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Chapter 4 Genetic manipulation techniques: potential of controlling post-harvest deterioration

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Genetic transformation
Selection of transformed cassava tissue
Regeneration of transgenic plants
Potential of breeding for resistance to deterioration

Advances in biology and biotechnology have enabled scientists to dissect biochemical pathways, to isolate genes of interest from different organisms and to transfer these back into the original or alternative host plants. In agriculture this can lead to the production of plants with novel character) sties that would not be achievable through conventional breeding. The use of genetic manipulation techniques in breeding for resistance to physiological deterioration in cassava may alleviate the problems encountered by the use of conventional techniques. These problems are largely due to the highly heterozygous nature of the crop (see Chapter Three) which makes generating the parental genotype highly improbable.

To allow the potential of genetic manipulation to be realized, techniques are needed to integrate new genes into the cassava genome through genetic transformation of individual cells or tissues and to regenerate whole plants from the transformed material.


Genetic transformation

The aim of transformation is to introduce foreign DNA into the plant genome without altering the desirable characteristics of the genotype that is being transformed. Several techniques have been developed for introducing DNA into plant cells and these have been adapted for use with a wide range of plant species (Figure 16). Direct methods of treating plant protoplasts (plant cells from differentiated tissue, free of their polysaccharide cell walls following enzymatic treatment) are the most efficient methodologies in terms of foreign DNA uptake by protoplast suspensions after treatment with polyetheleneglycol (PEG) or electroporation (Cabral et al., 1993). For many economically important plant species, including cassava, regeneration of plants from protoplasts is not as yet possible. The prolonged periods of tissue culture associated with this type of regeneration are prone to cause undesirable somaclonal variation of the original genotype which generally introduces undesirable new traits such as albinism.

The use of organized tissues or organs with regenerative potential to grow into full plants, such as somatic embryos (embryos derived by tissue culture from non-sexual tissues), is required to avoid the above problem. In these cases, DNA must be introduced across the cell wall, using methods such as particle bombardment with DNA-coated gold particles (Klein et al., 1987) or the use of the crown gall bacterium, Agrobacterium tumefaciens. Both these methods are suitable for the delivery of DNA into somatic embryos and other organized tissues capable of regeneration. However, with particle bombardment methodology, DNA is delivered with very low efficiency to the cells as compared to DNA uptake into protoplasts. Electroporation or PEG treatment of partially digested embryogenic tissue, using cell wall degrading enzymes, is a novel process that tries to combine the advantages of both methodologies (Yang et al., 1991).

FIGURE 16 Possible ways of introducing foreign DNA into intact cells

A large number of plants belonging to many families have been transformed by Agrobacterium but this technique has not been successful with some important crops, such as maize, soybean and cotton. In the last few years, however, particle bombardment has demonstrated wide applicability in the transformation of these otherwise recalcitrant crops. Notably, regeneration in these crops is only possible when organized tissues are used as the starting material (Christou et al., 1990; Finer and McMullen, 1990; Gordon-Kamm et al., 1991).

A recent improvement in transformation is the combined use of particle bombardment with Agrobacterium. This treatment produces a high number of microwounds in the plant tissue which in turn facilitate subsequent penetration by Agrobacterium (Bidney et al., 1992). Microwounded cells, into which Agrobacterium has delivered the gene construct, have a high probability of resuming multiplication after repair of the cell wall and membrane.

The development of particle bombardment devices that can target the site of bombardment more accurately should be capable of achieving higher transformation efficiency by directing blasts of microprojectiles into meristematic tissues and specific groups of cells of only a few square millimetres (Potrykus, 1992). To prevent tissue destruction while allowing particles to enter the cells, the blast power must be controlled by careful finetuning.

At present protocols which use organized tissues, such as somatic embryos, are the only practical regeneration methods for cassava. These embryos are thus preferentially used as recipients of DNA constructs in cassava using Agrobacterium and particle bombardment methods. Results using these protocols were reported by several groups working on cassava transformation at the International Cassava Biotechnology Network Meeting in Cartagena, Colombia, in 1992. Somatic embryos from cassava have been produced using various starting materials, such as young leaf lobes (Raemakers et al., 1993; Stamp and Henshaw, 1987b; Szabados, Hoyos and Roca, 1987), primary somatic embryos (Raemakers et al., 1991; Stamp and Henshaw, 1987a), shoot meristems and cotyledonary leaves from somatic embryos (Sarria et al., 1993) and embryos derived from the tissue of sexual embryos (Stamp and Henshaw, 1982).

