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BAN QUẢN LÝ KHU NÔNG NGHIỆP CÔNG NGHỆ CAO TP HCM TRUNG TÂM NGHIÊN CỨU VÀ PHÁT TRIỂN NÔNG NGHIỆP CÔNG NGHỆ CAO BÁO CÁO NGHIỆM THU KHẢO SÁT MỘT SỐ YẾU TỐ ẢNH HƯỞNG LÊN KHẢ NĂNG TĂNG SINH VÀ TỔNG HỢP SAPONIN CỦA RỄ BẤT ĐỊNH CÂY ĐẢNG SÂM Codonopsis javanica Blume ThS Huỳnh Thị Kim ThS Nguyễn Thị Điệp Thành phố Hồ Chí Minh, tháng 01/2018 BAN QUẢN LÝ KHU NÔNG NGHIỆP CÔNG NGHỆ CAO TP HCM TRUNG TÂM NGHIÊN CỨU VÀ PHÁT TRIỂN NÔNG NGHIỆP CÔNG NGHỆ CAO BÁO CÁO NGHIỆM THU (Đã chỉnh sửa theo góp ý Hội đờng nghiệm thu) KHẢO SÁT MỘT SỐ YẾU TỐ ẢNH HƯỞNG LÊN KHẢ NĂNG TĂNG SINH VÀ TỔNG HỢP SAPONIN CỦA RỄ BẤT ĐỊNH CÂY ĐẢNG SÂM Codonopsis javanica Blume CƠ QUAN CHỦ TRÌ (Ký tên, đóng dấu xác nhận) CHỦ NHIỆM ĐỀ TÀI (Ký tên) Thành phớ Hờ Chí Minh, tháng 01/2018 TĨM T T ng sâm (Codonopsis javanica c li u quý ch a nhi u h p ch t hóa h c có giá tr c coi nhân sâm c nghèo Trong nghiên c u nghiên c u y u t ng t o r b nh l c n ng lên s c m nh kh b K t qu nghiên c u cho th y, s c m ng t o r b i ng sâm ng sâm t tt t ng SH có b sung IBA 1,0 mg/l, saccharose 30g/l, agar g/l.T IBA thích h p cho s 100 vịng/phút, IBA 0,5 mg/l.Tr b t ng r kh r b ng l ng u thích h p cho s ng ni c y, ch s a ng cao nh t sau tu n nuôi c y 13,898 Saccharose 30 g/l thích h p cho s saponin triterpen r b triterpen 88,62 mg/g tr saponin triterpen n ng sâm, ch s ng khô mg/l v i th nh n sinh kh i r h mb t 89,91 mg/g tr th ng ng p chìm t m th ni c t 13,969 saponin ng l ng (210 mg/g tr c ngày thu ng khô H p 2,3 l n so v i ng khô) M CL C Trang Trang ph bìa i Tóm t tài ii M c l c iii Danh m c ký hi u, ch vi t t t vi Danh m c b ng vii Danh m c hình v th viii TÀI M U - T NG QUAN TÀI LI U 1.1 Gi i thi u v ng sâm 1.1.1 Phân lo i 1.1.2 Ngu n g c s phân b 1.1.3 m hình thái 1.1.4 Thành ph n hóa h c tác d 1.2 Các nghiên c u c lý c 1.3 Saponin trình sinh t ng h p 1.4 Nuôi c y r b 1.5 nh thu nh n h p ch t th c p (HCTC) 11 m ng h p ch t th c p (HCTC) nuôi c y mô th c v t 12 1.6 H th ng ng p chìm t m th i 13 ng V T LI U 2.1 N i dung 1: C m ng t o r b Thí nghi m 1: Kh o sát t or b ng r n 17 ng c a n IBA, NAA lên kh m ng ng sâm 17 18 2.2.1 lên kh Kh o sát ar b ng c a t l c n IBA thích h p ng sâm 18 2.2.2 Thí nghi m 3: Kh o sát ng c a tr nuôi c y lên kh ar b 2.2.3 Thí nghi m 4: Kh o sát ar b ng r u th i gian ng sâm 19 ng c a n vitamin B1 lên kh ng sâm 19 20 2.3.1 Thí nghi m 5: Kh o sát saponin c a r b ng c a n saccharose lên s ng sâm 20 2.3.2 Thí nghi m 6: Kh o sát ng c a n jasmonate (MeJA) lên s th i m b sung metyl ng saponin r b ng sâm 21 2.3.3 Thí nghi m 7: Kh o sát s ar b nh ng sâm h th ng ni c y ng p chìm t m th i Rita 22 - K T QU VÀ TH O LU N 3.1 ng c a n IBA NAA lên kh m ng t o r b nh ng r n 24 3.2 ng c a t l c n IBA lên kh ar b nh ng sâm 26 3.3 ng c a tr sinh c a r b 3.4 ng r u th i gian nuôi c y lên kh ng sâm 28 ng c a n vitamin