14 th August 2014 Introduction Cowpeas (Kunde) grown for seeds, - - PowerPoint PPT Presentation

In vitro regeneration and Agrobacterium mediated transformation of Kenyan cowpeas (Vigna unguiculata) Rachel Okeyo_Ikawa 3rd Annual Biosafety Conference 14th August 2014

Introduction

 Cowpeas (Kunde) – grown for seeds, leaves and pods.  Av. Production: Kenya – 0.3T / Ha, USA – 1.9T / Ha

(USDA 2010).

 Eastern province – 90% of national production

Drought Source:cherthollowfarm.com Bruchids Diseases (cowpea mosaic virus) Source: apsnet.org Maruca Source: iita.org

Cow pea production constraints

Source: storedgrain.com.au

Brief background on transformation

Crown gall disease

Source: wiki.bugwood.org

Mode of action of Agrobacterium tumefaciens

Source: Nepadbiosafety.net

• Bacterial DNA is transferred

to plant chromosome causing crown gall disease

Justification

 There is need to improve yields on existing cowpea varieties by enhancing tolerance to drought, diseases and insects / pests.  To accomplish this via transformation, an efficient plant regeneration and transformation protocol needs to be established.

Objectives

Main Objective To establish an efficient tissue culture regeneration and transformation protocol for Kenyan cowpeas. Specific objectives i. To establish a tissue culture regeneration protocol with various auxins and cytokinins. ii. Transformation of cowpea cotyledonary nodes with different strains of agrobacterium tumefaciens harboring the GUS reporter gene and nptll selectable marker gene.

• iii. To confirm the transfer and expression of the reporter

gene by GUS histochemical staining.

Methodology

Tissue culture

• ptimization

Seed varieties M66, K80 and KVU 271 Shoot initiation media with BAP at 0, 0.2, 0.5 and 1 mg/l Rooting media with IBA at 0, 0.2, 0.5 and 1 mg Acclimatization and potting in screen house Transformation (GUS) Agrobacterium strains EHA105 & LBA4404 Inoculation and co- cultivation with acetosyringone Selection on Kanamycin at 250mg/l GUS histochemical staining and PCR with nptll primers

Results – cont’d

Plasmid Map for pCAMBIA 2301

Source: www.snapgene.com

Results

Cotyledonary node explants – 1mg/l BAP

Germination Shoot initiation Shoot elongation 0 PGR, rooting 0.5mg/l IBA

4wks 3wks 3wks

Flowering plants after 8 weeks in soil

Direct organogenesis from cotyledonary node explants

Acclimatization in coco peat

1.8 2.5 2.3 2.2 1.5 1.6 1.6 1.8 1.2 2.6 2.8 2.7

0.0 0.5 1.0 1.5 2.0 2.5 3.0

K80 KVU-271 M66 Effect of BAP on shoot initiation in 3 cowpea varieties after 4 weeks of culture in MSB5 medium.

• No. of shoots per explant

0 0.2 0.5 1.0 BAP Concentration (mg/l)

Results – cont’d

1.0 4.4 3.5 2.8 2.4 3.4 7.0 4.4 2.9 6.6 3.4 8.0 4.2 4.8 5.6 4.7 3.9 3.4

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

0.2 0.5 1.0 MSO 0.5MSO K80 KVU-271 M66 Effect of IBA on root length of 3 cowpea varieties after 3 weeks in culture IBA concentration (mg/l) Root length (cm)

Results – cont’d

Regeneration of transformed plants in medium containing kanamycin at 250mg/l

Cotyledonary nodes Elongated shoots after 8 weeks in selection medium

Results – cont’d

Shoot initiation after 2 weeks in selection media

Results – cont’d

• Regeneration frequency after 2 weeks in selection medium

with 250mg/l kanamycin

Acetosyringone

• conc. (µM)

Inoculation time Days of co- cultivation Average regeneration (%) 100 30 mins 3 74.07 100 1 hr 3 91.07 100 30 mins 4 100.00 100 1 hr 4 66.81 200 30 mins 3 100.00 200 1 hr 3 81.90 200 30 mins 4 93.75 200 1 hr 4 45.60

• Best regeneration with 200µM acetosyringone, 30 minutes

inoculation and 3 days co-cultivation

Effect of Agrobacterium strain on regeneration after 2 weeks on selection medium

Results – cont’d

LBA4404 with pCAMBIA 1301 EHA105 with pCAMBIA 1301 A B

Transient GUS assay with x-gluc

Results – cont’d

• B. Leaves from plants after 4 weeks in selection media with 250mg/l

kanamycin expressing GUS gene.

• A. Cotyledonary nodes after 3 days Co-cultivation

Transformation frequency ≥ 90% Transformation frequency 17%

Results – cont’d

PCR of leaves after 8 weeks in selection medium with NPTll primers

1kb+ NT T1 T2 T3 P – NT = Non-transformed plant, T = Transformed sample, P = Plasmid DNA (+ve control), – = Water (-ve control) Thermo cycler conditions 950C – 3 min 950C – 45s 620C – 45s 720C – 2.5 min 720C – 10 min 35 cycles 600bp

• PCR analysis on 2 of the 3 plants sampled showed presence of

nptll gene – there are chances of escapes.

Conclusion

• The 3 varieties of cowpeas responded differently to growth

hormones.

• Variety M66 had the highest number of shoots per explant on

0.5mg/l BAP, while KVU 271 had the best rooting response in ½ strength MS with no growth regulators.

• A. tumefaciens strain EHA105 was more effective in

transformation in comparison to LBA4404

• Successful transformation protocol can be used to introduce

desirable agronomic traits e.g. those that confer drought or disease resistance into Kenyan cowpeas.

Acknowledgements

• The BecA – ILRI hub
• NACOSTI
• The University of Nairobi
• Supervisors – Dr. Amugune, Dr. Njoroge (UoN), Dr. Holton,
• Dr. Skilton (BecA)
• IITA , CIP and KARI