TAP CHl KHOA H p c DM HOC V A N LANG Ngo Thi XuyBii yi Igli X A C D I N H T H A N H P H A N H O A H O C V A H O ^ T T I N H S I N H H Q C T R O N G M A U L A C A Y X A O T A M P H A N (Paramignya Irimera (OUv.) Guill)
V A N G H I E N c i r u S i r P H A T S I N H H I N H T H A I T i r C A C N G U O N M A U K H A C N H A U
IDENTIFICATION OF THE CHEMCAL COMPONENTSAND BIOACTIVITIES OF Paramignya trimera (OUv.) Guill LEAF SAMPLES AND RESEARCH ON
MORPHOGENESIS FROM DIFFERENT EXPLANTS
fiGd THixvYEn'"', NGUYSN THI Bvpc'"', BANG THI KIM THVY^, NGVYSN THI HUYtN TRANG'"', DV'ONG B&C Hiii/"', Bd BANG GIAP'"', TRAN TSQNG TUAN'"' TOM TAT: Trong nghien cu-u nay, thanh phdn hoa hgc va hoat tinh sinh hoc cua cdy xdo lam phdn a ngoal vuon uom duoc khdo sat. Kit qud cho thdy mau Id duac chiet xudt bdng methanol co khd nang khdng oxy hoa vol gid tri IC^n la 6,4349mg/ml vd co khd ndng khdng chung vl khudn Gram (+) Staphllococcus aureus (duang kinh vong vo khudn 12mm). Ben canh do, cdc thi nghiem khdo sdt ve khd ndng tao mo seo vd choi cung duoc tien hdnh. Ty le mo seo duac hinh thdnh a nghiem thiec co sie dung TDZ vd dat ty le tot nhdt d nong do 0,6mg/l vai ty le la 76,19%. Ty le hinh thdnh chdi cao nhat dat 100% ct nghiem thicc su dung 3,Omg/1 BA. Tuy nhien, cdc choi ndy yeu vd de bi rung. Bekhdcphfc . tinh trang ndy, moi truang co bo sung 3mg/l BA vd nong do FeS04 thay doi (cao gdp 3 lan so vdl moi trudng MS ban ddu) dugc sir dung di khdo sdt tren mdu cdy cdy xdo tam phdn, kit qud buac ddu cho thdy cdc choi khong bl gay vd xanh han.
Tir khoa: FeS04, khdng oxy hod, khdng khudn, TDZ, xdo tam phdn.
ABSTRACTS: The study Investigates the chemical components and bioactivities of Paramignya trimera (OUv.) Guill grown In a nursery. The results show that the leaf samples extracted using methanol have antioxidant ability, IC50 value of 6.4349 mg/ml.and the capability of fighting Gram (+) Staphllococcus Aureus (I2mm diameter). In addition, the study conducts an experience on the ability of callus and shoot Induction. The calbis was formed In the treatment using TDZ, the best ratio was achieved at 0.6 mg/l at 76.19%. In particular, the highest shoot forming rate was 100% in the treatment using 3.0 mg/l BA; however, these shoop are weak and vulnerable. To overcome the situation, the scientists modified the environment by adding 3.0 mg/l BA and adjust the concentration of FeS04 The initial results show that the shoots In the new environment were greener and were not broken.
Key words: FeSOt^ antioxidant, bioactivities, TDZ, Paramignya trimera.
i..F°^'^^' '"'"'""S Bai liQc VSn Lang. [email protected]. Ma sd: TCKH09-07-20I8
" Vien Sinli lioc nliiet dcii, Vi6n Han lam Kiloa iipc va Cong ngh? Viet Nam
TAP CHI KHOA HQC DAI HQC V A N LANG So 09, Thang 5 - 2 0 1 8 I. GIOI THIEU
Xao tam phan cd ten khoa hpc la Paramignya trimera (OHv.) Guill, thugc hg cam chanh (Rutaceae), phan bd nhieu d khu vyc Nam Trung BO. Xao tam phan cd cdc thanh phdn nhu flavonoid, saponin, alkaloid, chu yeu la courmarin vd triterpenoid. Cac thi nghiem cho thay, xdo tam phan ed tac dyng gay dpc ddi vdi 5 ddng te bao ung thu: ung thu dai trang HTC116, ung thu vfl MDA MB231, ung thu budng trflng OVCAR-8, ddc bidt cd tdc dung manh ddi vdi dong te bdo ung thu gan Hep-G2 va ung thu cd ttt eung Hela [17]. Nam 2015, Van Tang Nguyen vd edng su da nghien cflru anh hudng cfla cdc phuong phdp va dung mdi tach chiit cac hogt chdt sinh hpc tu rd xao tam phan va khd nang chdng oxi hda cua chflng [12]. Ddn nam 2016, Van Tang Nguyen cdn khdo sdt sy anh hudng cfla cdc phuong phap sdy khdc nhau (thdng thudng, hut chdn khdng, Id vi sdng, khdng khi ndng) Ien tiianh phdn boat chdt sinh hoc va tinh chdt chdng oxi hda cua re cdy [13].
