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High frequency adventitious root and shoot regeneration in recalcitrant sweet potato cultivars
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High frequency adventitious root and shoot regeneration in recalcitrant sweet potato cultivars

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Presentation made by Abel Sefasi,

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

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    High frequency adventitious root and shoot regeneration in recalcitrant sweet potato cultivars High frequency adventitious root and shoot regeneration in recalcitrant sweet potato cultivars Presentation Transcript

    • High frequency adventitious root and shoot regeneration in recalcitrant sweet potato cultivars Abel Sefasi Department of Agricultural Production Makerere UniversitySupervisors:Dr. G. Ssemakula Regional RUFORUMDr. A. Kiggundu Biennial Conference,Dr. M. Ghislain 24-28 Sept. 2012,Dr. S. Mukasa Entebbe Uganda
    • 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)
    • 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
    • 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
    • ObjectivesMain objective: The main objective of this study is to genetically transform selected Ugandan sweetpotato cultivars with weevil resistance genesSpecific 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
    • 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
    • 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
    • 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
    • Results cont’dEffect of TDZ concentration (0.5, 2.0, 4.0 µM) on shootregeneration frequency from stem and leaf explants ofBwanjule and Kyebandula cultivars
    • 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 0.342Explant 0.312 <.001TDZ concentration (µM) 0.168 0.344Cultivar X Explant 0.498 1Cultivar X TDZ concentration 0.193 0.477Explant X TDZ concentration 0.512 0.943
    • Results cont’dEffect of TDZ concentration (0.5, 2.0, 4.0 µM) in thepresence of NAA (0.25 µM) on shoot regenerationfrequency from stem and leaf explants of Bwanjuleand Kyebandula cultivars
    • (a) Adventitious budResults 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.
    • 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
    • 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.
    • 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
    • Progress with papers and manuscriptPapers 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. 20122. Thidiazuron-induced efficient adventitious bud and shoot regeneration in recalcitrant sweetpotato. Afr. Crop Sci. Journ. Submitted Aug. 2012Papers 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
    • Thank you