KHOA HQC CONG NGHE
IXIUOI iXIHAIV tm VITRO P H O I V O TilXIH C A Y DIIXIH LAIXIG L A IXIHO [Palyscias fruticosa (L.) H A R M S ) TREIXI H E
THOIXIG I V U d i C A Y LOIVG L A C
Trinh Vi6t Nga\ Nguyen Diic Minh Hiing^ Bui Minh Tri^
NgO Thi Anh Khoi^ Trieu Thi BichS Pham Thi Mmh TSm^ Nguyen Huu Hd^
T6MTAT
Nuoi cay mo la mgt trong nhung bi&n phap nhan giong vo tinh im viet tao cay con dong dSu va sach benh.
De nhan gidng sd luong lon thi phuong phap nuoi cay tren moi truong long co nhieu uu di^m dang ke. Mot thi nghiem hai yeu to da duoc bd tri theo kieu hoan toan ngiu nhien de xac dinh khoi lirong nuoi cay ban dau, tdc do ldc thich hgp tren he thdng nuoi cay long ldc da duoc thuc hien. Yeu td T la hai khoi luong ph6i [(0,5 g ttrong ung 0,625% •-w/v) va 1.0 g tuong iing 1,25%-w/v)]; yeu to Kla ba tdc do ldc (80 vong/ phiit;
100 vong/phiit va 120 vong/ phiit). Ket qua cho thay phoi vo tinh dmh lang tang sinh tot nhat khi nhan nuoi vol khdi luong phoi nuoi cay ban dau 1 g voi tdc do ldc 100 vong/phiit tren he thong long ldc. Khdi lugng tang sinh da dat 7,5 g, he so nhan phoi dat 8,5 Idn tai thoi diem 30 ngay sau cay. Su gia tang sinh khdi phoi dua tren su hinh thanh phoi bat dinh tir phoi so cap de tao phoi thii cap. Duong bieu diSn tang truong, he sfl' tang truong sinh khdi phdi da duoc thuc hien. Phoi vo tinh tao cay hoan chinh vdi ty le cao, cay tang tmdng in vitro binh thudng va kha ddng deu v6 kich thuoc tr6n moi trudng 1/2MS khong bo sung chat dieu hda sinh trudng.
Tii khda: Phdivo tinb, dmh lang la nho, tang tmdng smh khdi, nuoi cay long lic.
Dinh lang la cay dugc lieu cimg ho voi cay nhan sam, todn bp cay tu cu, than, la, rh d^u co th^ su dung de 1dm thudc tri benh. Cay dtnh lang co chua hai nhom hop chat chmh quan trpng Id polyacetylene va saponm. Cac hop ehdt nay co tdc dung chdng ung thu, chdng oxy hoa, khang khuan va khdng nam (Huan va cs, 1998; Mitaine-Offer vd cs, 2004). Phdt sinh phoi vo tinh Id mdt trong nhung ky thuat nuoi cay in vitro moi mang lai hieu qua vuot trdi ve hem nang nhan giong. Ky thuat ndy ben canh viec cho he so nhan giong cao thi cay con con cd bd re tuong tu nhu cay tir hat co the hinh thanh rd cu ngay hr giai doan han ddu. Tuy nhien, cac nghidn cuu ve dinh lang truoc day cung mdi chi dimg lai b budc tao dugc phdi md chua di vao thu nghidm nhan nudi tren he thdng nudi cdy long ldc, trong khi bien phdp ndy cho thay tnen vgng to lon trong viec nudi so lugng Idn smh khdi phuc vu cho vi nhan gidng thuong mat.
Mdt khi tao thdnh phdi, cde te bdo tiep tuc tang sinh vd tao thdnh cum tidn phdi. Cac cum phdi tao thdnh
' Vien Sinh hoc nhiet ddi TR HCM
- Khoa Ndng hpc, Tru'dng Dai hgc Ndng Lam TP HCM Email: trinhvietnga@pmail,com
dugc duy tri va tang smh tren mdi trudng tuong tu nhu mdi trudng dung de phat sinh nd. Ngudi ta cd the nuoi cay tren mdi trudng ban ran, tuy nhidn d^
nhan giong so luong Idn thi phuong phap nudi cay huyen phii hoac mdi trudng long co nhidu uu diem vugt trdi. Tdc dd tang sinh cao va qua trinh nudi cay trb nen ddng bd hon. Trong qua hinh nudi cay long, cde te bao don vd cac cum te bao phat trien thanh cac can tnic neng le. Do vay, cac te bdo duoc tdch rdi dd dang bang lpc hoac ly tam va sau dd cd thd cay chuyen (Duong Tdn Nhut va cs, 2008). Mot sd yeu td anh hudng den su thanh cdng trong nhan nuoi tren he thdng long lac la khdi lugng nudi cay vd tdc dd ldc. Nudi cay long lac phdi vd tinh da dugc cdng bo tren nhieu loai cay trdng tren the gidi (CervelU and Senaratna, 1995; Moorhouse va cs, 1996; Timmis, 1998; Ibaraki va Kurate, 2001; Paek va cs, 2001; Paek va Chakraharty, 2003).