Do xao tam phdn ed gid trj dugc lieu to ldn nen hien nay cdc ca the xdo tam phdn ngoai ty nhidn dang bi khai thae qud mttc vd cd nguy ca bj tan dipt. Ngay nay, sy phdt tridn efla cdng nghd nudi cay md thuc vgt gdp phan rdt Idn trong vi$c bao tdn cdc cay thudc quy hiem va dang bi de dpa. Tuy nhien, nhung nghidn cflu vd nudi cay md cdy xao tam phan van cdn ban che, chfl ydu tap trung vdo nghidn cuu cdc hgp ehdt cd hogt tinh sinh hgc co trong cay. Nghien cflu dugc tiln hanh nhdm tao ngudn nguyen lieu ban ddu cho qua trinh nudi cay in vitro cay xdo tam phan.
2. VAT LIEU v A PHUO'NG PHAP 2.1. Vgtli^u
Ld vd ddt than cfla cdy xdo tam phdn (Ninh Didm, Ninh Hda) 02 ndm tudi trdng tgi vudn uom cua phdng Cong nghd te bdo thyc vdt - Vi§n Sinh hgc Nhiet ddi dugc stt dung de lam mdu cdy.
2.2. Noi dung va phircmg phap nghien cmi 2.2.1. Xdc dinh thanh phan hoa hpc va hogi tinh sinh hpc trong mdu Id cay xdo tam phan (Paramignya trimera (OUv.) Guill)
Dinh tinh HCTC trong mdu Id xdo tarn phan
Chudn bj dich chidt: Ty Id bdt mau dugc ehilt vdi EtOH 70% Id 1:50. Ngam mau 24 gid. Lgc vd thu djch chiet.
Alkaloid: Lay 5 ml djch chiet, bd sung 2 ml dung djch acid chlohidric va Iac nh?.
Them 1 mL thudc thtt Dragendorff. Thdy xudt hien tua cam hogc ndu do cho thdy sy hidn dien cua alkaloid [1}.
Saponin: Bd sung 5 ml nude cat vao dng nghiem chtta 10 ml djch chiet mau, Lac manh, tao bgt dn dinh lidn tue. Them vdo hdn hgp 3 gigt ddu olive vd ldc manh.
Quan sdt thdy bgt biln mdt vd xuat hien mdu trang stta [1].
Triterpenoid: 5 ml djch chiet dugc thdm vao 2 ml chloroform. Lde nhe hdn hgp, sau do them tu tu 3 ml acid sulfuric theo thanh dng nghiOm. Mdu nau do xudt hien cho thay sy hidn dien eua triterpenoid [1].
Flavonoid: 5 ml acid sulfuric dugc thdm vao dng nghidm chtta 5 ml djch chiet.
Ldp mdu vang (nau dd) xuat hien, va bidn mdt sau khi ldc hdn hgp cho thdy sy hien dien cfla flavonoid [5].
TAP CHI KHOA HQC DAI HQC VAN LANG NgoThjXuyEn vai Glycoside: Chudn bj 5 ml djch chidt
vao dng nghiem, nhd vai gigt acid acetic lanh. Sau dd bd sung vai FeCh va 5 ml acid sunfuric dam dac. Quan sat su tdch ldp cfla dung djch: ldp trdn cd mau xanh Id, ldp dudi mau nau do [5].
Steroid: Them 5 ml chloroform vao dng nghiem chua 1 ml djch chiet Them 1 ml acid acetic va nhd 3 4 gigt acid sulfuric theo thdnh binh. Dung djch tach Idp, ldp tren xudt hidn mdu vdng c6 huynh quanh xanh la cho thdy sy hien dien efla steroid [5].
Phenolic: Nhd vai gipt chi acetate 5%
vdo dng nghidm chua 2 ml dich chiet. Xudt hien tfla trang eho thdy sy hien dien efla phenolic [2].
Xdc dinh khd ndng bat g6c tu do DPPH
Kha ndng bat gdc ty do efla cao chidt la xdo tam phdn dugc tidn hanh theo phuong phap cua Mensor [7]. Ldy 5g bOt la xdo tam phan vd chiet vdi methanol. Cd quay d 40"C de dudi het dung mdi, thu dugc cao chiet methanol. Pha lodng cao trong methanol, sau do lpc dung djch qua than hogt tinh dl logi bd diep luc. Djch chidt Id tilp tyc duge co quay chdn khdng d 40''C ih logi dung mdi vd thu cao c h i k Cao chiit dugc pha lodng trong metiianoi thanh day 5 ndng do. Dung dich acid ascorbic 200 pg/ml dugc stt dung Idm chdt ddi chttng duang; metiianoi la ehdt ddi chflng dm.
