Report to The Maize Trust 21 April 2008 Prof. Jennifer A Thomson - - PowerPoint PPT Presentation

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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

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Report to The Maize Trust

21 April 2008

Prof. Jennifer A Thomson

University of Cape Town

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Hydrated Dehydrated

Xerophyta viscosa

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Genes being used

Xv SAP1 = Stress associated protein No similar genes recorded Codes for membrane protein Xv Ald1 = Aldose reductase converts glucose to sorbitol (osmoprotectant) Xv Prx2 = Peroxiredoxin ie an antioxidant

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Xv SAP1  dehydrationR in arabidopsis

Wild type 6K 21J 10C Heat treatment

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Xv SAP1  dehydrationR in tobacco

Controls Transgenics

Water

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Xv Ald1  dehydrationR in arabidopsis

Dehydrated transgenic Arabidopsis plants (bottom) Control (top) expressing aldose reductase

WT VT L8 L12

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Inducible promoter ie one that switches on the gene under dehydration

luc

nos

2 kb Promoter luc

nos

1.5 kb Promoter luc

nos

1kb promoter

Different lengths of Xv Sap promoter driving the luciferase (luc) reporter gene

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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

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A B C D E F

Three promoter constructs transformed into tobacco showing shoot emergence (A) to whole plant flowering (F)

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2 kb WT 1 kb 1.5 kb

Tobacco transformants growing under bialaphos selection

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1.2kb

100bp+ve -ve –veD 1 2 3 4 5 6 7 8 9 10 11 12

2kb 1kb 1.5kb

PCR-DNA screening for luciferase of positive transformants

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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

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Luciferase activity in transgenic tobacco transformed with 2 kb promoter under dehydration treatment

50 100 Day 0 Day 3 Day 6 Day 8 Dehydration period RWC 5 10 RLU/μg Relative Water Content Relative Light Unit (RLU/μl)

Luciferase activity highest with 2 kb promoter

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Luciferase activity in transgenic tobacco transformed with 1.5 kb promoter under dehydration treatment

50 100 Day 0 Day 3 Day 6 Day 8 Dehydration period RWC 1 2 3 4 RLU/μg Relative Water Content Relative Light Unit (RLU/μl)

Luciferase activity intermediate with 1.5 kb promoter

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Luciferase activity in transgenic tobacco transformed with 1 kb under dehydration treatment

50 100 Day 0 Day 3 Day 6 Day 8 Dehydration period RWC 0.5 1 1.5 2 RLU/μg Relative Water Content Relative Light Unit (RLU/μl)

Luciferase activity lowest with 1 kb promoter

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C D E A B

Transformation and regeneration of maize carrying 2 kb promoter driving luciferase reporter gene

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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)

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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

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Monsanto / CIMMYT / AATF Consortium

funded by Gates Foundation

Monsanto droughtR 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

nly subsistence farmers

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Problem no. 1 = maize streak virus (MSV) Problem no. 2 = biosafety regulations

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Maize Streak Disease

Leaf chlorosis
Deformed cobs
Stunting
Yield losses of

up to 100%

Death

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Klerksdorp, NorthWest; April 2005

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Non-transgenic Transgenic Comparison of asymptomatic plants with controls, Day 20 pi

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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?

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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

 1st biosafety analysis of African-developed

transgenic crop