Transgenic Cassava Research Articles

RNAi-mediated resistance to Cassava brown streak Uganda virus in transgenic cassava.

Cassava brown streak disease (CBSD), caused by Cassava brown streak Uganda virus (CBSUV) and Cassava brown streak virus (CBSV), is of new epidemic importance to cassava (Manihot esculenta Crantz) production in East Africa, and an emerging threat to the crop in Central and West Africa. This study demonstrates that at least one of these two ipomoviruses, CBSUV, can be efficiently controlled using RNA interference (RNAi) technology in cassava. An RNAi construct targeting the near full-length coat protein (FL-CP) of CBSUV was expressed constitutively as a hairpin construct in cassava. Transgenic cassava lines expressing small interfering RNAs (siRNAs) against this sequence showed 100% resistance to CBSUV across replicated graft inoculation experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed the presence of CBSUV in leaves and some tuberous roots from challenged controls, but not in the same tissues from transgenic plants. This is the first demonstration of RNAi-mediated resistance to the ipomovirus CBSUV in cassava.

Senescence‐Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz)

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.

Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava

Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop.

Dose-dependent RNAi-mediated geminivirus resistance in the tropical root crop cassava

Cassava mosaic disease is a major constraint for cassava production in Africa, resulting in significant economic losses. We have engineered transgenic cassava with resistance to African cassava mosaic virus (ACMV), by expressing ACMV AC1-homologous hairpin double-strand RNAs. Transgenic cassava lines with high levels of AC1-homologous small RNAs have ACMV immunity with increasing viral load and different inoculation methods. We report a correlation between the expression of the AC1-homologous small RNAs and the ACMV resistance of the transgenic cassava lines. Characterization of the small RNAs revealed that only some of the hairpin-derived small RNAs fall into currently known small interfering RNA classes in plants. The method is scalable to stacking by targeting multiple virus isolates with additional hairpins.

Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants

The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar "KU50" was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19-21% was observed. Among the total number of ESP explants, 32-38% regained normal extended shoot phenotype and 88-96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries.

Transgenic cassava resistance to African cassava mosaic virus is enhanced by viral DNA-A bidirectional promoter-derived siRNAs

Expression of double-stranded RNA (dsRNA) homologous to virus sequences can effectively interfere with RNA virus infection in plant cells by triggering RNA silencing. Here we applied this approach against a DNA virus, African cassava mosaic virus (ACMV), in its natural host cassava. Transgenic cassava plants were developed to express small interfering RNAs (siRNA) from a CaMV 35S promoter-controlled, intron-containing dsRNA cognate to the common region-containing bidirectional promoter of ACMV DNA-A. In two of three independent transgenic lines, accelerated plant recovery from ACMV-NOg infection was observed, which correlates with the presence of transgene-derived siRNAs 21-24 nt in length. Overall, cassava mosaic disease symptoms were dramatically attenuated in these two lines and less viral DNA accumulation was detected in their leaves than in those of wild-type plants. In a transient replication assay using leaf disks from the two transgenic lines, strongly reduced accumulation of viral single-stranded DNA was observed. Our study suggests that a natural RNA silencing mechanism targeting DNA viruses through production of virus-derived siRNAs is turned on earlier and more efficiently in transgenic plants expressing dsRNA cognate to the viral promoter and common region.

Production of the first transgenic cassava in Africa via direct shoot organogenesis from friable embryogenic calli and germination of maturing somatic embryos

The impact of cassava transformation technologies for agricultural development in Africa will depend largely on how successfully these capabilities are transferred and adapted to the African environment and local needs. Here we report on the first successful establishment of cassava regeneration and transformation capacity in Africa via organogenesis, somatic embryogenesis and friable embryogenic callus (FEC). As a prerequisite for genetic engineering, we evaluated six African cassava genotypes for the ability of a) induction of FEC b) hygromycin sensitivity and c) T-DNA integration potential by different Agrobacterium strains. FEC was induced in genotypes TMS 60444, TME 1 and TMS 91/02327. Potential tissues for FEC formation were induced in TMS 91/02324, TME 12 and TME 13. Pure and proliferating FEC was obtained and maintained only in TMS 60444. FEC growth and shoot organogenesis were completely suppressed when hygromycin was used at a concentration of 20 mg/l in all tissue types and genotypes. With somatic cotyledons, statistically significant differences (p
0.05) were observed between Agrobacterium strains and genotypes with respect to T-DNA transfer efficiency. Using somatic cotyledons, TME 8 was found to be the most amenable to transformation with maximum blue spots per GUS-positive explants, and Agrobacterium GV3101 proved to be superior to EHA105, LBA4404, and AGl-1 for T-DNA transfer based on transient assays with a reporter gene (GUS). With FEC, Agrobacterium LBA4404 was superior to other strains. This study also identified EHA105 as a new vir helper strain to recover transgenic cassava plants. PCR and Southern hybridization of genomic DNA of the hygromycin-resistant cassava plants to a hpt probe confirmed the integration of hpt with integration events varying between 1 and 2 insertions. The benefit of combining the FEC and shoot organogenesis systems for recovering transgenic cassava plants is described. The contributions of this report to enhancing the development and deployment of genetic engineering of cassava for agricultural biotechnology development in Africa are discussed.