6 trong nude cung da cd mdt sd nghien cuu cho thay nudi cay long ldc cho hieu qua tdt tren mdt so ddi tuong cay trdng cd gid tri kinh te cao nhu cay gd tdch, cay sa nhan, cay sam Ngpc Linh (Mai Trudng va cs, 2014). Tuy nhidn chua ghi nhan dugc cdng trinh cdng hd ve nudi nhan phdi vd tinh cay dmh lang bdng hd thdng nudi cay long lac. Do vay, muc tieu
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KHOA HOC CONG NGHE
c u a n g h i e n ciiu n a y Id x d c d i n h d u g c k h o i l u g n g phdi n u d i c a y b a n d a u , tdc d o ldc t h i c h h g p d e n h a n s m h k h d i p h d i vd t i n h b d n g h e t h d n g n u d i cay long ldc va b u d c ddu d d n h gia c h a t l u o n g c u a p h d i v a cay con tir p h d i d- didu k i e n n u d i cay in vitro.
2. VAT UEU VA PHUONG PHAP NGHIBU CUU 2 . 1 . V a t lidu, m d i t m d n g v a d i ^ u kidn n u o i cay P h d i vd tinh d m h l a n g d u o c lay sau giai doan b o a t h d a p h d i , c u m p h d i cd m a u v a n g nhat, kich t h u d c 0,6 ~ 0,8 c m (Hinh 1), c d n g u d n g d c tir nudi cay m a n h Id (Trinh Viet N g a vd c s , 2017).
H i n h 1. C u m p h d i d i n h ISng Id n h o dfing nudi l o n g ldc M d i t r u d n g nudi p h d i l o n g lac: M S cd b o s u n g B A d' n d n g d o 1,5 m g / L va NAA b n d n g dp 0,1 m g / L (BA, NAA c u a S i g m a ; d o tinh k h i e t > 98,5%), d u o n g saccharose 30 g/L. Mdi trudng tao cay, nudi cay tu phdi: MS/1/2MS (Murashige va Skoog, 1962), dudng saccharose 20 g/L. Mdi trudng dugc chinh pH 5,7 - 5,8; hap khii tning d 121''C hong 20 phtit, ap suat 1 atm.
Binh nudi cay Id loai 300 mL chua 80 niL mdi trudng- Qud trinh ldc dupe thuc hien tren may ldc trdn, tdc dd ldc Ididc nhau tiiy thi nghiem, quy dao ldc 16 mm (Model SSLl, STUAET (BIBBY-UK).
Phdng nudi cdy cd nhiet dp la 25 ± 2''C; cudng dp chidu sang ~ 3.000 lux, thdi gian chieu sdng 12 gid'/ngay.
2.2. B6 tri thi nghidm va each thiic theo ddi Thi nghiem hai yeu td dupe bd tri theo kieu hodn todn ngdu nhien, 3 Idn lap lai vdi 6 nghiem thiic.
Ydu td T Id hai khdi lugng phdi [(0,5 g tuong ung 0,625% - w/v) va 1,0 g hrong iing 1,25% - w/v)]; ydu td K Id ba tdc do ldc (80 vdng/ phiit; 100 vong/phut vd 120 vdng/phiit).
Phuong phdp thuc hien:
Can lugng phdi dinh lang theo timg nghiem thiic cay vdo binh tam gidc dung tich 300 mL cd chiia 80 mL mdi trudng va dat hen mdy ldc. Tdc dd ldc tuong ling vdi timg nghiem thtrc. Phdi dugc nudi hong 30 ngdy.
Khdi lugng tuoi cua phoi (g) duoc xac dmh haa^
each thu sinh khdi va can khdi lugng phdi thu a u o c | mdi bccjsb vao cac thdi diem 10, 20 vd 30 ngay sai cay (NSC).
He sd tang sinh phdi duoc xac dinh bang c a i diu sinh khdi va can khdi lugng phdi thu dugc amfi d CO so d 30 NSC. He sd tang sinh khdi = (Khdi liroag phdi 30 NSC/ Khdi lugng phdi truoc khi nudi cdy). \ Ghi nhan sd la, sd rd/ cay va chidu cao cay tir phdi crgiai doan 84 NSC.
