Header Page of 149 B GIO DC V O TO TRNG I HC S PHM H NI TRNG TRNG KIấN NGHIấN CU NH HNG CA IU KIN ex vitro N S BIN I HèNH THI V SINH Lí, HểA SINH CA PHONG LAN PHI IP TM (Dendrobium anosmum) NUễI CY Mễ GIAI ON RA NGễI LUN VN THC S KHOA HC SINH HC H NI, 2016 Footer Page of 149 Header Page of 149 B GIO DC V O TO TRNG I HC S PHM H NI TRNG TRNG KIấN NGHIấN CU NH HNG CA IU KIN ex vitro N S BIN I HèNH THI V SINH Lí, HểA SINH CA PHONG LAN PHI IP TM (Dendrobium anosmum) NUễI CY Mễ GIAI ON RA NGễI Chuyờn ngnh: Sinh hc thc nghim Mó s: 60.42.01.14 LUN VN THC S KHOA HC SINH HC Ngi hng dn khoa hc: TS.Cao Phi Bng H NI, 2016 Footer Page of 149 Header Page of 149 LI CM N hon thnh bn lun ny, tụi ó nhn c rt nhiu s giỳp ca cỏc quớ thy cụ, bn bố, ng nghip v gia ỡnh Trc ht, tụi xin by t lũng bit n sõu sc v kớnh trng ti thy giỏo hng dn l TS.Cao Phi Bng ó tn tỡnh ch bo, giỳp tụi sut quỏ trỡnh thc hin lun Tụi xin trõn trng cm n cỏc thy, cụ giỏo b mụn Sinh lớ thc vt, Khoa Sinh hc v Phũng Sau i hc - Trng i hc S phm H Ni ó to iu kin cho tụi c hc tp, nghiờn cu v hon thnh lun Tụi xin chõn thnh cm n th cỏc cỏn b, cụng nhõn viờn, cỏc thy cụ giỏo v cỏc em sinh viờn khoa Khoa hc T nhiờn - Trng i hc Hựng Vng, tnh Phỳ Th ó to mi iu kin giỳp tụi sut quỏ trỡnh thc hin lun Cui cựng, tụi xin gi li cm n chõn thnh ti gia ỡnh, ngi thõn v ton th bn bố, ng nghip ó c v, ng viờn, giỳp tụi sut quỏ trỡnh hc v nghiờn cu H Ni, ngy 15 thỏng 10 nm 2016 Tỏc gi Trng Trng Kiờn Footer Page of 149 Header Page of 149 LI CAM OAN Tụi xin cam oan rng s liu v kt qu nghiờn cu lun ny l trung thc v khụng trựng lp vi cỏc ti khỏc Tụi cng xin cam oan rng mi s giỳp cho vic thc hin lun ny ó c cm n v cỏc thụng tin trớch dn lun ny ó c ghi rừ ngun gc Tỏc gi Trng Trng Kiờn Footer Page of 149 Header Page of 149 MC LC LI CM N LI CAM OAN MC LC DANH MC BNG DANH MC HèNH DANH MC CC THUT NG VIT TT PHN M U 1 Lý chn ti Mc ớch nghiờn cu Nhim v nghiờn cu i tng v phm vi nghiờn cu Gi thuyt khoa hc PHN NI DUNG CHNG TNG QUAN TI LIU NGHIấN CU 1.1 Gii thiu chung v phong lan Phi ip tớm 1.1.1 V trớ phõn loi, phõn b 1.1.2 c im hỡnh thỏi 1.1.3 í ngha, tm quan trng ca phong lan Phi ip tớm4 1.2 Khỏi quỏt v nuụi cy mụ t bo 1.2.1 C s khoa hc ca nuụi cy mụ t bo 1.2.2 Cỏc giai on nuụi cy 1.3 Cỏc nghiờn cu nhõn ging phong lan v giai on ngụi 11 1.3.1 Cỏc nghiờn cu nhõn ging phong lan 11 1.3.2 Cỏc nghiờn cu v giai on ngụi 13 CHNG I TNG V PHNG PHP NGHIấN CU 16 2.1 i tng, phm vi v thi gian nghiờn cu 16 2.2 Phng phỏp nghiờn cu 16 Footer Page of 149 Header Page of 149 2.2.1 Phng phỏp nghiờn cu hỡnh thỏi .17 2.2.2 Phng phỏp xỏc nh cỏc ch tiờu sinh lớ, húa sinh 18 2.2.3 Phng