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Richard Mundembe

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Focus of the presentation The talk will focus on methods of plant transformation that have been used on non-model crops. - Cowpea, cassava, sweet potato and banana will be used as the main examples. - Crop and traits of interest - Method of transformation, efficiency and safety implications, - Other methods of plant transformation and other non-model plants will also be discussed in brief.

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Methods of Plant Transformation Agrobacterium- mediated transformation Microprojectile bombardment/ biolistics Direct protoplast transformation Electroporation of cells and tissues Electro-transformation The pollen tube pathway method Other methods such as infiltration, microinjection, silicon carbide mediated transformation and liposome mediated transformation

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Model plants Arabidopsis Nicotiana benthamiana, N. tabacum Tomato Rice Maize Highly optimized methods High transformation efficiencies

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Non-model Plants Cowpea (Vigna unguiculata) Cassava (Manihot esculenta) Sweet potato (Ipomoea batatas) Banana (Musa spp) Many monocotyledonous cereal crops e.g. sorghum (Sorghum bicolor; grain; sweet stem), pearl millet (Pennisetum glaucum, mhunga), finger millet (Eleusine coracana, zviyo). and more Recalcitrant to transformation and regeneration (efficient, reliable and reproducible)

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Cowpea transformation Importance of cowpea source of dietary protein in traditional diets partially replenishes the soil nitrogen is used as fodder Currency of trade/barter Grown mainly by women Cowpea production constraints Low yields of traditional varieties Viral, bacterial and fungal diseases in the field Post-harvest storage diseases and pests http://www.fao.org/fileadmin/

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Key dates of cowpea genetic transformation From Diouf, 2011

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Cowpea transformation Methods of cowpea transformation Electro-transformation Agrobacterium-mediated Biolistics Ivo et al., (2008), TF 0.9% Molecular approaches to virus resistance Coat protein-mediated resistance RNA-mediated resistance Cowpea aphid-borne mosaic virus (CABMV) Molecular approaches to contain post-harvest damage Bruchid resistance, trypsin inhibitors

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Illustration of the binary plasmids used for tobacco transformation by Agrobacterium -mediated transformation, and effectiveness of each approach in conveying virus resistance CP-MR Delayed symptom development RNA-MR Delayed symptom development Recovery Anti- sense RNA-MR Modified symptoms Delayed symptom development

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Electro-transformation DNA can also be delivered into cells, tissues and organs by electrophoresis (Ahokas 1989; Griesbach and Hammond, 1994; Songstad et al., 1995). This method is known as transformation by electrophoresis or electro-transformation. The tissue to be transformed is placed between the cathode and anode. The anode is placed in a pipette tip containing agarose mixed with the DNA to be used for transformation. The assembly is illustrated in the next slide.

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Diagrammatic illustration of the electro-transformation equipment and experimental set-up. Cowpea Transformation by Electro- transformation

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A common feature of the GUS positive plants is that the manipulations were carried out on plants that had straight stems, first true leaves open and cotyledons still attached to the seedling. No pre-treatment other than maybe punching the meristem appear to be necessary. Both DC and AC are effective in delivering DNA to the plant cells. The leaves of GUS positive plants had a sectored appearance; Kanamycin resistance was not an effective assay against germinating cowpea seedlings The mechanism of DNA integration is probably non- homologous recombination into sites on the genome that are undergoing repair or replication Has potential for marker-free transformation Efficiency less than 0.3% (4 in 1200) Electro-transformation of Cowpea

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Agrobacterium-mediated transformation In crown gall disease of dicotyledonous plants, caused by Agrobacterium tumefaciens and hairy root disease caused by Agrobacterium rhizogenes, the bacterium transfers part of the DNA of its Ti or Ri plasmid DNA respectively into the host plant where it becomes integrated into the host genome (Herrera-Estrella et al., 1983). The natural host range of the bacterium expanded Harnessed for use in Plant Biotechnology for in vitro plant transformation, using various modified versions of the Ti plasmid

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T.J. Higgins method (Popelka et al., 2006) Co-cultivation Agrobacterium strain containing pBSF16, in liquid medium (MS/MES/vits/BAP/GA3/acetosyringone/DTT/cys) Cotyledonary nodes of 3 different cultivars Co-cultivation of explants for 6 days Shoot initiation Shoots were initiated on MS/NTS/timentin. 12 days, no selection for multiple shoots to appear. Selection Selective medium (MS + 5 mg/l PPT), refreshed every 2 wk; remove dead tissue, 4 6 transfers Agrobacterium-mediated transformation of Cowpea

