A genotype independent transformation method of cassava

1. A genotype independent transformation method of cassava Kirsten Jørgensen1, Ivan Ingelbrect2, Susanne Jensen1, Evy Olsen1, Charlotte Sørensen1, Rubini Kannangara1 and Birger Lindberg Møller1.

2. Breeding traits Nutritional value: Vitamin A – has been done by breeding Protein content need to be enhanced – protein source wild cassavalines other sources Cyanogenic glucoside content – need to be treated correct controlled by molecular breeding Iron and zinc

3. VITAMINS IN CASSAVA ROOTS COMPARED TO OTHER FOOD PRODUCTS FAO – Cassava processing

4. Protein content in cassava tubers compared to similarfoodproducts Bradbury and Holloway, 1994  Need to enhancenutritionallevel in cassava tubers

5. Improving traits – need for applicable techniques Traditional breeding Time consuming Irregular flowering and few seeds complex genetics Molecular breeding Transfer of single traits consumer and legislative approval Transformation methods developed first for model lines – Mcol22 and TME60444 To be amenable for further breeding and the farmer – need to improve the transformation systems for all cassava lines Breeders selection and elite lines

6. Cassava lines from IITA for embryo culture List with african names from dixon

7. Cassava with enhanced carotenoid content in the tubers • Include cassava lines containing the precursof for vitamin A • Carotenoid tubers can be found in wild cassava - this trait has been breed into elite lines • Three lines from IITA: 01/1277;01/1371;94/0330 Mcol22 01/1371

8. 25x App. 3 weeks Induction of primary embryos Enlargement of nodes (10 mg BA/l) 15x 10x App.3 weeks Maturation of primary embryos Induction of secondary embryos (6 mg 2,4D/l) Transformation system based on cotyledons from secondary embryos App. 10 days (10 mg picloram/l) (0.1mg BA/l) (6 mg 2,4D/l) Maturation of cotyledons – ready for further culture or transformation

9. 1 week Start of transformation Induction of shoot selection, 2 weeks 3 weeks Shoots emerging 4 -16 weeks after transformation Cassava transformation

10. Focus points for the basic culture and transformation Time schedule 17 days for induction of embryos on 2,4D and 10 days for maturation of embryos to develop cotyledons Transfer of material need to be according to the developmental stage and cannot always follow a fixed time schedule. Cutting technique all lines cannot be cut into small pieces do not damage the tissue with the tweezers Sucrose content 4% sucrose is normally used but some lines are preferentially maintained at 2% sucrose because it reduces the amount of calli

11. Succesful transformation of chosen elite lines

12. Molecular breeding • Down regulation of cyanogenic glucosides • Meet the requirement of vitamin A • Enhance the protein content • Use patatin from potato • Identify a suitable candidate from wild cassava lines

13. Cyanogenic glucosides Derived from amino acids: valine, isoleucine, leucine, tyrosin, phenylalanin Are found in more than 2000 plant species, e.g.: Cassava, almonds, eucalyptus, barley, wheat, rice, sorghum, cherry and apple.

14. Synthesis of cyanogenic glucosides in cassava The youngest unfolded leaves are most active in cyanogenic glucoside synthesis Transport of cyanogenic glucosides from the top to the tubers Cyanogenic glucosides mainly stored in the tubers. The tubers synthesize cyanogenic glucosides in the outer cell layers.

15. BIOSYNTHESIS Bioynthesis of cyanogenic glucosides - linamarin and lotaustralin from valine and isoleucine, respectively • All genes encoding the enzymes in the pathway has been identified in our laboratorium • The entire pathway - the conversion of valine and isoleucine into cyanogenic glucosides takes place in a metabolon of two P450 enzymes and the last step is activated by a glycosyl transferase

16. 1.Generation RNAi construct RNA interference technolgy was used to downregulate the expression of CYP79D1 and CYP79D2. 300 bp from the distal and proximal end of EXON2,respectively, was used in antisense and sense direction. The intron used was isolated from CYP79D1. General structure of CYP79’s exon1 exon2 intron 35S D2 D1 intron D1 D1 D2 Suppression of cyanogenicglucosidesynthesis in cassava by RNA interference CYP79D1 CYP79D2 Putative CYP71E ortholog Putative UGT85B ortholog acetone cyanohydrin 2-hydroxy-2-methylbutyronitrile

