Tgp chi Cdng nghi Sinh hpc 1(1): 235-240, 2009 ANH HirONG CUA STRESS NACI LEN SlT ON DINH CUA cAu TRUC GENOME TE BAO LUA ORYZA SA TIVA NUOI CAY DAI HAN IN VITRO Nguyen Tbanb Thuy, D6 Quang Binh Viin Cdng nghi sinh hoc :• TOMTAT Nudi cay in vitro cac te bao thuc vat la mdt cdng cu hiiu ich cho cac nghien ciiu lien quan tdi chuyen hda sinh hpc tir mdt te bao dan le den cay hoan chinh in vitro. Sit dung cdng nghe in vitro nay, cac nha nghien ciru khdng chi cd the kiem soat cac chudi phan iing sinh hgc ma cdn tao ra nhieu san pham mdi khac vdi nhiing san pham cua cac vat lieu khdi dau. Tuy nhien, sinh trudng frong cac dieu kien nhan tao in vitro frong thdi gian dai, te bao thuc vat khdng franh khdi hien tugng bien di soma. Miic do bien di cua cac te bao nudi cay phu thupc vao nhieu yeu td khac nhau gdm cd kieu gen va frang thai sinh ly cua vat lieu thuc vat khdi diu, cac diSu kien nudi cay, thdi gian nudi cay Nghien ciiu nay trinh bay cac ket qua hen quan tdi nhiing bien ddi phan ttr frong genome ciia te bao liia (Oryza sativa) ma chiing da dugc nudi cay frong mdt thdi gian dM frong cac dieu kien cd sfress mudi (NaCI) khac nhau bang ky thuat RADP. So sanh vdi vdi vat heu khoi diu khdng bit ngudn tir nudi cay cac te bao phan lap in vitro, genome cua cac te bao nudi cay frong dieu kien khdng cd sfress NaCI mang nhieu bien ddi phan tu han genome cua cac te bao nudi cay lien tuc frong diSu kien cd sfress mudi. Mac dii cac te bao cua hai dang nudi cay nay chia se mdt sd sir bien ddi dac thu gay ra bdi nudi cay in vitro dai han, cac te bao thich nghi vdi sfress mudi chia se nhieu su tuang ddng chung vdi vat heu khdi dau han la cac te bao khdng bi sfress. Cac ket qua thu dugc tir nghien ciiu hien tai phu hgp vdi cac quan sat trudc day cua chimg tdi dupc tien hanh d miic dp te bao la tinh toan nang cua cac te bao liia phan lap dugc dn dinh frong dieu kien nudi cay cd stress mudi thich hgp tdt han frong dieu kien khdng cd yeu td sfress nay. Tie khoa: Bien di khde Met, nuoi cdy te bdo ddi hgn, Oryza sativa, stress NaCI su on dinh cua genome MdDAU Cvi \M Nhihig phat hien ca ban ve su sinh truang khong giai han cua te bao thuc vat frong nuoi cay in vitro va tinh toan nang cua chiing da tao ca so cho hang loat huong nghien ciiu nhu: te bao hpc, sinh ly, hoa sinh te bao, cac qua frinh sinh truang va phat frien cua te bao, nhan dong ca the, san xuat cac chat thii cap, loai bo benh virus, nghien cuu tuong tac giiia mo thuc vat va cac loai vi khuan tao u - not san o cay va cac nghien cuu di truyen ve bien di, dot bien soma frong te bao nuoi cay, iing dung kha nang tao cay dan boi, dong thuan frong chpn giong, dung hap protoplast tao dong lai soma hay lai te bao chat va chuyin DNA, (Gautheret, 1985). Tinh toan nang cua te bao cay liia tiong chau A (O. sativa L.) ciing da dupc nghien ciiu va chiing minh bang ky thuat nuoi cay mo, te bao in vitro. Sau thi nghiem tao mo seo tit npi nhii cua cay ngo do La Rue (1949) thuc hien thanh cong, Fujiwara va Ojima (1955) da tiSn hanh thi nghiem nuoi cac doan re lua tach rai. Nam 1956, Amemiya va dong tac gia tien hanh nuoi ciy phoi non cua loai cay luong thuc quan frong nay. Tuy nhien, tien bp frong nuoi cay mo te bao cdc loai cay luong thuc mpt la mam bat dau tir viec bd sung vao moi truang nuoi cay mpt lupng chat dieu hoa sinh trudng ngoai sinh (Stieet, 1957). Furuhashi va Yatazawa (1964) da tao duac mo seo tit dot than, tiong khi Yatazawa va dong tac gia (1967) tao duoc