Sd lieu fhi nghidm dugc thu thap tong hpp bli^
phdn mdm Microsoft Excel va xu ly thdng ke theo phuong phdp phan tich phuong sai (ANOVA), k6 qua phan hang theo Duncan bdng chuong trinh SAS 9.L
3 . KET QUA VA THAO LUAN
3.1. Su gia tang sinh kh6i vd ti'nh dim tr6n ca so phat sinh phdi bat djnh
Cac cum phdi ban dau vdi kich thuoc va kh6i lugng xdc dmh (Hinh 1) dugc cay vao binh tam gidc chiia mdi trudng long va nudi ldc vdi cde tdc do ldc khac nhau trong thdi gian 30 ngay. Ket qua cho Ihay sau thdi gian nudi, nhin chung phdi tang sinh khdi ro ret, cum phdi cd mau tir vang kem den vang hoi sam va vang sam (Hinh 2). Phdi phat sinh nhieu hinh flia khde nhau va lien tuc tang sinh khdi. Ket qua nay cung tuong tu nhu ket qua nghien ciiu cua Mai Truong vd cs (2014); Etienne va cs (1999);
Firoozabady vd Moy (2004). Trong dieu kien khdng cd anh sdng, phdi cd hien tugng chuyen sang mail vang sam va ra rd nhidu. 6 tdc do ldc 100 vdng/phiit vd 120 vdng/phiit do dugc cung cap khdng khi/Oj hda tan va dinh duong thuan Igi da giiip phdi phat trien tdt. Tuy nhien o tdc do ldc 120 vdng/phiit phoi cd hien tugng nhanh hda nau va dich nuoi cay hoa due. O tdc do ldc 80 vong/phut phdi cd mdu vang sam va hda gia nhanh chdng so vdi hai tdc do tren.
Vdi khdi lugng phdi nudi cay ban ddu 1 g (1,2596) gilip phdi nhanh chdng dat didu kien mat dp tdi hao va bdt ddu chu ky sinh trudng sdm hon. Phdi vd tinh tang sinh cham d giai doan 0 ddn 10 NSC vi mat do cdn thdp va ddn tang sinh de dat didu kien ir • do tdi ban bdt ddu chu ky smh hudng. Giai doan tu lo den 20 NSC, mat dd cd su t5ng nhanh chdng va dat dea mat dp tdi han tao didu kien tdt cho viec tang sinh phdi. Giai doan 20 NSC den 30 NSC, pho: vdn tang smh khdi tuy nhien phdi dan chuyen sang mau v n p 26
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 3 20^9
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sam vd hda gia.Hinh 2. Phoi vd tinh cay dinh lang la nho nudi nhan h^n hd thdng long ldc d thdi di^m 30 NSC a. Cic binh phoi vdi khdi luong phdi cay ban diu 0,5g, nudi a cic tdc do Uc khic nhau 80, 100, 120 vdng/phut, theo thd tu tu trii sang phii; b. Cic binh phdi vdi khdi luong phdi eay ban dau 1 g, nuoi d cic tdc do lie nhu trdn, theo thd tir td trai sang phai
Cau tnic da phdi (poly-embryos) cua cum de dang dupe ghi nhan sau khi lay mdu nudi ra khdi binh tam giac (Hinh 3a, b). Do tdc ddng ca hpc cua qud trinh ldc, cd the quan sdt dugc mdt sd it phdi don hien dien d day binh hoac chu dgng tdch thu sd lugng Idn cac phdi tdch rdi/don (isolated) bdng cdch dung que cdy ep nhe vao cum phdi (Hinh 3c, d).
Nhan thay, phdi thu dugc d cde giai doan khac nhau, tuong tu nhu d chu trinh phdt tridn phdi huu tinh vdn cd cua cay hai la mdm, nhu dang cdu, dang tim (Hinh 3c), chu yeu dang thtiy Idi va dang cd la mam, rd mam (Hinh 3d, e). Vdi vide dp dung ky thuat ndy vao diing thdi diem, cd the thu duoc qudn the phdi vd tinh don khd dong ddu vd mat kich thudc vd giai doan phdt trien.
Su gia tang sinh khdi phdi theo thdi gian nudi, trong mdi trudng cd chat didu hda sinh trudng thich hgp, theo chting tdi, Id do cd su phdt sinh phdt soma bat dinh (adventitious embryogenesis) lan 2, lan 3 (secondary/tertiary embryo) ttf phdi so cdp (primary embryo) (sau day goi chung la su phdt sinh phdi thii cap). Phdi thti cap cd the hinh thdnh trdn khap co the ctia phdi so cap, chii yeu la d phdn ngpn va than cua phdi, it gap d phdn rd phdi (Hinh 3f, g, h, i, j). Co chd hinh tiianh phdi vd tinh thii cap da duoc ghi nhan d lodi cay cimg chi - dinh lang la to Polyscias BUcifoUa (Sliwmska vd cs, 2008), d k h a nhieu tru'dng hgp khde nhu Panax ginseng (Arya vd cs, 1993; Kim vd cs.
2012), Panax quinqueiolius (Zhou S. va Brown, 2006), Panax japonicus (You va cs, 2007), Panax vietnamensis (Mai Trudng va cs, 2014).
Ndi chung, tao phdi va tao phdi vd tinh thir cap - hien tugng dugc ghi nhan d nudi cdy md in vitro mpt sd gidng cay trong, mang tinh dae tnmg cua nhieu lodi thuc vat thudc chi Panax (ho Sam - Araliaeeae) tao thuan Igi cho nghien ciru vi nhan gidng/nhan gidng bdng phuong phap tao vd nhan phdi vd tinh (Kishua va cs, 1992; Hussein va cs, 2006; Zhou vd Brown, 2006; You va cs, 2007, 2012; Mai Trudng va cs, 2014). Ngoat ra, he thdng nudi cay nay cdn cd tiem nang lon trong san xuat sinh khdi phdi thu hgp chat thti' cap.
