Strawberries tissue culture

1. Jetwell Mugabe
Generation and
testing of virus free
strawberries
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Dr Simango (MUAST)
Dr G. Zvobgo (TRB)

2. Presentation layout
01 Introduction
Objectives
Hypothesis
02
03
04 Materials and Methods
05
06 Budget and expected results
Data Analysis & Work plan

3. Background
 Strawberries help to lower blood pressure and protects against cancer.
 It's high in vitamins; fibre; antioxidants; manganese and potassium.
 Strawberries have higher vitamin C content than oranges.
 It has export opportunities to Europe, particularly the Netherlands.
 Strawberries are classified as berries in Zimbabwe, and their exportation has been steadily increa
sing.
 In 2018, totalled US $4 million, and has a potential to increase
Introduction

4. Introduction
Problem definition
 Production challenges and diseases, such as fungal, bacterial, and viral diseases,
are a major stumbling block.
 Adoption of strawberries as a cash crop has been slow.
 Grey mould, anthracnose, red stale, and leather stale are some the problematic
diseases.
 Diseases are passed down from one farmer to the next when sharing plant mat
erial
 Viruses are the most devastating
 In Zimbabwe, there is no tissue culture laboratory producing disease-free straw
berries.

5. Introduction
Justification
 In Zimbabwe, there isn't a lot of information about strawberry production.
 There is no meaningful research ever done in Zimbabwe on straberries.
 Plant tissue culture can provide bacteria- and fungal-free planting material via
micro-propagation.
 Strawberry plants can be grown virus- and disease-free using meristem tip cult
ure, a plant tissue culture technique.
 The majority of farmers purchase strawberries from growers who have no idea
what varieties they are producing.

6. Objectives
The study's main objective is to develop a tissue culture pr
otocol for strawberry regeneration.
 To investigate the prevalence and incidence of strawberry viruses in Zimbabwe's
major strawberry-growing regions.
 To develop a cost-effective tissue culture protocol for strawberries in Zimbabwe.
 To compare the performance of strawberries from farmers and those produced t
hrough tissue culture.

7. Hypothesis
 Locally available strawberries are infected by a wide array viruses
 There are differences in response to hormonal levels of meristem tip cultures of
strawberries cultured in vitro
 There is difference on time response to different protocols on meristem tip cult
ures of strawberries
 There is difference in performance of virus free and virus infested strawberries

8. Materials and methods
Seven Activities
Survey Germplasm
collection
Characteris
ation
Virus
Indexing
Meristem
Tip culture
Micro-
propagatio
n
Yield
Evaluation

9. Survey
The survey seeks to identify the following
 Individual farmers will be identified using snowballing.
 The electronic survey will be administered using Surveyheart.
 Farmers will be identified and visited individually.
 The geographical distribution of farmers and the challenges they face.
 Source of planting materials.
 The varieties they grow
 Agronomic challenges they face.
 Their understanding of plant tissue culture.
 Their markets and market demands are.

10. 1
2
3
4
This will be done at the same time with the survey
Known varieties will be collected and their agronomic
attributes listed
Collected varieties will also be characterized includin
g unknown varieties
Random samples will also be collected to use identify
farmers have challenges of viral disease
Germplasm collection
& Characterisation

11. Virus indexing
Double stranded RNA extraction
Add 50ml 5.8 M KoAc and
spin at 15 000g
04
Collect supernatant, add 0.8V
isopropanol and spin at 20,000g
05
06
. 01
Grind 10g tissue in Liquid
Nitrogen
.
02
Add 50ml extraction
buffer and shake at
room temperature
.
03
Using 30 samples collected randomly
around Zimbabwe
Resuspend Pellet cellulose in in 50ml STE/
18% ethanol & add 1g CF-11 cellulose

12. Virus indexing
Double stranded RNA extraction
Pour into chromatography col
umns, wash with 50ml STE/18
% ethanol and elute dsRNA in
3ml STE
10
dsRNA is precipitated in ethanol
11
12
. 07
Add 300ml of 2M MgCl2 and
incubate at 37oC with RNAse
and DNAse
08
Add 1ml 0.5 EDTA, etha
nol to 25%, 0.3g cellulos
e & shake at room tem
perature
09
Pellet cellulose at 15,000g, resuspend
in 50ml STE/18% ethanol and pair int
o chromatography columns
Each extracted dsRNA will be loaded on 2
% agarose gel

