Thi nghiém Sink hoe phan nit -t- Bién soan : Lé Ly Thuy Tram BAI 1: AYA 1.CAC THIET BI CO BAN CUA MOT PHONG THI NGHIEM NUOI CAY MO VATE BAO THUC VAT a. Phong rita va cét mtée ~ May cat nude 1 Ln ~ May ct nude 2 lin b. Phong hap — sdy - Autoclave - Tui say 60- 200°C c. Phong chudin bi méi truang - Can phan tich (chinh xdc dén 0,0001 g) - Can k¥ thuat (chinh xd dén 0,01 g) - pH ké - May khudy tit - Ti lanh - La vi séng (microwave) d. Phong thao téc nudi cdy - Tu cay v6 tring (laminar) = Quat thong gid - Dan tit ngoai treo tung ¢. Phang nudi cy - Cae gian ké ¢6 giin dén huynh quang - May digu hda nhiét d6 - May Lic nim ngang Ta dm f. Phang thi nghigm: (phdng nay dig dé tién hanh céc phan tich sinh h6a, phan ui va di truyén) ~ Kinh hién vi 2 mat (d6 phéng dai 1000 lan) ~ Kinh hip 2 mat (d6 phéng dai 75 lan) - Microtome ~ May anh ky thuat s6 - Hé théng dén chiéu = Quang phé kéThi nghiém Sink hoe phan nit -2- Bién soan : Lé Ly Thuy Tram 2.CAC NHAN TO DAM BAO THANH CONG TRONG NUOI CAY MO TE BAO THUC vAT C63 nhan t6 chinh: - Bao dam diéu kién v6 tring - Chon diing mdi trudng va chudin bi mdi trudng diing cach ~ Chon mé cay va xit ly mé cy thich hgp trude va sau khi cy. 2.1. ¥ nghia cia v6 tring trong nudi cdy mé va té bao thue vat M@i truding dé nudi cdy mé ya té bao thuc vat c6 chifa dudng, vitamin, mudi khodng. thich hgp cho céc loai nm va vi khudin phat trién, Do téc d6 phan bao ctia nam va vi khudin 1dn hon rat nhiéu so véi t& bao thuc vat, néu trong mdi trudng nudi cAy chi nhiém mot vai bao i ndm hoge vi khudn thi chi sau vai ngay dén m6t tun, toan b6 bé mi cy sé phi diy mé nhiéu loai nam va vi khudn. Thi nghiém phai bo di vi trong diéu kién nay m6 nudi cy sé khong phat trign va chét dan. 2.2 Nguon tap nhiél C63 ngudn tap nhiém chinh: ~ Dung cu thuy tinh, mdi tring nudi cdy va mit day khong duge vo hodic m trimg tuyét = Trén bé mat hoc bén trong mé cdy tn tai cdc si nam, bao tit nam hodc vi khuan - Trong qué trinh thao téc Lam ri nam hodc vi khudn theo bui lén bé mit mdi trudng 2.3 Ky thuat vo tring 2.3.1 V6 tring dung cu thuy tinh, mat day va mdi truding a. Dung cu thuy tinh ‘Thong thuding cdc dung cu thuy tinh ding trong ede thi nghiém thudng dude xt ly bing dung dich sulfocromate mét lin dau truéc khi dua vio sit dung: vé sau chi can nia sach bing xA phOng, tring sach bing nut that réo trude khi st dung. Trong wuding hyp cdc dung cu thuy tinh ding wong cdc thi nghiém nudi cy md va té bao thue vat ddi hoi v6 tring, c6 thé khit tring trong ti say d nhiét dé cao trong nhiéu phuit hodc i. Cae dung cu nay ludn dude gdi trong gidy nhém hoje hép kim loai dé tranh bi nhiém tré Lai sau khi da khit va tring Bang 1.1: Thoi gian khit tring dung cu thus tinh béing nhiét vet nhigt dé khet tring Nhiét d6 (.C) Thi gian khittriing(phiit) 160 45 170 1Thi nghiém Sink hoe phan nit -3- Bién soan : Lé Ly Thuy Tram 180, 15 190 LSThi nghiém Sink hoe phan nit “4. Bién soan : Lé Ly Thuy Tram b. Nuit day ‘Thudng dimg nhat 1a céc mit day Lim bing béng gon kh6ng tham nudc.Nut phai twang doi chit dé dam bao bui khong di qua dude, dong thai nude tit m6i truding khong bi boc hai qué dé dang trong qué tinh nudi cay. Bong khéng thdm nude 14 loai mit don gian nhat nhung 06 cde huge diém - Néu khi hp mit bong bi wét hode dinh mdi truding d 14 vi nhiing thi nghiém tién han trong mot thai gian dai - Thao téc 1am mit béng cham, khéng thudn tién khi nudi cy trén qui mid In ~ Chi ding dude vai lan 1a phai bo Hién nay ngudi ta sit dung nhiéu loai np day khac thay thé mit bong. Cac hang san xuat dung cy nudi cy mé cung cp logi nip Sng nghiém va binh tam gide bing nhya chiu nhiét thé hp vo uring 6 nhiét dO 1210C ma khong bi bién dang. M6t sé phong thi nghiém ding nip dng nghiém bing inox hode cao su rat thuan tién cho yiée v6 tring khé hole wét. Cling c6 thé sit dung gidy nhém dé 1am np day. c. Méi truiing ‘Méi truting nudi cdy thuding duge hap kh trimg trong ndi hp (autoclave), khut tring bing 4p suat hoi nude bio ha. Thai gian hp tit 15-30 phuit 6 dp sudt hoi mide bao hoa 1a 103.4 kPa (lam) tong duténg v6i nhidt d6 1210C. O nhiét dO 1210C, hau hét cdc sinh vat c6 trong mdi tring déu bi tiéu diét, ké ca 6 dang bao tit, Sau khi v6 tring cn phai lim kh n&p éng nghiém hoie mit bong dé tanh bi nhiém tr3 lai. vé sau sé rat dé bi nhiém ndm, nhat Bang 2: Thoi gian khit tring dung dich va cdc moi trading long bing noi hap (autoclave) 6 121. tai 103,4 kPa Thé tich moi trvding (mL) Thai gian hp khit tring(phiit) <50 15 5 20 250-500 25 1000 30 Vigc hap khit trimg bing ndi hp thi khéng thich hgp véi nhiéu hod chat nhay cém véi nhiét d6 nhut: céc acid amin, cdc vitamin, céc hormon ting trudng, va cdc chat khang sinh. Céc chat nhu vay thudng phai dude khit tring bing cdch loc v6 tring. Mang loc duge lam bing mang polyethylen hoe sof cellulose. Cac 16 trén mang siéu loc nay duge thiét ké higu qua cho vig gift lai cdc vi sinh vat gay nhiém. d. Loc va tringThi nghiém Sink hoe phan nit Bién soan : Lé Ly Thuy Tram Phuong phdp don gidn nhat 1a ding cdc mang loc Millipore hoe ding céc phéu loc thy tinh x6p s6 5, Mét sé loai mang loc v6 kh tring bing chiéu xa va chi dig 1 Tn Phuang phap sit dung mang loc Millipore: hang Millipore cung cp mang loc va gid d ing nhya hiu nhiét. Dudi diy mé ta mang loc loai Millipore Swinex c6 duving kinh 25mm. B6 loc gém cé gid dd bing loai nhya chiu nhiét gam np va dé, vong cao su va mang loc. Dit mang loc (c6 kich thudc 16 0.25pm) trén dé, dit vong cao su lén va vn chit np vao dé. Géi ton b9 bé loc vao trong mot t gidy nhOm va khit tring trong autoclave 6 121°C trong 15-20 phuit. Déng théi cing hip vé tring mét binh huy tinh dé hing dich loc. Dimng éng tiém hit dich loc va bom qua bé loc. ng cita hang Millipore. Bay la lai mang loc di duse 2.3.2 Khi tring mé thu vat ‘M6 cay 6 thé 1a hdu hét cdc bd phan khde nhau ciia thyc vat nhw hat gidng, phoi, non, dé ho: -tu¥ theo sip xtc voi mdi trudng ben ngodi, cde bo phn nay chia ohigu hay it vi khun va nam. Dang hia non khi con trong be, mé thit bén trong qua... thuréing ngude lai, 14, thn dic biét 6 cdc bé phan nam sau trong dat nhu ré, ci... 66 Iugng nm, khudn tap rat cao, Hu nhut khéng thé v6 trig mé cy dude Khun nim sau 6 céc té bao bén trong mé chit han ché bé mat. La khoai lang c6 thé v6 triing dé dang trong mita khé nhung khong thé 1am dutgc trong mba mua. Phuong phap vo tring mé cdy thong dung nl ién nay 18 ding cdc cht hod hoc cé hoat tinh dit nam khui Higu lye diét nm khudn ciia