B1 lên kh ar b nh ng sâm 29 3.5 ng c a n b 3.6 nh saccharose lên s ng sâm 30 ng c a n th m b sung metyl jasmonate (MeJA) lên s ng saponin triterpen r b 3.7 S saponin r ar b ng sâm 32 ng sâm h th ng ni c y ng p chìm t m th i Rita 33 - K T LU N VÀ KI N NGH 4.1 K t lu n 35 4.2 Ki n ngh 35 TÀI LI U THAM KH O 36 PH L C DANH SÁCH CÁC CH Vi t t t VI T T T Thu t ng ti ng vi t AAS a-amyrin synthase bAS b-amyrin synthase DDS Dammarenediol synthase HCTC H p ch t th c p SH ng SH (Schenk Hilderbrant) IBA Indole butyric acid LUS Lupeol synthase MeJA Metyl jasmonate MEP 2-C-methyl-D-erythritol4-phosphate NAA - Napthalene acetic acid PgSE Squalene epoxidase PNA Dammarenediol synthase-II PNX Cycloartenol synthase TIS Temporary immersion system DANH SÁCH B NG B ng T a 2.1 B ng kh o sát n ng sâm 2.2 B ng kh o sát c ar b 2.3 B ng kh o sát kh 2.4 B ng kh o sát nh h ng c a n sinh c a r b ng sâm 2.5 B ng kh o sát n IBA NAA lên s c m ng t o r b ng c a t ng sâm l c n IBA lên kh 21 ng sâm th m b sung metyl ng saponin r b nh ng c a IBA NAA lên s c m ng t o r b u ki n t i 3.2 ng c a t l c n ng sâm 3.3 ng c a tr sinh c a r b 3.4 ng c a n ng sâm 3.5 ng c a n lên r b 3.7 19 20 saccharose lên s 3.1 3.6 17 18 vitamin B1 lên kh B ng kh o sát ng c a n jasmonate (MeJA) lên s ng sâm r b nh ng c a tr ng r u th i gian nuôi c y lên ar b ng sâm c ar b 2.6 Trang ng sâm IBA lên kh ng r ng sâm a u th i gian lên kh vitamin B1 lên kh 22 25 27 29 ar b t saccharose lên s 30 31 ng sâm ng c ng sâm ng saponin triterpen r b t 32 ng c a h th ng nuôi c y ng b t n kh ng sâm 34 DANH SÁCH HÌNH Hình T a hình ng sâm Codonopsis javanica Blume 1.1 1.3 Quá trình t ng h p steroid triterpenoid 1.4 H th ng RITA® 3.1 3.3 3.4 ng sâm Codonopsis javanica Blume 1.2 3.2 Trang nhân sâm 11 14 ng c a NAA IBA lên s t o r b u ki n t i R b nh t R b nh tu n tu ng c a t ng sâm ng r ng r n ng l ng 26 ng l ng l c n ns 26 26 b nh 28 tài: Kh o sát m t s y u t ng lên kh t ng ng sâm Codonopsis javanica Blume h p saponin c a r b Mã s : tài: Nghiên c u ng d ng D Th i gian th c hi n: T n tháng 12/2017 T ng kinh phí: 141.014 ng a T c: 141.014 b T ngu Nghiên c a T ng ng b : ng xuyên xu t: t hàng) t hàng nghiên c u: tài: (n u có ch nhi Ch nhi Ch nhi thơng tin c ch nhi m) tài 1: H tên: Hu nh Th Kim 1986 H c v : Th Gi i tính: N Chun ngành: Cơng ngh sinh h c Ch c danh khoa h c: t h c v : 2014 c phong ch c danh: Ch c v : Chun viên phịng Cơng ngh t bào th c v t Trung tâm Nghiên c u Phát tri n Nông nghi p Công ngh cao a ch p 1, xã Ph n tho (028) 8862726 i, huy n C Chi, TPHCM Fax: (08) 37990500 a ch nhà riêng: n tho 0967355357 E-mail: kimhuynhpy@gmail.com Ch nhi tài 2: H tên: Nguy n Th p [10] Kim Ji Ah, Eung Jun Park and Yong Eui Choi (2012), Induction and Proliferation of Adventitious Roots in Codonopsis spp., Korean J Medicinal Crop Sci., 20(6): 493 499 [11] Hosakatte Niranjana Murthy, Eun Joo