Luận án Nghiên cứu thiết kế và chuyển gen agpopt tổng hợp nhân tạo vào cây sắn (manihot esculenta crantz)

es it was 82% in KM94 compared with 59.6% in KM140. On somatic embryo germination medium containing 0.3 mg/L BAP, there were 71 - 89% somatic embryo masses of KM140 and 79 – 79.2% of KM94 turned up into healthy plants. The regenerated plants produced strong roots (3-4 roots/shoot) on the MS medium without growth hormone. To develop the genetic transformation procedure for studied cassava varieties, Gus gene (in pBI121 vector) was transformed into KM140 using A. tumefaciens method. PCR using nptII_F/R primers indicated that 17 regenerated plants were putative transgenic plants. The gene transformation efficiency was 0.9%. Further modification was carried out in order to have more available initial explants for transformation. Green, hard and shiny cotyledons formed from 115 primary somatic embryos were separated, cut into 4 mm 2 pieces and inoculated with bacterial suspension for 15 minutes. The secondry cotyledon pieces were placed in co-culture medium CIM (MS + picloram 12 mg/L) containing acetosynringone at 100 mM concentration. After two days of co-culture, transgenic cotyledon pieces were washed with cefotacim antibiotics at concentration of 500 mg/L to remove extra A. tumefaciens. Then, they were transferred to a new CIM medium containing 500 mg/L cefotacim and 50 mg/L kanamycin (to select nptII). This modification in the transformation procedure obvious enhanced the performance/effectiveness of gene transfer to studied cassava varieties. Putative transgenic plant analysis using gus assay and PCR with gene specific primers indicated a high number of plant carring transferred genes.. Based on the data of the glgC protein of E. coli K12 (code number AKD93525.1) and the mutation G336D, a DNA sequence encoding an enzyme of 431 amino acid was designed. After optimizing codon usage for gene effective expressed in plants, DNA sequence was linked with a nucleotide segment encoding a signal peptide of rbcS protein (Arabidopsis) for transporting proteins into chloroplasts in the 5‘end and a segment of DNA coding for c-myc and KDEL tail at 3‘ end. Two recognition positions of restriction enzymes XbaI and SacI were added to the 5 'and 3' end of the gene in the respective order. Finally, the designed AGPopt gene encoding AGPase of 1527 bps long, was synthesized by Integrated DNA Technologies (USA) agent and cloned in the vector pUCIDT-AMP. Then AGPopt genes were inserted to pBI121 plant expression vector in and transferred in to A. tumefaciens CV58/pGV2260 for transformation, The constructed pBI121 vector harboring AGPopt gene has been tested using model tobacco plant. PCR using the primers G336F/G336KDEL R proved 32 out of 39 regenerated lines were positive; Southern blot resulted that all 8 tested lines shown AGPopt copies in their genomic. Further analysis of AGPase activity in the transgenic tobacco lines indicated the increase from 121% (L6) to 153% (L7) and 116 the starch contents also increased from 140.05% (L2) to 168.99% (L7) compared to the non-transgenic control. The transformation of AGPopt vector into cassava was conducted with 1018 explants in 6 experimental plots. Totally, there were 69 regenerated plants produced (6.78%), however only 6 plants form heathy roots. PCR analysis for the presence of transgene structure using nptII_F / R primers proved 5 out of 6 lines were positive. By using specific primers G336 F/G336KDEL R , PCR bands have been found at the size of 1.5 kb, corresponding to AGPopt gene. Following, double PCR concluded that 5 regenerated cassava lines are possible transgenic plants. Further experiments to prove the transgenic cassava lines are going on. Results of this study are leading to following conclusions: 1. In vitro tissue culture and plant regeneration via somatic embryogenesis using shoot apex and young leaves have been optimized for two local cassava varieties KM140 and KM94, suitable for genetic transformation. Somatic embryo was produced on MS medium supplied with 12 mg/lpicloram and germinated with the present of 0,3 mg/l BAP. 2. The genetic transformation procedure for studied cultivars was optimized using GUS gene with the efficiency of 0.9%. 3. Based on amino acid sequence of GlgC protein (Accession number AKD93525.1) of E. coli K12, an AGPopt gene has been synthesized, in which codon usage has been optimized for efficient expression of AGP in plant and glycine at position 336 was substituted by aspartate (G336D) for enhancing APG activity. AGPopt gene was then inserted into the plant expression vector pBI121 and this recombination vector was transferred into tobacco for evaluation before introducing into target species. 4. AGPopt gene was successfully transferred to cassava cultivar KM94 and transgenic plants were regenerated through somatic embryo development. Primarily, PCR amplification had proved there were 5 out of 6 obtained cassava plants containing nptII and AGPopt genes in their genomes. 117 TÀI LIỆU THAM KHẢO 1. Abhary M, Siritunga D, Stevens G, Taylor NJ, Fauquet CM (2011) Transgenic Biofortification of the Starchy Staple Cassava (Manihot esculenta) Generates a Novel Sink for Protein. PLoS ONE 6(1). 2. Abraham TE (1996) Dry extraction of starch from cassava tubers. In: Kurup GT, Palaniswami MS, Pottty VP, Padmaja G, Kabeerathumma S, Pillai SV, eds. Tropical Tuber Crops: problems, prospects and future strategies. Science Publisher INC., Lebanon, New Hampshire. 3. AgroMonitor (2016) Toàn cảnh thị trường sắn tháng 6/2016. Phân tích và dự báo thị trường AgroMonitor - Báo cáo thường niên. 4. Anderson S, Smith SM (1986) Synthesis of the small subunit of ribulose- bisphosphate carboxylase from genes cloned into plasmids containing the SP6 promoter. Biochem Journal 240: 709–715. 