mo seo tit re cua cay ma tiong moi truang co bo sung 2.4-D. Nam 1968 la nam danh dau su truang thanh quan frpng cua ky thuat nuoi cay mo te bao doi voi cay Ilia. Nhieu phong thi nghiem da tai tao duac cay Ilia hoan chinh tir mo, te bao c6 nguon goc tit viing re cua hat (Nishi et al, 1968), tii rl (Kawata, 1968) va tii phoi Ilia (Maeda, 1968; Tamura, 1968). Dac biet, Nuzeki va Oono (1968) da tai smh duoc cay lua don bpi tir nuoi cay bao phan, tao tiSn de cho kha nang ling dung mpt each huu hieu phuang phap nuoi cay don bpi frong viec nit ngan thoi gian tao dong thuan va chpn giong liia. Nhung nam tiep theo duoc danh dau bang cac tien bp frong viec chpn dong te bao va tai sinh cay liia tir nuoi cay te bao frong dung dich long (Abe et al, 1991; Binh, Heszky, 1990; Gobel et al, 1985). Nhiing thi nghiem ve nuoi cay te bao fran cua lua cung da dupc tien hanh tir dau nhung nam 1970 (Ham, 1973; Deka, Sen, 1976). Thanh cong frong viec tai tao cay lua hoan chinh tii nuoi cay te bao fran bat dau dat duac tir giiia nhirng nam 1980 (Abdullah et al, 1986; Datta et al, 1992; Fujamura et 235 Nguyin Thanh Thuy & D6 Quang Binh al, 1985; Jenes, Pauk, 1989). Ben canh do, nhiing nghien ciiu ve anh huang cua cac loai sfress khac nhau (NaCI, t°, ) len su tai tao lai thanh te bao ciia tl bao tian va jkha nang phan chia te bao cua chiing ciing da duac quan tam (Binh, 1995). Ky thuat nuoi cay mo te bao in vitro da duac ung dung mpt each co hieu qua tiong nghien cim te bao, chpn dong chong chiu cac loai sfress sinh hpc va cac loai sfress moi truang, tao cac giSng liia mai a Viet Nam (Phan Thi Bay et al, 2001; Nghiem Nhu Van, 1983; Phong et al, 2001) ciing nhu a mpt so nuac khac (Heszky et al. 1996; Lestari, 2006). Cimg vai thatih cong frong viec dieu khien qua trinh sinh trudng va phat trien cua te bao thuc vat trong dieu kien nuoi cay in vitro nhan tao, cac nha nghien cim con phat hien ra cac hien tuang bien di va dot bien di truyen 6 mirc dp te bao va cay tai sinh CO nguon goc nuoi cay mo in vitro (Kabir et al, 2008; Nishi et al, 1968) ciing nhu o miic dp phan tu DNA (Ngezahayo et al, 2007; Rasheed et al, 2005). Larkin va Scowcroft (1981) da dung thuat ngii bien di ddng soma cho cac cay tai sinh co nhiing su thay ddi nhat dinh so vdi nguyen lieu gdc ban dau. Scowcroft (1985) coi nhiing su thay ddi d miic dp nhiem sac the, hinh thai, sinh hoa hay phan tit la su bat dn dinh do nudi cay in vitro gay ra. Nghien ciiu nay trinh bay cac ket qua ve anh hudng cua stiess NaCI len su dn dinh cua cau true genome te bao lua Oryza sativa nudi cay dai han in vitro bang ky thuat RADP (Random AmpUfication of Polymorphic DNA). Ket qua thu dupc phii hop vdi cac quan sat trudc day cua chung tdi dupc tien hanh d miic dp te bao la tinh toan nang cua cac te bao Ilia phan lap dupc nudi cay tiong dieu kien cd stiess NaCI thich hpp da duy tri su dn dinh cua tinh tiang nay lau dai han so vdi cac te bao sinh trudng frong dieu kien nudi cay in vitro tuong ddng nhimg khdng cd yeu td stiess nay (Binh, 1995; Binh, Heszky, 1990; Binh e/a/., 1990, 1992, 1993). NGUYEN LIEU VA PHUONG PHAP ' Nguyen lieu Hai ngudn md liia nudi cay in vitro dai han frong mdi trudng cd va khdng cd sfress mudi (NaCI) cd ngudn gdc tir hat ciia mdt gidng liia faponica cd ngudn gdc tii Hungary (Binh, Heszky, 1990). Hat liia dupc bdc vd, khii tning, rdi duoc cay chuyin len mdi trudng MS (Murashige, Skoog, 1962) cd bd sung 2% saccharose, 2 mg/l 2.4-D, 0,5 mg/l kinetin va 1 