Effect of Temperature Shock on Soybean Microspore Embryogenesis

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1 537 Vol.47, n. 4 : pp , August 2004 ISSN Printed in Brzil BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY AN INTERNATIONAL JOURNAL Effect of Temperture Shock on Soyen Microspore Emryogenesis An Pul de Mores 1, Mri Helen Bondese-Znettini 1, Sídi Mri Cllegri-Jcques 2 nd Eline Kltchuk-Sntos 1 1 Deprtmento de Genétic; Universidde Federl do Rio Grnde do Sul; eline.kltchuk@ufrgs.r; C. P ; ; Porto Alegre - RS - Brzil. 2 Deprtmento de Esttístic; Universidde Federl do Rio Grnde do Sul; Porto Alegre - RS - Brzil ABSTRACT In this study the effect of cold nd het shock on ndrogenetic induction in soyen nther culture ws tested. Anthers of soyen were sujected to 4 nd 33ºC, while the nthers used for control were mintined t 25ºC. Cytologicl nlysis done during the 30 dys of culture showed tht frequencies of symmetricl inucleted nd multicellulr pollen grin did not differ mong temperture tretments. Multicellulr pollen grin could e formed y symmetricl division, s well s y ssymetricl division. In reltion to the emryo induction, the results of the tretments did not differ too. These results suggested tht these tretments did not induce sporophytic pthwy in soyen. Key words: Anther culture, soyen, ndrogenesis, symmetricl mitosis, temperture shock, stress INTRODUCTION The formtion of emryos nd hploid plnts from microspores represents fundmentl switch in development from norml gmetophytic to sporophytic pthwy. This phenomenon in which microspores chnge their ontogenetic route is clled ndrogenesis nd it ws firstly reported y Guh nd Mheshwri (1964). These reserchers verified the formtion of emryos on surfce of cultured nthers of Dtur innoxi. Since then, microspore emryogenesis hs een reported in more thn 200 species of ngiosperms (Indrinto et l., 1999). Doule hploid plnts otined from nther culture re incresingly used in plnt reeding for production of homozygous lines, ut its ppliction hs een limited in mny species minly ecuse ndrogenetic mechnism is lrgely unknown. Inductive stress tretment hs een identified s the mjor trigger in inducing microspore nd pollen grin emryogenesis for mny plnts. This inductive stress tretment hs n influence in the first sporophytic mitosis. Studies hve shown tht in severl species the first division is symmetricl in ndrogenetic emryos (Fn et l. 1988; Zki nd Dickinson, 1990, 1991, 1995). According to these uthors, symmetricl division is key step to susequent pollen emryogenesis. Then, pollen grins follow rpid cell divisions nd morphogenetic process, closely resemling zygotic emryogenesis, resulting in hploid plnts. Four stress tretments hve een shown to induce microspore emryogenesis: cold shock, het shock, crohydrte nd nitrogen strvtion nd Author for correspondence Brzilin Archives of Biology nd Technology

2 538 Mores, A. P. et l. colchicine. Het shock hs een reported to increse the emryogenic route in severl plnts like whet (Tourev et l., 1996c), tocco (Tourev et l., 1996 nd 1996) nd Brssic (Telmer et l., 1995; Binrov et l., 1997; Custers et l., 1994) while the cold shock nd strvtion induced microspore emryogenesis in mize, whet, rley nd rice (Indrinto et l., 1999). The developmentl stge of microspore t the time of culture initition is of crucil importnce, with lte vcuolted microspore to erly icellulr pollen grin eing the responsive stges to het stress (Pechn nd Keller, 1988). Since not ll microspores respond to the inductive tretment, the cultures lwys contin high percentge of gmetophytic pollen grin. This pper reports the effect of two inductive stress tretments, cold (4 C) nd het (33 C) shock, in six soyen cultivrs. The formtion of emryo-like structure nd cytologicl chrcteristics were oserved. MATERIAL AND METHODS Plnt mteril Soyen (Glycine mx (L.) Merrill; 2n=40) cultivrs IAS 5, Brgg, Décd, RS 7, BR 4, nd BRS 133 field-grown were used s source of inflorescences. Anther culture Florl uds of mm in length contining uninuclete microspores were collected nd kept t 4 C for 12h. The surfce of the uds ws sterilized with 70% ethnol for 30s, 2% NOCl solution with trce mount of detergent for 12 min nd then rinsed three times in sterile distiled wter. Emryo induction Anthers were cultured in induction medium I - B5 sl medium (Gmorg et l., 1968) enriched with 16 orgnic compounds - supplemented with Yeung s mino cids (Yeung nd Sussex, 1979), 9% sucrose, 0.25% Phytgel, 2.0mgL -1 2,4-D nd 0.5 mgl -1 BAP. For ech cultivr, eighteen smll Petri dishes with induction medium I were used, six dishes for tretment (4, 25 nd 33 C). Six cultivrs were used, totlizing 36 dishes in ech temperture. Fifty-five nthers were inoculted for dish. The dishes were incuted during four dys in the drk. After this period, the cultures were mintined t 25 C under 16h photoperiod of 22.5 µmol.m -2.s -1 provided y fluorescent light nd sucultured every 4 weeks. After 8 weeks, the nthers were trnsferred to n induction medium II (medium I with 1.0 mgl -1 2,4-D nd 1.0 mgl -1 BAP) with suculture on the sme fresh medium every 4 weeks. Emryos mturtion On the 75 th dy, hlf of emryogenic clli were trnsferred to MSM6 (Finer nd McMullen, 1991) nd the other hlf, to induction medium II. After 30 dys, clli nd emryos were counted, seprted, nd the histodifferentited trnsferred to MSO (MS slts, B5 vitmins, 3% sucrose, 0.25% Phytgel, ph 5.8) medium. In vitro Anlysis After 60 dys of culture the nthers were clssified into three clsses: no responsive nthers, rough clli nd emryogenic clli (green, smooth nd right clli). Cytologicl Anlysis Five nthers per Petri dish were fixed in ethnol:glcil cetic cid (3:1) t 0, 15 nd 30 dys of culture for cytologicl nlysis. The nthers were squshed in propionic crmine on glss slides nd seled under cover slips with ee s wx. Microspores were stged nd clssified under Zeiss Axiopln Universl microscope. Sttisticl Anlysis Dt on totl clli (rough nd emryogenic clli) nd emryogenic clli formtion were compred mong tretments nd cultivrs with the Kruskl- Wllis (KW) test, followed y Dunn s nonprmetric multiple comprisons (Zr, 1999). Dt on inuclete nd multicellulr pollen grins ws log trnsformed nd three-wy fctoril nlysis of vrinces ws conducted to evlute the effects of genotype (6 cultivrs), temperture (4, 25 nd 33 C), dys of culture (0,15 nd 30) nd interctions in the proportion of inuclete nd multicellulr pollen grin. As significnt heterogeneity mong vrinces were found, the Welch s test ws performed to check the significnce of the interction effects, followed y Dunnett s-t3, test for multiple comprisons tht do not ssume equl vrinces. Brzilin Archives of Biology nd Technology

3 Effect of Temperture Shock on Soyen Microspore Emryogenesis 539 RESULTS Emryogenic nd totl clli formtion The numer of emryogenic s well s of totl (rough plus emryogenic) clli formed in ech Petri dish ws vlited for ech tretment (N=36), not mtching distinction mong cultivrs (Tle 1). The vrege numer of emryogenic clli vried from 2.7 to 4.1, ut the differences mong tempertures were not sttisticlly significnt (χ 2 KW=4.251; df=2; p=0.119). The vrege for the totl clli formtion did not vry mong tempertures either (χ 2 KW=1.423; df=2; p=0.49). On the other hnd, significnt differences, independent of tretment, were found oth for the numer