céc cht nay phy thude vao thai gian xitly, nong kha ni xém nhdp ctia chiing vao cdc ké ngdch 16i 16m tran ba mit md cy, kha ning diy hat cée bot khf bam trén bé mat m6 cdy. Dé ting tinh linh dong va kha nung x4m nhap ciia chit diét khuan, théng thuting ngudi ta wif ly mé cy trong vong 30s trong migu ethylic 70% sau d6 méi xit ly dung dich diét khudn. Déng thai ngudi ta thém céc chit giam sife cing bé mit nhu Tween 80, Fotoflo, Teepol vao dung dich diét nam khuan. Dé c6 khai niém vé néng dé va thdi gian sit dung cdc chat diét nam khudn dé xit ly mo cAy, xin dan (i ligu nghién cifu cla Street (1974) 6 bang sau: ft bi mhiém vi sinh ‘Tae nhan v6 trang Nong 46 % ‘Thai gian xith Hiéu qua (phiit) Calci hypochlorit 9-10 5-30 Rat 6t Natri hypochlorit 2 5-30 Rit tot Hydro peroxid 10-12 5-15 Tét Nude Brom 1-2 2-10 Rat tt HgCh; 01-1 2-10 Trung binh Chat khang sinh 4—50 mg/l 30-60 Kha ttThi nghiém Sink hoe phan nit -6- Bién soan : Lé Ly Thuy Tram ‘Trong thai gian xit ly, md c4y phai ngdp hoan toan trong dung dich digt khudn, Déi véi cde 6 phan c6 nhigu bui eat, trude khi xit ly nén rita edn than bling nude xa phdng bot va nita sach Jai bling nude may. Khi wit ly xong, mé cy duge nta nhiéu lin bling mide cit v6 trimg (tdi thiéu 14 3 14n). Nhiing phan trén mé cay bi téc nhan v6 tring Jam cho tring ra can phai dude ct bé true khi dit m6 cdy lén méi tung. Dé tranh anh hudng ctia téc nhan v6 tring 1én md cy, nén chit ¥ dé lai mt lép boc ngoai khi ngam m6 vao dung dich diét khudn. Lop ngoai, cling nay sé duge 16t bo di rude khi dit mé cy 1én moi wudng. V6 wing md cdy 18 mdt thao tde kh6, tkhi thanh cong ngay tr lan dau tién. Tuy vay, néu kién ti dim dude néng dO va thoi gian v6 triing thich hop thi sau vai lan thit chic chin sé dat két qua. C6 thé ding khang sinh dé kiém sodt hod loai bé su nhiém nam trén m6 cdy. Hau hat cdc khéng sinh nhay cam vdi nhiét do dé khang thé hap v6 tring. Chiing ho’ tan dude trong nude hoe dung mdi thich hop Khéc. loc v6 tring va thém vao mdi trng v6 tring khi mdi truting nay di dude Lam ngudi cdn 45- 50°C. Khong phai tat ca cdc khdng sinh déu thich hgp cho sit dung trong nudi cay mé thye vat. Céc khang sinh nhut Streptomycin, Kanamycin va Neomycin thuting dc cho mé thy khong hoat dong tt wong mét pham vi pH nhat dinh. Tetracylin cting 1 ddc 06 th Khuynh hutng tic ché su ting trudng cia mé thyc vat sau khi bi xif ly trong mét thd giandai Chloramphenicol cé phé hoat dong rong nhung dée cho thu vat (va ngudi) 6 ndng dé thip. Céc khang sinh thuding duc ding trong nudi cay mé thute vat gdm Rifampicin (thing dude ding két hyp v6i cdc khang sinh khdc), cde polymicin va vancomycin.Thi nghiém Sink hoc phan tie Cac logi khéng sinh Aminoglycoside - Streptomycin - Kanamycin - Neomycin Quinolone - Nalidixic acid - Ofloxacin - Norfloxacin B-Lactam - Penicillin - Ampicillin ~ Carbenicillin ‘Trimehtoprim va sulphonamide Chloramphenicol Macrolide va lincosamide - Erythromycin -Lincomycin Glycopeptide - Vancomycin Polymixin - Polymixin B - PolymicinE, Rifampicin Bién soan : Lé L¥ Thuy Tram Khé ning hoat dong Uc ché su sinh téng hop protein bing céch téc déng lén cdc ribosome 30S hode 50S Gay trd ngai cho qué trinh sao chép DNA. hiing cach tic ché enzym DNA gyrase Uc ché sy tng hop vach té bao vi khuan ‘inh t6ng hop protein bing cach tae déng lén ribosome 308 Uc ché su sinh téng haptetrahydrofolate Uc ché sy sinh tng hyp protein bing céch tée dong lén Rbx 50S Ue ché su sinh téng hop protein bing cach tae dong lén Rbx 50S Tae dong lén su sinh tng hop vach té bao vi kin Gian lén mang 1é bao lam thay déi ddng ion dn dén su pha va té bao Tac dong lén RNA bing céch gin vio RNA polymeraseThi nghiém Sink hoc phan tie Bién soan : Lé Ly Tins} Tram yy m6 thye vat, cdc nha khoa hoe lam yiée trong ti cA v6 trig (laminar). Dé 1a céc tit cy c6 thiét bi théi khéng khf da loc vé tring vio ché thao tée cay. Tu cy v6 tring loai tit mét cach higu qua ngudn tap nhiém tit bén ngoai va tao diéu kién thoai mai chongudi cy, nén hién nay dude sit dung rat phé biéan trong cdc phdng thf nghié Khéng khi tit bén ngoai dutde quat hiit vao qua mt mang loc thé. 99% bui trong khéng khi due gitf lai d mang loc thé. Sau dé khéng khi dude théi qua nung loc tinh va phan phdi déu ra kp bé mit cia ti cdy, khong tao nhiing xody khong khf dua byi vao ché cdy. Mang loc tinh ngain can cdc phan ut 14n hon 0.3micron vi hiéu qua 99.99% va do cdc hang chuyén sin xudt mang loc cung cp. Tudi tho ciia mang loc thé ty theo sé Iugng bui nai Lam vide, thudng tit 6 thang dén 1 mim va cla mang loc tinh tr 2 dén 3 nim. Khi thay hiéu qua v6 wing ciia ui cay lantinar kém di thi cn phai thay mang loc mdi, Dé kéo dai tuéi tho ctia bd mang loc trong ti cy laminar- thuding rit dat tién - nén bé uf ti cy trong céc phdng riéng, cng ft bui cang tat. Dé wanh bi nhiém trong sudt thao téc Sm. 2.3.4 Khif tring ndi thao tac cAy va dung cu cay Negudn nhiém tap quan trong va thudng xuyén nhat 14 bui roi vao dung cu thuy tinh chita méi trudng trong khi mé nip hoc mit bong dé thao tac cdy. Ngudi ta dp dung nhiéu php khéc nhau dé chéng lai nguon nhiém tap nay. Phong cy thuding 1a budng cé dién tich hep, rng tit 10- 15m’, c6 hai lép cita dé tinh khong khi chuyén déng tit bén ngoai true tiép dua bui vao. San va tung lat gach men dé c6 thé lau chi thudng xuyén. Trude khi dua vao sit dung, phong c duge xif ly hoi formol bing céch r6t formaldehyde (formalin) 4% ra mét s6 np dia petri dé rai réc vai ngi trong phdng cho béc hai tt do. Déng kin ctla phdng cdy trong 24h, sau d6 bd % cling trong 24h. Bé formaldehyde di va khit hai formaldehyde cdn thita bling dung dich NIT mit nai chudn bi cy, bé mit bén trong va ngoai ti cdy phai duce kh trimg trudc khi cy bing céch lau sach cdc bé mit nay bing 7. Tia UV cling c6 thé dude sit dung dé khit tring mit phong cAy va ti cdy nhung né it hiéu qua hon va nguy hiém hon Ia sit dung cén. Tia UV chi tigu diét dude cdc mam vi sinh nim truc tiép ngay uén cac bé mit ma tia nay chiéu vao; nhung n6 Khong thé théim qua cde 1ép bui dé tiéu diét cde vi c 1ép bui nay. Tia UV con gay hai cho mat va gay ung thy da. Cac dung cu mang vao phOng cy déu vO tring tude: th do chodng, mii vai, khdu trang cita ngudi cdy dén dao, kéo, kep (forceps), gidy loc, binh dung nuée cat... Trén ban cay thudng xuyén c6 mot d&n cin dé sit dung trong khi cy va mot cde dung cén 90% dé nining céc dung cu lam vige. Tre khi cy, ngudi cy can rita tay bing xa phong va Jau ky dén khuyu tay