Chudn bj dTa 96 gidng, mdi gilng ngp 100^1 mdu thtt hoac chdt ddi chung vd lOOpI dung djch DPPH 300^M. Ldc nh? va u d 37''C trong 30 phflt. Sau dd, do mgt dp quang hgc d budc sdng >. = 517 nm.
Hogt tinh uc chd gdc ty do cua mlu dugc tinh theo cdng thttc:
Hogt tinh uc chi goe t{c do
(%)=/•/-
QD (meiii)-r
IOOOD ichihigy
Ttt dudng tuydn tinh hogt tinh uc chl gdc ty do d cac ndng do khdc nhau, tinh gia tri ICso cua mdu. Gia di IC50 cdng thdp thi khd ndng flc chd gdc ty do cang cao.
Kiim tra khd nt'tng khdng khudn Khd nang khdng khudn cua cao ehilt la xao tam phan dugc phdn tich theo phuong phap cfla Smdnia [ I I ] . Theo dd, mdu la dugc nghien nho thdnh bgt vd bdo quan d 4''C. Sau dd ta ldy 5g mdu va chidt vdi dung mdi methanol. Djch chidt dugc thu Igi va cd quay 6e logi dung mdi cdn khoang 1 ml cao chidt. Cao chiet methanol dugc pha lodng trong dung djch DMSO 5% din ndng dO lOOmg/ml. Cac dung dich mdu cung dugc pha lodng d l xdc djnh hogt tinh khang khudn.
Cdc chflng vi khudn dugc stt dyng dl khdo sat Id Salmonella typhimurium ATCC 29629, Bacillus subtilis NRRL B-354, Staphylococcus aureus NRRL B-313, Pseudomonas aeruginosa NRRL B-14781 va Escherichia coli ATCC 8739. Tdt ca cdc chung trdn se dugc nudi cdy ldc trong mdi trudng LB Idng, d 37°C trong 24 gid trudc khi kilm djnh.
Cdy trai 100 pi mdi chflng vi khudn kilm dinh (10^ CFU/ml) len dTa mdi trudng Mueller Hilton agar. Mdi dTa due 4 gilng vdi dudng kinh 6 mm. Hut 100 pl mdu cho vdo cac gilng tren dTa thgch. 100 pl dung djch DMSO 5% dugc stt dung Idm dli chiing dm. DTa cay sau khi bam mdu dugc u d 37''C trong 24 gid. Quan sdt, do vong khang khudn vd ghi nhan kit qua.
TAP CHI KHOA HQC DAI HOC V A N LANG S6 09, Thang S-2018 2.2.2. Nghien ctiu sir phat sinh hinh
thdi cay xao tam thin tij- cdc nguon mau khdc nhau
Bo tri thi nghiem: Anh hudng efla thdi gian khu triing mau den ty le sdng efla mdu xdo tam phdn
Ddt thdn non dugc chgn lam mdu cdy Id cac ngpn chdi non phat sinh ttt chdi ben khodng 2 thdng tudi. Mdu Id dugc chgn la nhttng Id khdng qua non va khdng qua gid giup dd thao tac, td bao it bj tdn thuang hon khi khtt trung. Mdu cdy dugc khu trung be mat bang xa phdng loang va sau dd dugc rtta sgch dudi vdi nude chdy khoang 30 phflt. Mdu dugc tdch ridng vd dua vao tfl cay vd trung de tiep tuc qua trinh khu trung.
Dung dich HgCb 0,1% cd bd sung vai gigt Tween 20 dugc stt dung de ngam mdu cdy trong cdc khoang thdi gian 4, 8 va 12 phut. Sau dd, mdu duge rtta lai 3-4 ldn bdng nude cat vd trung. Mdu cdy dugc cat thdnh cac ldp mdng theo tiet dien ngang (0,2-0,5 mm) va cdy vao cdc binh nudi cdy chtta 20 ml mdi trudng khoang co ban MS [8] cd bd sung 30g/l sucrose, 8g/l agar.
Sau 10 ngay nudi cay xdc djnh ty 1?
mdu nhiem (%) va ty Id mau sdng vd trung (%).