Hahn and Kee Yoeup Paek (2008), Adventitious roots and secondary metabolism, Chinese journal of biotechnology, 24 (5): 711 [12] 716 Kee-Won Yu, Hosakatte Niranjana Murthy, Eun-Joo Hahn, Kee-Yoeup Paek (2004), Ginsenoside production by hairy root cultures of Panax ginseng: influence of temperature and light quality, Biochemical Engineering 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Murthy, Kee-Yoeup Paek (2004), Adventitious root growth and ginsenoside accumulation in Panax ginseng culture affected by methyl jasmonate, Biotechnology letter, 26: 1619-1622 [28] Zhao, J., Davis, L.C., Verpoorte, R (2005), Elicitor signal transduction leading to production of plant secondary metabolites, Biotechnology Advances, 23: 283-333 Trang web [29] www.botanyvn.com [30] http://buixuanphuong09blogspot.blogspot.com/2014/04/1992-ang-sam.html PH L C Ph l c 1: r b ng c a t l c n IBA lên kh a ng sâm Descriptive Statistics Dependent Variable:CSTS IBA TDL 80 1.14800 047429 100 2.03120 072600 120 1.10180 053691 Total 1.42700 446004 15 80 3.26200 126568 100 3.96720 069582 120 3.15920 095816 Total 3.46280 383101 15 80 1.24860 047342 100 1.94240 122355 120 1.37520 093796 Total 1.52207 323950 15 80 1.88620 1.010782 15 100 2.64693 970763 15 120 1.87873 947451 15 Total 2.13729 1.021459 45 10 Total Mean Std Deviation N Tests of Between-Subjects Effects Dependent Variable:CSTS Type III Sum of Source Squares Partial Eta df Mean Square F Sig Squared Corrected Model 45.643a 5.705 772.681 000 994 Intercept 205.560 205.560 2.784E4 000 999 IBA 39.600 19.800 2.682E3 000 993 TDL 5.845 2.922 395.763 000 956 IBA * TDL 198 050 6.720 000 427 Error 266 36 007 Total 251.469 45 45.909 44 Corrected Total Descriptive Statistics Dependent Variable:CSTS IBA TDL 80 1.14800 047429 100 2.03120 072600 120 1.10180 053691 Total 1.42700 446004 15 80 3.26200 126568 100 3.96720 069582 120 3.15920 095816 Total 3.46280 383101 15 80 1.24860 047342 100 1.94240 122355 120 1.37520 093796 Total 1.52207 323950 15 80 1.88620 1.010782 15 100 2.64693 970763 15 120 1.87873 947451 15 10 Total Mean Std Deviation N a R Squared = 994 (Adjusted R Squared = 993) Post Hoc Tests Multiple Comparisons CSTS Tukey HSD 95% Confidence Interval Mean Difference (I) TDL (J) TDL 80 100 120 (I-J) Std Error Sig Lower Bound Upper Bound 100 -.76073* 031377 000 -.83743 -.68404 120 00747 031377 969 -.06923 08416 80 76073* 031377 000 68404 83743 120 76820* 031377 000 69151 84489 80 -.00747 031377 969 -.08416 06923 100 -.76820* 031377 000 -.84489 -.69151 Based on observed means The error term is Mean Square(Error) = 007 * The mean difference is significant at the 05 level Ph l c 2: ng c a tr sinh c a r b ng r u th i gian lên kh ng sâm Tu n 1: CHISOTS 95% Confidence Interval for Mean Std N Mean Deviation Std Error Lower Bound Upper Bound Minimum Maximum 0.51 3.62880 301073 134644 3.25497 