 1.X NLN liquid medium, respectively; however, the difference ws not significnt. The.5X NLN liquid medium hd the highest emryo formtion, with.. The MDE formtion in the.x nd.x NLN liquid medium ws low (Figure 3). The high concentrtions of mcro nd micro nutrient were not effective for MDE formtion. Therefore, reducing the concentrtion of mjor slt to one nd hlf in the NLN liquid medium seems to increse emryogenesis frequency in roccoli microspore culture. The nutritionl requirements for induction nd production of emryos vry widely from species to species. One of the most importnt medi components influencing emryogenesis is sl slt. For MDE formtion in roccoli, most experiments use the stndrd NLN-13 medi. In this study,.5x NLN liquid medium proved to e significntly etter thn the other medi strengths. Sto et l. (199) otined similr results in Brssic cmpestris ssp. Pekinensis, nd the sme result ws reported in somtic emryo formtion of Pimpinellrchycrp (N nd Chun, 9). A reduction in the concentrtions of some of the mcronutrients in NLN-13, minly NO 3, my e useful for promoting emryogenesis. Higher concentrtions of mcronutrients my e inhiitory to the induction of emryogenesis, s well s to emryo growth (N nd Chun, 9). The ddition of vrious mounts of micronutrients to the.5x NLN liquid medium ws less effective thn dding no micronutrients t ll. The medi to which micronutrients were not dded hd the highest formtion rte in MDE (Figure ) nd lso in rooted MDE (dt not shown). This finding differed from results for Chinese cge, which showed n increse in MDE formtion fter the ddition of micronutrients to.5x NLN medium (dt not shown). One of the most importnt medium components influencing the induction of emryogenesis is sucrose. The MDE formtion ws 7 nd 9 in the 13 nd 15% sucrose concentrtions, respectively. The difference in MDE formtion etween the two concentrtions ws not significnt, ut the MDE formtion in the 15% sucrose concentrtion ws the highest. A sucrose concentrtion less thn 1% decresed the emryo formtion rte

4 153 Afr. J. Biotechnol. Num er of m icrospore derived em ryo formtion 1 X.5 X.5 X1. X. X. Medium concentrtion Figure 3. Microspore derived emryo yields (numer of emryos/petri-dish) of Brssic olerce L.vr itlic of microspore Numer of microspore derived emryo formtion 1 X X.5 X.5 X 1. X. Micro elements concentrtion Figure. Microspore derived emryo yields (numer of emryos/petri-dish) of Brssic olerce L.vr itlic of microspore culture medium (.5X NLN) with vrious microelement strength of NLN liquid medium. Dt ws collected 3 dys fter culture. Ech vlue is the verge otined from ten replictions. Columns with the sme letters re not significntly different y Duncn s multiple rnge tests t P <.5.

5 N et l Num er of microspore derived em ryo form tion 1 c c Sucrose (g L -1 ) Figure 5. Microspore derived emryo yields (numer of emryos/petri-dish) of Brssic olerce L.vr itlic of microspore cultures treted with vrious concentrtion of sucrose. Dt ws collected 3 dys fter culture. Ech vlue is the verge otined from ten replictions. Columns with the sme letter re not significntly different y Duncn s multiple rnge tests t P <.5. remrkly, nd there ws no microspore formtion in the 5% sucrose concentrtion (Figures 5 nd ). Ferrie et l. (1999) found tht 13% sucrose hd higher emryo yield s compred to 1%. However, previous studies showed tht high level of sucrose is required for initil microspore survivl nd division, ut lower level is importnt for the continution of microspore division (Dunwell nd Thurling, 195). Additionl reserch on the different effects of pplied sucrose concentrtion ccording to MDE formtion phse is required. Microspore emryogenesis is induced y the het shock stress tretment. In B. npus, the most efficient induction is otined y incresing the culture temperture to 3 C for minimum of h (Custers et l., 199; Pechn et l., 1991). Binrov et l. (1997) reported tht DNA synthesis ws initited in oth genertive nd vegettive nuclei y the ppliction of het stress tretment. MDE formtion t the het shock tempertures of 5 nd 3.5 C in roccoli ws.5 nd 7.5, respectively; however, it ws merely.5 t 37 C, nd none t.5 C (Figure 7). The optimum het shock temperture for MDE formtion ws 3.5 C. The MDE formtion t het shock temperture of 3.5 C ws counted t het shock times of,, nd 7 h. At, nd 7 h, MDE formtion ws 1., 1.7 nd.9, respectively. The highest MDE formtion ws.9 t the het shock time of h (Figure ). Duijs et l. (199) estlished stndrd protocol for microspore culture using pre-tretment ( h t 3 C). MDE formtion ws significntly incresed in mny roccoli genotypes fter incuting t the het shock temperture of 3.5 C for 1 dy, s compred to the stndrd incution (Duijs et l., 199). The results of the polyploidy test for microspore-derived plntlets produced from the erlier experiments showed tht the men percentges of hploid, diploid, tetrploid, hploid + diploid, nd diploid + tetrploid nuclei were 5, 3,, nd %, respectively, indicting the existence of endopolyploid cells in the microspore-derived plntlet, which re considered to e mixoploid. These results were consistent with the reserch of Chen et l. (9), who otined vrious mixoploidy plnts from the protocormlike ody of Phlenopsis. This study descried methodology for chieving high frequency of microspore emryo formtion y controlling nutritionl fctors. Moreover, the efficient microspore culture protocols developed in this study could e useful in the production of homozygous line used to produce F 1 hyrids.