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Shoot elongation green shoots were transferred to shoot elongation medium (MS/GA3/Asp/IAA/timentin/5 mg/l PPT) sub-cultured every 2 wk until shoots were more than 1 cm long. (14 weeks!) Rooting Transfer from large culture jars for growth under selection. Some rooted, others needed to be grafted directly onto 10-day- old seedlings with the aid of a silicon ring (G, 17 weeks). Transfer to soil, high humidity chamber then to greenhouse. Transformation efficiency: Agrobacterium-mediated transformation of Cowpea

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Cassava Transformation Importance of Cassava Important source of dietary carbohydrate, food security Yields relatively well even under low rainfall and in poor soils, but there is need for improvement Has potential as an industrial crop biofuels and starch industries Cassava production constraints Viral (e.g. CMD), bacterial and fungal diseases in the field Does not store well once removed from the soil Methods of cassava transformation Agrobacterium-mediated transformation Microprojectile-mediated transformation / biolistics

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RNA interference (RNAi) This is the process that depends on small RNAs (sRNAs) to regulate the expression of the eukaryotic genome, including maintenance of genome integrity, development, metabolism, abiotic stress responses and immunity to pathogens. micro RNAs (miRNAs) and small interfering RNAs (siRNAs). siRNAs are derived from perfectly paired double stranded RNA (dsRNA) precursors, that are derived either from antisense or are a result of RNA-dependent RNA polymerase (RDR) transcription. Hairpin RNA is more effective at inducing RNAi

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History of Cassava Transformation Li et al., 1996 - Agrobacterium-mediated transformation of somatic cotyledons to then regenerate transgenic shoots by organogenesis. Schpke et al. 1996,- microparticle bombardment of embryogenic suspension-derived tissues and then regenerated transgenic plantlets by embryo maturation. (= FEC suspension cultures). Gonzalez et al., (1998), Zhang et al., (2000) and Shrueder et al., (2001), Agrobacterium-mediated transformation of FEC Optimisation by Bull et al, (2009) SE induction, FEC production, co-cultivation and selection.

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Agrobacterium-Mediated Transformation of Cassava Friable Embryonic Callus (FEC) FEC are a specialized totipotent cell clusters Methods for induction of FEC and Agrobacterium-mediated transformation were optimized by Bull et al. (2009). 1. Somatic embryo production 2. Production of FEC 3. Co-cultivation of FEC with Agrobacterium 4. Maturation and development of transformed FEC 5. Selection and regeneration of transgenic plants Bull et al., 2009

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Agrobacterium-Mediated Transformation of Cassava Friable Embryonic Callus (FEC) We have successfully used this method to transform TMS60444 (IITA model cultivar) and T200 (a commercial grown SA landrace) using pCambia-based construct designed to convey resistance to various CMD causing viruses by hp RNAi, replicase and antisense strategies. Transformation efficiencies are relatively high () Evaluation of levels of resistance is ongoing pCambia map

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Microprojectile Bombardment/ Biolistics A gene transfer method developed to transform crops that remained recalcitrant to Agrobacterium - mediated transformation The DNA construct, attached to a microprojectile (gold or tungsten), is delivered at high speed across the various plant cell barriers (cell wall, cell membrane, cytoplasm, nuclear envelop, to enter the nucleoplasm) transient expression or integration (whole or fragments) into the plant genome may occur.

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Microprojectile Bombardment Delivery into the nucleus results in 45 x higher likelihood of transient expression in cytosol, and 900 x higher than in vacuole (Yamashita et al., 1991). The mechanism of integration is thought to be (non- homologous integration) Efficiency of transformation is influenced by the stage of the cell cycle, higher expression if close to the time the nuclear membrane disappears at mitosis

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Microprojectile Bombardment Result in transformants with higher copy numbers, especially with amounts of bombarding Integration into the same or tightly linked loci, most likely in relation to replication forks or integration hot spots resulting from initial integration events Rearrangements (deletions, direct repetitions, inverted repetitions, ligation, concatamerization) may occur prior to, or during integration 90% of integrations are into random sites within transcriptionally active regions.

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Minimum cassette technology When only the required gene expression cassettes (promoter, coding region of interest, terminator) is bombarded into the plant cells Sometimes co-transformed together with marker genes to be removed before commercialization Screening and selection might be more difficult, probably depending on detection of the gene sequence or gene product of interest, But the approach is very attractive since absence of reporter genes and selection markers results in address the biosafety concerns of consumers and are safer for the environment Marker genes also limit options for gene stacking in an original transgenic line.

