Presentation made by Abel Sefasi, Dr. G. Ssemakula, Dr. A. Kiggundu, Dr. M. Ghislain, Dr. S. Mukasa at the Biennial Conference 2012

1. High frequency adventitious root and
shoot regeneration in recalcitrant
sweet potato cultivars
Abel Sefasi
Department of Agricultural Production
Makerere University
Supervisors:
Dr. G. Ssemakula Regional RUFORUM
Dr. A. Kiggundu Biennial Conference,
Dr. M. Ghislain 24-28 Sept. 2012,
Dr. S. Mukasa Entebbe Uganda

2. Background
 Sweetpotato is seventh among the food crops in
annual production in the world (FAO, 2007)
 Uganda is the biggest producer in Africa at 1.8
million tons (FAO, 2010)
 Weevils, Cylas puncticollis and C. brunneus cause
production losses over 28% (Kiiza et al., 2009)
 Heritability of the resistance trait is extremely low
and not well understood (Stevenson et al., 2009)
 Three Bt Cry proteins (Cry7Aa1, Cry3Ca1, ET33-
34) active against the two African weevil species
identified (Ekobu et al 2010)

3. Justification
 The genes expressing the Cry proteins have been
assembled into a plasmid gene construct
 Agrobacterium-mediated genetic transformation is the
option
 However production of transgenic sweetpotato requires
an in vitro protocol for regeneration nos promoter
nptII gSPOA1 promoter
3' 35S
LB
kanamycin (R) cry3Ca1
 Sweetpotato is considered pBR322 ori 3' gSPOA1
recalcitrant to both pBR322 bom site
pCIP84
transformation and in vitro
14602 bp
ß-amy promoter
pVS1-REP
regeneration
cry7Aa1
pVS1 Stab
3' ß-amy
RB

4. Justification
 It is necessary that there are very few ‘escapes’ and no
chimeric plants resulting from regeneration
 Two protocols that have been reported for regeneration of
some cultivars of sweetpotato
b
a
Meristems
excision Embryogenic calli 4–5 Transformation 1
4 – 5 weeks (6 weeks in weeks day in MIB medium
transformation (2
dark) days in dark) in dark, then 4 days
to medium with 2,4-
D
High transformation frequency
medium with 2,5-T
(2 months)
Short
duration
Medium with Kin or
Zea subculture
each 15 days per 2
Regenerants to months
Medium with Medium with ABA
Regenerants to MPB medium
GA3 (2 months) (3 months)
MPB medium

5. Objectives
Main objective:
 The main objective of this study is to genetically transform
selected Ugandan sweetpotato cultivars with weevil resistance
genes
Specific objectives:
 To develop a tissue culture protocol for regeneration of selected
Ugandan sweetpotato cultivars
 To develop a protocol for genetic transformation of Ugandan
sweetpotato cultivars to improve weevil resistance
 To study expression patterns of transgenes and their efficacy
against Ugandan sweetpotato weevils after genetic

6. Materials and methods
 Media: MS, myo-inositol (0.1 g l-1), sucrose
(30 g l-1) and 1 ml l-1 sweetpotato vitamin
stock, pH 5.8
 Cultivars: Kyebandula and Bwanjule
 Explant types: whole leaves and stem
internode
 Growth regulators: TDZ (0.5, 2.0, 4.0 µM)
and TDZ with NAA (0.25 µM)
 Completely Randomised Design

7. Data collection and management
 Three replicates with 30 explants per rep
 Each rep had 3 petri dishes each containing 10 explants
 Data on number of shoots regenerated was collected 12
weeks after culture initiation
 Statistical analyses done using ANOVA
 Means were compared using the LSD test at the P ≤ 0.05
level

8. Results
 Shoot regeneration was successfully achieved
 Regeneration through adventitious shoots / organogenesis
 Cultivar type, [TDZ], explant type significantly (P ≤ 0.05)
affected the no. of adventious buds formed per explants
(not presented).
 Both type of cultivar and concentration of TDZ did not affect
shoot regeneration efficiency
 Stem explants gave a significantly (p < 0.05) high
regeneration efficiency for all TDZ concentrations

9. Results cont’d
Effect of TDZ concentration (0.5, 2.0, 4.0 µM) on shoot
regeneration frequency from stem and leaf explants of
Bwanjule and Kyebandula cultivars

10. F-Test: Effect of TDZ concentration (µM), types of
explants of cultivar on bud induction and shoot
regeneration frequency
Explants forming Shoots per
shoots (Frequency) explant
(No.)
Cultivar 0.134 0.342
Explant 0.312 <.001
TDZ concentration (µM) 0.168 0.344
Cultivar X Explant 0.498 1
Cultivar X TDZ concentration 0.193 0.477
Explant X TDZ concentration 0.512 0.943

11. Results cont’d
Effect of TDZ concentration (0.5, 2.0, 4.0 µM) in the
presence of NAA (0.25 µM) on shoot regeneration
frequency from stem and leaf explants of Bwanjule
and Kyebandula cultivars

12. (a) Adventitious bud
Results cont’d
protrusion along the
length of petiole of leaf
explants after 2 weeks
(b) Elongation of
adventitious buds
leading to development
of shoots
(c) Development of multiple
adventitious shoots on
stem internode explants
(d) Growth of plants in soil
4 weeks after
acclimatisation in the
greenhouse.

13. Discussion
 We have regenerated cultivars that were recalcitrant to
common protocols used in in vitro regeneration
 The results achieved in this study are very important for
the breeding of I. batatas, particularly African cultivars,
which have been reported to be difficult to regenerate in
vitro.
 Only two cultivars were tested. It is interesting that cultivar
type did not show significant effect on regeneration
efficiency
 These results confirm that sweetpotato regeneration in
vitro is cultivar-dependent

14. Conclusions
• A reliable adventitious regeneration protocol has been
established for I. batatas cultivars which have not been
regenerated before
• This protocol has potential to be extended to the
regeneration of other economically important I. batatas
cultivars
• The ultimate goal is to apply this protocol in genetic
transformation for improvement of I. batatas traits,
especially for resistance to weevils.
• Most I. batatas cultivars that were thought to be recalcitrant
can be regenerated following optimisation of media
composition.

15. Recommendations
 Most adventitious buds failed to turn into shoots
 It is recommended that culture systems be developed for
efficient adventitious regeneration to be effectively induced
in a wide range of cultivars

16. Progress with papers and manuscript
Papers submitted to peer reviewed journals:
1. Induction of somatic embryogenesis in recalcitrant sweetpotato
(Ipomoea batatas L.) varieties. Afr. Journ. Biotech. Responded to
reviewers comments’ in Aug. 2012
2. Thidiazuron-induced efficient adventitious bud and shoot
regeneration in recalcitrant sweetpotato. Afr. Crop Sci. Journ.
Submitted Aug. 2012
Papers in early draft form:
1. Genetic transformation of recalcitrant sweetpotato through somatic
embryogenesis from stem internodes. In vitro cell Dev. Biol. Plant.
2. 2,4-dichlorophenoxyacetic acid confirms recalcitrance to somatic
embryogenesis in some important African sweetpotato cultivars.
Short communication. Plant Phys

17. Thank you