6 15 Afr. J. Biotechnol. Figure. Morphology of microspore derived emryo formed from microspores cultured in n NLN liquid medi with vrious concentrtions of sucrose. A, ; B, 5; C, 1; D, 13; E, 15; F, g L-1. Numer of microspore derived emryo formtion 1 c d Temperture ( C) ( o C) Figure 7. Microspore derived emryo yields (numer of emryos/petri-dish) of Brssic olerce L.vr itlic of microspore cultures treted with vrious het shock temperture for h. Dt ws collected 3 dys fter culture. Ech vlue is the verge otined from ten replictions. Columns with the sme letter re not significntly different y Duncn s multiple rnge tests t P <.5.

7 N et l. 151 Numer of microspore derived emryo formtion 1 hr hr hr 7 hr 7 Het shock time (hour) Figure. Microspore derived emryo yield (numer of emryos/petri-dish) of Brssic olerce L.vr itlic of microspore cultures treted with vrious het shock time t 3.5 C. Dt ws collected 3 dys fter culture. Ech vlue is the verge otined from ten replictions. Columns with the sme letter re not significntly different y Duncn s multiple rnge tests t P <.5. REFERENCES Chen WH, Tng CY, Ko YL (9) Ploidy douling y in vitro culture of excised protocorms or protocorm-like odies in Phlenopsis species. Plnt Cell Tissue Org. Cult. 9: 9-3. Custers JBM, Cordewener JHG, Nöllen Y, Dons JJM, Vn Lookeren Cmpgne MM (199). Temperture controls oth gmetophytic nd sporophytic development in microspore cultures of Brssic npus. Plnt Cell Rep., 13: Dis JCD (1999). Effect of ctivted chrcol on Broccoli microspore culture emryogenesis. Euphytic, 1: 5-9. Dis JCD (1). Effect of incution temperture regimes nd culture medium on roccoli microspore culture emryogenesis. Euphytic, 119: Duijs JG, Voorrips RE, Visser DL, Custers JBM (199). Microspore culture is successful in most crop types of Brssic olerce L. Euphytic, : Dunwell JM, Thurling N (195). Role of sucrose in microspore emryo production in Brssic npus ssp. oleifer. J. Exp. Bot. 3: Frnhm MW (199). Douled-hploid roccoli production using nther culture: effect of nther source nd seed set chrcteristics of derived lines. J. Am. Hort. Sci. 13: Ferrie AMR, Tylor DC, McKenzie SL, Keller WA (1999). Microspore emryogenesis of high sn- erucic cid Brssic olerce germplsm. Plnt Cell Tissue Org. Cult. 57: 79-. Gmorg OL, Miller RA, Ojim K (19). Nutrient requirements of suspension cultures of soyen root cells. Exp. Cell Res., 5: Keller WA, Armstrong KC (1979). Stimultion of emryogenesis nd hploid production in Brssic cmpestris nther cultures y elevted temperture tretments. Theor. Appl. Genet. 55: 5-7. Lichter R (19) Induction of hploid plnts from isolted pollen of Brssic npus. Z. Pflnzenphysiol. 15:7-3. Lichter R (199). Efficient yield of emryoids y culture of isolted microspores of different Brssiccee species. Plnt Breed., 13: Mishi K, Ando T, Mii M, Wtne H, Kokuun H, Hshimoto G, Mrchesi E (). Nucler DNA content s n index chrcter discriminting tx in the genus Petuni sensu Jussieu (Solncee). Ann. Bot N HY, Chun C (9) Nutritionl, chemicl nd physicl fctor ffecting somtic emryo formtion nd germintion in Pimpinell rchycrp. Kor. J. Hort. Sci. Technol. 7: -. Sto T, Nishio T, Hiri M (199) Plnt regenertion from isolted microspore cultures of Chinese cge (Brssic cmpestris ssp. pekinensis). Plnt Cell Rep., : -. Swnson EB, Erickson LR (199) Hploid trnsformtion in Brssic npus using n octopine-producing strin of Agrocterium tumefciens. Theor. Appl. Genet. 7: Swnson EB, Coumns MP, Brown GL, Ptel JD, Beversdorf WD (19). The chrcteriztion of hericide tolernt plnts in Brssic npus L. fter in vitro selection of microspores nd protoplsts. Plnt Cell Rep. 7: 3-7. Tkht Y, Keller WA (1991) High frequency emryogenesis nd plnt regenertion in isolted microspore culture of Brssic olerce L. Plnt Sci. 7: 35-. Tylor DC, Ferrie AMR, Keller WA, Gilin EM, Pss EW, McKenzie SL (1993). Biossemly of cyl lipids in microspore derived emryos of Brssic cmpestris L. Plnt Cell Rep. 1:

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