Biotechnological approaches to cassava protein improvement

Cassava starchy storage roots are an excellent source of carbohydrates but lacking in protein. To enhance their nutritional quality, here we discuss several biotechnological strategies that might be used to increase protein levels as well as improved essential amino acid content in transgenic cassava. Application of such strategies in this major crop could, in the long term, help to fight against malnutrition in those regions which depend heavily on cassava consumption.

Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes

African cassava mosaic virus (ACMV) is a major contributor to cassava mosaic disease (CMD), the economically most important and devastating disease of cassava in Africa. We have developed transgenic cassava plants with increased ACMV resistance using improved antisense RNA technology by targeting the viral mRNAs of Rep (AC1), TrAP (AC2) and REn (AC3). Viral DNA replication assays in detached leaves demonstrated that replication of two ACMV isolates was strongly reduced or inhibited in most transgenic lines. After ACMV infection of plants using biolistic inoculation, several lines remained symptomless at lower infection pressure (100 ng viral DNA/plant). Symptom development was reduced and attenuated even at higher DNA doses. Transgenic ACMV-resistant plants had significantly reduced viral DNA accumulation in their infected leaves. Short sense and antisense RNAs specific to AC1 were identified in transgenic lines expressing AC1 antisense RNA, suggesting that the short RNAs mediate interference by post-transcriptional gene silencing. Our results demonstrate that resistance to ACMV infection of cassava can be achieved with high efficacy by expressing antisense RNAs against viral mRNAs encoding essential non-structural proteins, providing a new tool to combat CMD in Africa.

Regeneration of transgenic cassava from transformed embryogenic tissues.

Production of transgenic plants is gradually becoming routine in cassava biotechnology. Green cotyledons of maturing somatic embryos (somatic cotyledons for short) and friable embryogenic suspensions (FES) are the target tissues for transformation by Agrobacterium or biolistics. Putative transgenic shoots develop from transformed somatic cotyledons via shoot organogenesis or from FES via somatic embryogenesis under selection. Maturation of transgenic somatic embryos is induced by transfer to maturation medium with reduced concentrations of selective agents. Mature somatic embryos can also develop directly from FES cells under selection. Transgenic plants are regenerated by the elongation of transgenic shootlets from organogenesis experiments and by the germination of or shoot development from transgenic mature embryos cultured without selection. beta-Glucuronidase (GUS) assays and rooting tests can be used to screen for escapes from selection, which improves the regeneration rate of truly transgenic plants.

Engineering cyanogen synthesis and turnover in cassava (Manihot esculenta)

Cassava is the major root crop for a quarter billion subsistence farmers in sub-Saharan Africa. It is valued for its ability to grow in adverse environments and the food security it provides. Cassava contains potentially toxic levels of cyanogenic glycosides (linamarin) which protect the plant from herbivory and theft. The cyanogens, including linamarin and its deglycosylated product, acetone cyanohydrin, can be efficiently removed from the root by various processing procedures. Short-cuts in processing, which may occur during famines, can result in only partial removal of cyanogens. Residual cyanogens in cassava foods may cause neurological disorders or paralysis, particularly in nutritionally compromised individuals. To address this problem and to further understand the function of cyanogenic glycosides in cassava, we have generated transgenic cassava in which cyanogenic glycoside synthesis has been selectively inhibited in leaves and roots by antisense expression of CYP79D1/D2 gene fragments. The CYP79D1/D2 genes encode two highly similar cytochrome P450s that catalyze the first-dedicated step in cyanogenic glycoside synthesis. Transgenic plants in which the expression of these genes was selectively inhibited in leaves had substantially reduced (60- 94% reduction) linamarin leaf levels. Surprisingly, these plants also had a greater than a 99% reduction in root linamarin content. In contrast, transgenic plants in which the CYP79D1/D2 transcripts were reduced to non-detectable levels in roots had normal root linamarin levels. These results demonstrate that linamarin synthesized in leaves is transported to the roots and accounts for nearly all of the root linamarin content. Importantly, transgenic plants having reduced leaf and root linamarin content were unable to grow in the absence of reduced nitrogen (NH3) . Cassava roots have previously been demonstrated to have an active cyanide assimilation pathway leading to the synthesis of amino acids. We propose that cyanide derived from linamarin is a major source of reduced nitrogen for cassava root protein synthesis. Disruption of linamarin transport from leaves in CYP79D1/D2 anti-sense plants prevents the growth of cassava roots in the absence of an alternate source of reduced nitrogen. An alternative strategy for reducing cyanogen toxicity in cassava foods is to accelerate cyanogenesis and cyanide volatilization during food processing. To achieve this objective, we have expressed the leaf-specific enzyme hydroxynitrile lyase (HNL) in roots. HNL catalyzes the breakdown of acetone cyanohydrin to cyanide. Expression of HNL in roots accelerated cyanogenesis by more than three-fold substantially reducing the accumulation of acetone cyanohydrin during processing relative to wild-type roots.