5. Andrew G (2003) Starch synthesis and its manipulation. Andrewgray.com. 6. Angov E (2011) Codon usage: Nature‘s roadmap to expression and folding of proteins. Biotechnol Journal 6:650–659. 7. Angov E, Hillier CJ, Kincaid RL, Lyon JA (2008) Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host. PLoS ONE 3:e2189. 8. Apio H, Alicai T, Baguma Y, Mukasa S, Bua A, Taylor N (2015) Production of friable embryogenic callus and regeneration of Ugandan farmer-preferred cassava genotypes. Africa Journal Biotechnology 14:1854–1864. 9. Arias-Garzon DI (1997) Genetic engineering approaches to improve agronomic traits in cassava (Manihot esculenta Crantz). PhD Thesis. Columbus: The Ohio State University. 10. Bae JM, Giroux M, Hannah LC (1990) Cloning and characterization of the brittle-2 gene of maize. Maydica 35:317–322e. 118 11. Baecker PA, Furlong CE, Preiss J (1983) Biosynthesis of bacterial glycogen: primary structure of Escherichia coli ADP-glucose synthethase as deduced from the nucleotide sequence of the glgC gene. Journal of Biological Chemistry 258: 5084–5088. 12. Ball SG, Morell MK (2003) From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule. Annual Review of Plant Biology 54: 207-233. 13. Barbara Pfister, Samuel C. Zeeman (2016) Formation of starch in plant cells. Cellular and Molecular Life Sciences 3:2781–2807. 14. Baroja-Fernández E, Muñoz FJ, Montero M, Etxeberria E, Sesma MT, Ovecka M, Bahaji A, Ezquer I, Li J, Prat S, Pozueta-Romero J (2009) Enhancing sucrose synthase activity in transgenic potato (Solanum tuberosum L.) tubers results in increased levels of starch, ADPglucose and UDPglucose and total yield. Plant Cell Physiology 50(9):1651-62. 15. Baroja-Fernández E, Muñoz FJ, Saikusa T, Rodríguez-López M, Akazawa T, Pozueta-Romero J (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants. Plant and Cell Physiology 44: 500-509. 16. Batard Y, Hehn A, Nedelkina S, Schalk M, Pallett K, Schaller H, Werck- Reichhart D (2000) Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems. Arch Biochem Biophys 379:161–169. 17. Beeching JR (2013) Manihot esculenta (Cassava). eLS:1–7. 18. Beyene G, Chauhan RD, Wagaba H, Moll T, Alicai T, Miano D, Carrington J, Taylor N (2015) Loss of CMD2-mediated resistance to cassava mosaic disease in plants regenerated through somatic embryogenesis. Mol Plant Pathol. 19. Bộ Nông nghiệp và PTNT (2016) Diện tích, năng suất, sản lượng sắn của Việt Nam năm 2016. Báo cáo thường niên. 119 20. Brand A, Quimbaya M, Chavarriaga P (2016) Identification and characterization of Arabidopsis LEC1 and LEC2 orthologous genes in cassava somatic embryo development (Manihot esculenta, Crantz). In: World Congress on Root and Tuber Crops, Abstract No. S09-03, January 18–22, 2016, Nanning, Guangxi, China. 21. Buchanan BB, Gruissem W, Russell LJ (2002) Biochemistry and Molecular Biology of Plant. Printed United State of America 637-643. 22. Bùi Lan Anh, Nguyễn Phan Cẩm Tú, Trần Nguyên Vũ, Bùi Văn Lệ (2008), Xây dựng quy trình biến nạp gien bar – gien kháng thuốc diệt cỏ vào cây khoai mì (Manihot esculenta Crantz) bằng phương pháp bắn gien. Tạp chí phát triển KH&CN 11(1). 23. Buléon A, Colonna P, Planchot V, Ball S (1998) Starch granules: structure and biosynthesis. International Journal of Biological Macromolecules 23:85– 112. 24. Carolin R, Carrol N (2003), Agrobacterium-mediated transformation of plants. CAMBIA Intellectual Property Resource GPO Box 3200 Canberra, ACT 2601 Australia. 25. Carvalho LJCB , Agustini MAV, Anderson JV, Vieira EA, de Souza CRB, Chen S, Schaal BA, Silva JP (2016) Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root. BMC Plant Biology. 16: 133 26. Chauhan RD, Beyene G, Kalyaeva M, Fauquet CM, Taylor N (2015) Improvementsin Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) for large-scale production of transgenic plants. Plant Cell Tiss. OrganCult. 121 591–603. 27. Chavarriaga-Aguirre P, Brand A, Medina A, Prías M, Escobar R, Martinez J, Díaz P, López C, Roca WM, Tohme J (2016) The potential of using biotechnology to improve cassava: a review. In Vitro Cell Dev Biol Plant 52(5): 461–478. 120 28. Chetty C, Rossin C, Gruissem W, Vanderschuren H, Rey M (2013) Empowering biotechnology in southern Africa: establishment of a robust transformation platform for the production of transgenic industry-preferred cassava. New Biotechnol 30:136–143CrossRefGoogle Scholar 29. Chin JX, Chung BK-S, Lee D-Y (2014) Codon optimization online (COOL): A webbased multi-objective optimization platform for synthetic gene design. Bioinformatics 30:2210–2212. 30. CIAT (2016) Genetic Resources Program, Available via F918DB953DD808, accessed 15 February 2016 31. D‘Hulst C, Mérida A (2010) The priming of storage glucan synthesis from bacteria to plants: current knowledge and new developments. New Phytologist 188: 13–21 32. Damba Y, Quainoo AK, Sowley ENK (2013) Effectiveness of somatic embryogenesis in eliminating the Cassava Mosaic Virus from infected cassava (Manihot esculenta Crantz). Plant Mater IJSTR 2:282–287 33. Daniel E, Onwukwe GU, Wierenga RK, Quaggin SE, Vainio SJ, Krause M (2015) ATGme: Open-source web application for rare codon identification and custom DNA sequence optimization. BMC Bioinformatics 16:303–309. 34. Danso KE, Elegba W (2017) Chapter 5. Optimisation of Somatic Embryogenesis in Cassava. In Jankowicz-Cieslak J, Tai TH, Kumlehn J, Till BJ (eds.), Biotechnologies for Plant Mutation Breeding. Springer International Publishing AG Switzerland. 35. Denyer K, Dunlap F, Thorbjornsen T, Keeling P, Smith AM (1996) The major form of ADP-glucose pyrophosphorylase in maize endosperm is extra- plastidial. Plant Physiology 112: 779-785. 36. Derde LJ, Gomand SV, Courtin CM, Delcour JA (2012) Characterisation of three starch degrading enzymes: Thermostable β-amylase, maltotetraogenic and maltogenic α-amylases. Food Chemistry 135(2): 713–721 121 37. Díaz PT, Bernal AG, López CC (2014) Transient GUS gene expression in cassava (Manihot esculenta Crantz) using Agrobacterium tumefaciens leaf infiltration. Rev.MVZ Córdoba 19(3):4338-4349. 