of emryogenic (χ 2 KW=32.251; df=5; p<0,001) nd the totl (χ 2 KW=14.517; df=5; p=0.013) clli produced. BRS 133 showed the lowest induction frequency of emryogenic clli in comprison with other cultivrs (Fig. 1). The sme pttern, however, ws not oserved for the totl clli production: BR 4 produced significntly more clli thn IAS 5 nd Brgg; the other cultivrs did not present sttisticl differences in reltion to these three cultivrs (Fig. 2). The emryos formed until the 60 th dy of culture showed high vriility in their morphology during development. Mny emryos were prtilly or completely fused (Fig. 3). Severl of these emryos were not le to develop into plntlet nd just smll numer survived in culture during mny months. These structures were mintined in the MSO, nevertheless, prt of them eventully lckened nd died. When trnsferred into MSO medium continig 1% sucrose, the plntlets further developed. A complete plntlet with leves ws oserved fter the 80 th dy of culture (Fig. 3c). Chromosome counts in this plntlet showed the expected hploid (2n=20) condition (Fig. 3d). Tle 1: Avrege (± stndrd devition) numer of emryogenic nd totl clli formed y soyen nthers (six cultivrs) mntined t three different temperture conditions (N=36 dishes for tretment). Temp. Numer of emryogenic clli Numer of totl clli formtion C Avrege ± SD Avrege ± SD ± ± ± ± ± ± 4.4 Avrege numer of emryogenic clli 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 IAS 5 Brgg RS 7 BRS 133 BR 4 cultivrs Figure 1 - Avrege numer (N=18) of emryogenic clli produced per cultivr, on the 60th dy of culture. Avrege followed y the sme letter do not differ significntly (=0,05). Brzilin Archives of Biology nd Technology

4 540 Mores, A. P. et l IAS 5 Brgg RS 7 BRS 133 Avrege numer of totl clli,,, BR 4 Cultivrs Figure 2 - Avrege numer (N=18) of totl clli produced per cultivr, on the 60th dy of culture. Avrege followed y the sme letter do not differ significntly (=0,05). c d Figure 3 - Structures formed in culture: nd - fused cotyledons (rs=1 mm), c- plntlet formed from emryo (r=1 cm), d- hploid cell with 2n=20 (r=10µm). Cytologicl nlysis In order to investigte whether het or cold shock tretment would e effective to induce the formtion of inucleted symmetricl nd multicellulr pollen grins in soyen (Fig. 4), oth with ndrogenetic potentil, two 3x3x6 fctoril nlysis of vrince ws performed. Two nd three fctor interctions were considered esides the min effects temperture, dys of culture nd cultivrs. As the three fctors Brzilin Archives of Biology nd Technology

5 Effect of Temperture Shock on Soyen Microspore Emryogenesis 541 interction ws sttisticlly significnt for oth vriles (F= 4.91, p< nd F=1.91, p=0.014 for symmetricl nd multicellulr pollen grins, respectively), tretments could not e compred without considering cultivrs nd dys of culture. Therefore, the effects of cultivrs nd temperture (s well s its interctions) were nlysed t ech dy of culture seprtely. Differences were detected only t dy 15 of culture for symmetricl inucleted nd multicellulr pollen grins (Tle 2). Cultivr RS 7 incuted t 25 nd 33 C showed more symmetricl inuclete pollen grins thn Décd incuted t 4 C, other differences eing not significnt. For multicellulr pollen grins, RS 7 incuted t 25 C showed higher frequencies of multicellulr pollen formtion thn Brgg/Décd incuted t 4 C, IAS 5 (33 C) nd Décd/BR 4 incuted t 25 C, which presented the sme verge numer of multicellulr pollen grins (0-0.2). c d e Figure 4 - Symmetricl nd multicellulr soyen pollen grins. - symmetricl pollen grin;, c nd e- multicellulr pollen grin; d- multicellulr pollen grin with two vegettive nd one genertive cell. (rs=10µm). Brzilin Archives of Biology nd Technology