phỏp nghiờn cu gii phu 24 2.3 Phng phỏp x lý s liu (4) 24 CHNG KT QU NGHIấN CU V THO LUN 25 3.1 c im hỡnh thỏi ca phong lan Phi ip tớm giai on ngụi 25 3.1.1 c im ca lỏ 25 3.1.2 c im ca thõn .26 3.1.3 c im ca r 27 3.2 Hm lng nc, hm lng cht khụ mụ lỏ cõy Phi ip tớm 28 3.3 Kh nng gi nc ca mụ lỏ 31 3.4 Cng thoỏt hi nc ca mụ lỏ 33 3.5 Hm lng sc t quang hp 35 3.6 Hunh quang dip lc 37 3.7 Hot catalase 39 3.8 Hot peroxidase 41 3.9 Hm lng prolin mụ lỏ 42 3.10 c im gii phu ca lan Phi ip tớm giai on ngụi 45 PHN KT LUN V KIN NGH 47 Kt lun 47 Kin ngh 48 TI LIU THAM KHO 498 Ti liu ting vit 499 Ti liu ting nc ngoi 49 PH LC NH Footer Page of 149 Header Page of 149 DANH MC BNG Bng 3.1 Hm lng nc, hm lng cht khụ mụ lỏ phong lan Phi ip tớm thi kỡ ngụi ex vitro .29 Bng 3.2 ng thỏi s mt nc ca mụ lỏ phong lan Phi ip tớm thi kỡ ngụi ex vitro 32 Bng 3.3 Cng thoỏt hi nc ca mụ lỏ phong lan Phi ip tớm thi kỡ ngụi ex vitro 34 Bng 3.4 Hm lng sc t quang hp mụ lỏ phong lan Phi ip tớm thi kỡ ngụi ex vitro .35 Bng 3.5 Hunh quang dip lc lỏ phong lan Phi ip tớm thi kỡ ngụi ex vitro 38 Bng 3.6 Hot catalase phong lan Phi ip tớm thi kỡ ngụi ex vitro 39 Bng 3.7 Hot peroxidase phong lan Phi ip tớm thi kỡ ngụi ex vitro 40 Bng 3.8 Hm lng prolin ca cõy phong lan Phi ip tớm thi kỡ ngụi ex vitro ..43 Footer Page of 149 Header Page of 149 DANH MC HèNH Hỡnh 2.1 Phong lan Phi ip tớm Hỡnh 3.1 Sinh trng tng i ca lỏ phong lan Phi ip tớm thi kỡ luyn ex vitro 25 Hỡnh 3.2 Sinh trng tng i ca thõn cõy phong lan Phi ip tớm thi kỡ luyn ex vitro .27 Hỡnh 3.3 Sinh trng tng i ca r cõy phong lan Phi ip tớm thi kỡ luyn ex vitro 28 Hỡnh 3.4 Hm lng nc v hm lng cht khụ mụ lỏ phong lan Phi ip tớm thi kỡ ngụi ex vitro 29 Hỡnh 3.5 Kh nng gi nc c lỏ cõy phong lan Phi ip tớm thi kỡ luyn ex vitro..33 Hỡnh 3.6 Cng thoỏt hi nc c lỏ cõy phong lan Phi ip tớm thi kỡ luyn ex vitro 34 Hỡnh 3.7 Hunh quang dip lc lỏ phong lan phi ip tớm thi kỡ ngụi ex vitro38 Hỡnh 3.8 Hot catalase phong lan Phi ip tớm thi kỡ ngụi ex vitro. .40 Hỡnh 3.9 Hot peroxidase phong lan Phi ip tớm thi kỡ ngụi ex vitro .41 Hỡnh 3.10 Hm lng prolin ca cõy phong lan Phi ip tớm thi kỡ ngụi ex vitro .43 Hỡnh 3.11 Lỏt ct ngang ca r cõy phong lan Phi ip tớm 45 Footer Page of 149 Header Page of 149 DANH MC CC THUT NG VIT TT STT Ch vit tt Tờn y /gii ngha AC Than hot tớnh APX Ascorbate peroxidase Car Carotenoid Chl Chlorophyll CNSH Cụng ngh sinh hc D Day/ Ngy C i chng PLB Protocorm like body SD lch chun 10 Trung bỡnh 11 TN Thớ nghim Footer Page of 149 Header Page 10 of 149 PHN M U Lý chn ti Phong lan Phi ip tớm (Dendrobium anosmum) thuc ging Hong tho (Dendrobium), h phong lan (Orchidaceae) õy