38. Doai NV, Ngoc LT, Ngoc PB (2015) Manihot esculenta isolate KM140 AGPase large subunit protein mRNA, complete cds. https://www.ncbi.nlm.nih.gov/nuccore/KU243122.1 39. Doai NV, Ngoc LT, Ngoc PB (2015) Manihot esculenta isolate KM94 AGPase large subunit protein mRNA, complete cds. https://www.ncbi.nlm.nih.gov/nuccore/ KU243121.1 40. Elisa Leyva-Guerrero (2011) Enhancement of the free amino acid and protein content of cassava storage roots and evaluation of root-specific promoters in cassava. PhD dissertation. The Ohio State University. 41. El-Sharkawy MA (2012) Stress-tolerant cassava: the role of integrative ecophysiology-breeding research in crop improvement. Open J Soil Sci 2(02):162–186. 42. Erwin G (2011) Plant Biochemistry. Translation of the 4th German edition. Theor Appl Genet 119: 815–825 43. FAO (2013) Save and grow cassava: a guide to sustainable production and identification. E-ISBN 978-92-5-107642-2 44. FAOSTAT (2015) Food and Agriculture Organisation of the United Nations. Accessed 10 February 2016 45. Fauquet C, Fargette D (1990) African Cassava Mosaic Virus: Etiology, Epidemiology, and Control. Plant Disease 74: 404-411. 46. Feher A (2015) Somatic embryogenesis—stress-induced remodeling of plant cell fate. Biochim Biophys Acta Gene Regulatory Mech 1849:385–402 47. Feitosa T, Bastos JLP, Ponte LFA, Jucá TL, Campos FAP (2007) Somatic embryogenesis in cassava genotypes from the northeast of Brazil. Brazilian Archives of Biology and Technology 50:201–206. 122 48. Fregene M (2002) Cassava Biotechnology. In R.J. Hillocks, J.M. Thresh, and A.C. Bellotti, eds. Cassava: Biology, Production and Utilization. CAB International, Wallingford, UK. pp. 179–207. 49. Fuglsang A (2003) Codon optimizer: A freeware tool for codon optimization. Protein Expression Purification 31:247–249. 50. Gámez-Arjona FM, Li J, Raynaud S, Baroja-Fernández E, Muñoz FJ, Ovecka M, Ragel P, Bahaji A, Pozueta-Romero J, Mérida Á (2011) Enhancing the expression of starch synthase class IV results in increased levels of both transitory and long-term storage starch. Plant Biotechnology Journal 9(9):1049-60. 51. Gaspar P, Oliveira JL, Frommlet J, Santos M, Moura G (2012) EuGene:Maximizing synthetic gene design for heterologous expression. Bioinformatics 28:2683–2684. 52. Geigenberger P, Stamme C, Tjaden J, Schulz A, Quick PW, Betsche T (2001) Tuber physiology and properties of starch from tubers of transgenic potato plants with altered plastidic adenylate transporter activity. Plant Physiology 125:1667–78. 53. Geigenberger P (2011) Regulation of starch biosynthesis in response to a Fluctuating Environment. Plant Physiology 155:1566–77 54. Grote A, Hiller K, Scheer M, Münch R, Nӧrtemann B, Hempel DC, Jahn D (2005) JCat: A novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Researh 33:526–531. 55. Guimaraes JC, Rocha M, Arkin AP, Cambray G (2014) D-Tailor: Automated analysis and design of DNA sequences. Bioinformatics 30:1087–1094. 56. Gustafsson C, Minshull J, Govindarajan S, Ness J, Villalobos A, Welch M (2012) Engineering genes for predictable protein expression. Protein Expression Purification 83:37–46. 123 57. Hakata M, Kuroda M, Miyashita T, Yamaguchi T, Kojima M, Sakakibara H (2012) Suppression of α-amylase genes improves quality of rice grain ripened under high temperature. Plant Biotechnol Journal 10:1110–7. 58. Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium 41:95-98. 59. Hankoua BB, Ng SYC, Fawole I, Puonti-Kaerlas J, Pillay M, Dixon AGO (2005) Regeneration of a wide range of African cassava genotypes via shoot organogenesis from cotyledons of maturing somatic embryos and conformity of the field-established regenerants. Plant Cell, Tissue and Organ Culture 82(2): 221-231. 60. Hankoua BB, Taylor NJ, Ng SYC, Fawole I, Puonti-Kaerlas J, Padmanabhan C, Yadav JS, Fauquet CM, Dixon AGO, Fondong VN (2006) Production of the first transgenic cassava in Africa via direct shoot organogenesis from friable embryogenic calli and germination of maturing somatic embryos. African Journal of Biotechnology 5 (19): 1700-1712. 61. Hartzenberg L (2016) Identification and Manipulation of Starch Synthases from Marama Bean and Potato. PhD Thesis. Stellenbosch University https://scholar.sun.ac.za 62. Hennen-Bierwagen TA, Liu F, Marsh RS, Kim S, Gan Q, Tetlow IJ (2008) Starch biosynthetic enzymes from developing maize endosperm associate in multisubunit complexes. Plant Physiology 146:1892–908. 63. Hiệp hội sắn Việt Nam (2016) Tổng quan ngành sắn Việt Nam - Báo cáo Hội thảo (tháng 12 2016) tại Yên Bái. 64. Hoàng Kim và Phạm Văn Biên (1995) Cây sắn. Nhà xuất bản Nông nghiệp TP. Hồ Chí Minh. 65. Hoàng Kim, Trần Ngọc Quyền, Võ Văn Tuấn (2011) Giống Sắn KM94 124 66. Holsters M, De Waele D, Depicker A, Messens E, Van Montagu M, Schell J (1978). Transfection and transformation of A. tumefaciens. Molecular & general genetics 163:181–187. 67. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278. 68. Huang H, Luo Y, Huang Q, Tian Y, Li H (2013) Time Course of Starch Biosynthesis Enzymes Activityand Root Tuber Starch of FourCassava Cultivars. Journal of Agricultural Science 5(1). 69. Iglesias AA, Barry GF, Meyer C, Bloksberg L, Nakata PA, Greene T, Laughlin MJ, Okita TW, Kishore GM, Preiss J (1993) Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli. Biology Chemistry 268: 1081–1086 70. Ihemere UE, Diana A, Susan L, Richard S (2003) Somatic embryogenesis and transformation of cassava for enhanced starch production. PhD Thesis. The Ohio State University, Columbus, OH 71. Ihemere UE, Diana A, Susan L, Richard S (2006) Genetic modification of cassava for enhanced starch production. Plant Biotechnology Journal 453– 465. 