A genotype independent transformation method of cassava Kirsten

A genotype independent transformation method of cassava Kirsten Jørgensen 1, Ivan Ingelbrect 2, Susanne Jensen 1, Evy Olsen 1, Charlotte Sørensen 1, Rubini Kannangara 1 and Birger Lindberg Møller 1. Cassava meeting Ghent july 2008 Slide

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 Cassava meeting Ghent july 2008 Slide 2

VITAMINS IN CASSAVA ROOTS COMPARED TO OTHER FOOD PRODUCTS Vitamin A Vitamin B I. U. /100 g Vitamin C mg/100 g Cassava tubers (peeled) 10 20 Gaplek 10 Tapioca flour Potatoes 40 30 -80 13 - 15 Potato flour Husked rice 100 -150 FAO – Cassava processing Cassava meeting Ghent july 2008 Slide 3

Protein content in cassava tubers compared to similar food products Bradbury and Holloway, 1994 Need to enhance nutritional level in cassava tubers Cassava meeting Ghent july 2008 Slide 4

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 – Mcol 22 and TME 60444 To be amenable for further breeding and the farmer – need to improve the transformation systems for all cassava lines Breeders selection and elite lines Cassava meeting Ghent july 2008 Slide 5

Cassava lines from IITA for embryo culture Elite lines Local name List with african names from dixon Amala TME 8 Cyanide /carotenoid Low to medium/no TME 12 Tokunbo Low to medium/no TME 13 MS-20 Low to medium/no TME 282 Alice local Low to medium/no TME 60444 - Medium to high/no I 95/0680 - Medium to high/no 92/0057 Slicass 6 (Sierra Leone) Low/no 96/0304 Low to medium/no I 91/02324 Nyerikobga Medium to high/no 96/0160 Nsansi Medium to high/no 97/4763 - Medium to high/no 01/1371 Medium to high/yes 01/1277 Medium to high/yes Cassava meeting Ghent july 2008 94/0330 Medium to high/yes Slide 6

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 Mcol 22 • Three lines from IITA: 01/1277; 01/1371; 94/0330 Cassava meeting Ghent july 2008 Slide 7 01/1371

Transformation system based on cotyledons from secondary embryos 25 x App. 10 days App. 3 weeks (10 mg picloram/l) (0. 1 mg BA/l) Induction of primary embryos Enlargement of nodes (10 mg BA/l) 15 x 10 x App. 3 weeks (6 mg 2, 4 D/l) Maturation of primary embryos Cassava meeting Ghent july 2008 Slide 8 Induction of secondary embryos (6 mg 2, 4 D/l) Maturation of cotyledons – ready for further culture or transformation

Cassava transformation 1 week Start of transformation Induction of shoot 2 weeks selection, 3 weeks Shoots emerging 4 -16 weeks after transformation Cassava meeting Ghent july 2008 Slide 9

Focus points for the basic culture and transformation Time schedule 17 days for induction of embryos on 2, 4 D 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 Cassava meeting Ghent july 2008 Slide 10

Succesful transformation of chosen elite lines Elite lines Transformation frequency TME 8 0. 20 % TME 12 3. 75 % TME 13 0. 45 % TME 60444 0. 75% 92/0057 1. 80% 96/0160 3. 50 % 97/4763 0. 50 % Cassava meeting Ghent july 2008 Slide 11

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 Cassava meeting Ghent july 2008 Slide 12

Cyanogenic glucosides Derived from amino acids: valine, isoleucine, tyrosin, phenylalanin Are found in more than 2000 plant species, e. g. : Cassava, almonds, eucalyptus, barley, wheat, rice, sorghum, cherry and apple. Cassava meeting Ghent july 2008 Slide 13

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. Cassava meeting Ghent july 2008 Slide 14

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 P 450 enzymes and the last step is activated by a glycosyl transferase Cassava meeting Ghent july 2008 Slide 15

Suppression of cyanogenic glucoside synthesis in cassava by RNA interference CYP 79 D 1 CYP 79 D 2 General structure of CYP 79’s exon 1 intron exon 2 1. Generation RNAi construct 5 S 3 Putative CYP 71 E D 2 D 1 intron D 1 D 2 ortholog RNA interference technolgy was used to downregulate the expression of CYP 79 D 1 and CYP 79 D 2. Putative acetone cyanohydrin 2 -hydroxy-2 -methylbutyronitrile UGT 85 B 300 bp from the distal and proximal end of EXON 2, respectively, was used in antisense and sense direction. ortholog The intron used was isolated from CYP 79 D 1. Cassava meeting Ghent july 2008 Slide 16