Development of a reliable and genotype-independent methodology for the transformation of cassava is particularly important because of the crop's agricultural importance in many developing countries (see Introduction). The introduction of any new trait into cassava by a transgenic approach will have to be carried out for many different genotypes that are suited to different agro-ecological zones and that fulfil diverse end uses and requirements.


Selection of transformed cassava tissue

The availability of a selection procedure designed to eliminate nontransformed plants or tissue and thereby reduce the number of plants to regenerate, is an important component of practically all transformation protocols. Gene constructs generally contain a marker gene in addition to the gene of interest. Screenable marker genes allow transformed cells to be visualized and therefore facilitate separation from non-transformed cells. There are two commonly used approaches to the use of marker genes. One method is to insert a gene coding for an enzyme that, with the appropriate substrate, generates a coloured product that can be visualized in transformed cells. This kind of gene is frequently used for studying transient expression. The other method of selecting marker genes is to code for antibiotic or herbicide resistance so that transformed cells can be separated by their ability to grow on media containing the corresponding antibiotic or herbicide.

In cassava, selection has been attempted with three selectable marker genes, nptll, hyg and bar. which confer resistance to the antibiotics kanamycin and hygromycin and to the herbicide phosphinothricin, respectively. Low sensitivity of embryos to kanamycin allows higher concentrations of the antibiotic to be used in the selection process, but this might interfere with the regeneration process. Phosphinothricin is more efficient at selecting embryos, as opposed to calli (C. Sch÷pke, unpublished results) and less inhibitory of their development (Sarria et al., 1993). Recurrent use of the selecting agent has led to the development of chimeric plantlets that express the gene construct in only certain cells or groups of cells (Raemakers, Jacobsen and Visser, 1993; Sch÷pke et al., 1993).

Studies on cassava have also been conducted utilizing the gusA gene (GUS) from Escherichia coli, which codes for a ▀-glucuronidase, as a screenable marker (Jefferson, Kavanagh and Bevan, 1987). The enzymatic activity of the glucuronidase is a measure of the number of embryos expressing the marker gene and indicates transformation efficiency. This reaction results in transformed cells staining blue (Figure 17). It is also a measure of promoter strength and is very useful in the study of gene cassettes for the efficiency of the different elements of the gene construct (promoters, enhancers and regulatory regions).

Transformation of cassava somatic embryos has been achieved using particle bombardment and several strains of Agrobacterium, including some that naturally infect cassava. Homogeneous GUS activity has been observed in only a few secondary embryos three months after excision and bombardment of the cassava meristem. Only about 1 percent of the GUSpositive embryos detected in the first few weeks displayed enzymatic activity after three months (Figure 18). However, their presence provided a strong indication of stable integration of the gene construct into the cassava genome (transformation efficiency) (Sarria et al., 1993).

The drawback to the GUS technique is that the enzymic reaction is destructive, killing the tissues in which it is active (Jefferson, Kavanagh and Bevan, 1987; Kosugi et al., 1990). Protocols for the detection of GUS activity in the surrounding growth media of seedlings and other tissues are now available (Martin et al., 1992). However, this is not a very sensitive method and its usefulness for evaluating transformed cassava embryos/ plantlets remains to be established. The development of non-destructive screenable markers to replace the existing GUS assays would greatly enhance selection procedures and allow positive embryos to be directly selected for regeneration. To avoid embryos being destroyed during the selection procedure, developments are being made towards a secreteable GUS and a novel enzyme, aryl sulphatase, which is not produced by plants (Richard Jefferson, personal communication).

The most commonly used promoter sequence in transgenic studies is the cauliflower mosaic caulimovirus 35S promoter. Transient expression of gene constructs containing 35S promoter has demonstrated the utility of this promoter in cassava ( Franche et al., 1991). The activity of the promoter can be increased by including enhancer sequences (Cabral et al., 1993; Franche et al., 1991). Expression in cassava has been demonstrated for mesophyll cells but the constructs were not functional in root cells. This failure may be due to different 35S promoter specificities (Benfey and Chua, 1990; Benfey, Ren and Chua, 1990a; 1990b) or DNA degradation by nucleases present in the root (Arias-Garzˇn and Sayre, 1993).

In some plant species, the production of chimeric plants could lead to pollen or egg cells being transformed. However, these events do not offer a viable alternative in cassava because of its high heterozygosity, low rates of seed germination and lack of flowering in certain genotypes (see Chapter Three). In cassava, transformed patches from regenerable tissue could only give rise to solid transformants if it is possible to excise and regenerate such groups of cells.