72. Ihemere UE, Narayanan NN, Sayre RT (2012) Iron biofortification andhomeostasis in transgenic cassava roots expressing the algal iron assimilatorygene, FEA1. Frontiers of Plant Science 3: 171 73. Ikeuchi M, Sugimoto K, Iwase A. (2013) Plant Callus: Mechanisms of Induction and Repression. Plant Cell 25:3159–3173. 74. ITIS (2012), ITIS Standard Report Page: Manihot esculenta, Taxonomic Serial No.: 503688. Integrated Taxomonic Information System, 75. Jefferson R (1987) GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. European Molecular Biology Organization Journal 6(13): 3901-3907. 125 76. Jelili TO, Oluyemisi AA (2016) Faithful transmission and expression of transgenes via somatic embryos of transgenic cassava plants at the sixth cycle of vegetative propagation. BioTechnologia 97(2): 65-77 77. Jennings DL, Iglesias C (2002) Breeding for Crop Improvement. In: Hillocks, R.J., J.M. Thresh and A.C. Bellotti, eds. Cassava: Biology, Production and Utilization. CABI Publishing, New York, USA, ISBN-13: 149-166. 78. Jensen L, Olsen O, Kops O, Wolf N, Thomsen K, von Wettstein D (1996) Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)- beta-glucanase during germination. Proc Natl Acad Sci USA 96:3487–3491. 79. Kawabata A, Sawayama S, Nagashima N, del Rosaria RR, Nakamura M (1984) Some physico-chemical properties of starches from cassava, arrow- root and sago. Journal of Japanese Society of Starch Science 31: 224-232. 80. Kawano K (2003) Thirty years of cassava breeding for productivity- biological and social factors for success. Crop Science 43: 1325-1335. 81. Keeling PL, Bacon PJ, Holt DC (1993) Elevated temperature reduces starch deposition in wheat endosperm by reducing the activity of soluble starch synthase. Planta 191: 342–8. 82. Kim SY, May GD, Park WD (1994) Nuclear protein factors binding to a class I patatin promoter region are tuber-specific and sucrose-inducible. Plant Molecular Biology 26: 603–615. 83. Kim Y, Hendrickson R, Mosier N, Hilaly A, Ladisch MR (2011) Cassava Starch Pearls as a Desiccant for Drying Ethanol. Industrial & Engineering Chemistry Research 50, 8678–8685. 84. Kleczkowski LA, Villand P, Luthi U, Olsen OA, Preiss J (1993) Insensitivity of barkey endosperm ADP-glucose Pyrophosphorylase to 3-phosphoglycerate and orthophosphate regulation. Plant Physiology 101: 179-186. 85. Koehorst-van Putten H, Sudarmonowati E, Herman M, Pereira-Bertram I, Wolters A, Meima H, De Vetten N, Raemakers CJJM, Visser R (2012) Field 126 testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia. Transgenic Research 21:39–50. 86. Konan NK, Sangwan RS, Sangwan BS (1994) Somatic embryogenesis from cultured mature cotyledons of cassava (Manihot esculenta Crantz). Plant Cell, Tissue and Organ Culture 37: 91–102. 87. Konan NK, Schiipke C, Ircamo RC, Beachy RN, Fauquet C (1997) An efficient mass propagation system for cassava (Manihot esculenta Crantz) based on nodal explants and axillary bud-derived meristems. Plant Cell Reports 16: 444-449 88. Konstantin K, Shalini M, Belay TA (2016) Extraction and Assays of ADP- glucose Pyrophosphorylase, Soluble Starch Synthase and Granule Bound Starch Synthase from Wheat (Triticum aestivum L.) Grains. Bio-protocol 6(18). 89. Kossmann J, Lloyd J (2000) Understanding and Influencing Starch Biochemistry. Critical Reviews in Plant Science 19: 171-226. 90. Kosugi S, Ohashi Y, Nakajima K, Arai Y (1990). An improved assay for B- glucuronidase in transformed cells: Methanol almost completely suppresses a putative endogenous p-glucuronidase activity. Plant Science 70:133-140. 91. Kumar A, Ghosh P, Lee YM, Hill MA, Preiss J (1989) Biosynthesis of bacterial glycogen: determination of the axit amin changes that alter the regulatory properties of a mutant E. coli ADP-glucose synthase. Journal of Biology Chemistry 264:10464-10471 92. Lanza AM, Curran KA, Rey LG, Alper HS (2014) A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae. BMC System Biology 8:33–43. 93. Lee SK, Hwang SK, Han M, Eom JS, Kang HG, Han Y, Choi SB, Cho MH, Bhoo SH, An G (2007) Identification of the ADP-glucose pyrophosphorylase isoforms essential for starch synthesis in the leaf and seed endosperm of rice (Oryza sativa L.). Plant Molecular Biology Reports 65: 531–546 127 94. Legg J, Lava-Kumar P, Makeshkumar T, Tripathi L, Ferguson M, Kanju E, Ntawuruhunga P, Cuellar, W (2014) Cassava Virus Diseases: Biology, Epidemiology, and Management. In: Gad L, Nikolaos IK, eds. Advances in Virus Research 91: 85–142 95. Leggewie G, Kolbe A, Lemoine R, Roessner U, Lytovchenko A, Zuther E (2003) Overexpression of the sucrose transporter SoSUT1 in potato results in alterations in leaf carbon partitioning and in tuber metabolism but has little impact on tuber morphology. Planta 217:158–67. 96. Li HQ, Sautter C, Potrykus I, Puonti-Kaerlas J (1996) Genetic transformation of cassava (Manihot esculenta Crantz). Nature Biotechnology 14: 736–740. 97. Li J, Baroja-Fernández E, Bahaji A, Muñoz FJ, Ovecka M, Montero M (2013) Enhancing sucrose synthase activity results in increased levels of starch and ADP-glucose in maize (Zea mays L.) seed endosperms. Plant Cell Physiology 54:282–94. 98. Li KT, Moulin M, Mangel N, Albersen M, Verhoeven-Duif NM, Ma Q, Zhang P, Fitzpatrick TB, Gruissem W, Vanderschuren H (2015) Increased bioavailable vitamin B6 in field-grown transgenic cassava for dietary sufficiency. Natural Biotechnology 33:1029–1032 99. Li XQ,Wei JZ, Tan A, Aroian RV (2007) Resistance to root-knot nematode in tomato roots expressing a nematicidal Bacillus thuringiensis crystal protein. Plant Biotechnol Journal 5:455–464. 100. Lin TP, Caspar TS, Preiss CR (1988) Starch deficient mutant of Arabidopsis thaliana with low ADPglucose pyrophosphorylase activity lacks one of the two subunits of the enzyme. Plant physiology 88(4): 1175-81 101. Liu X, Deng R, Wang J, Wang X (2014) COStar: A D-star Lite-based dynamic search algorithm for codon optimization. J Theor Biol 344:19–30. 