13. Meristem tip culture & Micropropagation
01
02
03
04
Shoots that regenerate
will be sub-cultured and
micro-propagated in
solid M&S
Four different hormonal
variations will be used in
solid M&S media:
Dissect the apical and
axillary meristem under
a stereo microscope
Surface sterilization
using 2% sodium
hypochlorite and rinsing
in sterile water
Hormonal treatments
No hormones;
1mg/l IAA;
1mg NAA;
1mg/l IAA+ 20mg/l GA3,
 2mg/l BAP+1mg/l IAA

14. Yield evaluation
The yield of the collected varieties will be based on..
 The rate of establishment
 Regeneration capabilities
 The number of runners produced per plant
 The rate at which that plant spreads and covers the ground
 The number of fruits per plant
 Weight per fruit
 And total weight of fruits per plant
 Fruit quality
 This will be done under green house conditions

15. Experimental design
01 02
03 04
The collection of virus
indexing samples will be
done randomly (3 reps
for each sample
The yield evaluation will
be set up in a
randomised complete
block design (3 blocks
and 30 plants per
treatment)
The meristem tip culture
will be arranged as 4x5
randomised factorial
treatment (30 meristems
for each treatment)
The survey will be done
using snow balling to
identify strawberry
farmers

16. Data Analysis
All the experiments will be analyzed
using SPSS Version 26
Analysis of variance (ANOVA)
Least Square Difference (LSD) will be
used for data analysis at 95% level of
significance
P value = 0.5

17. Work plan
Activity Nov Dec Jan Feb Mar Apri May June
Proposal
Survey
Germplasm Collection
Characterisation
Virus Indexing
Meristem tip Culture
Micro-propagation
Yield evaluations
Data collection
Thesis writing

18. Budget
Item Unit cost Quantity Amount
Stationary 100.00
$ 1 100.00
$
Tissue culture jars 4.00
$ 1000 4,000.00
$
Test tubes 2.00
$ 600 1,200.00
$
Virus indexing kit 150.00
$ 1 150.00
$
Diesel litres 300.00
$ 1.2 360.00
$
Ammoniium Nitrate 50kg 36.00
$ 1 36.00
$
Compound L 50kg 30.00
$ 2 60.00
$
M&S media 1kg 50.00
$ 5 250.00
$
Agar 1kg 50.00
$ 1 50.00
$
Data per month 20.00
$ 8 160.00
$
IAA 200g 200.00
$ 1 200.00
$
BAP 200g 300.00
$ 1 300.00
$
NAA 200g 400.00
$ 1 400.00
$
2.4D 200g 80.00
$ 1 80.00
$
Kinetin 200g 300.00
$ 1 300.00
$
Total 7,646.00
$

19. Expected Results
 Information on challenges faced by strawberry farmers
 Five known strawberry varieties and their descriptors
 The presence of viruses in local strawberry cultivars
 A protocol for meristem tip culture for strawberries
 Virus free strawberries
 Field performance of various virus free strawberry varieties

20. References
Belkengram, R. O., & Miller, P. W. (1962). Culture of apical meristems of Fragaria vesca strawberry plants as a method
of excluding latent A virus. Plant Disease report, 46: 119-121.
Boxus, R. O., Jemmali, J. M., Terzi, & Arezki, O. (2000). Drift in genetic stability in micropropagation: t
he case of strawberry. Acta Horticulture, 530: 155-161.
Danyl, G., Echeverria, D., Golino, D., & Sim, T. S. (2008). Guide to the strawberry clean plant program.
Retrieved from Foundation Plant Services: fps.ucdavis.edu/WebSitePDFs/Articles/FPSStrawberry
Brochure08
Miller, P. O., & Belkengren, R. O. (1963`). Elimination of yellow edge, crickle and vain banding virus a
nd certainother virus complexes from strawberries by excision and culturing of epical meriste
ms. Plant Diseases, Rep 47: 298-300.
NCAGAR. (2017). Strawberry tissue analysis. Retrieved from N.C. Dept of Agriculture and consumer s
ervices: www.ncagar.gov/agronomi/document/StrawberryArticle2017.pdf
Swartz, H. J., Galletta, G. H., & Zimmerman, R. H. (1981). Field Performance and phenotypic Stability
of Tissue Culture-propagated Strawberries. Horticultural Sciences, 105(2):667-673.

21. Acknowledgements
 TRB management and staff
 Dr G. Zvobgo
 Dr Simango

22. Thank You