bang con 90%. Dé dam bao nic d6 v6 tring cao trong phong cay an c6 mot dén tit ngoai 40W treo wan. Chi cho dén nay lam vigc khi khong c6 ngudi trong phong cAy. Nén bat dén tit ngoai 30 phuit trudc khi cay. Can giam sy chuyén dong ciia khong khf trong phong cdy dén nue ti thiéu, vi vay tit cd cdc dung cu phuc vy cho vige cdy du phai chudin bi day dii dé trong théi gian cAy trénh di lai ra vao phdng cay qua nhieu. Cac dung cu bing kim loai nhu kep Cong Nghé Sinh Hye Vidt Bee Biotechnol inh vat nim bén duéiThi nghiém Sink hoc phan tie -9- Bién soan : Lé Ly Tins} Tram cdy, dao m6, que cy vong, kim mii nhon c6 thé dugc khit tring bing céch dét dudi ngon lita dén edn, Nhiing dung cy nay trude hét phai duge nhting vao cén tuyét ddi rdi mdi dét. Nhé dé réo di cdc giot cén thita rdi mdi dua vao ngon lia dan cén. Khi mé nip chai hodc nip éng nghiém mdi tniding nudi cay, thi ding ngén tay ké vit vA ngén tay wit cam lay nap gon, va khong cham tay vio bé mit bén trong cia np gon cing nhw khéng tha nap gon xudng bat ctf be mat nao cho dén khi gan n6 tré lai chai mi trvdng. BAI 2: : : ra 7 KY THUAT PHA CHE MOI TRUONG AYA .vAN DE LUA CHON MOI TRUONG Khi khdi sue nudi cdy m6 va té bao déi vdi mt s6 déi wrong nt dinh, van dé dat ra 1a chon méi trudng nao va trén co sé ndo dé phdi hyp ty 18 cde chit dinh dudng. Caéch thuding 1am 12 qua cde tai ligu da xuat ban, cde cong winh di nghién ctfu vé doi twang d6 hod cing ho tong duong xem cic te gid da sit dung mdi truting loai ndo. Bute dau c6 thé gid’ nguyén mdi truding ciia tie gid dé holc trén co sé dé ma cai tién cho phi hdp qua céc thi nghiém thm dd, Trong hang trim mdi tering do rit nhiéu téc gia dé nghi cho nhiéu loai cay khdc nhau, nhiéu miuc dich nudi cy khée nhau. Vé co ban c6 thé chia ra lam 3 loai: - Mai tung nghéo chat dinh dudng: dién hinh 18 moi trudng White.Knop va Knudson C... - Méi trdng c6 ham Ivong chat dinh dudng trung binh: dign hinh am6i wudng BS cia Gamborg - Mai tring gidu dinh dung: din hinh 1a mdi tring MS (Murashige-Skoog).. Vi vay khi bit dau nghién ctfu nudi cdy mé mot sé déi tusng mdi, chua c6 ti Liéu trude thi nén Joai méi trading trén xem doi twang nghién citu phit hdp vdi loai moi tring nao nhat. Sau dé can thif tim ti 1é NO,/ NH,’ thich hgp. Cac téc gia phwong Tay lam viée vdi cay wong can thudng khong dua NH,’ vao mdi trudng. Nhung doi véi nhiing cay dinh dudng NH," manh nhv cay hia, viée thém yao méi uwGng nudi cdy mot ti 18 NH," thich hgp chic chin s& ¢6 gi. Vige sit dung mang tinh kinh nghiém chi nghta déi véi mt s6 mdi truding da can wé kha nhiéu su tién bé ctia cong téc 6 mt sd phdng thi nghiém nudi cdy m6 thue vat. Thudc 14 va carét 122 Logi e@y kinh dign cia nudi cy mé thue vat. Mdi truting nudi cay 2 loai cay nay da dude xAy dung khé hoan chinh, Tuy vay, khong thé ding nguyén cdc mdi trudng dé dé nghién citu cdc cay hoa thao hodc cae cay ho dau ma khéng cé sy cai tién, sita déi, Diéu nay giai thich sy tién bd cham chap cia nudi cay tham dé so sénh m6 mét sé cay hoa thao so véi cay 2 14 mam. Cong Nghé Sinh Hye Viet Biotechnolo;Thi nghiém Sink hoc phan tie -10- Bién soan : Lé Ly Tins} Tram Hién nay, mOi wrung MS dude coi nhw 14 mdi tudng thich hyp vi nhigu loai cay do gidu va cn bling vé mit dinh dutng. Vi vay, nhiing ngudi tap sit lam nudi cdy mé thuding bit diu véi méi trudng nay true khi tim duge mdi trudng ctia riéng minh. 2.CHUAN BI MOI TRUONG Dé thudn tign cho vide pha cdc mdi tuding nudi cdy ngudi ta khong cn hod chat mdi ln pha ma thuting chudn bj tude dudi dang cdc dung dich dim dic (cdn goi [8 cdc stock), sau d6 chi cn pha loang khi sit dung. Céc stock nay thuting duge bao quan dai ngay trong ti lanh thuing hod tit Janh sau. 2.1 Thanh phan mdi trudng dinh dudng Nutée cit Chat hitu co - Duiing- Acid amin- Vitamin (B1, Bs, H, PP...) Cac chai thutc vét (Auxin, Cytokinin, Gibberellin...) Chat v6 co Da lugng NP K Ca Mg S Vi lw ong Fe Zn B Co N Mn Cu Al Mo Cac hop chat khong biét ré thanh phan tic dita, nude khoai tay, mic chuéi, casein hydrolysalt, trypton, pepton. ‘u hoa sinh trudng -10- Cong Nghé Sinh Hye VitThi nghiém Sink hoc phan tie He Bién soan : Lé Ly Tins} Tram THANH PHAN MUOI KHOANG C6 BAN CUA MOI TRUONG MS (Murashige va Skoog, 1962) SKOOG I SKOOG II SKOOG II NHiNOs FeSO:.7H20 KI 1650 mg/L 278 mg/L 0.83 mg/L. KNO3 MnSO« Na2Mo0s.2H20 1900 mg/L. AERO 0.25 mg/L. KELPOs 22,3 mg/L. CuSO15H20 170 mg/L. HsBO3 0,025 mg/L. MgSO..7H20 6,2 mg/L. CoCh.6H20, 370 mg/L. Zn$O1.7H20 0,025 mg/L. CaCh.2H2 8,6 mg/L. 440 mg/L. NawxEDTA 37.3 mg/L. THANHPHAN VITAMIN CUA MOREL Morel's Vitamin Pirydox Biotin Meso- Nicotini Thiami Pantotat ine (B6) ay inositol c acid n-HCl eCalei I mgit 0.01 100 (PP) B) I mgf. mg/L mg/L mg/l. I mg/L. 2.2. Cach pha cée dung dich me (stock) * Stock da lrgng MS : SKOOG I (x10) (Pha thanh LLvéi ndng dé sit dung khi pha [Lméi trating MS 1a 100mL/L) NHiNOs KH2PO. CaCh.2H20 16500 mg 1700 mg 4400 mg KNO3 MgSO:.7H20 19000 mg 3700 mg Cén va ding nude ct hod tan Hin Iuat ting cht trong bécher cho tan hodn toin. Ding éng dong 11 diéu chinh cho ii lit. Cong Nghé Sinh Hye Vidi ii BiotechnologyThi nghiém Sink hoc phan tie -12- Bién soan : Lé Ly Tins} Tram * Stock st MS: SKOOG TI (x100) (Pha thanh 200miL. vdi nong d6 sit dung khi pha LL mdi trwdng MS 18 2mL/L) NaxEDTA FeSO:.7H20 3730 mg 2780 mg (Can va ho’ tan ting chat bing 100ml nude ct trong mdi bécher ring. Dit 2 bécher dung dich lén bép va gia nhigt cho dung dich 4m én vita cé hoi néng boc lén la dude. Khudy déu va dé dung dich NaxEDTA vao éng dong 200 mL, rdi cho tir tit dung dich FeSOuvao, vita cho vita khudy déu, Dé ngudl rdi cho vao binh t6i bao quan trong ti lanh. * Stock vi haong MS: SKOOG III (x10) Tnitde tién cdn chudin bi céc stock con: Dung dich KI: (83mg/mL) Can 8300 mg KI hoa tan véi mide ct cho dit 10OmL Dung dich Na2MoOs.2H20: (25mg/mL) C4n 2500mg Na2MoOs.2H20 hoa tan vi nude ct cho di 100mL Dung dich CuSOs.5H20 (2,5mg/mL.) Can 250mg Cu$Os.5H20 hda tan véi muée cét cho di 100mL, Dung dich CoCl2.6H20 (2.5 mg/mL) Cén 250 mg CoCh.SH20 hda tan vi nude clit cho dt 100 mL. SKOOG ILI (x100): pha thanh 500mL véi ndng dé sit dung khi pha 1L mai tring MS Ia SmL/L MnS0s.410 KI 2230 mg 83 mg/l mL. HBO: NasMoO«.21 620 mg 25 mg/l mL. ZnSOs.7E2O CuSO..5H20 360 mg 2.5 mg/l mI CoCh.6420 2,5 mg/l ml CAn 3 chat dau tién va hoa tan véi nude cit trong ting bécher riéng.Cho ting dung dich theo thit ty vao 6ng dong 500ml. Sau dé hit LmL dungdich trong stock con ciia 4 chat tiép