102. Lloyd JR, Springer F, Buléon A, Müller-Röber B, Willmitzer L, Kossmann J. (1999) The influence of alterations in ADP-glucose pyrophosphorylase 128 activities on starch structure and composition in potato tubers. Planta 209(2): 230-238. 103. Luong HT, Shewry PR, Lazzeri PA (1994) Gene transfer to cassava somatic embryos via tissue electroporation and particle bombardment. In: Second International Scientific Meeting of Cassava Biotechnology Network 11. Bogor. Indonesia. 303-314. 104. Ma Guohua, Xu Qiusheng (2002) Induction of somatic embryogenesis and adventitious shoots from immature leaves of cassava. Plant Cell, Tissue and Organ Culture 70: 281–288. 105. Ma Q, Zhou W, Zhang P (2015) Transition from somatic embryo to friable embryogenic callus in cassava: dynamic changes in cellular structure, physiological status, and gene expression profiles. Frontiers of Plant Science. 6: 1-14. 106. Manyong VB, Douthwaite O, Coulibaly, Keatinge JDH (2000) Participatory impact assessment at IITA: Functions and mechanisms. Paper presented at the Workshop in the Future of Impact Assessment in the CGIAR: Needs, Constraints and Options. Organized by the Standing Panel on Impact Assessment (SPIA) of the Technical Advisory Committee (TAC) of the CGIAR. 3-5 May 2000, FAO, Rome. 107. Martin C, Smith AM (1995) Starch Biosynthesis. The Plant Cell 7: 971- 985. 108. Martin T, Schmidt R, Altmann T, Frommer WB (1992) Non-destructive assay systems for detection of -glucuronidase activity in higher plants. Plant Molecular Biology Reports 10: 37-46. 109. Mason-Gamer RJ, CF Weil, EA Kellogg (1998) Granule-bound starch synthase: structure, function, and phylogenetic utility. Molecular Biology and Evolution 15:1658–1673. 110. Meyer CR, Bork JA, Nadler S, Yirsa J, Preiss J (1998) Sitedirected mutagenesis of a regulatory site of Escherichia coli ADPglucose 129 pyrophophorylase: the role of residue 336 in allosteric behavior. Archives of Biochemistry and Biophysics 353:152–159. 111. Mongomake K, Doungous O, Khatabi B, Fondong VN (2015) Somatic embryogenesis and plant regeneration of cassava (Manihot esculenta Crantz) landraces from Cameroon. SpringerPlus 4:477. 112. Moorthy SN, Ramanujan T (1986) Variation in properties of cassava starch in relation to age of crop. Starch/Starke 38: 77-78. 113. Moreno F, Fernandez T, Fernandez R, Herrero P (1986) Hexokinase PII from Saccharomyces cerevisiae is regulated by changes in the cytosolic Mg 2+ - free ATP concentration. European Journal of Biochemistry 161(3):565-9. 114. Morillo Y, Sánchez T, Morante N, Chávez AL, Morillo AC, Bolaños A, Ceballos H (2012) Preliminary study of inheritance of the carotenoids content in roots of cassava (Manihot esculenta Crantz) segregating populations. Acta Agronómica 61:253–264. 115. Mukherjee A (1994) Embryogenesis and regeneration from cassava Galli of anther and leaf. The Cassava Biotechnology Network. Proceeding of the Second International Scientific Meeting. Bogor, Indonesia 375-377. 116. Munyikwa TRI, Chipangura B, Salehuzzaman SNIM, Jacobsen E, Visser RGF (1994) Cloning and characterization of cassava genes involved in starch biosynthesis. In: The Proc. 2nd International Scientific of Cassava Biotech. Network, Bogor Indonesia 639-645 117. Munyikwa TRI, Langeveld S, Salehuzzaman SNIM, Jacobsen E, Visser RGF (1997). Cassava starch biosynthesis: new avenues for modifying starch quantity and quality. Euphytica 96: 65-75 118. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco cultures. Physiologia Plantarum 15: 473-497. 119. Nagai YS, Sakulsingharoj C, Edwards GE, Satoh H, Greene TW, Blakeslee B (2009) Control of starch synthesis in cereals: metabolite analysis of transgenic rice expressing and up-regulated cytosplasmic ADP-glucose 130 pyrophosphorylase in developing seeds. Plant Cell Physiology 50:635–43. 120. Nakamura Y, Umemoto T, Takahata Y, Komae K, Amano E, Satoh H (1996) Changes in structure of starch and enzyme activities affected by sugary mutations in developing rice endosperm. Possible role of starch debranching enzyme (R-enzyme) in amylopectin biosynthesis. Physiologia Plantarum 97: 491-498. 121. Narayanan N, Beyene G, Chauhan RD, Gaitán-Solis E, Siritunga D, Grusak MA, Taylor N, Anderson P (2016) Biofortification of iron and zinc in cassava storage roots to nutritionally significant level. In: World Congress on Root and Tuber Crops, Abstract No. S15-06, January 18–22, 2016, Nanning, Guangxi, China. 122. Narayanana N, Beyenea G, Chauhana RD, Gaitán-Solisa E, Grusakb MA, Taylora N, Donald PA (2015) Overexpression of Arabidopsis VIT1 increases accumulation of iron incassava roots and stems. Plant Science 240: 170–181. 123. Narayanaswamy TC, Ramaswamy NM, Sree R (1995) Somatic embryogenesis and plant regeneration in cassava. In: The Cassava Biotechnology Network. Proceeding of the Second International Scientific Meeting. Bogor, Indonesia 24-329. 124. Ng NQ, Asiedu R, Ng SYC (1994) Cassava genetic resources programme at IITA, Ibadan. In: Report of the First Meeting of the International Network for Cassava Genetic Resources. International Crop Network Series No. 10. International Plant Genetic Resources Institute, Rome, Italy 71-76. 125. Nguyễn Hữu Hỷ (2016) Cây sắn Việt Nam, hiện trạng và phát triển. Viện Khoa học Kỹ thuật Nông Nghiệp Miền Nam. viet-nam-hien-trang-va-phat-trien-a15438.html. 126. Nguyễn Văn Đồng, Nguyễn Anh Vũ, Lê Tiến Dũng, Tống Thị Hường, Lê Thị Ngọc Quỳnh, Lê Thị Lý, Vũ Anh Thu, Vũ Hoàng Nam, Vũ Thế Hà, Lê Huy Hàm (2014) Kết quả tạo mô sẹo phôi hóa phục vụ cho chuyển gien vào cây sắn. Tạp chí Nông nghiệp và phát triển nông thôn 8(1): 29-35. 131 127. Nkaa FA, Ene-Obong EE, Afuape SO, Okwuonu IC, Kahya SS, Taylor NJ (2015) Evaluation of Agrobacterium-mediated Transformation of Two Nigerian Cassava (Manihot esculenta Crantz) Cultivars TME 419 and ―Okwuoto‖. Journal of Advances in Biology & Biotechnology 4(2): 1-10. 