Hinh 3. Cdu tnic da phdi vd cde dang phat trien khde nhau ciia phdi vd tinh dinh lang Id nhd, 30 NSC
a. Cic cum da phdi; b. Can cinh cac cum da phdi tidu bieu; c. d, e. Can eanh phdi d eac giai doan phit trien khac nhau (Idnh ehup dudi kinh hien vi soi ndi, dd phdng dai lOX); i, g, h,ij. Su hinh thanh phdi thu cap tuphdi sa cap (mui ten chi vi tii phdi tiiu cap) vdi phdi hinh thanh ti-en khip ca the phdi ($, ti'dn than phdi (g), ddn phan ngon vi than phdi (h), trdn phan than va rSpbdi (i) vi chi trdn phin ngon cua phdi (j) (hinh ehup dudi kinh hien vi soi ndi. do phong dai 20X)
Da cd nghien ctiu nhan phdi vd tinh thanh cdng trdn mdi trudng thach ddi vdi dinh lang Id to Polyscias dhciioha (Sliwmska va cs, 2008) nhtmg den nay chua ghi nhan duoc cdng trinh cdng bd vd nudi nhan Idng ldc phdi dinh lang Id nhd.
3.2. Anh hudng ciia khdi lugng phdi nudi cay han ddu vd tdc do ldc ddn kha nang tSng sinh khdi phdi trdn hd thdng nudi cay long ldc
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 3/2019
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Thuc td nghien ciiu cho thay khi nhan nudi phdivd tinh cay dmh lang la nhd tren he thdng nudi cay long ldc, dnh hudng ciia khdi lugng phdi nudi cay ban ddu va tdc do ldc den kha nang tang sinh phdi rat Idn, the hien d ket qua khdi lugng phdi thu hoach d cudi giai doan nudi cay. Nudi cay phdi vdi khdi lugng phdi nudi cay ban dau 1 g (1,25%) da gia tang khdi lugng phdi dang ke d 20 NSC (3,8 g) vd 30 NSC (6,3 g) va khac biet rat cd y nghia thdng ke so vdi khdi luong phdi nudi cay ban ddu 0,5 g (0,625%) d cimg thdi diem theo doi (Bang 1). Kdt qua nay phu hgp vdi kdt qua cua Ducos va cs (2007) khi cho rdng nudi cay phdi cd phe vdi khdi lugng nudi cay ban ddu tir 0,5 - 1 g/L la thich hgp nhat.
Tdc do lde cflng tdc ddng rat Idn den den khd nang tang smh phdi. Thdi gian ddu nudi cdy, toe dp lac Idn 100 -120 vdng/phut giiip phdi tang sinh manh Bang 1. Anh hudng cfia khdi lugng phdi nudi cay ban
tinh cdy dinh lang Id nhd trdn
lan lugt la 0,9 g d 10 NSC va 2,9 - 3.2 g d 2 ' NSC va khde biet rat cd y nghla thdng ke so vdi khdi lugng phdi d tdc dp ldc thap (80 vdng/phtit). Tuy nhien 6 30 NSC, tdc dp ldc 100 vdng/phiit giup khd nang tang sinh phdi tdt nhat, khdi lugng phdi thu dugc dat cao nhat Id 5,8 g, khac biet rat cd y nghla thdng kd so vdi khdi luong phdi d tdc dd ldc thdp 80 vdng/phiit hay tdc do ldc cao nhdt 120 vdng/phiit (Bang 1). Ket qua nay cd thd ly giai la luong phdi thu nhan giam 6 tde do lde cao (120 vdng/phiit) d 30 ngdy do sd lupng phdi lon cong vdi tdc dp ldc cao xdy ra nhieu va cham giua cde phdi lam phdi bi tdn thugng, han che tang trudng.
Su tuong tac giiia khdi lugng phdi nudi cay ban ddu va tdc dp ldc chua thdy tao ra su khac biet cd y nghia thdng kd den kha nang tang sinh khdi phdi otA ca cde thdi diem theo doi trong thi nghiem (Bang 1).
dau va tdc dp lac ddn kha n ^ g tang sinh khdi phdi vo hd thdng nuoi c ^ long ldc fe)
Thai gian theo doi (NSC)
10
20
30
Khoi luong phoi nuoi cay ban
ddu (g) (A) 0,5 (0,625%)
1,0 (1,25%) TB(B)
Tdc do lac (vong/ phut) (B) 80
0,5 0,8 0,7»
100 0,6 1,1 0,9*
120 0,7 1,0 0,9*
TB(A) 0,6"
1,0*
CV - 12,5%; F(A) - 64,8"; F(B) - 8,5"; F(A*B) -1.5°' 0,5 (0,625%)
1,0 (1,25%) TB(B)
1,0 2,9 2,0»
1,6 4,2
2,9«
2,1 4,2
3,2*
1,6=
3,8*
CV - 9,3%; F(A) - 344,7"; F(B) - 38,9"; F(A*B) = 3,2°' 0,5 (0,625%)
1,0 (1,25%) TB(B)
2,5 5,2 3,8^
4,1 7,5 5,8*
3,5 6,2 4,9=
3,3=
6,3*
CV - 8,7%; F(A) - 258,7"; F(B) = 37,2"; F(A*B) = 1 7">
Trong cimg mdt nhom gii tii tivng binh, cac bi sd ed cung ky tii di kem khic biet khdng eo y ngbm thdng ke.ns: khac biet khdng cdy nghia tiidng ke;**: khic bidt rat edy nghia tiidng ke(a<0,01).