thea cho vao 6ng dong, vita cho vita khuay déu va thém mide c4t cho dit 500mL ‘Cong Nghé Sinh Hye Vidi -12- BiotechnologyThi nghiém Sink hoc phan tie “Be Bién soan : Lé Ly Tins} Tram * Thanh phén vitamin Pha cdc stock con Dung dich pirydoxine (Bs) (10mg/mL) Can 1000mg Bshda tan véi nude ct cho dit 100mL Dung dich Biotin (H) (Img/nmL) Can 100mg biotin hda tan véi mde ct cho dit 100 mL Dung dich Nicotinic Acid (PP) (10mg/mL) Can 1000 mg nicotinic acid hda tan vdi nude c4t cho di 100mL. Dung dich Thiamin-HCl (Bi) (10mg/mL) Cn 1000 mg Bi hod tan vdi nude ct cho ati 100 mL. Dung dich Pantotheate-Ca (10mg/ml.) Cn 1000 mg Pantotheate-Ca hda tan vdi nutée ct cho dit 100mL Vitamin Morel (x 100) Meso-inosito 110 g/102 Thiamin - Pirydoxine (B6)100g/10mL. Nicotinic acid(PP) HCKB:)100mg/10ml. Biotin (H)1 mg/l mL. 100mg/10mL. Pantotate Calci100 mg/10 mL C4n meso-inositol r6i hoa tan véi nude cat. Dong tig chat theo thit ur cho vao dng dong cé chita nude cAt , vita cho vita khudy déu va thém nuée cho dit 200mL. * Thanh phan cc chat diéu hoa sinh trudng thie vat (tinh theo mg) Dung dich BAP (1mg/mL) Can 100 mg Benzylaminopurine (BAP) hoa tan trong SmL NaOH 1N.Cho thém adc edt cho dit 100 mL. Méi mL dung dich c6 chita Img BAP Dung dich [AA (Img/mL) Can 100mg Indolacetic acid (IAA) hda tan trong 5 mL NaOH IN. Cho thém mude ct cho dit 100ml, Mai mL dung dich c6 chita Img IAA. Dung dich IBA (Img/mL) Can 100mg Indolebutyric acid (TBA) hda tan trong SmL NaOH LN. Cho thém mide ct cho dit 100mL., Mai mL dung dich c6 chita Img IBA Dung dich Kinetin (mg/mL) Can 100mg Kinetin (KIN) hda tan trong 5 mL NaOH IN. Cho thém nudc c&t cho di 100mL. Méi mL dung dich cé chita Img KIN. Dung dich NAA ( Img/niL) Can 100mg Naphthaleneacetic acid (NAA) hda tan tong SmL NaOH IN. Cho thém nude cdt cho dit 100m. Méi mL dung dich c6 chia Img NAA. Dung dich 2,4-D (1mg/mL)Can 100mg 2.4-D (2,4-dichlorophenoxyacetic acid) hda tan trong 50m, ethanol 50%. Cho thém mute ct cho di 100mL. Mai mL dung dich eéchita Img 2,4-D. * Cée dung dich chat diéu hoa sinh truting the vét (tinh bang mol/l) Cong Nghé Sinh Hye Vidi a ts BiotechnologyThi nghiém Sink hoc phan tie “He Bién soan : Lé Ly Tins} Tram BAP cé trong lugng phan tt (MW) 1a 225.26 ~ Hoa tan 225.26gr BAP trong 11 nude cat tic ta c6 11 dung dich BAPIM hay Lmol/L - Cn 225.26 mg BAP hoa tan trong 11 nude eft tte ta c6 11 dung dich BAP [mM hay Immol/1 - Cin 225.26x10" mg BAP hoa tan trong 1] nude ct tic ta c6 11 dung dich BAP 1M hay Ipmol/l Dung dich BAP ( MW 225.26) (10mM) Can 225.26 mg BAP hoa tan trong 5 ml. NaOH IN. cho thém nude cAt cho di 100 mL. , tic ta 6 100 mL dung dich BAP 10M. Védu: Cho ImL dung dich BAP 10mM yao mdi truding MS dé pha duge 1L méi tudng. Nhw vay tacé IL mdi trating MS c6 chia 10HM BAP. Mudn pha ILmdi trating MS cé chia 1yM BAP. ta sit dung 0.1mL dung dich BAP 10 mM. Tuong tu ohu vay pha cho céc loai kich thich t6 cdn lai, biét: - IAA (MW 175.19) -NAA (MW 186.21) - IBA (MW 203.24) -2.4-D (MW 221.04) - Kinetin (MW 215.22) 3. THUC HANH - Tién hanh pha Skoog I, II va III cua méi wudng MS ~ Pha stock vitamin Morel, glycin va inosytol - Pha céc stock cht diéu hoa sinh trudng nhu da hudng din phan wén Cong Nghé Sinh Hye Vidi tia BiotechnologyThi nghiém Sink hoc phan tie “1. Bién soan : Lé Ly Tins} Tram BAI 3: GIEO HAT IN-VITRO AYA LCHON MO CAy VA XU LY MO CAY 1.1 Chon Iva mé cay Khong cé nhiing hudng dan cu thé trong viéc chon mé cy. Vé nguyén tic, trit nhitng mé cy di héa g6, cdc mé khée trong cu thé thuc vat déu c6 thé ding lam mé cy. Tuy vay c6 thé nhan xét chung 18 cde m6 dang phat trién, thit qua non, 14 non, cudng hoa, dé hoa, mé phan sinh... khi dit vao mdi trudng c6 chita mOt lnong chat sinh trudng thich hgp déu cé kha ming phan chia va phan héa. Dé bit dau nghién citu nhan giéng v6 tinh mot cay nhat dinh, trude tién ngudi ta chi ¥ dén céc chdi nach va mé phan sinh ngon. Can biét rng tuy mang mét hrgng théng tin di truyén nhu ohau, céc mé khdc nhau trén ciing mét cay c6 thé sinh trudng va phat trién v6i kha ning tai sinh chi, 18 hay cay hon chinh rét khéc nhau. Vi vay, khi khdi sy chon giéng, nhan giéng mot cay cu thé bingphuong phap nudi cay mova té bao thyc vat, trudc hét cn thf nghiém tim hiéu phan (ing clia cdc bO phn Khéc nhau cita cdy trong nudi ey 6 cée ndng 46 chat sinh truding kha hau. Sau khi cy, mo ey cf duge dit trong digu kign nhigt dO va dnh séng On din. Tuy vao ede muc dich nghién citu ma cé ede ché d6 chiéu sang khae nhau, ching han qué trinh tao mé seo c6 thé cin béng t6i hay anh sing. nhung qué trinh tai sinh va nhan giéng v6 tinh nit thiét phai c6 nh sang. Nhiét d6 phdng nudi nén git Gn dinh 2542°C bling may diéu hda nhiét d6. Cudng do chiéu sang khoang tit 2000 -3000 lux. 1.2 Xtly v6 trimg mau cay Mau dua vio nudi cay invitro c6 nhiéu nguén gc khéc nhau, C6 thé 1 nhitag mau cay di vo tring nhu cy con invitro cho ny mam trong diéu kién v6 tring; cing c6 thé dé 1a cdc mau chua v6 tring, lay truc tiép tir bén ngoai ty nhién nhw ch6i non, 14, than, cu, ré... Thuan Idi nhat 1 stt dung cdc mau cy da v6 trig bdi lé cdc phuong phap v6 tring mau cAy true tiép thudng ft nhiéu gay hai cho mau cy do cht khif wing gay ra. C6 nhiéu phuong thife v6 ting mau cy: a. Vo tring hat: - Rita hat bing x8 phong, lic déu 2-3 phuit. Voi cac loai hat nho, thing dung hat trong tii vai ming. - Rifa sach xa phdng bing nude may duéi voi nude chay manh - V6 tring so bé bing cén 70%, lc déu 1-2 phit - Rita sach con bing nude v6 tring - Cho hat vio dung dich khit trig NaOC! 1-15%. Thém vai giot bam Cong Nghé Sinh Hye Vit -15-Thi nghiém Sink hoc phan tie -16- Bién soan : Lé Ly Tins} Tram dinh Tween20. Khif ing trong 15-20 phit wy mau. - Rifa sach chat khit wimg nhiéu lin bing mvc v6 thing (khodng 5 in) cho dén khi hét mii javel - Hat v6 tring da c6 thé nudi cdy trén mdi trudng tao mau v6 tring. b. Cée phutong thite khde v6 tring hat ~ Khit tring Jn thit nbdt vi NaOCl 5,25% - Khit tring Hin thit hai véi HgC 20,1 —1%, thém vai giot HC10,5% trong 10 phuit. - Rita mau véi H20> 6% v6 trimg truéc khi nfa lai bing nude v6 tring ~ C6 thé khif triing 66 b6 hat biing acid sulfuric trong 2-10 phiit - Voi hat c6 v6 cxing, c6 thé ding phutong phdp dat dé khit trimg so bd. ¢. V6 tring chéi non, Id va than co: - V6 tring so bé bing cach ngim mau vao cén 70% trong 1-3 phuit, thal gian xif ly nay cing con tuy thude vao dé cing hay mém ciia mau ~ V6 tring mau vdi cht khit tring NaOCl 1% hay vdi javel thuong