Development and application of transgenic technologies in cassava

The capacity to integrate transgenes into the tropical root crop cassava (Manihot esculenta Crantz) is now established and being utilized to generate plants expressing traits of agronomic interest. The tissue culture and gene transfer systems currently employed to produce these transgenic cassava have improved significantly over the past 5 years and are assessed and compared in this review. Programs are underway to develop cassava with enhanced resistance to viral diseases and insects pests, improved nutritional content, modified and increased starch metabolism and reduced cyanogenic content of processed roots. Each of these is described individually for the underlying biology the molecular strategies being employed and progress achieved towards the desired product. Important advances have occurred, with transgenic plants from several laboratories being prepared for field trails.

Over‐expression of hydroxynitrile lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification

Cassava (Manihot esculenta, Crantz) roots are the primary source of calories for more than 500 million people, the majority of whom live in the developing countries of Africa. Cassava leaves and roots contain potentially toxic levels of cyanogenic glycosides. Consumption of residual cyanogens (linamarin or acetone cyanohydrin) in incompletely processed cassava roots can cause cyanide poisoning. Hydroxynitrile lyase (HNL), which catalyses the conversion of acetone cyanohydrin to cyanide, is expressed predominantly in the cell walls and laticifers of leaves. In contrast, roots have very low levels of HNL expression. We have over-expressed HNL in transgenic cassava plants under the control of a double 35S CaMV promoter. We show that HNL activity increased more than twofold in leaves and 13-fold in roots of transgenic plants relative to wild-type plants. Elevated HNL levels were correlated with substantially reduced acetone cyanohydrin levels and increased cyanide volatilization in processed or homogenized roots. Unlike acyanogenic cassava, transgenic plants over-expressing HNL in roots retain the herbivore deterrence of cyanogens while providing a safer food product.

Generation of cyanogen-free transgenic cassava.

Cassava ( Manihot esculenta Crantz.) is the major source of calories for subsistence farmers in sub-Saharan Africa. Cassava, however, contains potentially toxic levels of the cyanogenic glucoside, linamarin. The cyanogen content of cassava foods can be reduced to safe levels by maceration, soaking, rinsing and baking; however, short-cut processing techniques can yield toxic food products. Our objective was to eliminate cyanogens from cassava so as to eliminate the need for food processing. To achieve this goal we generated transgenic acyanogenic cassava plants in which the expression of the cytochrome P450 genes ( CYP79D1 and CYP79D2), that catalyze the first-dedicated step in linamarin synthesis, was inhibited. Using a leaf-specific promoter to drive the antisense expression of the CYP79D1/ CYP79D2 genes we observed up to a 94% reduction in leaf linamarin content associated with an inhibition of CYP79D1 and CYP79D2 expression. Importantly, the linamarin content of roots also was reduced by 99% in transgenic plants having between 60 and 94% reduction in leaf linamarin content. Analysis of CYP79D1/ CYP79D2 transcript levels in transgenic roots indicated they were unchanged relative to wild-type plants. These results suggest that linamarin is transported from leaves to roots and that a threshold level of leaf linamarin production is required for transport.