Ngodi ghi nhan gia tii khdi lugng tiroi tai cac thdi diem nudi, viec xay dung dudng cong tang trudng sinh khdi phdi qua qud trinh nudi long ldc dudi anh hudng eua hai thdng sd nudi cay phd bien la khdi lugng phdi nudi cay ban ddu va tdc dp nhu da neu cung da dugc thuc hien. Khdi lugng phdi duoc ghi nhan d cac thdi diem 10, 20 va 30 ngay sau eay.
Thuc hien thu sd hdu d thdi diem ddu - la 10 NSC va cudi - la 30 NSC vi thuc te cho thay tiirdc thdi diem
NSC vi sau thdi diem nay str tang sinh khdi phoi chung lai do dang d pha cudi (stationary phase). Kdt qua duoc trinh bay d hinh 4 va hinh 5.
Dtmg dudng cong tang tiudng sinh khdi md t^
bao theo thdi gian la thi nghiem ludn dugc cac nha nghien ciiu quan tam thuc hien vi nd thd hien siic va khuynh hudng tang trudng h-ong mdt quang thdi gian nudi cay nhat dinh (chu ky nudi) - vira cd y nghia ly luan va thuc tidn. Da ghi nhan du(x mdt sd 10 ngdy su tang sinh phdi chua rd, do phdi dang d cdng trinh cdng bd ve dudng cong tang trudng te pha khdi diem (\ag phase) vd thdi diem cudi la 30 bao, phdt sinh phdi vd tinh theo thdi gian tren ddi 28
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tuong Chd la (date palm) Phoenbc dactyhfera (Al-Khayri, 2012), dudng cong tang trubng sinh khdi phdi vd tinh Corydahs ambigua, phdi vd tinh sam Siberia (Siberian ginseng) Bleutiierococcus senticosus phuc vu cho nhidu muc dich khde nhau trong do cd muc dich nhan gidng (Hiraoka va es, 100 v/r 120 v/r
NSC
2004; Shohael vd es, 2014). Nhin chung, ket qua dung dudng cong tang trudng d dinh lang Id nhd cho thay cd su tuong hop vdi edc ket qua nghidn ctru neu trdn d xu hudng phdt ti-ien sinh khdi the hien d cac pha tang trudng dae trung.
Ig]
Hinh 4. Dudng bi^u didn kdt qua tang sinh khdi (g) trudng hgp khdi lugng phdi nudi cdy ban ddu 0,5 g (0,625%) d edc tdc dO lac vd thdi didm
ghi nhan khde nhau
3.3. Anh hirdng cua khdi lugng phdi nudi cdy ban dau va tdc dp lac ddn hd sd tang sinh khdi phdi trdn hd thdng nudi cdy long lac
He sd tang sinh khdi phdi Qdn) cung da dugc ghi nhan d thi nghidm anh hudng cua khdi luong phdi nudi cay ban ddu va tdc do ldc den su gia tang sinh khdi, ket qua dugc trinh bdy d bang 2. Qua nghien Clin, nhan fhay he sd tang sinh khdi phdi dat cao nhat khi nhan nudi phdi vdi khdi lugng phdi nudi edy ban ddu 0,5 g (0,625%) d 10 NSC la 2,2 lan va 4,8 lan (d 20 NSC), khde bidt rat cd y nghia thdng ke so vdi he sd tang sinh phdi khi nhan nudi phdi vdi khdi luong phdi nudi cay ban ddu 1 g (1,25%). Tuy nhidn, den 30 NSC, khdi luong phdi nudi eay ban ddu da khdng tac ddng nhieu vd khac bidt khdng ed y nghla thdng ke den he sd tang sinh khdi phdi.
Tdc do lac cung tdc dpng rdt Idn den he sd tang sinh khdi phdi. Thdi gian ddu nudi cay, tdc dp ldc Idn 100 - 120 vdng/phut giiip phdi tang sinh manh ddn den he sd tang sinh phdi nhanh lan luot la 2,2 lan d 10 NSC vd 4,7 - 5,2 lan d 20 NSC vd khac biet rat cd y nghia thdng ke so vdi he sd tang sinh phdi d tdc do lac thdp (80 vdng/phut). Tuy nhien d 30 NSC, tdc dp ldc 100 vdng/phiit giiip he sd tang sinh phdi nhanh nhat (8,8 lan) do kha nang tang sinh phdi tdt nhat, khac bidt rat cd y nghla thdng ke so vdi he sd tang smh phdi d tdc do ldc thap 80 vdng/phut hay tdc do ldc cao nhat 120 vdng/phtit (Bang 2).