pham theo ti lé 1:4 hay 1:5 trong thdi gian 5-20 phuit tuy theo mau ~ Ria lai bing nude v6 tring cho sach hoan tan chat khit tring a. V6 tring cit, ré = Mau nuéi c4y phai dude nia duéi voi nude chay manh cho that sach dat cét bam trén mau, sau dé ngam trong nude xa phdng loding 10 phit - V6 tring so b6 bing cén 70% trong 1-5 phuit - V6 tring véi chit khit wing c6 néng dé va thdi gian thay déi wy mau - Rifa lai bing nude v6 ting cho that sach chat khit tring ¢. Trénh hogi mau ‘Trong qué tinh nudi cay thudng xuat hién nhiém 1a do trong qué tinh nudi c4y da dé bao ut trong khdng khi ri vio hay mu cy chya duge v6 tring hodn tan, Sau mdr thai gian thi bao a phat trién nhanh chéng va cd thé sé 1am chét mau hole canh tranh chat dinh dudng lam mau khong phat trién. Hoai mau thudng do nam mde (khudn lac c6 dang sgi) hode do khudn (khudin lac c6 dang van nhay). nam hodic khudn chi moc t6en bé mit mdi truding, dé thuving 1 nhiém do thao tac cay chwa tot; néu c6 khép trong mdi tring 14 nhiém do mdi tntving chuta dutde tét tring hoan ton; con néu khudn va nm xuat phat tit g6c mau lan ra thi dé 1a do mau chua dugc khif trimng triét dé. Tuy theo quan sat va dya vao kinh nghiém cua ngudi cdy ma e6 nhan dinh chinh xée vé nguyén nhan gay hoai mdu dé dim céch khdc phuc tinh wang nay. Trong truding hgp cdc binh nudi cy da bi nhiém, cin dugc hap b6 tude khi dem di rita sach dé tanh lay lan nguén nhiém trong khu vuc cdy. 2.THUC HANH. Cong Nghé Sinh Hye Viet ge Biotechnolo;Thi nghiém Sink hoc phan tie -17- Bién soan : Lé Ly Tins} Tram 2.1 Mue dich Gitip cho sinh vién lam quen véi thao téc v6 tring mau cay va ky thuat cAy v6 tring 2.2 Dung cu, f va héa chat 2.2.1 Dung cu: - Binh tam gide (250mL) - Bécher (cdc dét thuy - Forceps (kep), dao cay, - Gidy cdy vo wing tinh) 500m, 1000m. dén con - Dia petri v6 tring 2.2.2 Thiét biz - Autoclave - Ti lanh - May Khudy tit -Ti sdy - Cn k¥ thuat - pH ke -Tii cay (laminar) - May cat nude 2 lan 2.2.3 Hod chat - Dung dich Skoog I, II - Dut: - Dung dich hypochloride va TIT cia méi rudng MS ~ Agar calcium 10% - Stock vitamin Morel - Nutic eft v6 img - XA phing bot - Stock glycin - Cin 90%, 70% 2.3 Cac bude tién hanh 2.3.1 Chudn bi moi trudng (thanh phan cho 1L mdi tong) ~ Skoog I (MS):100mL ~ Glycine: 2mL, - Skoog I (MS): 2ml. - Suerose: 30g ~ Skoog I (MS): Sm. - pH 5.8 = Vitamin Morel: 2m, ~ Agar:7g 2.3.2 Nguyén ligu thie véi ~ Céc hat cam chanh cn nguyén ven, khong bi hut hai 2.3.3. Céc butée thute hién ~ Cho hat vio bécher ¢6 chita nude xa phdng loding, rita hat cho sa hat h chat nhét bam xung quanh ~ Rifa hat cho sach xa phong bing nutéc ct. Ngdm hat 2phit trong cn 70% Cong Nghé Sinh Hye Viet S -17- BiotechnolThi nghiém Sink hoc phan tie “18. Bién soan : Lé Ly Tins} Tram - Rifa hat sach etanol bing nude ct, Sau dé ngam hat 1Ophuit trong dung dich hypochloride calcium 10% - Trong ti cy vé tring, rita hat cho sach chit khif tring bing edch Vic hat trong nude cat vo tring (3 lin) - Tién hanh tach do hat bing kep va dao mé vé triimg. Cho céc hat vita tach bo vé do vio dia petri v6 tring cé chifa gidy thim v6 tring va lam dm gidy thim bing mot it nudc ct v6 tring. - Gieo hat: gieo 4-5 hat vao cdc binh tam gide chita mdi tntting nudi cay da duge hap khit tring. - Day nip binh cAy va ghi rd ho tén, ngay cy, s6 nhém, tén gidng va mdi trudng 2.4 Yeu cau - Thy hign pha mdi trung gieo hat - Thye hign céc thao te v6 tring trong nudi cdy va ky thudt gieo hat - Thu duge cy con v6 triing trong dng nghiém ‘Cong Nghé Sinh Hye Viet ae Biotechnolo;Thi nghiém Sink hoc phan tie -19- Bién soan : Lé Ly Tins} Tram _ BALA NUOI CAY DINH SINH TRUONG AYA 1.G161 THIEU Trong mudi cy initro, mot phung thie don gin va thuting hay due sit dung dé ta sinh chdi invirro Fa nudi cy dinh sinh trudng. Higu mot cach ding nghia thi nudi cdy dinh sinh trading Ta st dung phdn md phan sinh ngon véi 3-4 tidn phét khdi 14, te 12 cdc dinh sinh trudng c6 kich thude tit 0.1 -0.15mum tinh tit chép sinh wudng. Ky thudt nay khd phite tap, phai thye hign dudi kinh hip va kha niing song s6t ctia mau cy c6 kfch thude nhé nhu thé thudng khong cao, do dé chi due tién hanh khi can nuéi cdy véi mue dich tao cdc cay con invitro sach virus. Trén thyc té ngudi ta thing nudi cA dinh chdi non véi kich thutde khodng vai mm. Dé c6 thé 1a dinh chéi ngon hoic dinh chéi néch. Méi dinh sinh trudng nudi cy 6 diéu kién thich hop sé tao ra mot hay nhiéu chdi va mdi chéi sé phat trign thanh cay hoan chinh. Xét vé nguén géc cia cdc cay dé, c6 thé c6 ba kha ning: - Cay phat tit chdi ngon - Cay phat trign tit chdi ndch pha ngit - Cay phat trign ti choi mdi phat sinh, vi du: mudi cdy doan wu ha diép ctia cay mang cau (Annona squamosa) sé cho rat uhiéu mam (buds) tén md cdy va mdt sé mam sau dé sé phat wién thanh chdi va sau dé sé thanh cay invitro hoan chink. Tuy nhién thong thung rat kho phan biét chdi phd ngi va chéi mdi phat sinh. Cac phuing thife phat tién c4y hodn chinh ti dinh sinh uténg mudi cdy nhu sau: * Phat trién cay truc tiép. Chit yéu 6 cde d6i tung hai 14 mam (dicotyledon) nhw khoai tay, thudc 14, cam chanh, hoa Vi dy: Khoai tay (Solanum tuberosum): Mam (dinh sinh trudng) — Chdi néch + Cay * Phat trién cay thong qua giai doan protocorm Chi yéu gp 6 cde déi tugng mét 14 mam (monocotyledon) nhut phong lan, difa, hug... Cimg mot hic dinh sinh trudng tgo hang loat protocorm (proembryo) va cdc protocorm nay c6 thé tiép tuc phan chia thanh céc protocorm mdi hodc phat trién thanh cay hoan chinh, Bang phuong thite nay trong mot thdi gian ngn ngudi ta cé thé thu dudc hang trigu c4 thé Vi du:Hoa lan (Orchidaceae): Dinh sinh trudng — Protocorm — Cay Theo Champagnat (1977) va Fast (1980), cde chéi non dang ting wudng dai 10-15em, cita mdi nhii lé thuding duge ding lam vat ligu cho viée nudi cy dinh sinh trudng. Dat ban duge ddi séich Cong Nghé Sinh Hye Vit ni ae BiotechnokThi nghiém Sink hoc phan tie -20- Bién soan : Lé Ly Tins} Tram dudi voi nu chay va Ia dude 16t séch cho dén khi thy rd cde chdi ban. Chdi non hic nay duge nhning vio cén 70% va duge khit tring trong dung dich khit tring 10%(v/v). Cée chdi ban duge lay ra va nia trong nude cat v6 tring. Sau d6, ching dude khit lai thém 10 phuit nifa trong dung dich khit tring 3% c6 chita Tween80 va dude nia lai trong mrde cat v6 tring. Trong ti cay v6 tring, phan géc cia nhimg chéi oho nhft duce cét bé va viée cy dude tién hanh. Trén cdc chdi lén hon, wdc hit tach bo cdc 14 va phan géc bi chét do téc dong cia