mg/l thiamine, pH 5,6 - 5,7 va 0,8% agar dl tao md so cap trudc khi dua vao nudi cdy dung dich long. Trong qua tiinh nudi ciy dai han, 2 nam dau tien te bao liia dugc nudi ciy tiong mdi trudng dung dich ldng khdng cd sfress mudi NaCI (Binh, Heszky, 1990). Sau nam tiep theo, mdt phan te bao nay dupc cay chuyen vao mdi trudng dung dich ldng cd bd sung 1,5% NaCI va phin cdn lai vin tilp tuc duoc duy tii tren mdi trudng ldng khdng cd stress mudi nhu cii. Nhumg nam sau cua giai doan nudi cay tiong dieu kien dung dich long cd va khdng cd sfress mudi nay, cac cum te bao duoc chuyen len nudi cay fren mdi trudng thach (0,8% agar) voi ndng dp mudi tuong tu nhu trong mdi trudng dung dich ldng da dung trudc do (Binh, 1995). Md dupc cay chuyen sau 4 tuan va sinh trudng d nhiet dp 27 - 28°C trong dieu kien anh sang khuech tan yeu, khoang 100 lux/lO h/ngay. Cay ddi chiing cd ngudn gdc tir hat cua gidng liia ban dau dupc duy tri d dang cay non in vitro. Pbuong pbap Phan ling RAPD dupc dung tiong nghien ciiu bien dpng genome cua te bao lua nudi cay in vitro dai han. Phan iing nay dupc tien hanh frong the tich dung dich hdn hpp la 25 |^1 vdi thanh phan ciia phan ling: 10 mM Tris-HCl (pH 8,3), 50 mM KCl, 1,5 mM MgClz, 0,01% gelatin, 200 \iM moi loai dNTP, 0,5 don vi enzyme Taq DNA polymerase (Promega), 0,25 |ag mdi oligo ngau nhien (Bang 1) va 20 ng DNA tdng sd cua mdi mau. Phan iing dupc thuc hien theo chu trinh nhiet: bien tinh ban dau d 96°C frong 3 phut, lap lai 35 lan chu trinh co ban (bien tinh d 96°C/45 giay, gan mdi vao khudn d 42°C/1 phiit, keo dai d 72°C/2 phiit), keo dai d 72°C/8 phiit d giai doan cudi cua phan iing. San pham cua cac phan iing RAPD nay dupc bao quan d -20°C de dung cho nhiing nghien ciiu tiep theo. Bang 1. Cac doan moi oligonucleotide dung trong phan u'ng RAPD. STT Trinh tiF base (5' - 3') gaggcctacgccccatagaa aatgcgttgaggcgcagcag cggaagcaatttgcttggct gcaattactatggctcggca ctccctcatgattcttggga ttgttcctgaccctggttca caattcgaggatccagagac tgccttgatccacttggcta 236 Tgp chi Cong nghe Sinh hpc 1(2): 235-240, 2009 ^ Cac phan iing duac lap lai 3 lin, cac bang hinh on dmh dupc ghi nhan va so lieu duac phan tich bang phan mem NTSYS-pc 1.80 (Rohtf, 1993). KET QUA VATHAO LUAN Nhiing bien ddng trong ciu tiiic cua genome tl bao lua nudi ciy dai han frong cac dilu kien cd va khdng cd sfress mudi NaCI dupc khao sat bing ky thuat RAPD vdi 8 mdi oligo (Bang 1). San phim cua phan ling RAPD vdi hai mlu tl bao tren duoc so sanh vdi san phim tuong ddng cua cay ddi chiing. Kit qua dien di cac san phim RAPD cho thiy cac mlu DNA cd ngudn gdc md, tl bao khac nhau tao ra cac san phim cd mlu bang da hinh DNA khac nhau (Hinh 1, 2). Hinh 1. Bien di (gel agarose 1%) cac san pham RAPD vdi doan mdi oligo so 3 (1 - 3) va 4 (4 - 6) cua: genome cay ddi chiing (1, 4), ti bao nudi cly in vitro dai han trong mdi trudng khdng cd stress mudi NaCI (2, 5) va cd stress mudi NaCI (3, 6). M: thang DNA chuan 123 bp (Gibeo). Trong sd cac bang da hinh, mdt sd bang DNA chi dac trung cho md cua cay liia ddi chiing, hay cac bang dai dien chung cho cac loai tl bao lua nudi ciy in vitro ma khdng phu thudc vao dieu kien nudi ciy co hoac khdng cd stress mudi NaCI cua chiing. Mpt sd bang khac dac trung cho cac tl bao nudi ciy trong mdi trudng khdng cd stress mudi, hay cac bang chi cd mat trong san pham ciia phan iing RAPD vdi DNA cua te bao nudi cay cd stress mudi va mo cua cSy ddi chiing, Tdng hop ket qua ciia phan iing RAPD vdi 8 mdi oligo va ba loai miu md, tl bao thi nghiem, chiing tdi thu duoc tdng s6 bang DNA la 50, trong sd do 20 bang la bang da hinh, chiim ty le 40% (Bang 2), Tren co sd cdc sd lifu thu duac tii phan ling RAPD, cay lidn ket phan tii nhirng "viing genome duoc khao sat bang 8 mdi oligo diing trong thi nghiem nay, ciia ba loai md, te bao khac nhau ndi tren duoc xay dung theo chuang trinh NTSYS-pc 1.80 (Hinh 3). 