PIG-mediated cassava transformation using positive and negative selection

In order to develop new selection systems for production of transgenic cassava (Manihot esculenta Crantz), two different selection regimes were assessed for their efficiency on regeneration of transgenic cassava plants: positive selection using mannose and negative selection using hygromycin. Explants from somatic cotyledons and embryogenic suspensions were used as target tissues in the transformation experiments and bombarded using the particle inflow gun. Different culture and selection strategies were assessed to optimise the selection protocols. For the first time transgenic plants could be obtained using positive, and in the case of embryogenic suspensions, hygromycin-based negative selection. The stably transformed nature of the regenerated cassava plant lines and the expression of the transgenes were verified with PCR, RT-PCR, Southern and northern analyses. A rooting test for transgenic plants on a medium supplemented with mannose was developed to further improve the efficacy of the positive selection system. Our results demonstrate that it is possible to obtain transgenic cassava plants using non-antibiotic positive selection.

Cyanogen reduction in transgenic cassava

Cyanogen reduction in transgenic cassava

Efficient production of transgenic cassava using negative and positive selection.

In order to improve the efficiency of cassava (Manihot esculenta Crantz) transformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one based on hygromycin resistance encoded by an intron-containing hph gene. Transgenic plants selected on mannose or hygromycin were regenerated for the first time from embryogenic suspensions cocultivated with Agrobacterium. After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic. A system allowing plant regeneration from only transgenic lines was designed by combining chemical selection with histochemical GUS assays. In total, 12 morphologically normal transgenic plant lines were produced, five using mannose and seven using hygromycin. The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis. RT-PCR and northern analyses confirmed the transgene expression in the regenerated plants. A rooting test on mannose containing medium was developed as an alternative to GUS assays in order to eliminate escapes from the positive selection system. Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micro-organisms or an antibiotic-free positive selection system.

Regeneration of transgenic cassava plants (Manihot esculenta Crantz) through Agrobacterium-mediated transformation of embryogenic suspension cultures.

A protocol was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. The bacterial strain ABI containing the binary vector pMON977 with the nptII gene as selectable marker and an intron-interrupted uidA gene (encoding β-glucuronidase) as visible marker was used for the experiments. Selection of transformed tissue with paromomycin resulted in the establishment of antibiotic-resistant, β-glucuronidase-expressing lines of friable embryogenic callus, from which embryos and subsequently plants were regenerated. Southern blot analysis demonstrated stable integration of the uidA gene into the cassava genome in five lines of transformed embryogenic suspension cultures and in two plant lines.

Pinpointing towards improved transformation and regeneration of cassava ( Manihot esculenta Crantz)

Friable embryogenic callus (FEC) of the cassava genotype 60444 was transformed by particle bombardment with DNA from the plasmid constructs pHB1 and pJIT100. Both plasmids contained the luciferase ( luc) marker gene under the control of the CaMV 35S promoter. In addition pJIT100 had the CaMV35S driven phosphinothricin acetyl transferase ( pat) gene, while pHB1 contained the cassava cDNA coding for the small subunit of ADP glucose pyrophosphorylase (AGPase B) in antisense orientation under the control of a double CaMV35S promoter. A total of 2 weeks after bombardment, luciferase (LUC) positive FEC units (spots) were isolated and subcultured separately for further proliferation. A total of 4 weeks later, those cultures having at least four positive LUC spots were subjected to three different selection regimes namely: stringent LUC selection, non stringent LUC selection and combined LUC/phosphinothricin (PPT) selection. A total of 16 weeks after bombardment, stringent LUC selection gave rise to cultures in which 92% of the FEC units were LUC positive. Within the same time period non stringently LUC selected cultures and LUC/PPT selection had only 1 and 41% of the units that were LUC positive, respectively. The number of LUC positive mature somatic embryos formed was similar to the percentage of LUC positive FEC units, within a culture, found with each selection method. Stringent LUC selection enabled transgenic plants to be produced in 28–36 weeks compared to 32–41 weeks for LUC/PPT selection and 53–78 weeks for non stringent LUC selection. This indicates that stringent selection is a more efficient method for obtaining transgenic cassava plants. Southern blot analysis of transgenic cassava plants revealed that they had between one to seven copies of the pHB1 and pJIT100 construct. The production of the first cassava plants carrying an agronomically important trait affecting starch biosynthesis is reported. Expression of the antisense AGPase B gene resulted in cassava plants within which the AGPase mRNA steady state levels was greatly decreased or even absent. The plants with no AGPase mRNA expression also had extremely low levels of starch, compared to control plants, as shown by iodine staining of in vitro thickened stems. In plants exhibiting the highest AGPase B antisense effect, starch formation was limited only to the epidermal layer of in vitro thickened stems.