Hinh 5. Dudng bidu didn kdt qua tSng sinh khdi (g) hudng hgp khdi lupng phdi nudi cdy ban dau 1 g (1,25%) d cde tdc dd lac va thdi didm ghi
nhan khde nhau
Su ttrong tac giua khdi Itrgng phdi nudi cay ban dau vd tdc do ldc chi anh hudng khae biet rat cd y nghia den he sd tang smh phdi d 20 NSC. Khdi lugng phdi nudi cay ban ddu tii 0,5 g (0,625%) den 1 g (1,25%) ket hgp vdi tdc dd lae cao 120 vdng/phut va khdi Itrgng phdi nudi cay ban dau 1 g (1,25%) ket hpp vdi tde dd ldc 100 vdng/phiit cho he sd tang sinh khdi phdi nhanh nhat dao ddng 5,2 Idn - 5,3 lan (Bang 2).
Trong linh vuc nudi cdy md te bdo, ngodi xay dtmg dudng cong tang trudng, he sd tang trudng sinh khdi md te bao (%, lan) ciing dugc de cap vi nd phan anh nhanh mirc do tang trudng d mdt thdi diem nhdt dinh dae bidt d pha cd sue tang trudng manh nhdt. He sd tang sinh khdi te bao chudi Musa acuminata sau thdi diem nudi cay nhdt dinh (14 ngay) tang 165-210% dudi anh hudng ciia pH mdi trudng khde nhau trong hioreactor; nang suat sinh khdi tang tir 2,2 den 3 Idn so vdi khdi luong mau cay ban ddu klii duge nudi lan luot trong binh tam giac va bioreactor sue khi (Chin va cs, 2014). He sd tang sinh khdi phdi vd tinh Eleutherococcus senticosus theo thdi gian nudi cay (den 63 ngay) cung nhu he sd tang sinh dudi anh hudng cua loai bioreactor str dung, lugng khi cung cap, khdi lugng phdi nudi cay ban dau; d cac nhidt dp, ndng do acid gibberelUc, lugng O2, CO2, C2H,|, ndng dd tdc nhan kich hoat hgp chat thii cap methyl jasmonate khac nhau qua nudi cay bdng he thdng bioreactor sue khi V 3 Kt cung da duoc edng bd (Shohael va cs, 2014).
NONG NGHIEP VA PHATTRIEN NONG THON - KY 1 - THANG 3/2019 29
KHOA HOC CdNG NGHfi
Nhin chung, he sd tang sinh khdi md te bdo quanudi eay phu thudc vao nhidu ydu td khac nhau nhu loai vat Ueu dimg nudi cdy, mdi trudng nudi, dieu kien nudi, ddi tugng thuc vat nghien ciiu vd tat nhien cung phu thupe vao cac nghiem thirc khde nhau bd
tri b mdi yeu td anh hudng. O nghien ciiu trdn dinl lang la nhd, vdi khdi lugng phdi nudi ciy ^^ ^^^ ^^' g (1,25%) va nudi d tdc dp ldc 100 vdng/phut, hd so tang smh khdi phdi dat 8,5 lan tai thdi diem 30 n g | sau cay.
Bang 2. Anh hudng cua khdi lugng phdi nudi cdy ban ddu va tdc do lac ddn h§ sd tang sinh khdi phdi vo tinh Thdi gian
theo ddi (NSC)
10
20
30
cay dinh lane Id nhd hdn hi Khdi luong phdi
nudi cay ban ddu (g) (A) 0,5 (0,625%) 1,0 (1,25%) TB(B)
thdnE nu6i cay ong lac Oan) Toe do Mc (vong/ phiit) (B) 80
2,0 1,8 1,9=
100 2,2 2,1 2,2*
120 2,4 2,0 2,2*
TB(A) 2,2*
2,0=
CV = 7.62%: F(A) - 9.06'; F(B) = 6,42'; F(AB) - 1,02"
0,5 (0,625%) 1,0 (1,25%) TB(B)
3,1' 3,9' 3,5=
4,3' 5,2' 4,7*
5,3"
5,2"
5,2*
4,8*
4,2=
CV . 6,7%: F(A) . 17,2"; F(B) - 54,2"; F(AB) - 4,7"
0,5 (0,625%) 1,0 (1,25%) TB(B)
5,9 6,2 6,1'
9,1 8,5 8,8*
8,0 7,2 7,6=
7,7 7,3 CV - 7,7%; F(A) - 1,6"; F(B) - 34,6"; F(AB) - 1,3"
Trong cimg mdt cot, nhtrng 0a tn theo sau cd cung ky tir su khac biet cd y nghia trong thdngke **: coy nghia dmdc P<0,01.
