_Leta Tulu Bedada presentationFinal.pptx

Genetic engineering of maize for drought tolerance

1. Gene Transfer Promising to Enhance Drought Resilience in Tropical Maize in East and Central Africa Leta Tulu Bedada , Miccah S. Seth, Steven M. Runo and Richard O. Oduor National Conference on Economic Research for Development June 10-11, 2017, Axum Hotel Mekele , Ethiopia

2. Maize is a staple food crop for millions of Africans More than 300 million people in Eastern and Southern Africa depend on maize (Edmeades, 2008). Potential of producing more than 10 t/ha, actual mean productivity not more than 2 t/ha Drought has been a major limitation to higher productivity of maize in the tropics. Grain yield losses of as high as 70% has been reported while complete crop losses are also common depending the severity of drought Farmers loose return to the investments he did in productivity enhancing inputs

3. Increasing frequency, intensity, and duration of drought expected to continue with the current situation of climate change causing crop failures and hunger crisis.

4. We need to develop drought resilient varieties adapted to the changing environment. Contribution of conventional breeding : inadequate because of limited genetic diversity in maize for tolerance to drought. Limited genetic diversity and the complex biochemical response plants have to drought stress (Anna Hardy, 2010). Introgression of genomic portions (QTLs) involved in stress tolerance: Genetic drag is a problem ( Bhatnagar-Mathur , et al ., 2008).

5. 4 Genetic engineering : Makes better option to develop drought tolerant crop circumventing limitation of conventional breeding Insertion of a single gene that codes for a specific protein involved in drought stress response path-ways. Damaging effect of drought: interfering with different physiological aspects of the plant in two ways Contribute to the decline in cytokinin level interfering with its biosynthesis in the roots which triggers leaf senescence(van Staden et al. , 1988). Plants produce large quantities of reactive oxygen species (ROS) as a response to drought stresses.

6. 05/03/2025 5 Drought induces pre-mature leaf senescence Maize launches leaf senescence as an adaptation strategy by reducing canopy size. The earliest significant change: Breakdown of the chloroplast Carbon assimilation is replaced by catabolism of chlorophyll and macromolecules It is undesirable in food crops

7. 05/03/2025 6 Leaf Senescence is genetically regulated SDGs : Senescence down regulated genes, Include genes involved in photosynthesis, SAGs : Senescence activated genes, genes encoding RNases , proteinases, lipases are up regulated ( Gan and Amasino , 1997 )

8. 05/03/2025 7 Depends on physiology of phytohormone : Cytokinin Exogenous application of cytokinin retards senescence Cytokinin promotes cell division, leaf expansion, accumulation of chlorophyll Directly or indirectly inhibits transcription of SAGs encoding RNases , proteinases, lipases (Buchanan-Wollaston et al ., 1997 ). Approaches to delay leaf senescence

9. 05/03/2025 8 Discovery of ipt gene in Agrobacterium ( Akiyoshi et al. 1984) Tumer inducing plasmid of Agrobacterium bearing gene for cytokinin biosynthesis on the T-DNA . Molecular-Genetic approach to delay drought induced leaf senescence in maize To enable it defend itself by enhancing endogenous cytokinin production through genetic engineering using ipt gene

10. PATHWAY OF CYTOKININ BIOSYNTHESIS

11. IPT gene delays leaf senescence Over expression of the ipt gene in plants led to elevated foliar cytokinin concentrations and delayed leaf senescence ( Rivero et al ., 2007; Peleg et al ., 2011). This created interest to investigate if ipt gene can be useful in enhancing drought tolerance in farmers preferred and locally adapted tropical maize genotypes .

12. Plants produce large quantities of reactive oxygen species (ROS) as a response to drought stresses. Plants can protect themselves against harmful ROS by undergoing a variety of biochemical and physiological responses. One of such responses is the generation of antioxidant enzyme like peroxiredoxin2, which scavenges ROS and convert them to harmless molecules, thereby protecting plant cell membranes and DNA from damage.

13. Xerophyta viscosa , a resurrection plant native to South Africa Can be dehydrated to an air dry state but can be rehydrated upon rewatering , mechanism controlled by a number of genes

14. The XvPrx2 gene is stress-inducible in response to abiotic stresses and it has been revealed that XvPrx2 homologues exist within the X. viscosa proteome ( Govender et al., 2016). In vitro DNA protection assay showed that, in the presence of XvPrx2 DNA protection occurred . In vitro assays have also revealed maximum activity of the XvPrx2 with DTT(DL- Dithiothreitol ) as electron donor and H 2 O 2 as substrate ( Govender et al., 2016), implying that the gene is responsible for managing the ROS generated by plants under stress. Objective: To transform tropical maize inbred lines, CML144 and CML216, using XvPrx2 and ipt gene constructs, respectively, and to evaluate the performance of transformed maize lines against their non-transgenic counterparts under drought stress condition.