4.00263 3.300 4.122 1.01 2.81020 170987 076468 2.59789 3.02251 2.627 3.026 1.51 85500 084820 037933 74968 96032 745 937 2.01 19260 059555 026634 11865 26655 132 261 2.51 10740 043178 019310 05379 16101 061 164 Total 25 1.51880 1.473183 294637 91070 2.12690 061 4.122 ANOVA CHISOTS Sum of Squares Between Groups Mean Square 51.557 12.889 530 20 026 52.086 24 Within Groups Total df F Sig 486.428 Homogeneous Subsets CHISOTS Subset for alpha = 0.05 NONG DOIBA Duncana N 2.51 10740 2.01 19260 1.51 1.01 0.51 Sig 85500 2.81020 3.62880 418 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 5.000 1.000 1.000 000 Tu n 2: CHISOTS 95% Confidence Interval for Mean Std N Mean Deviation Std Error Lower Bound Upper Bound Minimum Maximum 0.52 8.90160 657455 294023 8.08526 9.71794 7.770 9.432 1.02 7.12040 291528 130375 6.75842 7.48238 6.830 7.526 1.52 2.47380 081478 036438 2.37263 2.57497 2.384 2.567 2.02 1.48660 141670 063357 1.31069 1.66251 1.363 1.658 2.52 1.13620 081174 036302 1.03541 1.23699 1.042 1.248 Total 25 4.22372 3.253222 650644 2.88086 5.56658 1.042 9.432 ANOVA CHISOTS Sum of Squares Between Groups Within Groups Total df Mean Square 251.801 62.950 2.202 20 110 254.003 24 F Sig 571.719 000 Homogeneous Subsets CHISOTS Subset for alpha = 0.05 NONG DOIBA Duncana N 2.52 1.13620 2.02 1.48660 1.52 1.02 0.52 Sig 2.47380 7.12040 8.90160 111 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed Tu n 3: CHISOTS 95% Confidence Interval for N Mean Std Deviation Std Error Mean Minimum Maximum Lower Bound Upper Bound 0.53 1.38988E1 682628 305281 13.05121 14.74639 12.966 14.734 1.03 1.02176E1 475862 212812 9.62674 10.80846 9.762 10.876 1.53 4.37060 305386 136573 3.99141 4.74979 4.003 4.711 2.03 2.99020 160615 071829 2.79077 3.18963 2.792 3.204 2.53 2.12720 120618 053942 1.97743 2.27697 2.020 2.286 Total 25 6.72088 4.676049 935210 4.79070 8.65106 2.020 14.734 ANOVA CHISOTS Sum of Squares Between Groups Mean Square F 521.466 130.367 3.304 20 165 524.770 24 Within Groups Total df Sig 789.113 000 Homogeneous Subsets CHISOTS Subset for alpha = 0.05 NONG DOIBA Duncana N 2.53 2.03 1.53 1.03 0.53 Sig 2.12720 2.99020 4.37060 1.02176E1 1.38988E1 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 5.000 Tu n 4: Descriptives CHISOTS 95% Confidence Interval for Mean N Mean Std Deviation Std Error Lower Bound Upper Bound Minimum Maximum 0.54 1.54104E1 1.091388 488083 14.05526 16.76554 13.892 16.4 1.04 1.06904E1 199883 089390 10.44221 10.93859 10.483 11.0 1.54 4.48460 312809 139892 4.09620 4.87300 4.048 4.8 2.04 3.04380 190077 085005 2.80779 3.27981 2.843 3.2 2.54 2.19940 085319 038156 2.09346 2.30534 2.076 2.2 Total 25 7.16572 5.210945 1.042189 5.01475 9.31669 2.076 16.4 ANOVA CHISOTS Sum of Squares Between Groups Within Groups Total df Mean Square F 646.205 161.551 5.489 20 274 651.695 24 Sig 588.599 000 Homogeneous Subsets CHISOTS