128. Nkaa FA, Ene-Obong EE, Taylor N, Fauquet CM, Mbanaso ENA (2013) Elimination of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) from cassava (Manihot esculenta Crantz) cv. ‗Nwugo‘ via somatic embryogenesis. AJBMS 3:33–40. 129. Ntui VO, Kong K, Khan RS, Igawa T, Janavi GJ, Rabindran R, Nakamura I, Mii M (2015) Resistance to Sri Lankan Cassava Mosaic Virus (SLCMV) in Genetically Engineered Cassava cv. KU50 through RNA Silencing. PLoS One 10:e0120551. 130. Nweke FI, Spencer DSC, Lynam JK (2002) The Cassava Transformation: Africa’s Best Kept Secret. East Lansing: Michigan State University Press. 131. Nyaboga E, Njiru J, Nguu E, Gruissem W, Vanderschuren H, Tripathi L (2013) Unlocking the potential of tropical root crop biotechnology in east Africa by establishing a genetic transformation platform for local farmer- preferred cassava cultivars. Frontier of Plant Science 4:526. 132. Nyaboga EN, Njiru JM, Tripathi L (2015) Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14. Frontiers of Plant Science 6:411. 133. OECD (2014) Consensus Document on the Biology of Cassava (Manihot esculenta Crantz). OECD environment, health and safety publications. 134. Ohdan T, Francisco PB, Sawada T, Hirose T, Terao T, Satoh H (2005) Expression profiling of genes involved in starch synthesis in sink and source organs of rice. Journal of Experimental Botany 56(422):3229-44. 135. Olufemi OO, Jelili TO, Adebola AR, Ingelbrecht IL (2015) A cassava vein mosaic virus promoter cassette induces high and stable gene expression in 132 clonally propagated transgenic cassava (Manihot esculenta Crantz). South African Journal of Botany 97: 184–190. 136. Opabode JT, Akinyemiju OA (2015) Tissue- and Organ-Specific Promoters for Expression of Heterologous Genes in Transgenic Cassava (Manihot Esculenta Crantz) Plants. Gene Technology 4:3. 137. Opabode JT, Akinkunmi JA, Akinyemiju OA (2013) Influence of Marker Genes on Physicochemical Properties of Starch Produced by Transgenic Cassava (Manihot esculenta Crantz) Plants . Canadian Center of Science and Education. 138. Osakabe Y, Watanabe T, Sugano SS, Ueta R, Ishihara R, Shinozaki K, Osakabe K (2016) Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants. Scientific Reports 6: 26685. 139. Ovalle T, Perea C, Pizarro M, Morante N, Ceballos H, Dufour D, Meike A, Becerra López-Lavalle A (2016) Elucidating high beta-carotene accumulation in cassava based on next generation sequencing. In: World Congress on Root and Tuber Crops, Abstract No. SP06-16, January 18–22, 2016, Nanning, Guangxi, China. 140. Paul C, Alejandro B, Adriana M, Mónica P, Roosevelt E, Juan M, Paula D, Camilo L, Willy MR, Joe T (2016) The potential of using biotechnology to improve cassava: a review. In Vitro Cellular & Developmental Biology - Plant 52:461–478. 141. Perlak FJ, Fuchs RL, Dean DA, McPherson SL, Fischhoff DA (1991) Modification of the coding sequence enhances plant expression of insect control protein genes. Proceedings of the National Academy of Sciences of USA 88:3324–3328. 142. Phạm Thị Nhạn, Nguyễn Hữu Hỷ (2015) Nghiên cứu chọn tạo giống sắn bằng phương pháp xử lý đột biến. chon-tao-giong-san-bang-phuong-phap-xu-ly-dot-bien-7294.html. 133 143. Preiss J (1998) Biosynthesis of starch and its regulation. In: Preiss J., ed. The biochemistry of plant 181-254. 144. Puigbò P, Guzmán E, Romeu A, Garcia-Vallvé S (2007) OPTIMIZER: Aweb server for optimizing the codon usage of DNA sequences. Nucleic Acids Research 35: W126–W131. 145. Raemakers CJJM (1993) Primary and cyclic somatic embryogenesis in cassava Manihot esculenta Crantz. PhD Thesis. Agricultural University Wageningen, The Netherlands 119. 146. Raemakers CJJM, Amati M, Staritsky G, Jacobsen E, Visser RGF (1993) Cyclic somatic embryogenesis and plant regeneration in cassava. Annals of Botany 71:289-294. 147. Raemakers CJJM, Bessembinder J, Staritsky G, Jacobsen E, Visser RGF (1993) Induction, germination and shoot development of somatic embryos in cassava. Plant Cell Tissue and Organ Culture 33:151-156. 148. Raemakers CJJM, Schavemaker CM, Jacobsen E, Visser RGF (1993) Improvements of cyclic somatic embryogenesis of cassava (Manihot esculenta Crantz). Plant Cell Reports 12:226-229. 149. Raemakers CJJM, Sofiari E, Jacobsen E, Visser RGF (1997) Regeneration and transformation of cassava. Euphytica 96: 153–161. 150. Raemakers CJJM, Sofiari E, Taylor NG, Henshwa E, Jacobsen E, Visser RGF (1996) Production of transgenic cassava plants by particle bombardment using luciferase activity as a selection maker. Molecular Breeding 2:339-349 151. Ral JP, Bowerman AF, Li Z, Sirault X, Furbank R, Pritchard JR (2012) Down-regulation of glucan water-dikinase activity in wheat endosperm increases vegetative biomass and yield. Plant Biotechnol Journal 10:871–82. 152. Regierer B, Fernie AR, Springer F, Perez-Melis A, Leisse A, Koehl K (2002) Starch content and yield increase as a result of altering adenylate pools in transgenic plants. Nature Biotechnology 20:1256–60. 153. Ren JS (1996) Cassava products for food and chemical industries: China. 134 In: Dufour DO, Brien GM and Best R, eds. Cassava floor and starch: progress in research and development. CIAT, Cali Colombia 48-54. 154. Roldán I, Wattebled F, Mercedes LM, Delvalle D, Planchot V, Jimenez S (2007) The phenotype of soluble starch synthase IV defective mutants of Arabidopsis thaliana suggests a novel function of elongation enzymes in the control of starch granule formation. Plant Journal 49:492–504. 155. Rösti S, Rudi H, Rudi K, Opsahl-Sorteberg HG, Fahy B, Denyer K (2006) The gene encoding the cytosolic small subunit of ADP-glucose pyrophosphorylase in barley endosperm also encodes the major plastidial small subunit in the leaves. Journal of Experimental Botany 57: 3619–3626. 156. Rudder S, Doohan F, Creevey CJ, Wendt T, Mullins E (2014) Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes. BMC Genomics 15:268. 