To date it has not proved possible to regenerate cassava plants from transformed cassava tissues. Transient gene expression in tissues that are not regenerative is, however, considered to be useful as a tool to assess the potential functionality of gene constructs. Agrobacterium transformation of cassava with constructs coding for coat proteins of cassava common mosaic virus and African cassava mosaic virus has been undertaken. High levels of expression of the viral protein coat have been detected in transformed call), thus confirming the potential of this approach. The use of such sequences to protect plants from virus infection cannot be fully assessed, however, until whole plants have been regenerated (Fauquet et al., 1993). In other plant species this technique has been found to be very successful in conferring virus resistance (Wilson, 1993).


Regeneration of transgenic plants

The regeneration of plants from transformed cassava cells has not as yet been achieved, although major progress has been made at the different stages of this technology. Once transformation protocols have been developed, transgenic plants could be initially multiplied through the efficient production of somatic embryos. Secondary embryos can be produced in a cyclic fashion, utilizing primary embryos as the starting material, a process called cyclic somatic embryogenesis (Raemakers et al., 1993). The development of embryonic suspension cultures might further accelerate the process and improve transformation protocols.

Somatic embryogenesis is the only well documented regeneration method for cassava and several cultivars from South America, Africa and Indonesia have been successfully used. Some previously recalcitrant cultivars have also been regenerated after the hormonal composition of the media was adjusted (Sudarmonowati and Henshaw, 1993; Taylor, Clarke and Henshaw, 1992). The fine-tuning of the transformation, selection, embryogenesis and regeneration media is often fortuitous and one component in the wrong concentration might inhibit the success of the whole process.

Evidence exists that cassava primary embryos are of multicellular origin, while secondary embryos are of unicellular origin (Tello, 1988). Further histological studies are being undertaken to clarify this point which indicates that solid transformants are likely to arise through secondary embryogenesis from transformed sectors of primary embryos. This fact, if correct, would facilitate the clonal propagation of cassava. Further evidence is suggested from homogeneous GUS expression from transformed secondary embryos.

Recently a significant improvement in the regeneration of somatic embryos, which results in germination of 85 percent of the embryos, has been reported (Mathews et al., 1993). This is based on a technique that uses activated charcoal in the medium and a mild desiccation treatment of the embryos. Culture metabolites and free phytohormones, which might have an inhibitory effect on specific development, are probably absorbed by the charcoal treatment, facilitating morphogenic development. Desiccation of embryos might also trigger morphogenic responses, by mimicking seed desiccation processes, which eventually lead to embryo germination. This technique is being considered as a rapid micropropagation system for cassava because a single leaf could produce hundreds of seedlings. The technique has been demonstrated with one South American cultivar and is currently being repeated with other African and South American cassava cultivars.

The use of Agrobacterium has been found to inhibit the production of secondary embryos and plant regeneration even when bacteria are not obviously present, such as when following antibiotic treatment with cefotaxime and carbenicillin (Sarria et al., 1993). This might be attributed to various causes, such as hormonal imbalance or the different antibiotics used for the elimination of bacteria or as a selection agent. Some improvements have recently been obtained after compositional changes to the growth media (Rodrigo Sarria, personal communication).


Potential of breeding for resistance to deterioration

To enhance the storage potential of cassava would have substantial impact on resolving the deterioration-related constraints associated with cassava marketing and utilization (see Chapter Five). A molecular genetic approach could envisage an increase in the storage potential of cassava roots to a minimum of two weeks' without the use of post-harvest treatments. This could be achieved by genetic manipulation to suppress the processes involved in physiological deterioration (see Chapter One) and to enhance the woundhealing response to prevent the onset of microbial deterioration (see Chapter Two).

Physical damage is an inevitable consequence of harvesting cassava roots. This initiates the chain of events leading to physiological deterioration, which usually precedes the opportunistic invasion of the root by microorganisms. The phenylpropanoid pathway is indicated as being actively associated with localized (curing) and non-localized (physiological deterioration) wound responses in cassava (see Chapter Two). The products of the phenylpropanoid pathway are ubiquitous in flowering plants and have diverse functions related to pathogen detence (Moerschbacher et al., 1990; Taniguchi et a/., 1984), wound healing (Lagrimini, 1991) and cell wall strengthening (Dixon and Harrison, 1990). Because of their implication in the processes associated with physiological deterioration and wound healing, the genes and enzymes responsible for the synthesis of phenylpropanoids, such as coumarins, lignin, anthocyanins, flavanoids and phenolic components of suberin (see Chapter Two), are principal targets for study and manipulation (Hahlbrock and Scheel, 1989).