chat khit tring, sau dé cdy vao mdi trudng. Phai mat mit thé gian twang déi dai thi protocorm mdi duge than lip. Céc protocorm nay dude ct ra va cdy chuyén vio méi trudng mdi. Ngdy nay, ngudi ta thudng cAy ede dinh chdi 16n hon (mang 3-4 tién phat khdi 14) vi dé thanh cong hon 1a cdy cdc dinh choi chi ¢6 2 tién phat khdi 14, New protocorm khéng dude cit khéi mau c4y thi né phat trién thanh ci va ra ré; néu durge téch ra khéi miu cy thi sé c6 su thanh lap cdc protocorm bit dinh mdi tit céc peotocorm ban dau. Khi cy dinh sinh wudng cia Cymbidium chi cé ving xung quanh tién phat khdi 1d u lén va cudi cing tao thanh protocorm. Sy thanh lap protocorm c6 thé due tao ra ma khong can cé dinh sinh tung ngon. Khi cy dinh sinh trudng cita Cattleya, eae mo thuting nhanh chéng hod nau. Vi ly do dé ma dinh sinh tudng dude cit wong mdi trudng léng howe nude cat vo tring va duce vay trong méi truding lng, nhdi dé cde chat nau dé Khuyéch tan vao trong méi trading va it gay anh hudng dén mé cay (Fast, 1980). O Cartleya, ngudi ta thing tach mot chéi (3-Smm) véi nhieu tién phat khdi 14. Mi truting cdy Cartleya thuding phitc tap hon méi truding cay Cyrabidium (Fast, 1980 va Champanat,1977) d6i khi c6 chifa auxin, cytokinin, nuéc dita va peptone. Sy thanh lap protocorm 6 Cartleya mat nhiéu thi gian va ludn dude tao ra é phan géc ctia 14 gid mht; thute t& dinh sinh trudng ngon khéng déng vai trd gi ca va mat di, Viée cy cae tién phat khdi 1d Cattleya cing cé thé dap ting cho sv thanh lap protocorm. M6i trvéng mudi c4y dinh sinh trudng tung déi dongian va rat gidng mdi trudng gieo hat cho nhiéu gidng lan kahe nhau. Cymbidium thuting dudc nudi cdy wén moi trtdng khodng Knudson C hoe Vacin & Went Dinh sinh tung thuding duce cy én mdi trudng dic (ngoai tit Cartleyaynhung protocorm thuting due nhan lén trong méi tring Ing, va protocorm chi ting tntdng thanh chéi con khi duge cy trén mdi ting dic, thanh phn méi tring thuting khdc nhau trong ting giai doan. Moi truting c6 pH trong Khoang tit 4,8 -5,8. Nong do duting saccharose tit 1- 3% (wiv) hode di khi 18 1,5% glucose + 1.5% fructose. Mét s6 loai lan don than thi khéng can dudng trong gi dinh sinh wudng vi su hign dign ca dung c6 thé 1am dinh sinh trudng va va chét. Trong giai doan tao protocorm, thudng duding va nude dita (10-15%) dude them vao mdi tring dé kich thich su thinh lap protocorm, Chat diéu hoa sinh tnrdng thye vat néi chung kh6ng edn thiét, sy hign dign ctia chting trong méi trudng c6 thé 1am co hdi cho dét bién 1dn han, Nhiét d6 ti wu cho nhan ging tit 22-28°C. doan nudi Cong Nghé Sinh Hye Viet one Biotechnolo;Thi nghiém Sink hoc phan tie -2)- Bién soan : Lé Ly Tins} Tram Anh sang dén huynh quang thing dude sit dung vdi 12-16 gid chiéu sing/ngay. C6 thé nudi thap nhung cn gia ting anh sang trong giai doan tao chdi tit protocorm, N6i chung cé thé thuc hién vige nhan giéng lan tit dinh sinh trudng theo 2 céch: hode tén moi trfdng dic hodc tn mdi trudng long. Trong mdi tung long cn lic vong vai van téc rit khdc nhau tuy theo loai nhung hau hét cdc nha nghién cau thudng thye hign vdi van téc thép 2-5 vong/phtit, Su nhan ging trong moi trudng Jéng thuting t6t hon wrén moi trudng dc bai vi khi lic sé cung cp Or va chat dinh duding cho mau cdy higu qua han. Khi cay con cao khong 5- Jem véi 3-4 14 06 thé mang ra trong trong vutin vom, Cc d6i tudng hoa lan di mang Jai hiéu qua kinh té dic biét cao. Sau nhiing két qua dau tién 6 chi Cymbidium ctia Morel (1966) agudi ta da thu dude két qua rat t6t 6 6 di nhan gidng v6 tinh hoa lan dat duye thanh cong Idn va duge ting dung rong rai nhu vay 18. vi hoa lan c6 phuong thie sinh sin qua protocorm. Nhd cé phuong thife nhan giéng nhanh va ré tién ma hoa lan vOn dit td nén cé gid ca phai ching va duce nhiéu nguii va chudng. Nhiing thanh cong ho Lan kh6ng nhiing chi 1a bing chig ma cdn mé dung cho vige ting dung ky thuat nay déi véi cdc loai cay khac. cy cuting dé chiéu san chi khéc nhau cia ho 2.THUC HANH 2.1 Mue dich - Khit tring mau dinh chéi - khao st su tdi sinh cay true tiép tit nudi cdy dinh chéi - Khao sat su thanh lap protocorm tit mudi cay dinh chdi 2.2 Nui phat trign thanh cay true ti 2.2.1 Neuyén vat li Doan than non cay 2.2.2 Mai trutding nudi cdy Méi truding MS bé sung BA 2ppm va NAA 0,2ppm 2.2.3 Tién hanh: - Céc canh mau non lay tit vutin wom vé dude cit bé hét 1a, ct thanh doan 2~3em, cho vio bécher - Rita sach canh mau bing mide x8 phng loang, sau dé rita sach xa phdng bing nhigu ln dudi vdi nude chay manh, - Dua canh m y v6 tring, ngdm trong cn 70% trong 2-3 phiit - Ria sach a wing 1 1n - Xttly mau bing Ca(OCl)26% trong 10 phnuit sau dé thay bing dung dich Ca(OCl)25% trong 5 phiit m hoe chanh mic may Cong Nghé Sinh Hye Vit aoe BiotechnokThi nghiém Sink hoc phan tie -22- Bién soan : Lé Ly Tins} Tram - Rita ~ Cah mau duge cit bé cudng 14 va 2 dau ctta phan than da bi cht kit ting tdy wing. Chia canh mau thanh ede 46 - Cm cae dét vio méi truding nudi cy da chun bi, cho phan cudng 1d hudng lén tén, chdi ngu phai nim trén mat thodng ctia mdi tring. - Nudi mau trong diéu kién séng 2000lux/16h/ngay 3 25°C. 2.3 Nudi cay phat trién cay thong qua giai doan protocorm +h mau bing nude ct vo tring 6-7 lin cho hét mii javel lem 2.3.1 Nguyén vat ligu Cae doan chéi con Dendrobium cao 10cm 2.3.2 Moi trung nudi cA y dinh sinh truéng (cho IL mdi truéng) -Khodng da ltgng Knudson - Vitamin Morel: 2mL. - Nutée dita: 150mL 100mL. - Glycin :2mL - Khoai tay: 60g ~ Khodng vi lugng Heller = Inositol : SmL - Than hoat tinh: 0.25g Sm. - Vitamin B1: SmL. - pH: 5,5 - Skoog II MS : 2mL. - During: 30g ~ Agar: 6g * Khodng da ligng cita KnudsonC (Morel,G.M - NHiNOs: 500mg/L. - NHA(SO.)e: 500mg/L. - KH2PO«: 250 mg/L = Ca(NOs)o: 241,3mg/L. - MgSO«: 122,15mg/L. * Khodng vi lugng ciia Heller (1953) ~ AICLs.6H20 : 0,054 mg/L. - Mn$O«.H20 : 0.08 mg/L. ~ CuSOs.5HbO : 0,03 mg/l. -NiCh.6H20 : 0,03 mg/L. - HsBOx: 6,2 mg/L. - ZnSOs.THLO :1,0 mg/l. - KI: 0,01 mg/L. 2.3.3 Cac bude tién hanh: ~ Rita sach chdi Dendrobium dui voi midic chay. Ding dao mé 1 hét cc 14 non bao xung quanh chéi cho dén khi dé 16 ra cdc ch6i bén va chdi ngon - Ngan chdi trong made xa phng loang va ding gon lau nhe lén than chéi. Rita cho sach x phng dudi vdi nudic chay - Lic chdi trong cn 70% trong | phuit. Sau dé xit ly vdi dung dich javel thuong pham theo ti 1é 1:5 wong vong 25-30 phiit - Trong ti cy, ding kep v6 tring cho chdi vio céc bécher co chita nude cat v6 wi thé 3-5 lan cho het