15. MATERIALS AND METHODS

16. Association for Strengthening Agricultural Research in East and Central Africa (ASARECA) embarked on enhancing drought tolerance in farmers’ preferred and locally adapted maize germplasm in East and central Africa through genetic engineering. Temperate maize adapts poorly to tropical environment Important genes known for conferring drought tolerance were accessed Locally adapted germplam were collected from five East and Central African countries (Ethiopia, Sudan, Kenya, Tanzania and Uganda) Brought to the Biosafety Level II Plant Transformation facility at Kenyatta University in Nairobi, Kenya to be targeted for genetic transformation Five Ph.D students, one from each of the five countries, were recruited to work on genetic transformation of the maize germplasm from their respective countries.

17. A grobacterium- mediated transformation of CML216 and CML144 maize inbred lines

18. Molecular cloning and selection of selectable marker Limitations to Genetic Engineering : The presence of antibiotic resistance marker genes may complicate future commercialization Selectable markers selected for safety to the environment and consumers. We, sub-cloned the expression cassettes to the binary vector, pNOV2819,

19. 05/03/2025 18 Sub-cloning ipt and XvPrx2 gene gene in to P NOV 2819

20. E. colie colonies transformed with ligation product

21. Plasmid DNA from five transformant colonies digested with Asc I and Hind III 2Kb 3 4 5 7 8 1Kb ladder

22. 05/03/2025 21 Transformation of Agrobacterium strain EHA 101 with plasmid DNA M C 1 C 2 C 3 C 4 C 5 C 6 C 7 C 8 2 kb Analysis of 8 EHA101 colonies transformed with pNOVIPT1 construct through colony PCR. M; 1 kb marker, C 1 to C 8 are EHA101 colonies ranging from 1 to 8 .

23. PNOV2819IPT1 map drawn using VectorNTI software

24. PSARK RB IPT NosT CMPS PMI NosT LB Linear map of the expression cassettes (TDNAs) XvPsap1 XvPrx2 osTN CMPS PMI NosT LB RB

25. 05/03/2025 24 Mannose based selection system as used to select for transformed cells Biochemical reaction catalyzed by phosphomannose isomerase coded by the manA gene

26. 05/03/2025 25 A-G: Transformation and regeneration profile of transgenic tropical maize using Agrobacterium C : Selection D : Regeneration A : Cocultivation B : Resting E : Acclimatization F : Putative in peatmoss G: P SARK ::IPTCML216

27. Molecular analysis of regenerated putative transgenic plants PCR: to confirm presence of the transgene Southern blot: to determine number of copies of the transgene RT-PCR: to confirm expression of the transgene

28. RESULTS AND DISCUSSSION

29. 05/03/2025 28 M E 1 E 2 E 3 E 4 E 5 E 6 E 7 E 8 E 9 +Cr WT W 2Kbp PCR analysis of putative T P SARK ::IPTCML216 plants using primers specific to the P SARK ::IPT::NOST expression cassette.

30. PCR detection of transgenic CML144 maize plants using PMI gene specific primers

31. 05/03/2025 30 PCR analysis of pSARK ::IPT T0 plants using primers specific to pmi gene in 19 independent events of Melkassa-2 M E1 E2 E3 E4 E5 E6 E7 E8 E9 M E10 E11 E12 E13 E14 E15 E16 E17 E18 E19 +Cr WT W 0.5kbp 0.5kbp

32. 05/03/2025 31 Southern blot analysis. Ten micrograms of genomic DNA extracted from young leaf tissues of T1 plants from nine independent PSARK::IPT CML216 events (E1-E9) was digested with Hind III restriction enzyme and hybridized with the pmi probe. WT: G enomic DNA from non-transformed maize inbred line CML216 plants taken as a negative control. +Cr: 20 ng of plasmid DNA digested with HindIII used as positive control. E 1 E 2 E 3 E 4 E 5 E 6 E 7 E 8 E 9 WT +Cr

33. 05/03/2025 32 Implications of molecular analysis Previous studies showed tropical maize is resistant to Agrobacterium infection and recalcitrant for regeneration. The study practically demonstrated that Agrobacterium -mediated transformation technique can be applied to elite tropical maize genotypes having desirable agronomic traits for commercial production and breeding purposes.