The uncontrolled intervention of a key enzyme in the phenylpropanoid pathway, could interfere with the production of vital components of the plant. The introduction into cassava of discrete gene constructs by genetic manipulation offers the unique advantage of adding new traits to elite genotypes without altering other functional properties or desired characteristics. At present there is no conclusive information available on the genes involved in the biochemical pathways associated with physiological deterioration in cassava.

There are well characterized wound-response model systems for other species that have been shown to be fairly universal among flowering plants. A number of the genes that encode key steps of the pathways associated with wound-induced responses have already been cloned and sequenced from several plant families (Hahlbrock and Scheel, 1989). Regulatory mechanisms of genes involved in the phenylpropanoid pathway seem to be conserved among different species (Fritze et al., 1991; Staiger, Kaulen and Schell, 1989).

Plant wound responses are triggered by signals (see Chapter Two) that initiate different types of reactions (Trewavas and Gilroy, 1991) and responses at this level can be observed within minutes of the external stimulus without the need for de novo synthesis (Dietrich, Mayer and Hahlbrock, 1990). Identification of wound-response elicitors activated in cassava and the genes induced by these substances will need to be characterized in order to understand the processes involved in physiological deterioration. Subsequent identification and isolation of the genes involved in these processes will be necessary to enable potential genetic modification of the wound responses.

To differentiate between desirable (localized) and undesirable (nonlocalized) post-harvest wound responses, phenylpropanoid pathway genes can be isolated from cassava using heterologous probes derived from other plants, such as petunia or tobacco. Genes being expressed de novo during the wound response can be isolated by subtracting complementary DNA (cDNA) libraries derived from wounded tissue with DNA from non-wounded tissue (Logemann et al., 1988). The cDNA libraries represent all the genes actually being expressed in the tissue from where the messenger RNA was isolated. It is possible to produce genetic libraries that represent the momentary status of expression in wounded/non-wounded tissues or localized/non-localized wound responses.

Gene expression patterns can also be analysed by immuno-histochemistry or in situ hybridization with antisense RNA (complementary to the gene sequence). Visualization of specific proteins or transcripts at the subcellular level can be performed on tissue sections by hybridization with radioactive or fluorochrome labelled antibodies or RNAs. The spatial distribution of the ▀-glucosidase transcript, which codes for an enzyme involved in cyanogenesis in cassava, has successfully been characterized using riboprobes (Pancoro and Hughes, 1992). This technique can be used to study the expression of selected genes during wound responses in cassava roots.

Additional information on the identification of genes expressed during the development of physiological deterioration can also be obtained by analysing the chemical changes following wounding. Compounds specific to the deterioration process can be distinguished and characterized. This could lead to the identification of the genes involved in their metabolism, allowing for the synthesis of DNA probes to isolate the corresponding genes from genomic or cDNA libraries.

The understanding of the molecular mechanisms associated with cassava wound responses will help in designing a transgenic approach to increasing the storage potential of cassava. The desired result could be obtained if the appropriate tissue-specific and temporally regulated promoters were provided. Some of the promoter genes with relevance to physiological deterioration may have to be: root specific; starchy root specific; wound specific; or spatially restricted in the xylem vessels, periderm, root tip or parenchyma. There are several tissue-specific genes that have been isolated, such as root-specific genes from tobacco (Conkling et al., 1990), tuber-specific genes from potato (Rosahl et al., 1986), genes specific to the shoot apical meristem (Medford, Elmer and Klee, 1991) and stamen-specific genes (Smith et al., 1990).

Successful transgenic plants have been obtained with genetic modifications to the phenylpropanoid pathway. The chalcone synthase promoter from petunia has been introduced into tobacco, where it maintained its ultraviolet-light inducibility (Kaulen, Schell and Kreuzaler, 1986). The phenylpropanoid pathway of petunia has been manipulated by the introduction of the maize dihydroflavonol-4-reductase gene, producing orange-flowered transgenic plants (Meyer et al., 1987). Ripening of climacteric fruits, such as tomato, has been blocked by suppressing the synthesis of amino-cyclopoppane carboxylate synthase, thereby inhibiting the production of ethylene, the phytochrome responsible for the ripening process (Oeller et al., 1991).

Enzymatic pathways have already been successfully manipulated in different plant species in different ways through the introduction of foreign or homologous genes. The molecular genetic approach to controlling physiological deterioration of harvested cassava roots therefore seems reasonably promising. An integrated approach directed at solving the deterioration problem of cassava will also deliver useful tools, such as gene constructs containing tissue-specific and temporally regulated promoters, for the rest of the research community. To achieve this goal, two main constraints will have to be solved, genetic transformation of cassava and the identification of key target genes for manipulation of the physiological deterioration process.

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