Report to The Maize Trust 21 April 2008 Prof. Jennifer A Thomson University of Cape Town
Xerophyta viscosa Hydrated Dehydrated
Genes being used = Stress associated protein Xv SAP1 No similar genes recorded Codes for membrane protein Xv Ald1 = Aldose reductase converts glucose to sorbitol (osmoprotectant) Xv Prx2 = Peroxiredoxin ie an antioxidant
Xv SAP1 dehydration R in arabidopsis Wild type 6K 21J 10C Heat treatment
Xv SAP1 dehydration R in tobacco - Water Controls Transgenics
Xv Ald1 dehydration R in arabidopsis WT VT L12 L8 Dehydrated transgenic Arabidopsis plants (bottom) Control (top) expressing aldose reductase
Inducible promoter ie one that switches on the gene under dehydration 2 kb Promoter luc nos nos 1.5 kb Promoter luc 1kb promoter nos luc Different lengths of Xv Sap promoter driving the luciferase ( luc ) reporter gene
Promoter contains the following elements: meristem activation = CCGTCC endosperm expression = GTCAT auxin responsive = AACGAC circadian control elements = CAANNNATC drought inducibility = CAACTG heat stress responsiveness = AAAAAATTTC
C A B E F D Three promoter constructs transformed into tobacco showing shoot emergence (A) to whole plant flowering (F)
2 kb 1.5 kb 1 kb WT Tobacco transformants growing under bialaphos selection
100bp+ve -ve – veD 1 2 3 4 5 6 7 8 9 10 11 12 2kb 1.5kb 1kb 1.2kb PCR-DNA screening for luciferase of positive transformants
1 2 3 4 5 2kb 1kb 2kb 1 2 3 4 5 6 2kb 1kb 1 2 3 2kb 1kb 1kb Southern blot DNA screening for luciferase of positive transformants
Luciferase activity in transgenic tobacco transformed with 2 kb promoter under dehydration treatment 100 10 RLU/μg RWC Relative Water 50 5 Content 0 0 Relative Light Unit Day 0 Day 3 Day 6 Day 8 (RLU/μl) Dehydration period Luciferase activity highest with 2 kb promoter
Luciferase activity in transgenic tobacco transformed with 1.5 kb promoter under dehydration treatment 100 4 RLU/μg 3 RWC Relative Water Content 50 2 1 0 0 Relative Light Unit (RLU/μl) Day 0 Day 3 Day 6 Day 8 Dehydration period Luciferase activity intermediate with 1.5 kb promoter
Luciferase activity in transgenic tobacco transformed with 1 kb under dehydration treatment 100 2 RLU/μg 1.5 RWC Relative Water 50 1 Content 0.5 0 0 Relative Light Unit (RLU/μl) Day 0 Day 3 Day 6 Day 8 Dehydration period Luciferase activity lowest with 1 kb promoter
C A B E D Transformation and regeneration of maize carrying 2 kb promoter driving luciferase reporter gene
1 2 3 4 5 6 7 8 9 1.2kb PCR-DNA analysis of transgenic maize plants (lane 1 = positive control, lanes 3 & 4 non-transgenic negative controls)
Future work Analysis of Black Mexican sweet corn tissue culture carrying 2 kb promoter driving luciferase Stress treatments of transgenic maize plants Transformation of maize with 2 kb promoter driving Xv Sap1 , Xv Ald1 and Xv Prx2 Stress treatments of transgenic maize plants
Monsanto / CIMMYT / AATF Consortium funded by Gates Foundation Monsanto drought R maize CIMMYT (Africa) for introgression into African maize varieties Intellectual property managed by AATF All farmers / seed companies in sub- Saharan Africa except in South Africa only subsistence farmers
Problem no. 1 = maize streak virus (MSV) Problem no. 2 = biosafety regulations
Maize Streak Disease • Leaf chlorosis • Deformed cobs • Stunting • Yield losses of up to 100% • Death
Klerksdorp, NorthWest; April 2005
Comparison of asymptomatic plants with controls, Day 20 pi Non-transgenic Transgenic
Biosafety Regulations No African developed transgenic crops tested for biosafety approval to date Will be unable to commercialise any such crops without this approval How to proceed?
Conclusions Use of genes from resurrection plant unique Xv SAP1 and its promoter are unique Xv Prx2 is unique Single gene MSV resistance unique – can be used to introgress into drought tolerant lines Applying for permission to conduct field trials (ca. US$10,000) – funded by DST and ? Gates Foundation 1 st biosafety analysis of African-developed transgenic crop
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