l loi lan cú hoa p, phự hp vi mc tiờu trang trớ, lm cnh, nờn c nuụi trng rng rói v cú giỏ tr kinh t cao ỏp ng c nhu cu ca ngi vi s lng ln, vic nhõn ging loi lan ny bng cụng ngh nuụi cy mụ t bo thc vt (in vitro) c thc hin nhiu ni Hin nay, nhiu nghiờn cu xõy dng quy trỡnh nhõn ging loi lan ny ó c bỏo cỏo [16] i vi cụng ngh nhõn ging in vitro, giai on ngụi (hun luyn cõy chuyn t giai on ng nghim mụi trng t nhiờn) úng vai trũ rt quan trng Trong giai on ny, cõy chu nh hng ca mụi trng sng thay i t mụi trng nhõn to n mụi trng t nhiờn mt thi gian ngn thớch nghi, c th chỳng s cú nhng bin i v hỡnh thỏi, gii phu cng nh sinh lý rt ỏng chỳ ý cú th phự hp vi mụi trng mi [24; 38; 39] Nhng nghiờn cu v nhng ng thỏi trờn thc vt cũn ớt c thc hin, c bit vi loi lan Phi ip tớm Nhng kt qu nghiờn cu v cỏc bin i hỡnh thỏi, gii phu v sinh lý ca lan Phi ip tớm giai on ngụi cú ý ngha ln, cung cp cỏc thụng tin khoa hc b ớch, ng thi giỳp ngi cỏc bin phỏp k thut luyn cõy mt cỏch cú hiu qu T nhng lớ trờn, chỳng tụi xut ti Nghiờn cu nh hng ca iu kin ex vitro n s bin i hỡnh thỏi v sinh lý, húa sinh ca phong lan Phi ip tớm (Dendrobium anosmum) nuụi cy mụ giai on ngụi Footer Page 10 of 149 Header Page 53 of 149 44 giỏ th thi im D12 (ngy th chuyn khay) ú giỏ tr t l 596,48àg/g Sau ú hm lng prolin gim mnh thi im D28 (t giỏ tr 336,51àg/g) v tng nh vo thi im D56 (t giỏ tr 378,21àg/g) Tuy nhiờn, hm lng proline mụ lỏ thi im D28 v D56 cao hn so vi thi im D0 v D7 Hm lng prolin lỏ phong lan Phi ip tớm tng nh t D0 v tng mnh vo thi im D12 sau ú gim cỏc thi im sau ú (D28, D56) cú th liờn quan n s mt nc nhiu ca cõy, quỏ trỡnh sinh tng hp prolin c hot húa theo c ch t v trc iu kin mt nc ó c bit thi kỡ cui ca quỏ trỡnh ngụi, cõy ó thớch nghi vi iu kin sng mi, cú th cỏc hot ng sinh lớ cõy din bỡnh thng, hm lng nc cõy mc cao cú th l nguyờn nhõn hm lng prolin cõy gim xung so vi thi im D12 Mc dự vy, hm lng prolin cỏc thi im ny cũn cao, nhiờn s bin ng ny khụng mu thun vi hm lng nc quỏ trỡnh ngụi, cú th cỏc thi im cui ca quỏ trỡnh ngụi cõy ó hỳt c nhiu nc, nhng li phi chu tỏc ng ca cỏc iu kin t nhiờn khỏc, ú hm lng prolin cao hn so vi thi im D0 v D7, õy l hai thi im m cõy bỡnh nuụi cy in vitro nờn cú hm lng nc cao Cú th chuyn cõy mụi trng t nhiờn, s mt nc ó kớch hot c ch t v bng cỏch tng sinh tng hp prolin ging nh nhiu loi thc vt ó bit khỏc b t iu kin hn [21; 53] Rt gn õy, nghiờn cu v quỏ trỡnh luyn cõy Pitcairnia encholirioides (h Da, Bromeliaceae) in vitro ca Resende et al (2016), hm lng prolin lỏ cõy ex vitro (180 ngy sau luyn cõy) cao hn so vi cõy in vitro (150 ngy ng nghim (khụng úng np) cha mụi trng dinh dng nhõn