Anh hu&ng cua NAA, TDZ riing le len khd ndng hinh thdnh mo seo cua mdu cdy Id
Cae mdu la khdng nhiem dugc cdy chuyen vao cac binh nudi cay chfla 20 ml mdi trudng MS ca ban [8] cd bd sung 30 g/1 sucrose, 8 g/I agar, NAA vdi cdc ndng dp 0,0; 0,5; 1,0; 1,5; 2,0 mg/l va TDZ d cdc ndng dg 0,0; 0,2; 0,6; 1,0 mg/l.
Sau 60 ngay nudi cay, cdc chi tieu theo doi dugc ghi nhdn nhu ty le mau hinh thanh md seo (%), hinh thai md seo.
Anh Jtu&ng cua BA len khd ndng tdi sinli ehoi tie mdu cdy dot thdn
Cde mdu cay ddt than khdng nhiem dugc cdy chuydn vdo cdc binh nudi cay chua 20 ml mdi trudng MS co bdn [8] cd bd sung 30g/I sucrose, 8 g/1 agar vd BA vdi cdc ndng dd khde nhau (0, 1, 2, 3, 4 mg/I).
Theo ddi kit qud sau 60 ngay nudi cay dya tren cac chi tieu: ty Id mdu hinh thanh chdi (%), hinh thai chdi, sd chdi trung binh/mau, chieu eao chdi (mm).
2.2.3. Phietfng plidp xie li so liiu Sd lieu dugc thu nhan bang phdn mdm Microsoft Excel vd dugc xu li theo phucmg phdp thdng ke bdng phan mem SPSS 16.0, theo phuong phdp Duncan [4] vdi dO tin cay P < 0,05.
3. KET QUA VA THAO LUij.N 3.1. Xdc dinh cdc nhdm hop chat c6 hoat tinh sinh hgc trong Id cdy xao tam phan (Paramignya trimera (Oliv.) Guill)
Dinh tinh nhom hap ehdt thic cap co trong mdu
Mdt sd phan umg vd thay ddi mau sac cua timg phdn flng cu the dugc sfl dung de Ige nhdm hgp chdt hod hgc ttt cao chidt methanol efla bdt la cay xdo tam phan. Cdc xet nghiem hda hgc dinh tinh cho thdy sy hien didn efla nhdm hod hgc khac nhau nhu glyeosid, phenolic, hgp chat alkaloid va saponin (Hinh 1). Ngodi cdc hgp chat, chiit xuat methanol chtta flavonoid vd cdc hgp chdt triterpenoid nhilu hon. Cac tinh chat ttt cac hgp chat dugc tim thdy cho rang chflng CO thi dugc stt dyng trong dugc phdm.
TAP CHl KNOA HOC DAI HOC VAN LANG NgoThjXuyEn valgk Bang I. Ket qua dinh tinh mot so hop chat thii
cap tir dich chiet bpt la tu nhieii CLia cay xao tam phan Paramignya inmera a tiong do dung moi
ethanol 70%
STT 1 2 3 4 5 6 7
Nhom chat Alkaloid Saponin Triterpenoid
Steroid Glycoside Flavonoid Phenolic
EtOH (70%) + + ++
+ +++
+ Ghi chu: (-). khong fiien man dgc tnmg, (+). hien mail dac Irung, (++) hien man dam, (+++) hi^n mau rat dam
Hinh 1 Ket qua dinh tinh mot so hgp chat thiJr cap tij dich chiet bgit la ttr nhien ciia cay xao tam phan Ghi chti a. alkaloid, b.Jlavonoid, c. glycoside, d.phenolic, s saponin,/, steroid, g. interpenoid Khd ndng klidng oxy hda khu cua mdu Id xdo tam phdn nuoi trdng ngoai vir&n team
Kha nang khang oxy hda eua mdu la cay xao tam phdn duge thyc hien bdng khao nghiem DPPH. Kha nang nhat rdc cfla chiet xuat methanol tren DPPH gdc tu do da dugc kiem tra trong phgm vi ndng dp 32,5-2,03125 mg/ml. Kit qua cho thay rdng chidt xudt methanol cd kha nang khang oxy hoa (Bieu dd 1) vdi gia trj IC50 la 6,4349 mg/ml.
Bang 2. Ket qua do OD Nong dp
(mg/ml) 32,5 16,25 8,125 4,0625 2,03125
ODI 0,165 0,287 0,485 0,699 0,865
OD2 0,15 0,264 0,478 0,713 0,84
%
Inliibitory 85,9375 75,40179 57,00893 36,96429 23,8393
SD 0,947011 1
1,45209 1 0,44192 ! 0,88388: • 1,57836:
Bieu do 1. Do thj tuang quan giiJa % ire chS g6c ty do va nong d6 cao chiit Khd ndng khdng khudn eua eao chiet tie Id xdo tam phdn
Cac chflng vi khudn dugc khdo sat de xem xet kha nang khdng khudn cua mlu djch chiet la xao tam phdn. Ket qua d hinh 2 cho thay mdu chi ed kha nang ddi khang vdi chflng vi khudn Gram (+) Staphllococcus aureus vdi dudng kinh vdng yd khudn 12mm va khdng cd klid nang khang cac chflng vi khudn con lai.