17. Cassava with enhanced carotenoid content in the tubers • Carotenoid tubers has been found in wild cassava and breed into elite lines at IITA • To prevent loss of carotenoids due to processing for cyanogenic glucoside removal – These lines have been transformed to downregulate the synthesis of cyanogenic glucosides. Mcol22 01/1371

18. Acyanogenic carotenoid lines • Content of cyanogenic glucosides in a line containing a high carotenoid content measured by LC-MS in leaves • The content in the first transformed lines varies from 0% to 180% of the wildtype content

19. Characterization of the first selected transgenic lines based on Mcol22 Tubers Leaves • Mcol22 –South American line • Linamarin content measured by LC-MS in 90 selected transgenic independent lines

20. Discrepancy shoot and tuber Downregulation is more easily achived in leaves compared to tubers Could be due to: Transport – restricted amounts of cyanogenic produced in leaves are effectively transported to the tubers and accumulates Transcribtional regulation – cassava regulates the content of cyanogenic glucosides – and does it differently in leaves and tubers Promotor specificity – 35S is generally considered as a constitutive expressed promotor, but this is not always the case.

21. 2 3 1 B C Apex 5 4 6 D 7 9 8 10 A E Transport Girdling experiments demonstrate transport of cyanogenic glucosides from the shoot apex down the stem

22. 0 10 11 12 15 12 12 14 19 20 25 25 28 31 32 28 42 36 Cell specificity of the E35S promotor in cassava tubersGus expression driven by E35S in transgenic cassava Mcol22. Numbers represent the linamarin content in % of the wildtype measured by LC-MS. Synthesis of cyanogenic glucosides proceeds in the outer layer of the tuber.

23. Choice of a new specific promotor for cassava Specific promotor • The down-regulation in tubers is not as high as in the shoot tissue Isolated CYP71 promotor • Higher expressed than CYP79A1 in Sorghum

24. A B C Characterization of the 1.generation of acyanogenic cassava linesPhenotype in vitro The highly downregulated lines exibit a specific phenotype on low salt media The stem is long and slender, the number of leaves reduced, and short, thick roots Transgenic 0,3% of wildtype Wildtype Transgenic 0,4% of wildtype

25. No phenotype in green house The highly downregulated lines has a wildtype phenotype when grown in soil Growth chararistics identical to wild-type both in pure sand and soil – indicating that even without nutrients in the growth medium - they convert to wild-type growth in greenhouse

26. Cyanogenic glucosides Ancient defence compound Primarily an phyto-anticipin Role in primary metabolism Nitrogen storage Nitrogen recovered as ammonia Examples from Sorghum – nitrilases has a role in turnover of cyanogenic glucosides in intact cells Brassica – the glucosinolate system – the modifiers known are to direct the formation of different degradation products

27. Apoplast,Laticifers Linamarase (b-glucosidase) Glucose Cyanohydrin Hydroxynitrile lyase pH>5, T>35°C Ketone Cyanide (poisonous gas) Cyanogenic glucosides act as defense compounds Plant cell Vacuole Linamarin/Lotaustralin (Cyanogenic glucosides)

28. Dhurrin degradation in Sorghum Wounding Turnover Detoxification Piotrowski et al., PNAS 2007

29. Glucosinolate/myrosinase system TFP TFP ESP TFP

30. Linamarase activity is localised differently in tubers of high- and low-cyanide cassava varieties No substrate With 6-BNG Orange Flesh: High CN variety Mbundumali: Low CN variety Visualisedwith Fast BB assay - see the poster of RubiniKannangara

31. Biofortification of cassava – Transformation Current constructs

32. CYP79D1 og D2 CYP71 Regulatoric elements UGT Biosynthesis Tissue localisation Microarray Cassava Cyanogenic glucosides Degradation - Bioactivation Transport Other degradation products -glucosidase – linamarase nitrilases Modifying proteins

33. Acknowledgement Faculty of Life Sciences Institute of Plant Biology Birger Lindberg Møller Susanne Jensen Evy Olsen Charlotte Sørensen Søren Bak Steen Malmmose Institute of Natural Sciences Carl Erik Olesen CIAT Martin Fregene IITA Alfred Dixon Ivan Ingelbrecht DSMZ Stephan Winther Funded by Danida and the Research council for technique and production