to cú b sung GA3 Tuy nhiờn, loi ny, iu kin ng nghim c úng np, hm lng prolin hu nh khụng thay i gia cõy ex vitro so vi cõy in vitro Cng cựng nghiờn cu, nu mụi trng Footer Page 53 of 149 Header Page 54 of 149 45 dinh dng nhõn to cú b sung NAA thỡ hm lng prolin lỏ cõy in vitro li cao hn so vi lỏ cõy ex vitro [42] 3.10 c im gii phu ca lan Phi ip tớm giai on ngụi Trong quỏ trỡnh ngụi ca cõy lan Phi ip tớm, nghiờn cu s bin i hỡnh thỏi r, thõn, lỏ, chỳng tụi thy rng r cú s thay i kớch thc mnh nht Do vy chỳng tụi la chn nghiờn cu c im gii phu ca r Bng k thut ct, nhum mu xanhmetylen v quan sỏt di kớnh hin vi vi phúng i 400 ln chỳng tụi ó thu c hỡnh nh lỏt ct ngang ca r cõy lan Phi ip tớm cỏc giai on D0, D28 v D56 (hỡnh 3.11) Hỡnh 3.11 Lỏt ct ngang ca r cõy phong lan Phi ip tớm; D = ngy (day) Thanh t l = 500 mm (hỡnh trờn) hoc t l = 50 mm (hỡnh di), ve = v la (cn mc, velamen), ex = ngoi bỡ (exoderrmis), co = cortex, en = ni bỡ (endodermis), vc = tr dn (vascular cylinder) Qua hỡnh 3.11 cho thy c im gii phu ca r gia cỏc thi im khỏc ca quỏ trỡnh luyn cõy cú s khỏc v khỏc vi thi im ban u Footer Page 54 of 149 Header Page 55 of 149 46 in vitro Khi quan sỏt lỏt ct ngang hon chnh r cõy phong lan Phi ip tớm cỏc thi im D0, D28 v D56 cú th nhn thy rng r cõy in vitro cha phỏt trin lp v la hay cn mc (velamen) c trng cho cỏc cõy phong lan Lp v la phỏt trin ngy cng mnh r cõy ex vitro, thi im D28 v D56 ca quỏ trỡnh luyn cõy c im gii phu ny rt ỏng chỳ ý vỡ lp cn mc c bit cú nhiu vai trũ i vi i sng ca cõy phong lan, c bit l vai trũ bo v ca chỳng [15; 35] Lp ngoi bỡ gm cỏc t bo cú kớch thc bộ, thnh t bo mng thi im D0 nhng cú kớch thc ln hn, thnh t bo dy hn cỏc thi im D28 v D56 Lp ni bỡ vi mc cellulose húa thp r cõy D0 v cellulose húa mnh dn thi im D28 v c bit l D56 Tr dn cng ngy cng phỏt trin, cỏc bú tr dn phỏt trin mnh thi im D56 so vi thi im D0 Nh vy, cỏc t chc ca r cõy phỏt trin mnh hn thi im ex vitro so vi thi im in vitro mt s loi, hin tng tng t cng cú th c quan sỏt S phỏt trin ca r cú vai trũ ln, tng cng kh nng hp thu CO2, dinh dng khoỏng v c v d tr nc, chng li iu kin thiu nc [33] Footer Page 55 of 149 Header Page 56 of 149 47 PHN KT LUN V KIN NGH Kt lun Trong nghiờn cu ny, cỏc c im hỡnh thỏi, sinh lý v húa sinh ca cõy phong lan Phi ip tớm cú ngun gc in vitro thi kỡ ngụi ex vitro ó c phõn tớch: Hỡnh thỏi Trong quỏ trỡnh ngụi, cú s tng lờn v s lng lỏ v kớch thc cỏc c quan thõn, lỏ v r ca cõy cỏc thi im D28 v D56 so vi thi im D0 Sinh lớ, húa sinh So vi thi kỡ in vitro, hm lng nc mụ lỏ ca cõy lan Phi ip tớm gim mnh thi kỡ u ngụi ng thi, hm lng cht khụ bin ng ngc vi hm lng nc mụ lỏ Mụ lỏ cõy