Hinh 2. Kha nang khang khuin ciia dich chiet tir mau la cay xao tam phan
TAP CHI KHOA HQC D ^ I HQC V A N LANG SQ 09, Thiing 5 - 2 0 1 8 3.2 Nghidn cuu s y phat sinh hinh thdi
cay xao tam thdn trong dieu kidn nuoi cay In vitro
Anh htfdng ci'ia thoi gian khir triing mdu din ty li song cua mau cay xdo tam phdn
Cac logi mdu cdy ddt than va Id dugc khu trung bang HgCl2 0,1% trong thai gian 4, 8 vd 12 phflt. Sau 2 tuan nudi cdy, ket qua dugc ghi nhdn d bdng 3. Cdc binh nudi bat ddu xudt hidn cac trudng hgp nhiem nam va khuan sau 5 ngay nudi cdy. Ket qua thi nghi?m cung cho thay thdi gian khu trung 8 phflt la tdt nhat ddi vdi mau cay Id vd 12 phut ddi vdi mdu ddt thdn non.
Bang 3. Ket qua khii triing mau cay x&o tam phan ThM
gian khu- trijng mau (phUl)
4 S 12
M i u 1^
T y l e nliiim
(%)
86,67a 16,67b 13,33b
T y l e mSu song
23,33b 76,67a 63,33a
M 3 U att tlian T y l e nhilm
(%)
100,00a 55,S6b 22,22b
T y l e man s6ng
(%)
0,00b 33,33ab
66,67a Anh hu&ng cua NAA, TDZ rieng le lin khd ndng hinh thdnh mo seo cua mdu cdy Id
Qua trinh phat sinh hinh thai trong nudi cay in vitro thyc chat la kdt qua cfla qua trinh bidt hda va phdn biet hda. Qud trinh ndy phu thugc vdo ty Id cfla hai nhdm chdt DHSTTV. Ngudn auxin thudng dugc stt dyng cho sy phat sinh hinh thai d cac loai thuc vat la NAA. NAA cd hogt tinh manh nen ket hgp chat che vdi cac thanh phdn dinh duang trong nudi cay md de kich thich su tdng trudng cua te bdo va didu hda sy phat sinh hinh thai cfla md seo.
6 thi nghiem nay, anh hudng efla NAA rieng le Idn su phdt sinh hinh thai cfla mdu cay Id dugc khao sat. Tuy nhidn, sau 60 ngdy nudi cay, ket qua thu thap (bang 4) khdng cho thdy su dap ung cua mdu ddi vdi mdi trudng nudi cdy. Kdt qud ndy khdc vdi kdt qud nghien cuu eua Trdn Trgng Tudn va cgng su (2014) khi nghien cuu trdn cay ba bdnh [14]. Nghien cflu cho thdy d cdc nghidm thttc su dyng NAA, cac mdu Id deu tgo md s?o va nghiem thttc stt dyng 2,5 mg/l NAA eho hidu qud tdt nhdt vdi 61,2%
ti Id mdu tgo seo trong didu kidn nudi cdy tdi. Didu ndy cd thd do cdc nhdm auxin dnh hudng khdc nhau ddi vdi tttng loai cdy khdc nhau.
Bang 4. Anh huong ciia NAA va TDZ len s\r phat sinh hinh thai ciia mlu la xao tam phan NAA
(mg/l)
0,5 1,0 1,5 2,0
TDZ (mgfl) 0,0 0,2 0,6 1,0
Ty If hinh th^nh m6 s?o(%)
0,00c*
38,10b 76.19a 57,14ab
Hinh thai Mau hoa nau, khong hinh thanh m5 s^o M6 seo xfip CO mau vang, hinh thanh ir gSn li M6 s?o xop c6 mau vang, hinh thanh a hau het la M6 seo x6p c6 mau vang, hinh thanh 6 hiiu hit la Mau van con xanh, nhung khdng dap ung Mau van con xanh, nhung khdng dap img Mau van con xanh, nhung khdng dap iing Mau van con xanh, nhung khdng Hip ling
*Cdc mdu ky tif khac nhau (a, b,...) biiu thf s^f khdc biet CO ;^ nghia P < 0,05 bang phep ihu Duncan TDZ la chdt dilu hda sinh trudng thyc v^t thudc nhom cytokinin cd kha nang kich thich phan chia te bao, thay ddi im thd ngpn
TAP CHi KHOA HOC BAI HQC VAN LANG Ngo Thj Xuyen \k tgk va phan hda chdi trong nudi cdy md, va dgc
biet Id trong nhiing nghien cttu ve qua trinh tdi sinh chdi bdt djnh d cay ddu tdy [9].