157. Rybicki EP, Williamson A-L, Meyers A, Hitzeroth II (2014) Vaccine farming in capetown. Hum Vaccin 7:339–348. 158. Saithong T, Rongsirikul O, Kalapanulak S, Chiewchankaset P, Siriwat W, Netrphan S, Suksangpanomrung M, Meechai A, Cheevadhanarak S (2013) Starch biosynthesis in cassava: a genome-based pathway econstruction and its exploitation in data integration. BMC Systems Biology 7:75. 159. Salvador E, Steenkamp V, McCrindle CME (2014) Production, consumption and nutritional value of cassava (Manihot esculenta Crantz) in Mozambique: an overview. Journal of Agricultural Biotechnology and Sustainable Development 6:29–38. 160. Sambrook J, Fritsch E, Maniatis J (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 161. Sarria R, Torres E, Angel F, Chavarriaga P, Roca WM (2000) Transgenic plants of cassava (Manihot esculenta) with resistance to Basta obtained by Agrobacterium-mediated transformation. Plant Cell Reports 19:339–344. 135 162. Sarria R, Torres E, Balcazar N, Destefano L, Roca WM (1995) Progress in Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz). In: The cassava biotechnology network: proceedings of the second international scientific meeting, Bogor, Indonesia (August 1994) 241. 163. Sayre R, Beeching JR, Cahoon EB, Egesi C, Fauquet C, Fellman J, Fregene M, Gruissem W, Mallowa S, Manary M, Maziya-Dixon B, Mbanaso A, Schachtman DP, Siritunga D, Taylor N, Vanderschuren H, Zhang P (2011) The BioCassava Plus Program: Biofortification of Cassava for Sub-Saharan Africa. Annual Review of Plant Biology 62: 251–272. 164. Schöpke C, Franche C, Bogusz D, Chavarriaga P, Fauquet C, Beachy RN (1993) Transformation in Cassava (Manihot esculenta Crantz). In: Bajaj YPS, ed. Biotechnology in agriculture and forestry Vol. 23: plant protoplasts and genetic engineering IV. Springer, Berlin Heidelberg New York 273-289. 165. Schünemann D, Borchert S, Flügge U-I, Heldt HW (1993) ATP/ADP translocator from pea root plastids: comparison with translocators from spinach chloroplasts and pea leaf mitochondria. Plant Physiology 103:131–137. 166. Scofield GN, Hirose T, Gaudron JA, Upadhyaya NM, Ohsugi R, Furbank RT (2002) Antisense suppression of the rice sucrose transporter gene, OsSUT1, leads to impaired grain filling and germination but does not affect photosynthesis. Functional Plant Biology 29:815–26. 167. Shewmaker CK, Boyer CD,Wiesenborn DP, Thompson DB, BoersigMR, Oakes JV (1994) Expression of Escherichia coli glycogen synthase in the tubers of transgenic potatoes (Solanum tuberosum) results in a highly branched starch. Plant Physiology 104: 1159–66. 168. Singh P, Punjabi M, Jolly M, Rai RD, Sachdev A (2013) Characterization and expression of codon optimized soybean phytase gene in E. coli. Indian Jounal of Biochemistry & Biophysics 50:537-547. 169. Siritunga D, Sayre RT (2003) Generation of cyanogen-free transgenic cassava. Planta 217: 367–373. 136 170. Smidansky ED, Clancy M, Meyer FD, Lanning SP, Blake NK, Talbert LE, Giroux MJ (2002) Enhanced ADP-glucose pyrophosphorylase activity in wheat endosperm increases yield. Proceedings of the National Academy of Sciences of the United States 99: 1724–1729. 171. Smith AM, Neuhaus HE, Stitt M (1990) The impact of decreased activity of starch branching enzyme on photosynthetic starch synthesis in leaves of wrinkled-seeded peas. Planta 181(3):310-5. 172. Sofiari E (1996) Regenration and transformation in cassava Manihot esculenta Crantz. PhD Thesis. Agricultural University Wageningen, The Netherlands 136. 173. Stamp JA (1982) Somatic embryogenesis in cassava. Zeitschrift für Pflanzenphysiologie 105:183-187. 174. Stamp JA, Henshaw GG (1987) Secondary somatic embryogenesis and plant regeneration in cassava. Plant Cell Tissue and Organ Culture 10:227-233. 175. Stark DM, Timmerman KP, Barry GF, Preiss J, Kishore GM (1992) Regulation of the amount of starch in plant tissues by ADP-glucose pyrophosphorylase. Science 258: 287–292. 176. Su WW (2002) Cell culture and regeneration of plant tissues. In: Khachatourians GG, McHughen A, Scorza R, Nip WK, Hui YH, eds. Transgenic Plants and Crops. Marcel Dekker, New York: 151–176. 177. Szabados L, Hoyos R, Roca W (1987) In vitro somatic embryogenesis and plant regeneration of cassava. Plant Cell Reports 6:248-251. 178. Szydlowski N, Ragel P, Raynaud S, Roldán I, Montero M, Lucas MM (2009) Starch granule initiation in Arabidopsis requires the presence of either class IV or class III starch synthase. Plant Cell 21:2443–57. 179. Taylor N, Gaitan-Solis E, Moll T, Trauterman B, Jones T, Pranjal A, Trembley C, Abernathy V, Corbin D, Fauquet CM (2012) A high-throughput platform for the production and analysis of transgenic cassava plants. Tropical Plant Biology Journal 5 127–139. 137 180. Taylor NJ, Chavarriaga P, Raemakers K, Siritunga D, Zhang P (2004) Development and application of transgenic technologies in cassava. Plant Molecular Biology 56: 671–688. 181. Taylor NJ, Edwards M, Henshaw GG (1995) Production of friable embryogenic calli and suspension culture system in two genotypes of cassava. In: Second International Scientific Meeting of Cassava Biotechnology Network 11. Bogor. Indonesia: 229-240. 182. Taylor NJ, Munyaradzi VM , Rosa C, Thao H, Schöpke C, Fauquet CM. (2001) Production of embryogenic tissues and regeneration of transgenic plants in cassava (Manihot esculenta Crantz). Euphytica 120: 25–34. 183. Tessy J, Hock-Hin Y, Chiang-Shiong L (2001) Linamarin content and genetic stability of cassava plants derived by somatic embryogenesis. Euphytica 120: 7–13. 184. Tiessen A, Hendriks JH, Stitt M, Branscheid A, Gibon Y, Farré EM, Geigenberger P (2002) Starch synthesis in potato tubers is regulated by post- translational redox modification of ADP-glucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to the sucrose supply. Plant Cell 14(9): 2191-213. 