phong lan Phi ip tớm iu kin ex vitro cú kh nng gi nc cao hn cõy iu kin in vitro Hm lng cỏc sc t quang hp mụ lỏ tng quỏ trỡnh ngụi so vi cõy in vitro Hiu sut quang húa ca quang h II (Fv/Fm) thi kỡ in vitro cao hn so vi cõy ex vitro Ch s ny gim mnh nht thi im D12 cõy mt nhiu nc nht Hot catalase v peroxidase ca cõy ex vitro cao hn so vi cõy in vitro, cao nht thi im D12 Hm lng prolin cõy in vitro tng i cao v tng lờn rt mnh cõy c chuyn mụi trng ex vitro, sau ú li gim xung cỏc thi im cui ca giai on ngụi Gii phu Footer Page 56 of 149 Header Page 57 of 149 48 R cõy cú s bin i v c im gii phu phn tr gia cỏc thi im D28 v D56 so vi thi kỡ in vitro (D0) Kin ngh - Tip tc m rng hng nghiờn cu ny trờn cỏc i tng thc vt khỏc xõy dng quy trỡnh ngụi hiu qu, cú t l sng cao - Tip tc nghiờn cu c ch sinh lớ, húa sinh ca s chng chu vi iu kin mt nc thc vt CAM Footer Page 57 of 149 Header Page 58 of 149 49 TI LIU THAM KHO Ti liu ting vit [1] [2] [3] [4] [5] [6] [7] [8] [9] Trn Hp (1998) Phong lan Vit Nam TP H Chớ Minh: NXB Nụng nghip Nguyn Nh Khanh, Cao Phi Bng (2013) Sinh lý hc thc vt H Ni: NXB Giỏo Dc V Ngc Lan, Nguyn Th Lớ Anh (2013) Nhõn ging in vitro loi lan bn a Dendrobium nobile Lindl Tp Khoa hc v Phỏt trin, 11(7), 917-925 Nguyn Vn Mó, La Vit Hng, Ong Xuõn Phong (2013) Phng phỏp nghiờn cu Sinh lý hc thc vt H Ni: NXB i hc Quc gia H Ni Hong Th Sn (2008) Hỡnh thỏi - Gii phu hc thc vt H Ni: NXB i hc S phm H Ni Nguyn Th Sn, Nguyn Th Lớ Anh, V Ngc Lan, Trn Th Mai (2012) Nhõn ging In vitro loi lan Dendrobium Fimbriatum Hook.(Hong tho long 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Propag Ornam Plants, 9, 107-112 Footer Page 64 of 149 Header Page 65 of 149 ix PH LC NH Phong lan Phi ip tớm c s dng cỏc giai on: Hỡnh P1 Kim tra mu cõy phũng thớ nghim Hỡnh P2 Cõn mu vi cõn k thut Footer Page 65 of 149 Header Page 66 of 149 x Hỡnh P3 Xỏc nh hm lng dip lc Hỡnh P4 Thc nghim gii phu r lan Footer Page 66 of 149 Header Page 67 of 149 xi Hỡnh P5 Lan Phi ip in vitro (trỏi) v ex vitro (phi) Footer Page 67 of 149 ... trên, Ďề xuất Ďề tài Nghiên cứu ảnh hƣởng điều kiện ex vitro đến biến đổi hình thái sinh lý, hóa sinh phong lan Phi điệp tím (Dendrobium anosmum) nuôi cấy mô giai đoạn ngôi Footer Page 10 of... DỤC VÀ ĐÀO TẠO TRƢỜNG ĐẠI HỌC SƢ PHẠM HÀ NỘI TRƢƠNG TRỌNG KIÊN NGHIÊN CỨU ẢNH HƢỞNG CỦA ĐIỀU KIỆN ex vitro ĐẾN SỰ BIẾN ĐỔI HÌNH THÁI VÀ SINH LÝ, HÓA SINH CỦA PHONG LAN PHI ĐIỆP TÍM (Dendrobium anosmum). .. rễ) phong lan Phi Ďiệp tím có nguồn gốc nuôi cấy mô tế bào thực vật giai Ďoạn - Nghiên cứu Ďộng thái sinh lý, hóa sinh phong lan Phi Ďiệp tím ảnh hưởng môi trường tự nhiên giai Ďoạn - Nghiên cứu