TDZ duge cho Id cd tde dung Idm tang ham lugng cytokinin npi sinh bang cdeh tte chd boat ddng cua cytokinin oxydase.
Trong mdi trudng MS cd bd sung TDZ ndng dO ttt 0,2-1,0 mg/l, md seo bdt ddu hinh thanh chgm sau khodng hon 30 ngdy nudi cdy. Ket qud thu dugc sau 60 ngdy nudi cdy, ty Id mo seo dugc hinh thanh d nghiem thttc cd su dyng TDZ va dat hieu qua tdt nhdt d ndng do 0,6 mg/I vdi ty Id mdu tao seo Id 76,19% va khdng co sy hinh thanh md seo d nghiem thttc ddi chflng. Kdt qua nay phu hgp vdi nghien cuu efla Trdn Trpng Tuan vd cdng sy (2014) [14]. Khi cac mau cay la cdy bd b^nh duge nudi cdy tren mdi trudng cd bd sung TDZ, ty Id hinh thanh md s?o dat mflc cao vd cao hon khi stt dyng NAA. 0 nghiem thttc cd bd sung 0,6 mg/l TDZ, ty le hinh thanh md seo cua mdu Id cdy bd benh dgt cao nhdt (32,6%) so vdi cdc khdo sat eon Igi. Trong nhdm cytokinin, TDZ cd hogt tinh sinh hgc mgnh nen ehi cdn hien dien d ndng do thdp trong mdi trudng nudi cay, ddc biet trong vi nhdn gidng. Mgt khac, TDZ chdng Igi sy phan hfly gay ra do cde tdc nhan oxy hda cytokinin, do dd TDZ bin vttng trdn mdi trudng nudi cdy [6]. Vl m^t hinh thai, hdu het md s?o deu xop vd co mdu vang dam.
Anh hu&ng cua BA dSn khd ndng tdi sinh chSi tie mdu cay dSt thdn
Skoog va Miller cho rdng cytokinin tac ddng din qua trinh hinh thanh chdi thdng qua sy kilm soat cdc qud trinh tdng hgp mOtsd Ioai protein [10].
Hinh 3. Anh huong ciia NAA, TDZ len sy phSt sinh hinh thai ciia mau
Ghi chii: a. Moi tru&ng bo sung !VAA; b. 0.2 mg/l TDZ: c. 0.6 mg/l TDZ; d. 1.0 mg/l TDZ;
f-l. hinh thai mo s?o Khi bd sung cytokinin ngoai sinh vao mdi trudng nudi cdy.sy thay ddi gradient ndng do chat didu hda sinh trudng dien ra ddng thdi thidt lap mdt gradient ndng do chat dieu hda sinh trudng mdi. Dieu nay giflp phd vd trang thdi ngfl va kich thich sy phat triln chdi.
Trong nghien cuu nay, khao sat anh hudng cua BA len khd nang tdi sinh chdi tii vide nudi cdy dot thdn dugc thyc hidn. Sau 6 tuan nudi cay, kit qud vd sy hinh thanh chdi dugc ghi nhdn d bang 5.'Ket qua nghiem thttc stt dyng BA vdi ndng dp tit 1,0 - 4,0 mg/l dlu cd sy hinh thanh chdi. Tj- Id hinh thanh chdi cao nhdt dgt 100% cr nghiem tiifl-e stt dung 3,0 mg/I BA, tilp theo la nghiOm thflc stt dyng 4 mg/l BA, tj"
le hinh thdnh chdi dat 83,33%. CJ cac nghi$m thuc stt dung ndng dO BA thap (1,0-2,0 mg/I), ty I? hinh tiianh chdi tii§p (trung binh khoang 50%). Trong nghiin cuu cfla Dd Ddng Gidp vd cgng su (2012) trdn cay chudi Musa spp., kdt qud cung cho thdy khi su dung BA d ndng dO cao thi ty I?
1 ^ 1 - t^J-ll KHUA HQC O^l HUU VAN LANG SoQ9, Thang5-2Uia hinh thdnh chdi tdt hon so vdi su dung BA
d ndng dO thdp. Tren mdi trudng cd ndng do 3,0-5,0 mg/l BA, sd lugng chdi hinh thanh cao han so vdi cdc nghidm thflc edn lgi [3]. Ket qua nay cung tuang ty vdi khdo sat dang thyc hien. Khi stt dung ndng dg BA cao thi sd chdi hinh thanh cao (2,67 chdi ddi vdi nghiem thue su dung 3,0 mg/l BA va 1,92 chdi ddi vdi nghiem thttc su dyng 4,0 mg/l BA).