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 – Mcol 22 These lines have been transformed to downregulate the synthesis of cyanogenic glucosides. Cassava meeting Ghent july 2008 Slide 17 01/1371

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 Cassava meeting Ghent july 2008 Slide 18

Characterization of the first selected transgenic lines based on Mcol 22 Tubers • Mcol 22 –South American line • Linamarin content measured by LC-MS in 90 selected transgenic independent lines Cassava meeting Ghent july 2008 Slide 19 Leaves

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 – 35 S is generally considered as a constitutive expressed promotor, but this is not always the case. Cassava meeting Ghent july 2008 Slide 20

Transport 2 3 5 8 1 6 B Apex 7 9 10 C Girdling experiments demonstrate transport of cyanogenic glucosides from the shoot apex down the stem Cassava line 4 AS 17 A AS 17 B Mcol 22 μmol HCN per g fresh weight D Cassava meeting Ghent july 2008 Slide 21 ND 59 0. 5 0. 6 15 Stem below incision E 26 Stem above incision A 5 th leaf 0. 3 0. 2 6 th leaf 29 19 55

Cell specificity of the E 35 S promotor in cassava tubers Gus expression driven by E 35 S in transgenic cassava Mcol 22. 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. 0 10 11 12 12 12 14 15 19 20 25 25 28 28 31 32 36 42 Cassava meeting Ghent july 2008 Slide 22

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 CYP 71 promotor • Higher expressed than CYP 79 A 1 in Sorghum Cassava meeting Ghent july 2008 Slide 23

Characterization of the 1. generation of acyanogenic cassava lines Phenotype 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 Cassava meeting Ghent july 2008 Slide 24 A Transgenic 0, 3% of wildtype B Wildtype C Transgenic 0, 4% of wildtype

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 soil – indicating that even without nutrients in the growth medium - they convert to wild-type growth in greenhouse Cassava meeting Ghent july 2008 Slide 25

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 Cassava meeting Ghent july 2008 Slide 26

Cyanogenic glucosides act as defense compounds Plant cell Vacuole Linamarin/Lotaustralin (Cyanogenic glucosides) Linamarase (b-glucosidase) Hydroxynitrile lyase Glucose Cyanohydrin p. H>5, T>35°C Ketone Cyanide (poisonous gas) Cassava meeting Ghent july 2008 Slide 27 Apoplast, Laticifers

Dhurrin degradation in Sorghum Wounding Turnover Piotrowski et al. , PNAS 2007 Cassava meeting Ghent july 2008 Slide 28 Detoxification

Glucosinolate/myrosinase system TFP Cassava meeting Ghent july 2008 Slide 29 TFP ESP TFP

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 Visualised with Fast BB assay Cassava meeting Ghent july 2008 Slide 30 - see the poster of Rubini Kannangara

Biofortification of cassava – Transformation Current constructs Constructs African lines Acyanogenic lines RNAi antisense CYP 79 D 1 & D 2 E 35 S: RNAi CYP 79 D 1 & D 2 01/1277 01/1371 CYP 71 promotor with GUS CYP 71 E 7: GUS 01/1277 01/1371 CYP 71 promotor in front of the RNAi antisense CYP 79 D 1 & D 2 CYP 71 E 7: RNAi CYP 79 D 1/D 2 01/1277 TME 12 01/1371 96/0160 Enhanced protein content Patatin promoter in front of GUS TME 12 96/0160 Patatin promoter in front of patatin 2 PB 33: PAT 2 TME 12 96/0160 Patatin promoter in front of patatin targeted to the amyloplast PB 33: GUS PB 33: GBSS-PAT 2 TME 12 96/0160 Patatin promoter in front of patatin targeted to the amyloplast and harboring a starch binding domain PB 33: GBSS-PAT 2 -SBD TME 12 96/0160 Cassava meeting Ghent july 2008 Slide 31

CYP 79 D 1 og D 2 CYP 71 Regulatoric elements UGT Biosynthesis Tissue localisation Microarray Cassava Cyanogenic glucosides Degradation Bioactivation -glucosidase – linamarase Cassava meeting Ghent july 2008 Slide 32 Transport Other degradation products nitrilases Modifying proteins

Faculty of Life Sciences Institute of Plant Biology Acknowledgement 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 Cassava meeting Ghent july 2008 Slide 33 Funded by Danida and the Research council for technique and production