6 542 Mores, A. P. et l. Tle 2 - Averge (± stndrd devition; N=6) of the numer of inucleted nd multicellulr pollen grins t dy 15 of culture. Cultivrs Temp. Binuclete pollen grins Multicellulr pollen grins C Men ± SD Men ± SD IAS ± 8.0 AB 0.3 ± 0.8 AB ± 5.0 AB 1.8 ± 4.5 AB ± 1.7 AB 0.2 ± 0.4 A Brgg ± 1.4 AB 0 ± 0 A ± 3.6 AB 1.8 ± 2.4 AB ± 1.8 AB 0.8 ± 1.0 AB Décd ± 0.8 B 0 ± 0 A ± 1.0 AB 0.2 ± 0.4 A ± 1.0 AB 0.8 ± 1.2 AB RS ± 19.0 AB 10 ± 17.3 AB ± 7.1 A 2.7 ± 1.2 B ± 1.9 A 0.8 ± 0.7 AB BRS ± 4.6 AB 3.3 ± 3.3 AB ± 5.6 AB 3.3 ± 4.0 AB ± 2.8 AB 0.8 ± 1.0 AB BR ± 7.7 AB 4.3 ± 7.4 AB ± 4.5 AB 0.2 ± 0.4 A ±1.5 AB 0.8 ± 1.6 AB Mens followed y the sme letter do not differ significntly (α=0,05). DISCUSSION Dt otined in the present study showed tht emryogenic clli formtion otined fter 4 dys t 4 nd 33 C did not differ from the control. Keller nd Armstrong (1979) tested tretment t 30 nd 35 C in Brssic cmpestris. The first tretment decresed emryo yield, ut the second stimulted the frequency of emryogenesis. Smýkl (2000) showed tht in B. npus, two conditions were importnt to trigger emryogenesis: ) in vitro temperture should e ner to the physiologicl limit of the plnt nd ) temperture of microspores in vitro should e C higher thn the temperture of donor plnt. In the present study, only one high temperture, 33 C, ws tested which might not e sufficiently elevted to increse the frequency of emryogenesis. In spite of gret vriility in the morphology of emryos formed in vitro, they resemled the somtic ones induced in high 2,4-D medium from immture cotyledons (Buchheim et l., 1989; Sntos, 1997). A previous study showed tht puttive ndrogenetic emryos of soyen were histologiclly similr to zygotic emryos (Kltchuk-Sntos et l., 1997). According to some uthors, induction of cell division is n independent process in the determintion of emryogenesis. Tourev et l. (1996) showed tht multicellulr structures could e formed fter symmetric mitosis indicting tht symmetry of microspore in vitro is not essentil for emryogenic induction. In the present nlysis it ws oserved tht multicellulr structures were formed from vegettive cell fter symmetric mitosis (Fig. 4d). Our results did not provide ny evidence to conclude wht ws the est temperture to induce symmetricl divisions Brzilin Archives of Biology nd Technology

7 Effect of Temperture Shock on Soyen Microspore Emryogenesis 543 nd/or multicellulr pollen grins formtion in soyen, since it detected interctions etween cultivrs, tretments nd dys of culture. ACKNOWLEDGEMENT This study ws supported in prt y CNPq, FAPERGS nd PROPESQ. RESUMO Neste estudo form testdos os efeitos do choque térmico n indução ndrogenétic em cultur de nters de soj. Prte ds nters formm sumetids o choque térmico de 4 ou 33 C, enqunto s nters controle formm mntids 25 C. Análises citológics formm relizds o longo dos 30 primeiros dis de cultur mostrndo que freqüênci de grãos de pólen inucledos simétricos e multinucledos não difere entre os trtmentos e que os grãos de pólen multinucledos podem ser formdos tnto prtir de um mitose simétric inicil como pós um mitose ssimétric. Em relção à indução emriogênic, os trtmentos tmém não diferem. Estes resultdos sugerem que o trtmento de choque térmico não induz rot esporofític nos micrósporos de soj. REFERENCES Binvor, P.; Huse, G.; Cenklová, V.; Cordewener, J. H. G. nd; Vn Lookeren Cmpgne, M. M. (1997), A short severe het shock is required to induce emryogenesis in lte icellulr pollen of Brssic npus L. Sex. Plnt Reprod.,10, Buchheim, J. A.; Colurn, S. M. nd Rnch, J. P. (1989), Mturtion of soyen somtic emryos nd the trnsition to plntlet growth. Plnt