Bang 5. Anh huong ciia BA len kha n3ng hinh th^nh ch6i xao tam phan BA
(mg/l) 1 2 3 4
S6 chM hinh thanh 1,40b' 1,17b 2,67a l,92ab
Ty ie hinh thanh choi
(%)
50,00 58,33 100,00 83,33
Hinh thai Choi bi vang la
va rgng Choi bl vang la
va rung Choi bj vang ]&
va rung Choi bi vang la
varung
*Cdc mdu ky tu khdc nhau (a, b,...) bieu thi sukhdc bi$l CO y nghta P < 0,05 bang phep thie Duncan Ve mat hinh thai, chieu cao chdi hau het ehi dgt ttt khoang 3,0 mm trd xudng.
Cac chdi nudi cay tren mdi trudng BA vdi cdc ndng dp 1,0-4,0 mg/l, hau hit eac chdi Id ddu bj vdng va rung. Trong nghien cttu cfla Huong va cgng su tren cdy mdm xdi dd (Rubus alcearfolius Poir.), cae mau nudi cdy trdn mdi trudng cd bd sung BA cung cd hien tugng chdi bi vdng la dan den ryng [15].
Hinh 4. Anh huong ciia BA l^n sir hinh th^nh choi vol cac nong d? Ian lugt la: a L 1,0 mg/l, a2. 2,0 mg/I, a3. 3,0 mg/l, a4. 4,0 mg/l; b,c,d,e. choi dugc cay tren moi truang cai tign c6 n6ng dp FeS04 tang
gip !,5 lln; f,g. Nhan nhanh choi.
Dd khdc phuc hidn tugng nay, tac gia cung dd khdo sat anh hudng efla nong do FeS04 khdc nhau len ehdt lugng chdi hinh thdnh. Ket qud cho thay khi nudi cay chdi mdm xdi dd tren mdi trudng MS dd thay ddi ndng dp FeS04 (cao gdp 3 Idn so vdi mdi trudng MS co ban) thi sd lugng chdi hinh thdnh cao hon, cae chdi khde manh va Id cd mau xanh. Do do, de khdc phyc vide gay choi va rung Id d chdi cay xao tam phan, mdi trudng cd bd sung 3,0 mg/l BA vd ndng dO FeS04 thay ddi (cao gdp 3 ldn so vdi mdi trudng MS ban ddu) dugc stt dyng dd khao sdt tren mdu c4y cdy xdo tam phdn, ket qua budc ddu cho thay cde chdi khdng bj gay va xanh hon (Hinh 4 b,c).
Ben canh do, khi quan sat v l mat hinh thdi, ede mau chdi c6 sy hinh thdnh md s?o vdng d vlt cat (Hinh 4d, 4e). Theo Zawadzka vd Orlikowska (2006), sdt ddng vai trd quan trgng trong viec tdi sinh cay [16]. Sdt la
TAP CHi KHOA HOC DAI HQC VAN LANG Ngo Thi Xuyen va lfil(
nMn to xuc tac trong viec hinh thanh diep 4. KET LUAN
luc va van chuyen oxy. ThiSu sit, ]i biin Chift xuat methanol ciia mlu la cay thanh mau ving hoac nSu lam giam silc xao tam phan co kha nSng Idiang oxy hoa s6ng ciia cay, voi gia tri ICso la 6,4349 mg/ml. MSu chi Trong nghien cim nay, liha nang nhan c6 kha nang ddi khang voi chung vi khuin nhanh ch6i cung da duoc khao siit duoi tac Gram (+) Staphllo-coccus aureus (ducmg dpng cua BA, tuy nhien khi cM cac ch6i va kinh vong vo khudn 12 mm),
cdy chuyin qua moi tradng nhan nhanh thi Thoi gian khit triing 8 phiit bjng HgClj hdu hSt mdu bj chgt, cac phdn tigp xuc vdi 0,1% li tot nhdt doi vdi mau cay l i ia va 12 m6i trucmg bj hoa nau ho^c khong phat phut doi vdi mau dot than non. Ty le mo triSn (Hinh 4f). Mot s6 mau cd sy hinh s?o duoc hinh thanh 6 nghiem thirc co sir thanh mo seo (Hinh 4g) va chdi nhd, tuy dung TDZ va dat ty 16 tdt nhdt d ndng dJ nhi6n, nhiing chdi nay khdng phat triSn chi 0,6 mg/l vdi ty le la 76,19%. Ty Ie hinh khoang 2 mm trd xudng. thanh chdi cao nhat dat 100% d nghiem
thirc str dung 3 mg/l BA.