3.4. T?io cay tir phdi
Hinh 6. Tao cay tDt phdi vd tinh dinh lang la nhd sau giai d o ^ nudi long lac
a. Cic cum phdi phit trien tao cum cay con trdn mdi trudng thach, 30 NSC; b. Can cinh cum cay con tuphdU c, d. Cay con, cay to phit trien tuphdi (mui tdn chi vi tri hai li mam va rd coe dien hinh cua cau true phdi); e. Cay to tii phdi ehuan bi dua ra trong d vuon uom (mdi ten chi vi trird coc); f. Hinh thii eay td nudi cay choi ngon khic bidt so vdi cay tir phdi khdng cd la mam, chi cd rd bat dinh
Sau qua trinh nudi long ldc, cde cum phdi duoc
cay nudi hdn mdi trudng thach MS hoac 1/2MS d^
tao cay. Sau khoang 1 thdng, edc cum phdi phat tiih thdnh cum cay con vdi rat nhidu Id mdm, la that va rl mam (Hinh 6a, b). Tiep theo, cae cay con dugc tdch nudi ridng cflng vdo mdi trudng nhu tren eho ddn khi dat tieu chuan dua ra bdu dat (Hinh 6c, d, e). Cd M nhan thay cde can tnic dae tnmg cua phdi cdn tdn tai d cay con nhu Id mdm (cotyledon), rd mdm/r§
cpc/rd hu (tap root) - tuong hr nhu cay tii phdi him tinh/phdi hat. Khde vdi cay tu' phdi, cay tir nudi cay chdi ngpn, chdi ngii doan than khdng cd cde can tnic dien hinh nhu da neu (Hinh 6f).
(y mdi trudng neu tren, budc ddu ghi nhdn ty I^
phdi phdt tiien thdnh cay cd kidu hinh binh thtrdng vdi day du rd, than, Id - khoang 95% (quan sat, ghi nhan sd Udu tren 300 phdi) do d ddi tuong cay trdng nay tdc ddng tao su trudng thdnh (maturation) cho phdi va sau do phdi tao chdi (germination) • nanh cay hodn chinh (plant conversion) la kha thuan lpi; cung nhan thay edc cay kha ddng ddu ve kich ihudc (Hmh 7a, h). 6 nghien ciiu ndy, cd mdt sd it truong hop (- 5%) phdi di dang/bat thudng (abnormal) nhu ph6i cc
30 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 3/2019
KHOA HQC CdNG N G H |
lid mam dinh rihau (fused) - khd tao chdi nhung rdmdm phdt tridn (Hinh 7c); phdi dinh ngpn, cd than imdm va rd mam kem phdt trien (Hinh 7d) da duoc ghi nhdn. Phdi di dang la phdi thudng gap trong qua trinh nudi cay nhu ddi vdi trudng hop phdi Panax quinquefotius (Zhou vd Brown, 2006).
Hinh 7. Su phdt tnin binh tiiudng, ddng ddu cua cay dinh lang la nhd tir phdi vd tinh vd mgt vdi dang bdt
thudng cfia phdi
a, b. Cay eon vdi kidu hinh binh thudng sau 30, 60 ngay nudi cay, theo thd tu;c. Phdi dinh li mim, rd mam phat trien; d. Phoi dinh ngon, tiian mam vi ri mam kem phit tridn
Theo bang 1, ttr khdi luong phdi nudi eay ban dau 1 g (1,25%), sau 30 ngay nudi long lde (100 vdng/phiit), khdi lugng phdi dat 7,5 g. Budc ddu nghidn cuu cho thay cd thd tao dugc -- 30 cay con tir 1 g phdi, nhu vay thu duge khoang 200 cay hoan chinh til 7,5 g phdi (da tiTJ hao hut do phdi bat thudng va do thao tac ky thudt tach cay va nudi cay).
He sd nhan cay ~ 7 lan. Tren thd gidi, ddi vdi sam Panax ginseng, ty le tao cay dat khoang 80% (Arya va cs, 1993), la 85% ddi vdi sam Bdc My {Panax
'' (So la/cay)
quinqueiolius) (Zhou va Brown, 2006); la 15,3% ddi vdi Panax Japonicus (You va cs, 2007).
Nhu da trinh bay, do anh hudng cua cac chat dieu hda sinh trudng ndn md phdi Uen tuc tang sinh khdi khi nudi ldc trong mdi trudng long, didu nay cd lpi khi nudi cdy phdi dd tdeh chiet hop chat thii cap, nhung khdng cd Igi cho viee tao cay (Merkel vd cs, 1995; Paek va es, 2005; Duong Tdn Nhut va cs, 2008;
Paul va cs, 1994). Do vay, thudng khdng sir dung mdi trudng ed chat didu hda smh tnrdng d giai doan nudi tao cay hoac ed bo sung nhung vdi ndng do rdt thap hoac bd sung than boat tinh va nudi trong vdi chu ky de than hap phu chat dieu hda sinh trudng tdn du giup lam giam su hinh thdnh phdi thir cap, tang khd nang dap ling tao cay hodn chinh.
Mdi tiudng MS, 1/2MS khdng bd sung chat dieu hda sinh trudng da dugc sir dung hong tao cay d trudng hgp dinh lang Id to (Sliwinska va cs, 2008) va mdt sd loai thuge ho Sam {Panax ginseng, Panax notoginsen^ (Kishira vd cs, 1992; You vd cs, 2012) hoac mdi trudng cd bd stmg than boat tinh ddi vdi Panax quinqueioh'us (Zhou va Brown, 2006).