Subset for alpha = 0.05 NONG DOIBA Duncana N 2.54 2.04 1.54 1.04 0.54 5 2.19940 Sig 3.04380 4.48460 1.06904E1 1.54104E1 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 5.000 Ph l c 3: ng c a n vitamin B1 lên kh ar b t ng sâm CHISOTS 95% Confidence Interval for Mean Std N 0.5 Mean 1.3342 4E1 Deviation 972113 Std Error 434742 Lower Bound 12.13536 Upper Bound 14.54944 Minimum 12.508 Maximum 14.920 1.5 5 Total 20 9.7552 9.6968 8.7760 1.0392 6E1 1.614785 722154 7.75018 11.76022 8.772 12.610 1.410348 630727 7.94562 11.44798 8.036 11.200 546333 244328 8.09764 9.45436 8.124 9.618 2.107456 471241 9.40628 11.37892 8.036 14.920 ANOVA CHISOTS Sum of Squares df Mean Square Between Groups 61.026 20.342 Within Groups 23.360 16 1.460 Total 84.386 19 F Sig 13.933 000 Homogeneous Subsets CHISOTS Subset for alpha = 0.05 NONGDOvita Duncana N 2 8.77600 1.5 9.69680 9.75520 0.5 13.34240 Sig .242 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 5.000 Ph l c 4: saponin lên r b ng c a n nh c saccharose lên s ng sâm CHISOTS 95% Confidence Interval for Mean Std N Mean Deviation Std Error Lower Bound Upper Bound Minimum Maximum 20 6.77400 477081 213357 6.18163 7.36637 6.206 7.500 30 1.39696E1 716033 320220 13.08053 14.85867 13.056 14.922 40 8.33400 492839 220404 7.72206 8.94594 7.778 9.070 50 5.04920 474533 212217 4.45999 5.63841 4.356 5.474 60 6.33360 1.028033 459751 5.05713 7.61007 5.462 8.106 25 8.09208 3.243733 648747 6.75313 9.43103 4.356 14.922 Total ANOVA CHISOTS Sum of Squares Between Groups Within Groups Total df Mean Square 243.462 60.866 9.061 20 453 252.523 24 F Sig 134.347 000 Homogeneous Subsets CHISOTS Subset for alpha = 0.05 SACCH ARO Duncana N 50 60 6.33360 20 6.77400 40 30 Sig 5.04920 8.33400 1.39696E1 1.000 313 1.000 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 5.000 Ph l c 5: S ar b ng sâm h th ng ni c y ng p chìm t m th i Rita CHISOTS 95% Confidence Interval for Mean N Mean Std Deviation Std Error Lower Bound Upper Bound Minimum Maximum 1.43360 167726 075010 1.22534 1.64186 1.286 1.679 5 1.39390 235206 105187 1.10185 1.68595 1.179 1.786 10 1.46570 226620 101347 1.18431 1.74709 1.286 1.845 15 1.47550 195665 087504 1.23255 1.71845 1.286 1.802 20 1.62830 335928 150232 1.21119 2.04541 1.284 2.024 25 1.47940 232977 046595 1.38323 1.57557 1.179 2.024 Total ANOVA CHISOTS Sum of Squares Between Groups df Mean Square 159 040 Within Groups 1.144 20 057 Total 1.303 24 F Sig .695 604 Homogeneous Subsets CHISOTS Subset for alpha = 0.05 NGAPCHIM Duncana N 5 1.39390 1.43360 10 1.46570 15 1.47550 20 1.62830 Sig .178 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 5.000 Ph l c 6: K t qu saccharose phân tích saponin triterpen t r b nh có b sung Ph l c 7: K t qu phân tích saponin triterpen t r b sung MeJA ng sâm có b Ph l c 8: K t qu phân tích saponin triterpen t r b h th ng ng p chìm t m th i ng sâm nuôi