1107 Hinh 2. Dien di (gel agarose 1%) cac san pham RAPD vdi doan mdi oligo sd 2 (1 - 3), 7 (4 - 6) va 8 (7 - 9) cua: genome cay ddi chiing (1, 4, 7), td bao nudi cay in vitro dai han trong mdi trudng l<hdng cd stress mudi NaCi (2, 5, 8) va cd stress mudi NaCI (3, 6, 9). M: thang DNA chuan 123 bp (Gibeo). Bang 2. Ty l# bang da hinh dua tren chi thj RAPD cua genome cd ngudn gdc tCr cay ddi chiing, te bao nudi ciy in vitro dai han trong mdi trudng khdng cd stress mudi NaCi, va cd stress muoi NaCI. STT 1 2 3 4 5 6 7 8 Tong Mdi 1 2 3 4 5 6 7 8 8 Tdng 9 7 8 5 7 5 5 4 50 s6 bang S6 bang da hinh 7 3 4 2 1 0 2 1 20 Qua hinh cay lien ket dua tren cac chi thi phan tir RAPD, chiing tdi nhan thay nhung vimg genome dupe khao sat cua te bao liia nudi cay trong dilu kien cd stress mudi NaCI cd mirc dp tuang ddng vdi 237 Nguyen Thanh Thuy & Dd Quang Binh nhiing viing genome tuong iing cua md cay ddi chiing ldn hon nhiing viing genome tuong tu cua te bao nudi cay frong mdi trudng binh thudng khdng cd stiess mudi NaCI. Ket qua thu dupc frong thi nghiem nay phan anh nhiing bien ddng frong cau tnic phan tir xay ra tai nhung viing nhat dinh frong genome dupc khao sat cua cac loai te bao liia nudi cay dai han in vitro trong cac dieu kien cd va khdng cd stiess NaCI la khac nhau. -Genome cua quan the cac te bao sinh trudng frong dieu kien cd stress mudi NaCI cd miic dp dn dinh cao hon so vdi genome cua quan the cac te bao sinh trudng frong mdi trudng co ban khdng cd stiess mudi NaCI. Kit qua nay phu hpp vdi nhung ket qua da dupc cdng bd trudc day cua chimg tdi (Binh, 1995; Binh, Heszky, 1990; Binh et al, 1990, 1992, 1993) ve tinh dn dinh cao tiong sinh trudng va tiem nang tai sinh cay hoan chinh ciia cac te bao tien phdi Ilia da thich nghi vdi dieu kien nudi cay in vitro dai han cd sfress muoi NaCI thich hpp. Bien ddng tiong cau tnic genome cua te bao lua nudi cay in vitro cung da dupc ghi nhan qua phan img RAPD vdi cac ddng liia tai sinh tii nhiing ddng md nudi cay ngin han hon frong cac dilu kien chpn lpc khac (Ngezahayo et al, 2007; Rasheed et al, 2005; Phan Thi Bay et al, 2001; Phong et al, 2001). Co chi cua hien tuong biln di soma hien cdn chua duoc hieu biet day du. Tuy nhien, nhiing nguyen nhan cd kha nang dan tdi bien di soma (Barr, Jain, 1998) bao gdm: (1) Thay ddi d miic dp nhiem sac the; (2) Dot bien diem; (3) Su bat cheo va trao ddi doan cua nhiem sac the chi em; (4) Su tai sap xep vi tri cua gen; (5) Su bdi nhan DNA; (6) Hoat ddng cua cac "gen nhay"; (7) Su methyl hda DNA; (8) Nhiing thay ddi tiong DNA cua co quan tii; (9) Bien di chiic nang cua gen, Nghien cim nay ghi nhan dupc nhiing bien ddng phan tu tiong genome cua cac te bao liia nudi cay in vitro dai han phu thudc rat nhieu vao dieu kien nuoi cay frong dd cd stiess mudi NaCI. Co che cua hien tupng bien di soma trong nudi cay in vitro hien van dang dupc tiep tuc nghien cim. Ksh Cn Co Hinh 3. Cay lien ket dua tren cac chi thj phan tu (RAPD) cua hai ddng te bao nudi cay in vitro dai han trong mdi trudng khdng cd stress muoi NaCi (Co) va cd stress muoi