Physiol., 89, Custers, J. B. M.; Cordewener, J. H. G.; Nöllen, Y.; Dons, H. J. M. nd vn Lookeren Cmpgne, M. M. (1994), Temperture controls oth gmetophytic nd sporophytic development in microspore culture of Brssic npus. Plnt Cell Reports, 13, Fn, Z.; Armstrong, K. C. nd Keller, W. A. (1988), Development of microspores in vivo nd in vitro in Brssic npus L. Protoplsm, 147, Finer, J. J. nd McMullen, M. D. (1991), Trnsformtion of soyen vi prticle omrdment of emryogenic suspension culture tissues. In Vitro Cell. nd Developmentl Biology, 27P, Gmorg, O. L.; Miller, R. A. nd Ojim, K. (1968), Nutrient requirements of suspension culture of soyen root cells. Exp. Cell. Res., 50, Guh, S. nd Mheshwri, S. C. (1964), In vitro production of emryos from nthers of Dtur. Nture, 204, 497. Indrinto, A; Herele-Bors, E. nd Tourev, A. (1999), Assessment of vrious stresses nd crohydrtes for their effect on the induction of emryogenesis in isolted whet microspores. Plnt Science, 143 : (1), Kltchuk-Sntos, E.; Mrith, J. E.; Mundstock, E. nd Znettini, M. H. B. (1997), Cytologicl nlysis of erly microspore divisions nd emryo formtion in cultured soyen nthers. Plnt Cell, Tissue nd Orgn Culture, 49, Keller, W. A. nd Armstrong, K. C. (1979), Stimultion of emryogenesis nd hploid production in Brssic cmpestris nther cultures y elevted temperture tretments. Theoricl nd Applied Genetics, 55, Pechn, P. M. nd Keller, W. A. (1988), Identifiction of potentilly emryogenic microspores in Brssic npus. Physiol. Plnt, 74, Sntos, K. G. B. (1997), Emriogênese somátic em soj (Glycine mx (L.) Merrill): efeito do genótipo, do etileno e ontogeni dos emriões. Dissertção (Mestrdo em Genétic), Universidde Federl do Rio Grnde do Sul, Rio Grnde do Sul, Brsil. Smýkl, P. (2000), Pollen emryogenesis - the stress medited switch from gmetophytic to sporophytic development. Current sttus nd future prospects. Biologi Plntrum, 43 : (4), Telmer, C. A., Newcom, W. nd Simmonds, D. H. (1995), Cellulr chnges during het shock induction nd emryo development of cultured microspores of Brssic npus cv. Tops. Protoplsm, 185, Tourev, A.; Ilhm, A.; Vicente, O. nd Heerle-Bors, E. (1996), Stress s the mjor signl controlling the developmentl of tocco microspores: towrds unified model of induction of microspore/pollen emriogenesis. Plnt, 200, Tourev, A.; Ilhm, A.; Vicente, O. nd Heerle-Bors, E. (1996c), Stress-induced microspore emryogenesis in tocco: n optimized system for moleculr studies. Plnt Cell Reports, 15, Tourev, A.; Indrinto, A.; Wrtschko, I.; Ilhm, A.; Vicente, O. nd Heerle-Bors, E. (1996), Efficient microspore emryogenesis in whet (Triticum estivum L.) induced y strvtion t hight temperture. Sex. Plnt Reprod., 9, Yeung, E. C. nd Sussex, I. M. (1979), Emryogeny of Phseoleus coccineus: the suspensor nd the growth of the emryo-proper in vitro. Z. Pflnzenphysiol., 91, Brzilin Archives of Biology nd Technology

8 544 Mores, A. P. et l. Zki, M. A. M. nd Dickinson, H. G. (1990), Structurl chnges during the first divisions of emryos resulting, from nther nd free microspore culture in Brssic npus. Protoplsm, 156, Zki, M. A. M. nd Dickinson, H. G. (1991), Microsporederived emryos in Brssic: the significnce of division symmetry in pollen mitosis I to emryogenic development. Sex. Plnt Reprod., 4, Zki, M. A. M. nd Dickinson, H. G. (1995), Modifiction of cell development in vitro: the effect of colchicine on nther nd isolted microspore culture in Brssic npus. Plnt Cell, Tissue nd Orgn Culture, 40, Zr, J. H. (1999), Biosttisticl Anlysis. New Jersey : Prentice Hll. Received: Ferury 03, 2003; Revised: July 02, 2003; Accepted: Novemer 24, Brzilin Archives of Biology nd Technology