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Microprojectile bombardment of Cassava Results from our lab (Poster) Optimisation of parameters for biolistic transformation of cassava FEC Linear and circular constructs Gold particle size Helium pressure Minimum cassettes GUS assay, Hyg re-rooting assay, PCR GUS, Hyg, Insert Southern analysis - pending

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Sweet Potato (Ipomoea batatas) Transformation Importance of sweet potato Important source of food crop roots and foliage Controversial alternative biofuel substrate Can be stored in the soil until needed Sweet potato production constraints Viral (e.g. SPFMV), bacterial and fungal diseases in the field Low yields from recycled disease-infested planting material, and poor farming practices Nematodes - Stem nematode (Ditylenchus destructor) Insect damage in the field and in storage - weevils (Cylas formicarius), Water stress -

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Sweet Potato Transformation Regeneration relatively easy, from protoplasts, via shoot organogenesis, from leaves, roots and stem internodes. Somatic embryogenesis can be induced from axillary bud shoot tips, apical and bud meristems, and leaf, petiole, stem and root explants Methods of sweet potato transformation Electroporation of protoplasts (Nishiguchi et al., 1992) Agrobacterium-mediated transformation of leaf and stem explants. Efficiency can be higher than 2% Biolistics

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Sweet Potato Transformation Viruses SPFMV, CPMR Nematodes oryzacystatin-I gene, OC1 (Gao et al., 2011),, Insect resistance - weevil (Cylas formicarius) Garcia et al., 2007, field trials, cowpea trypsin inhibitor (CpTI), snowdrop lectin (GNA) Other traits: - granule-bound starch synthaseI (GBSSI), - tobacco microsomal -3 fatty acid desaturase (NtFAD3), - starch branching enzyme II (IbSBEII) - bar gene - Xerophyta viscosa peroxiredoxin 2, XvPrx2, gene conferring drought stress tolerance (Kamwendo, P.M., 20xx)

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Banana and Plantain (Musa spp) Transformation Importance of Banana Important source of dietary carbohydrate and income Banana production constraints Viral (e.g. Banana bunchy top virus), bacterial (banana Xanthomonas wilt, BXW) and fungal (Fusarium wilt by Fusarium oxysporum) diseases in the field; Nematodes Radopholus similis natural resistance identified - Pratylenchus - Helicotylenchus

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Banana Transformation Methods of Banana transformation Target tissue is embryogenic cell suspension (ECS) establishment is not routine, because of low embryogenic response, long time needed, somaclonal variation, and contamination. Agronomic traits Quality traits Molecular pharming Banana ECS, Ramirez- Villalobos and de Garcia, 2008.

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Banana Transformation Methods of Banana transformation Protoplast electroporation Agrobacterium-mediated transformation Microprojectile-mediated transformation / biolistics GUS, Hyg Variable transformation frequencies, depending on cultivar. Agro. better than biolistics in a wider range of cultivars. Maize cystatin and synthetic repellent genes (plantain, Tripathi et al., 2011) Bacterial over-expression of sweet pepper plant like ferredoxin protein, Pflp, and hypersensitive response assisting protein, Hrap. (Abubaker et al, 2011) Fungal resistance pathogenesis-related protein genes as candidates for GE (FW) van der Berg et al., 2011)

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PEG-mediated transformation of protoplasts Plant cell walls are removed by enzymatic degradation to produce protoplasts. Polyethylene glycol (PEG) causes permeabilization of the plasma membrane, allowing the passage of macromolecules into the cell. Electroporation of Protoplasts Aelectric pulse permeabilizes the plasma membrane of the protoplasts. The cell wall and whole plants can be regenerated, if procedures exist. The transgenic plants generated have characteristics similar to those of plants derived from direct transformation methods. Carrier DNA (usually ~500 bp fragments of calf thymus DNA) included in the transformation mixture increases transformation efficiency, but increases prevalence of transgene rearrangements and integration of superfluous sequences. Protoplast cultures are not easy to establish and maintain Regeneration of whole plants is unreliable for some important species.

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Other methods

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Non-model Plants (cont.) Many monocotyledonous cereal crops e.g. sorghum (Sorghum bicolor; grain; sweet stem), pearl millet (Pennisetum glaucum, mhunga), finger millet (Eleusine coracana, zviyo). Ginger, Zingiber officinale Roscoe (Zingiberaceae), Bambara groundnut Indigenous vegetables such as Okra (Corchorus tridens/olitorius; derere, idelele), Spider flower (Cleome gynandraruni; runi/nyeve, elude) Not all are candidates for transformation.

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Acknowledgements Prof M.E.C. Rey Cassava transformation Prof I. Sithole-Niang Cowpea transformation Sweet potato transformation - Banana transformation - MCB Plant Biotechnology Group

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Thank you