34. 05/03/2025 33 EVALUATION OF TRANSGENIC LINES FOR TOLERANCE TO DROUGHT STRESS

35. 05/03/2025 34 Drought assay Transgenic Wild type Wild type

36. 05/03/2025 35 Drought assay: Optimization experiment A Transgenic Wild Type Wild Type

37. 05/03/2025 36 Responses of 8 weeks old transgenic and wild type plants to 3 weeks drought stress

38. Six week old transgenic and wild type CML144 maize plants under different stages of drought stress. 1 week stress 3 weeks stress 2 weeks stress 6 hours after rewatering 4 days after rewatering

39. RT-PCR on transgenic and wild type CML144 maize .

40. 05/03/2025 39 L eaf relative water content measured in 8 weeks old transgenic and wild type plants during drought stress assay in the glasshouse (CML216) sult a a a a a b b b b b Means labelled with different letters are significantly different according to LSD test at 5% probability level

41. Leaf RWC as affected by drought stress and recovery after re-watering in transgenic maize and wild type CML144 maize plants.

42. 05/03/2025 41 A B C D Chlorophyll level measured during watering, drought and rewatering experiments in the glass house, A : Total chlorophyll, B : Chlorophyll a, C : Chlorophyll b, D : Total carotenoids . Values are mean±SE (n=12) a a b b a a a b b b a a b b a b a b

43. Effect of drought stress and recovery re-watering on chlorophyll content in transgenic and wild type CML144 maize plants.

44. 05/03/2025 43 Four months old t ransgenic and wild type plants growing in the glass house 0.5kb M +Cr RT- P 1 P 2 P 3 P 4 WT 0. 5kb RT-PCR analyses of ipt gene expression in four drought stressed PSARK::IPT CML216 transgenic plants(P1-P4). The same cDNA amplified with Actin primers (lower panel) used as internal control Analysis of gene expression Wild Type Transgenic

45. 05/03/2025 44 Phenological and agronomic characters of wild type and transgenic plants recorded after watering/drought/ rewatering in the glasshouse Mean values followed by different letter for a particular parameter are significantly different from each other according to LSD test at 5% probability level. Values are mean±SE (n=4)

46. 05/03/2025 45 Seed yield and major yield components of wild type and transgenic plants recorded after watering/drought/ rewatering in the glasshouse *Weight of 32 seeds produced per plant Mean values followed by different letters for a parameter are significantly different from each other according to LSD test at 5% probability level. Values are mean±SE (n=4)

47. 05/03/2025 46 Ears harvested from wild type (A) and PSARK::IPTCML216 transgenic plants (B) after watering/drought/ rewatering treatments. . Wild type plants suffered from drought which resulted in wide anthesis-silking interval and poor receptivity of the late coming silks which caused poor seed set.

48. By 2050 crop yields diminish further by 10-20% owing to higher temperature and reduced rainfall in Africa (Jones and Thornton, 2003). By this time the African population is expected = 2 billion, and food production is projected to increase by more than 70%. Maize has been identified as a priority crop for enhancing adaptation to this changing environment ( Lobell et al ., 2008 and 2011). Demand of maize will be doubled Drought tolerant maize ranks higher in the list of technologies that are expected to overcome the challenges of climate change. CONCLUSION AND RECOMMENDATION

49. Conventional breeding P layed significant role in developing improved crop varieties which have contributed to better food production. Insufficient to develop drought tolerant variety because of limited genetic diversity (Hardy, 2010; Shiferaw et al ., 2011) and lack of suitable selection criteria for tolerance to drought stress ( Nigussie et al ., 2002). We cannot totally rely on conventional breeding as it cannot yield faster solutions to the problems ahead. Conventional breeding cannot stand alone to meet the challenges ahead. It has long been concluded that maize has to undergo genetic modification to adapt to the changing environment ( Shiferaw et al ., 2011).

50. 05/03/2025 49 Gene transfer promising to enhance drought resilience in tropical maize Given proper attention genetic engineering can be applied under African condition by African scientists to complement conventional breeding. Much expected from Ethiopian Universities Trained manpower Building training facilities (Laboratories) Universities are expected to be centre for scientific discoveries and inventions

51. 05/03/2025 50 THANK YOU FOR LISTENING