mii javel. ic nhu Cong Nghé Sinh Hye VitThi nghiém Sink hoc phan tie -23- Bién soan : Lé Ly Tins} Tram - Dit cdc chéi Ién gidy vo wing.ding dao cit riéng ting chdi bén va ch ngon ra khéi chéi me ban du. Nhé cit bo hét ede mé chét da bi cht kh ting lam wing 6 phan géc chéi. Cy ting chdi vao dng nghiém ¢6 chtia moi truting da chudin bi - Nudi trong phing sing - Ghi 16 tén nhém, ngay cy, giéng cay vao tat ca cdc Ong nghiém 2.4 Yeu cau = Thue hién tat thao téc cy v6 trang va khit tring mau BAIS: NUOI CAY MO SEO ALA L_ GIGI THIEU Nudi cy mé seo 1a khau rat quan trong trong nudi cy mé té bao. Mé seo 1a nguyen Tigw khdi dau cho cée nghién citu quan trong khéc nh: phan héa m6 va té bdo, chon dong té bao, nudi té bao tran, nudi cay té bao don, nudi cy phéi soma, san xuat cdc chat thit cp 6 hoat tinh sinh hoc...M6 seo 12 mét khdi t& bao khéng cé t6 chiéc, hinh thanh tit cdc md va ede eo quan phan héa dudi cde diéu kién dic biét (c6 vét thuang, xut ly ede chit diéu hoa sinh trudng thu vat...). Cac 1 bao thude cée mé hodc co quan nay phai chiu m6t su phan phan héa trude ln phan chia dau tién, Nhin chung stf tao m6 seo invitro (ahd auxin téc dong) do 3 qué tinh: - Su phan phan héa té bao nhu mé (it nhiéu 6 sau bén trong co quan) bao gom cac té bao nhu m6 méc va libe, nhu m6 v6 hay Idi. - Su phan chia ctia cdc tudng tang: cdc t& bao tugng tng ciia phan lén STD dé dang phan chia dui téc dng ciia auxin thim chi khéng cdn auxin ngoai sinh nhu é cdc loai cay cé hay day leo. - Sy xéo trén ciia cdc mé phan sinh so khéi (chi hay ré) qué winh nay duge wu tién 4p dung 6 DTD, vi cdc cay nay wong ting thiéu va nhu md khé phan phan hod so voi STD Mau sic ciia mo seo khéng gidng nhau trén céc méi tring nudi cy khéc nhau hay trén céc b6 phan khde nhau va ching thudng cé mau vang, tring, ndu hay tring xanh. Nong dé va loai kich thich t6 sit dung trong m6i trudng nudi cdy 1a nhiing yéu t6 c6 anh hudng én sy hinh thanh va phat trién m6 seo, Thuding mé seo duge hinh thanh trén mdi trvdng gidu auxin; c6 thé ding auxin ring r& hay két hgp vdi nhau hoe c6 thé két hgp vai cytokinin tuy ting loai cay. Ham twang hormon ndi sinh va chiéu di chuyén ctia céc hormon nay trong mu cy e6 anh hudng dén su phat sinh mé seo. Vi vay nguén mau cay, viée lay mau cay, céch dit mau cdy trén méi trudng nudi cdy sé anh hudng dén su phat sinh mé seo dan dén nhiing phan tng khéc nhau cila mau cay. Cong Nghé Sinh Hye Viet Biotechnolo;Thi nghiém Sink hoc phan tie -24- Bién soan : Lé Ly Tins} Tram ‘ay thi vin dé nay khong quan trong nhung cting c6 mét sé cay chiu anh hudng rat Voi mo 16n. THUC HANH 2.1, Mue dich Khao sat suf phat sinh mé seo tit ede bd phn khde nhau 6 cay thude 1a 2.2-Val 2.2.1 MOi trudng nudi cay MS (20g/ dutting) c6 bd sung 0.1pM 2,4-D va IM 2.4-D 2.2.2 Nguyén ligu thuie vat Cay con thude 1d in- vitro 2.2.3 Hod chat va dung cu - Nude cit v6 wing - Dao, kep, dia cy, gidy cdy... y con in-vitro ra khoi binh nudi cdy. Tranh kep qué manh lam dap mu cAy (hinh A) - Ditng dao cy eft doan ré, Ing than va 1d chuyén qua mét dia cy khée dé xit ly mau (hinh B) ~ La: cit bé gan 1d va ria 14. Phan 1d cdn lai duge cit thinh nhiéu manh. nhé véi kich thude 0.8 -Lmm x 8 10mm, Dit cée manh 1é may nudi trén ede dia petri chita mdi tring MS + 1M 2,4-D BC - L6ng than: chon céc doan Iéng than e6 dudng kinh 2-2,5mm duge cit lat mong 0,05 ~ 0,Lmm. bling ludi dao that sic. Cac lat clit duge dit nim trén cdc dia petri chita mdi trating MS + pM 24D RE: Rita sach agar bing nutéc cat v6 trimg, cit nhé thanh timg doan 1-1,5mm dat lén céc dia petri cé chia mdi wudng MS + 0.1yM 2.4-D - Ding nhwa nylon cuén quanh mép dia petri dé dam bao su v6 tring trong thdi gian nudi c: - Ghi ré s6 nhém, tén mau cy, tén mai trudng va ngay cdy. - Dit nudi trong ti 4. Yéu cau - Thao tée xf ly mau cy t6t, Lat ct ditt khodt cang mong cang Wt; trdnh dip m - Ghi nha inh thi gian phat sinh seo, hinh dang va mau sic khéi ‘m6 seo, vi trf phat sinh seo tit cdc mau c4y khdéc nhau Cong Nghé Sinh Hye Vit -04-Thi nghiém Sink hoc phan tie -25- Bién soan : Lé Ly Tins} Tram BAI 6: KHAO SAT SU PHAT SINH CHOI TU CAC BO PHAN KHAC NHAU CUA CAY THUOC LA AYA 1.6161 THIEU Nhan gidng cay trong bing phuong phdp nudi cdy mé té bao invitro dude goi la vi nhan gidng. C6 nhiéu phuong phap vi nhan gidng khéc nhau dé tao chdi tit dé tao cay con invitro hoan chinh Tuy thude vio ting doi ting khéc nhau ma sit dung phutong phép phir hgp va tit nhién méi phucing phap sé c6 nhitng wu va nhutge diém riéng * Vi nhdn giéng bing phuong phap - Hé sé nhan thip - Thung tg dung dcdc déi wong kh6 tao cum chéi (vi du nhw lan - * Vi nhdn gidéng bang cach tach chdi tit cum choi it dit gif canh - Hé s6 nhan cao - Phife tao han trong vige kich thich chéi phat trién cao thinh cay con hoan chinh Cum chéi c6 thé duoc hinh thanh nv nhiéu con dudng khde nhau: inh tir m6 seo (kha ning d6t bién cao hon nén thuding dude siz dung wong nudi cdy chon mdi) - Phat sinh chdi bat dinh tit cde c6 quan khong sinh san cita cay nhw léng than, 14, cudng 14, 18, . cdinh hoa... (mau cy wal qua giai doan phan phan héa dé tao ede t8 bao sinh m6; tiép dén 1a giai doan tao co quan vdi giai doan trung gian tao mé seo ma ta c6 thé quan sat thy hodc khéng r6i ti dé mdi phat sinh chdi bit dinh) ~ Pha trang thai ti chéi ngit 6 mau cy 1a 1 t6 che nhw dét tudng. Cin kiém sodt hormon ting trudng dé chin ditng s (cum chdi) Vé nguyén tc c6 thé kich thich sy thanh lap chdi bat dinh tir tt ca ede co quan khong sinh sin ctia thuc vat. Tuy nhién trén thye t& thudng chi thanh céng tren di tugng 1a 1 va cudng 14 ngoai trit 6 mot s6 di twang kinh dién, dé 1am nhut thude 14, c& chua.... Ham lung va loai kich thich t6 bé sung vio mdi trudng c6 anh hudng quyét dinh dén su thanh lip chéi (thudng sit dung kich thich t6 nhém cytokinin vdi néng d6 cao kéat hyp vi nhém auxin c6 néng dé thp). Kha ming tdi tao chéi cdn phu thudc vao kfch thudc clia miu cy. Mau c4y qué nhé cé thé khéng ddp ving dude véi cdc diéu kién nudi cdy va sé hod nau. 2. THUC HANH Cong Nghé Sinh Hye Vit 1 sinh cu n, dinh sinh phat trign cita chdi dé tao nhigu chdi -25-Thi nghiém Sink hoc phan tie = 26- Bién soan : Lé Ly Tins} Tram 2.1. Mue dich Ching minh nguyén tic vi nhan giéng va kha ning tai tao chdi bat dinh tit ede bé phn khéc nhau ciia thye vat 2.2, Vat ligu 2.2.1 Méi trudng MS (20g/1 dudng) cé bé sung 10uM BA 2.2.2 Nguyén ligu thue vat Cay con thude 1A invitro 2.2.3 Hod chat va dung cu - Nude cit v6 wing - Dao cy, kep, da cly va gidy cy v6 trig. 2. 