NaCi (Cn) vdi cay doi chiing (Ksh). KET LUAN Mudi NaCI, d ndng dp thich hpp nhu 1,5% NaCI dung tiong nghien cim hien tai vdi md, te bao cd ngudn gdc tir hat lua, cd nhiing anh hudng tich cue nhat dinh len su dn dinh ciia genome te bao - mdt frong nhiing yeu td quyet dinh de duy fri tinh toan nang ciia chiing frong nudi cay in vitro dai han. Loi cam on. Cdng trinh dupc hodn thdnh vai su hd tra kinh phi mpt phdn cua di tdi nghien cieu cdp ca so Viin Cdng nghe sinh hpc vd Quy hpc bdng UNIDO/ICGEB, Italy. TAI LIEU THAM KHAO Abdullah R, Cocking EC, Thompson JA (1986) Efficient plant regeneration from rice protoplasts through somatic embryogenesis. BioTech 4: 1087-1090. Abe T, Futsuhara Y (1991) Regeneration of rice plants from suspension culture. In: Bajaj YPS (Ed.) Biotechnol AgricultFores 14: 3S-45. Barr DS, Jain SM (1998) Somaclonal variation: mechanism and application in crop improvement. In: Jain SM, Barr DS, Ahloowalia BS Eds. Somaclonal Variation and Induced Mutation in Crop Improvement. 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Proc Jpn Acad 44: 543-548. 239 Nguyen Thanh Thiiy & Dd Quang Binh EFFECTS OF NACI STRESS ON THE GENOME STRUCTURE STABILISATION OF RICE ORYZA SATIVA CELLS LONG-TERM CULTURED IN VITRO Nguyen Tbanb Tbuy, Do Quang Binb* Institute of Biotechnology SUMMARY In vitro culture of plant cells is a useful tool for studies relating the biological fransformation from a single cell to the whole plant in vitro. Using the in vitro technology, researchers are able to not only confrol the biological reaction chains but also to create a number of new products other than those ofthe initial materials. However, growing in the in vitro artificial conditions for a long time, plant cells could not avoid the phenomenon of soma variation. The level of variation of the cultured cells depends on various factors, including the genotype and physiological state of initial materials, the culture conditions, the culture time, etc. This study presents results relating the molecular changes in the genome of rice (Oryza sativa) cells that have been cultured for a long time in different salt (NaCI) sfress conditions by RAPD technique. In comparison with the initial material, not derived from the culture in vitro of isolated cells, genome of cells cultured in a condition without NaCI sfress bears more molecular changes than that of cells continuously cultured in the salt sfress condition. Though cells of the two culture conditions shared several specific changes, induced by the long-term culture in vitro, the salt sfress adapted cells shared more common similarities with the initial material than the non sfressed cells. Results obtained from the present study are in accordance with our previous observations, carried out at the cell level, that the totipotency ofthe isolated rice cells has been stabilized much better in the suitable salt sfress culture condition than in the condition without the sfress factor. Keywords: Differential variation, genome stability, long-term cell culture, NaCI stress, Oryza sativa Author for correspondence: Tel: 84-4-38363470; Fax: 84-4-38363144; E-mail: dqbinh&.ibt.ac.vn 240 . cay in vitro va tinh toan nang cua chiing da tao ca so cho hang loat huong nghien ciiu nhu: te bao hpc, sinh ly, hoa sinh te bao, cac qua frinh sinh. cd stress mudi NaCI cd mirc dp tuang ddng vdi 237 Nguyen Thanh Thuy & Dd Quang Binh nhiing viing genome tuong iing cua md cay ddi chiing