185. Trần Công Khanh, Hoàng Kim, Nguyễn Hữu Hỷ (2011) Giống Sắn KM140 186. Vanderschuren H, Moreno I, Anjanappa RB, Zainuddin IM, Gruissem W (2012) Exploiting the combination of natural and genetically engineered resistance to cassava mosaic and cassava brown streak viruses impacting cassava production in Africa. PLoS ONE 7(9): e45277. https://doi.org/10.1371/journal.pone.0045277. 187. Waltz E (2016) Gene-edited CRISPR mushroom escapes US regulation. Nature 532:293. 138 188. Wang Z, Chen X, Wang J, Liu T, Liu Y, Zhao L (2007) Increasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic maize plants. Plant Cell Tissue Organ 88:83–92. 189. Webster GR, Teh AYH, Ma JKC (2017) Synthetic Gene Design—The Rationale for Codon Optimization and Implications for Molecular Pharming in Plants. Biotechnology and Bioengineering 114: 3. 190. Weichert N, Saalbach I, Weichert H, Kohl S, Erban A, Kopka J (2010) Increasing sucrose uptake capacity of wheat grains stimulates storage protein synthesis. Plant Physiology 152:698–710. 191. Weng L, Deng L, Lia F, Xiao G (2014) Optimization of the Cry2Aa gene and development of insect-resistant and herbicide-tolerant photoperiod- sensitive genic male sterile rice. Czech J. Genet. Plant Breed. 50: 19–25. 192. Wenham JE (1995) Post-harvest deterioration of cassava - A biotechnology perspective. Natural Resources Institute Chatham, UK FAO Plant Production and Protection: 130. 193. Wheatley CC, Chuzel G (1993) Cassava: the nature of the tuber and use as a raw material In: Macrae R, Robinson RK, Saddler MJ, eds. Encyclopedia of Food science, Food technology and Nutrition. Academic Press, San Diego, CA: 734-743. 194. Wiwithaphon S, Suttangkakul A, Leelapon O, Teerakathiti T, Boonseng O, Duerasor O, Paktanadechanon U, Chanvivattana Y (2013) Study of the Role of Cassava MFT Members in Storage Organ Development. International Graduate Research Conference. 195. Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Natural Biotechnology 33:1162–1164. 139 196. Wu G, Bashir-Bello N, Freeland SJ (2006). The synthetic gene designer: A flexible web platform to explore sequence manipulation for heterologous expression. Protein Expression and Purification Jounal 47:441–445. 197. Xue FY, Guo H, Hu Y, Liu R, Huang L, Lv H, Liu C, Yang M, Ma L (2016) Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production. BioMed Research International. 198. Zainuddin I, Schlegel K, Gruissem W, Vanderschuren H (2012) Robust transformation procedurefortheproduction of transgenic farmer-preferred cassava cultivars. Plant Methods 8:24. 199. Zeeman SC, Kossmann J, Smith AM (2010) Starch: Its Metabolism, Evolution, and Biotechnological Modification in Plants. Annual Reviews Plant Biology 61:209-234. 200. Zhang P, Phansiri S, Puonti-Kaerlas J (2001) Improvement of cassava shoot organogenesis by the use of silver nitrate in vitro. Plant Cell, Tissue and Organ Culture 67: 47–54. 201. Zhang P, Potrykus I, Puonti-Kaerlas J (2000) Efficient production of transgenic cassava using negative and positive selection. Transgenic Research 9:405–415. 202. Zhang Y, Chen X, Wang H, Xia Zhiqiang, Ling Peng, Wang Wenquan (2016) Annotation and validation of genes involved in photosynthesis and starch synthesis from a Manihot full-length cDNA library. Frontiers of Agricultural Science Engineering 3(4): 308–320. 203. Zhu ZP, Hylton CM, Rossner U, Smith AM (1998) Characterization of starch debranching enzymes in pea embryos. Plant Physiology 118: 561-590. 204. Zrenner R, Salanoubat M,Willmitzer L, Sonnewald U (1995) Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.). Plant Journal 7:97–107. 1 PHỤ LỤC 1. Sơ đồ cấu trúc vector pBI121 2. Cấu trúc vector tổng hợp pUCIDT-AMP:AGPopt AGPopt pUCICT-AMP: AGPopt Ref ID: 64938955 2,674,781,9 g/mole 2 3. Trình tự nucleotide của gen AGPopt tổng hợp nhân tạo 3 4. Sơ đồ biểu diễn bản đồ gen tổng hợp nhân tạo AGPase 4 5. Sơ đồ các bƣớc xác định hoạt tính enzim AGPase 5 6. Quy trình tái sinh cây sắn phục vụ chuyển gen Vật liệu: đỉnh chồi, thùy lá non Môi trường cảm ứng tạo phôi soma MS bổ sung: Picloram 12 mg l Môi trường cảm ứng tạo chồi MS bổ sung: BAP 0,3 mg l Môi trường tạo rễ MS không bổ sung kích thích sinh trưởng 6 7. Quy trình chuyển gen chỉ thị gus v o cây sắn 1. Chủng vi khuẩn/vector A. tumefaciens CV58/pGV2260/pBI121 2. Phương pháp và vật liệu lây nhiễm Lắc mẫu với huyền phù vi khuẩn với tốc độ 50 v/p; mảnh lá mầm 3. Thời điểm lây nhiễm và mật độ vi khuẩn Mật độ khuẩn tại OD 600 0,8 Thời điểm: sau khi cảm ứng trên môi trường tạo phôi soma 2 ngày 4. Nồng độ AS và thời gian lây nhiễm AS 100 μM; Thời gian lây nhiễm 15 phút 5. Thời gian đồng nuôi cấy thích hợp AS 150 μM; Thời gian đồng nuôi cấy 2 ngày 6. Loại kháng sinh và ngưỡng chọn lọc thích hợp Kanamycin 50 mg/l 7 8. Hoạt tính AGPase ở lá cây thuốc lá K326/AGPopt Hoạt tính AGPase Tỉ lệ % so với đối chứng ĐC 0.1 100% L1 0.137 137% L2 0.145 145% L3 0.137 137% L4 0.144 144% L5 0.140 140% L6 0.121 121% L7 0.153 153% L8 0.146 146% 9. Thành phần các môi trường nuôi cấy in vitro cơ bản Loại môi trường Thànhh phần MS lỏng MS (I-V) + Sucrose 30 mg/l; pH 5,8 MS đặc MS lỏng + 7,5 g Agar CIM MS (I - V) bổ sung picloram 12 mg/l, sucrose 30 g/l và 7,5g agar, pH 5,8 CEM MS (I - V) bổ sung BA 0,3 mg l, sucrose 30 mg l và 7,5g agar, pH 5,8 10. Thành phần các môi trường nuôi khuẩn Môi trường Thành phần YEB đặc 1 g/l yeast extract + 5 g/l beef extract + 5 g/l NaCl + 5 g/l tryptone + 5 g/l sucrose 15 g/l bacto agar, pH = 7,0 YEB lỏng 1 g/l yeast extract + 5 g/l beef extract + 5 g/l NaCl + 5 g/l tryptone + 5 g/l sucrose, pH = 7,0 LB lỏng 10 g/l Tryptone+ 5 g/l yeast Extract + 10 g/l NaCl, pH = 7,0 LB đặc 10 g/l Tryptone+ 5 g/l yeast Extract + 10 g/l NaCl, 15 g/l bacto agar, pH = 7,0 8 11. Thành phần sol I, sol II, sol III Sol I: 50 mM glucose, 10 mM EDTA, 25 mM Tris (pH 8,0) Sol II: 0,2 N NaOH, 1 % SDS Sol III: 3 M KOAc (pH 6,0)