TAI LIEU THAM KHAO
[1] AyoolaG., Coker H., Adesegun S., Adepoju-Bello A., Ezennia E., and Atangbayila T., (2008), Phylochemical screening and antioxidant activities of some selected medicinal plants used for malaria therapy In southwestern Nigeria, Tropical Joumal of
Pharmaceutical Research, 7 (3).
[2] Ben I., Woode E., Abotsi W., and Boakye-Gyasi E. (2013), Preliminary phylochemical screening and in vitro antioxidant properties of Trlchilla monadelpha (Thonn.) J. J. de
Wilde (Mellaceae), Joumal of Medical and Biomedical Sci, 2 (2).
[3] Dd Dang Giap, Pham Ngoc Vinh, Trdn Trgng Tudn, Nguyln Thi HuySn Trang, Phjm Ngd Anh Thu, Thai Xuan Du (2012), Tang h^ s6 nhdn nhanh chdi chudi laba (Musa sp.) nuoi cdy In-vitro bang cdch su dung dnh sdng, myo-inosltol vd adenln sulphate, Joumal of Biology, 34 (3se).
[4] Duncan D.B. (1955), Multiplerangeand multiple F test, Biometrics, 11.
[5] Jaradat N., Hussen F., and A. Ali. (2015), Preliminary phylochemical screening, quantitative estimation of total flavonoids, total phenols and antioxidant activity of Ephedra alata Decne, J. Mater. Environment. Sci., 6 (6).
[6] Liberman R., Liat S., AdaN.L., DaliaE., MosheR., and O.S. Michal. (2001), Shoot regeneration from leaf explants ofBrunfelsla calyclna. Plant Cell, Tissue and Organ Culture, 100 (3).
[7] Mensor LL, Menezes PS, Leitao GG, Reis AS, dos Santos TC, Coube CS, et al., (2001), Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH
TAP CHI KHOA HQC DAI HQC VAN LANG So 09,Thang 5-2018 free radical method, Braz Dent J 23(1) 2012 DPPH assay of solutions used for bleached
teeth 27 PhytOtherRes;15.
[8] Murashige T., and F. Skoog. (1962), A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiology Plant 15.
[9] Passey A.J., Barrett K.J., and D.J. James. (2003), Adventitious shoot regeneration from seven commercialstrawbery cultivars (Fragaria x ananassa Duch.) using a range of explant types. Plant Cell Rep, 21.
[10] Skoog F., and Miller C O . (1957), Chemical regulation of growth and organ formation in plant tissue and culture in-vitro, Symp. Soc. Biol, 11.
[11] Smania A.Jr., Delle Monache F., Smania E.F.A., and Cuneo R.S. (1999), Antibacterial activity of steroidal compounds isolated from Ganoderma applanatum (Pers.) Pat, (Aphyllophoromyce-tideae) fruit body, Intemational journal of Medicinal Mushrooms, 1.
[12] Tang N.V, Michael C.B, Quan Van Vuong, Van Altena I.A., Scarlett C.J., (2015), Phytochemicals and antioxidant capacity of Xao tam phan (Paramignya trimera) root as affected by various solvents and extraction methods. Industrial Crops and Products, 65.
[13] Tang N.V., Ngoc Minh Quynh Pham, Quan V.V., Michael C.B., Van Altena I.A. &
Scarlett C.J., (2016), Phylochemical retention and antioxidant capacity of xao lam phan (Paramignya trimera) root as prepared by different drying methods. Drying Technology, 34(3).
[14] Trdn Trgng Tudn, Nguyen Thi Huydn Trang, Dd Qudc Trudng, Nguyen Thj Kim Loan, Dd Dang Giap, Nguydn Hai Son, Vuu Nggc Dung, Thai Xuan Du (2014), Ung dung ky thuat nuoi cdy lap mong te bdo de cam ung tgo mo seo ciia mdu la cdy bd benh nuoi cay in-vitro. Journal of Science and Technology 52 (4D).
[ 15] Trinh Thi Huong, Tran Trong Tuan, Do Dang Giap (2016), The influence of factors to the regeneration of red rasperry (Rubus aleeaefolius Poir.), Proceeding of the 2" national scietific conference on biological research and teaching in Vietnam, 1059-1067.
[16] Zawadzka M. and Orlikowska T., (2006), Factors modifying regeneration in vitro of adventitious shoots in five red raspberry cultivars, J. Fruit Omam. Plant Res., 14, http://duocIieunamviet.com/tin-tuc/cong-van-vien-duoc-Iieu-bo-y-te_998.html.
Ngaynhanbai: 10-3-2017. Ngay bien tap xong: 27-3-2018. Duyet dang: 19-5-2018.