3. Cac buic tien hanh Xtrly mau cy asong ww bai 5 va cy vao cdc dia petri c6 chita mdi trudng 1) da chuan bi (Ré, Lng than, Phi 4.Yéu cau - Thy hign tt thao téc xit ly mau Jéng than can mong dén ~ Quan sét va ghi nan két qua vé su thinh lap chdi , théi gian va sé Intong ch cy, trén mdi mau BAI7: KHAO SAT SU TAI SINH CHOI TU MO SEO AYA LGIGI THIE Qué trinh hinh thanh co quan trong mé xay ra qua 2 giai doan: - Giai doan thi nhdt 1a t4i phan héa. Trong giai doan nay xay ra qué uinh chuyén céc té bao biét héa thanh m6 seo - Giai doan thit hai 1a hinh thanh cde mim médng co quan. Bing phuing phdp phdng xa té bio da thay ring nhiing t& bao ctta cdc mim méng nhu m6 ma 6 dy dugc hinh thinh mim méng co quan, t6ng hgp DNA va protein xay ra rt manh, ham ludng duding cling ting. Trong qué trinh phan héa, é cc mé seo khéng c6 t6 chtte dude hinh thanh céc cu tric hinh thai dan dén viéc tao chéi, 18, cnh, hoa va cdy hoan chinh, Qué tinh phan héa nay e6 thé thyc hién bing céch thay d6i chi hda sinh wudng trong mdi trudng nudi moi va ci y Doi vai md seo, xu the tao. ‘Cong Nghé Sinh Hye Viet ) ieee BiotechnokThi nghiém Sink hoc phan tie -27- Bién soan : Lé Ly Tins} Tram 6 quan giam dan khi mo cdy chuyén nhiéu lan vi khi cdy chuyén nhiéu Min nhu thé thudng hinh thanh cdc té bao da bai va léch bi, ngoai ra cé thé mat ede yéu té di truyén. Theo Vii Vin Vu (1999) mé seo khi hinh thanh thudng c6 2 loai: + Loai x6p: chifa nhiéu té bao x6p véi nhan nhé, té bao chit 16ng va khdng bao to. + Loai cing: Cac t& bao citing, chic thanh khi Dang m6 seo ciing cé anh hutdng dén kha nang tai sinh co quan cia khOi mo. Kha ning tai sinh 1 nhan to, té bao chat dim dic va khong bao oho. chdi sém mat di 6 mo seo xp nhumg vn duy tri 6 md seo cting. Nguyén nhan c6 thé do cic bao mé seo sé mat di kha nang téng hgp mot sé chat thiét yéu cho sur ti sinh cita n6 khi s6 cy chuyén ting Ién ( Gautht, 1962). Vi vay khi nudi cay mé seo nhim nue dich tai sinh chdi, nhat thiét phai c6 ging tim diéu kién mdi trading thich hgp cho su hinh thanh cdc khdi m6 seo cling, chic: cdc mé seo x6p can dude loai bé trong cdc lin cy chuyén vi déi khi dang mé seo nay phat trién rat nhanh va lan at ca cdc mé seo cttng c6 kha ning tai sinh phdi. 2.THUC HANH 2.1, Mue dich: Chitng minh kha niing tai sinh cl 2.2.1 Méi trudng MS (30g duding) + BA 1,5uM + NAA 0,5 pM 2.2.2 Nguyén ligu thue vat M6 seo tit l6ng than, 14 va ré ciia bai 3 sau 4 tuan nudi cay 2.2.3 Hod chat va dung cu - Nuféc c&t v6 trimg - Dao cay, kep, dia cy va gidy cy v6 tring... 2. 3. Cae bude thy hi - Ding kep gip mé seo tit binh mu cho vao dia petri ~ Ding dao cy lay sach phan agar con bam vao mau - Néu mé seo qua dn thi cit nhé thanh timg mau Khoang 14 léng tay - Cay vio mdi trading tai sinh chéi da chudn bi rd tén nhom, tén mdi trudng, tén gidng, ngay cdy - Dit mu nudi trong phdng séng 4, Yéu cau: - Thut hién tot thao tac cy chuyén Cong Nghé Sinh Hye VitThi nghiém Sink hoc phan tie = 28- Bién soan : Lé Ly Tins} Tram - Ghi nbn ¢1é mau tao chdi, théi gian tai sinh chdi, sé chdi wén mT mau, kich thude chi bién déi ctia miu - So sdnh su phat trién ciia ede loai mé seo tit ede bé phan khéc nhau. Ruit ra nhan xét. RE Phiénki M6 seo Long than sinh BAL 8: DUA CAY RA VUON UOM AIA 1.6161 THIEU Cac ky thudt chiét canh, gidm cinh va ghép 1 cde phutng phap nhan gidng v6 tinh thuc vat hay cdn goi 1A nhan gidng in vivo. Ngoai ra cdn 6 ky thuat nhan gidng in vitro hay nudi cay mé thuat trén Cay con in vitro khi di phat wién hoan chinh cé day du than, 14 va 18 sé duoc chuyén ra yudn uom. aay 18 giai dogn kh6 khdin nhat trong ky thugt nhan giéng vO tinh bling nudi cy mé. Cay in virro duge nudi cy trong digu kign dn di chuyén ra dit vi ede diéu kién tr nhién hoan ton khéc hin nhu dinh duving thap, anh sing ¢6 cuting d6 manh, nhiét d6 cao, am d6 thap.... cdy con dé bi stress, dé mat mde vi mau bi héo, Mat khdc trong mi tng ty nhién c6 rat nhiéu vi khudn va nm gay bénh lam thdi va chét cay. Dé khic phuc nh trang nay, ngudi ta c6 cde bién phép nhw sau - Vuin wom cay cy mé phai mat, cuding dé chiéu sang th4p, nhiét d6 khong khé thdp, din 40 thip - Cay con dude trong trén ludng wom cay 6 co chat dé thoat mudec, tai xp, giit dude do am. Co 14 mét phugng phap nhan gidng vé tinh thuc vat cho hé sé nhan cao hon céc ih vé dinh dudng, anh sang, nhiét dO, Am dé... nén khi cht va cay con cn dude xtt ly vai thude chéng nm hoc thude tim dé phong ngita nam bénh cho cay - Trntéc khi dua cay ra vutin wom nén dé céc binh mau ngoai vn wom trong 1-2 tuan cho cay thich nghi dan vdi diéu kién ty nhién - Trong nhiing ngay dau dua c4y ra vudn vom, lung vom cay cdn dugc phut nylon dé gidm qué tinh thodt hoi nude 6 14 ( thuing 7-10 ngay sau khi ra cy). Mi ngay nén edi nude 2 lin k&m theo phun suong thudng xuyén khi tri nding nhim duy «i dé aim. -Cay con sau khi wong 7-10 ngay can phai bén phan dé cay phat trién déng thdi phun thud ngita nam bénh, 2. THUC HANH 2.1, Muc dich Lam quen cac k¥ thuat dua cay ra vuGn vom Cong Nghé Sinh Hye Viet 1o0e Biotechnolo;Thi nghiém Sink hoc phan tie -29- Bién soan : Lé Ly Tins} Tram 2.2, Vat ligu 2.2.1.D6i (wong cay giéng: - Cay con Lan Hoang Thao ( Dendrobium sp.) in-vitro = Cay con Thude ld in-vitro 2.2.1 Nguyén ligu va dung cu - Xo dita - Day thun - Thaw nha - Tro trau - Titi wom 12x17em - R6 nha - Cat xay dung -Kep - Binh phun 2. 3. Cac bude thy hién -Cho mot it nude vao cdc binh mdi trudng c6 cay gidng, Hic manh dé m6i tuding vo ra. -Ding kep gp nhe nhang cay gidng ra Khoi binh tranh 1am ditt,dip than, 14 hole ré cay -Rifa sach agar bam 4 ré cy bang | tim nhon; can thao tac nhe nhang dé khong 1am ditt va dap ré ; néu khong khi dem trong 6 vutim wom cay sé dé bj nhiém nam bénh va chét. - Ngam cay trong dung dich thudc tim 1o/no trong 1 phit sau dé rita lai bing nudc sach dé ro. - Doi voi Thudc 1d (gid thé trong 14 h6n hgp xo dita, tro tau va y dung): Ding | cay dia xam_céc 16 wom trén vii wom GT dé sdu khoang Sem. Dt gon ré cay con yao 16 wom va ding tz ém chit dat lai dé gitt cay dig viing. Uom 5 cay cho | tii wom véi Lan hoang thao (gid th ¢ 1a x6 dita): Géi gon phan ré trong miéng xo dita, ding thun bude nhe dé dinh hinh cay. Xép vao r6 nhya, tudi phun sung nhe true khi dita ra vutin uom, 4. Yeu ca = Thye hanh thao téc dua c4y con ra vudn wom, tranh 1am dap va duit r& qua nhiéu - Quan sdt ty 18 cy con s6ng sét sau khi ra vudn wom. Cong Nghé Sinh Hye Viet o0e Biotechnolo;