Kdt qua nghien eiiu tao cay eua chiing tdi trdn ddi tijgng dinh lang la nhd dimg mdi trudng MS, 1/2MS khdng chat didu hda sinh trudng phit hgp vdi kdt qua cua cac tac gid ndu tren xet d khia canh tao eay va su tang trudng tidp theo eua cay.
Khao sat didn bien tang trudng cay theo thdi gian vd do dem sd Id/cay, sd rd/cay va chieu eao cay eon cd ngudn gdc phdi vd tinh da dugc thtrc hien. Kdt qua cho thay, d thdi didm 84 ngdy sau cay, trung binh sd Id/cay, trung bmh sd rd/cay, tinmg bmh chidu eao cay hen mdi trudng (MS, 1/2MS) la (7; 7,8 la), (5,3; 6,8 rd), (2,2; 2,6 cm), tiieo thu' hi (Hinh 8, Hmh 9, Hinh 10).
' (Sd re/cay)
Hinh 8. Kdt qua anh hudng ciia mdi hirdng ddn Hinh 9. Kdt qua anh hudng cua mdi hudng ddn sd sd Id/cay dinh lang tir phdi, 84 NSC rd/cay dinh lang hi phdi, 84 NSC
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 3/2019 31
KHOA HOC CONG NGHE
(Chidu cao cay-cm)
Hinh 10. Kdt qua anh hudng ctia mdi trudng den chidu cao (cm) cay dinh lang tir phdi, 84 NSC Cac cay (Hinh 7b) dugc cdy chuyen sang binh
nudi Magenta cung chira mdi trudng nhu ti-en ddn khi dat tieu chudn (cao khodng 5 cm) de dua ra ti-dng vudm uom. Viec tinh toan hieu qua kinh te eua phuong phdp nhan cay bdng phdi vd tinh so vdi phuong phdp nhan cay bdng phuong phap cdt nudi doan than mang chdi se duoc trinh bay d bai bao khac.
4 . KET LUAN
Phdi vd tinh nudi long lde in vido tang sinh khdi do su hinh thdnh phdi bat djnh thii cap. Phdi vd tinh dinh lang la nhd tang trudng sinh khdi tdt khi duoc nhan ui viti-o bdng hd thdng nudi long ldc vdi khdi lugng phdi nudi eay ban ddu la 1 g (1,25% - w/v), tdc dp ldc 100 vdng/phut; khdi lupng tang sinh dat 7,5 g, hd sd tang sinh khdi phdi dat 8,5 Idn tai tiidi diem 30 ngdy sau cay. Da x^y dtmg dugc dudng bieu didn tang tiirdng khdi lugng, he sd tang tiordng sinh khdi phdi dudi dnh hudng cua khdi lugng phdi nudi cay ban dau vd tdc dp ldc.
Phdi vd tinh dinh lang la nhd cd cau tnic dien hinh, trudng didnh vd tao cay hoan chinh vdi ty le cao (95%) khi dugc nudi cay tren mdi ti-udng 1/2MS khdng bd sung chat didu hda sinh trudng. Cay ed kieu hinh va sinh trudng binh thudng tren mdi trudng nudi cay.
TAI UEU 1HAM KHAO
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NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 3/2019 33
KHOA HOC CONG NGHE
IN W7S(9 BIOMASS MULTIPUCATION OF SOMATIC EMBRYOS OF Pofysdas 6vticosa (L.) HARMS BY SHAKING CULTURE
Trinh Viet Nga, Nguyen Due Minh Hung, Bui Minh Tri, Ngo Tlii Anh Khoi, Tn. - Th: Bich, Pham Thi Minh Tam, Nguyen Huu Ho Summary
In vitro mulUplicaUon of somatic embryos biomass is one of the most effective methods for plant vegetative propagation, especially by shaking culture method. In this study, material for cultunng was somatic embryos of Polyscias d-uticosa (L.) Harms onginated from leaf explants culhired in viUo. Two-fector expenments were laid out m randomized complete block design. T factor - inoculum size of somatic embryos (0.5 and 1.0 g - 0.625 and 1.25% w/v, respectively) and K factor - shakmg speed (80,100,120 rpm) were exammed. The results showed that biomass multiplication was best in the case of inoculum size of 1 g, shaking speed of 100 rpm; fresh weight was 7.5 g, embryos multiplication ratio was 8.5-fold after 30 - day of culture. The increase of somatic embryo biomass based on the formation of advenUUous somaUc embryos for producing secondary embryos. The curve of growth and growth rate of somatic embryo suspension cultiues were carried out. Plant conversion from germinated embryos led to weU-devetoped plants with high raUo, plants grew m viPo with normal phenotype and relatively equal size on the 1/2MS medium without plant growth regulators. This is the first report on in vitro biomass multiplication of somatic embryos of Polyscias duUcosa (L.) Harms by shakmg culture.
Keywords: Biomass growth, Polyscias duticosa, shaking culture, somatic embryo.
Ngudi phan bi$n: PGS.TS. Nguydn Van Ddng Ngdy nhan bdi: 13/11/2018
Ngdy thdng qua phdn bidn: 13/12/2018 Ngay duydt dang: 20/12/2018
