AGRE-0001 The Research Development and Production of Low Temperature Storage Tolerant Chipping (Crisping) Potato Cultivars

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Biotechnology : ECLAIR Cluster III - Carbohydrates : Starch

CURRENT STATE OF THE PROJECT

In March 1989 a project to research, develop and produce low temperature storage tolerant chipping potato cultivars by means of recombinant DNA techniques was submitted to the European Commission for assistance within the ECLAIR programme. This project was programmed to take place over 5 years.

From the two previous annual reports to the ECLAIR authorities and the most recent reports of the subprojects, progress can be measured against the time action work plan and the milestone criteria laid down. In the original ECLAIR application the milestone criteria were:

Years I to 3

- the extent to which the various enzyme genes have been sequenced and characterised;

- the stage at which transformation begins;

Years 4 to 5

- the level and quality of expression of the transform and modifications;

- the level of reduction in tuber hexose concentrations, and hence the level of discoloration after processing, in the resulting modified cultivars when stored at low temperature.

AGROTECHNOLOGICAL RESEARCH INSTITUTE

At the Agrotechnological Research Institute (ATO), significant progress has been made by cloning a full length PFK gene from a psychrophilic bacterium, thus possibly enabling the introduction of coldstable PFK activity in potato. Work has progressed to tailor the bacterial PFK gene to be more 'plantlike' and to produce suitable promoters for potato tuber expression.

Examination of the G + C content of the B.macquariensis PFK gene revealed three stretches of AUrich sequences that might function as introns. Therefore, the gene has been subjected to extensive modifications to study the effects of these potential introns on in plant a expression. In addition, a gene was constructed in which all stretches of 6 or more consecutive deoxyadenosines (dA) and/or deoxythymidines (dT) were modified by replacing one or more of them with deoxycytidine (dC) or deoxyguanosine (dG). All of these mutations have been incorporated without changing the amino acid sequence. Transgenic shoots containing constructs with these modified genes are currently being rooted and will subsequently be micropropagated and analyzed with respect to PFK expression.

DANISCO GROUP

The Danisco Group at Copenhagen has isolated the alphaamylase gene that is expressed in tubers and which increases with sprouting. To obtain the expected phenotype the gene should be turned off in the tubers by the use of antisense

technology. Antisense constructs have been made and put into plants under the control of a generally expressed (constitutive) promoter and been shown to be active (ie to produce mRNA). Some of the transformed potato plants have produced tubers which will be analyzed in due course. From the tuber specific alphaamylase it should be possible to identify the required tuber specific sequences for this promoter.

The Danish group are working with starch phosphorylase, and enzyme implicated in starch metabolism, and are seeking to turn this enzyme off using an anti sense approach. However, other research groups have already reported that although such anti sense constructs decreased the activity of the gene considerably, they do not bring about any change in cold sweetening. It is still worthwhile to continue the experiments at a low level of effort since there is a possibility that it is necessary to achieve a total suppression of activity before an effect is seen, this was not achieved by the other group.

MAX PLANCK INSTITUTE

The Max Planck Institute's approach to invertase control; that of cloning and expressing an invertase inhibitor, has run into some difficulties. It is not uncommon at the present state of the art for proteins to copurify. This can lead to wrong antibodies and amino acid sequences being generated from the impurity rather than the desired protein which has all the appearances of being an inhibitor of enzymes that degrade proteins (pro tease inhibitor). However, there are also cases where proteins have two activities and it is not yet totally ruled out that the protein may have an effect on the activity of invertase. Constructs to express the gene in potato have been made and transformation begun so some definitive results might be expected in the future. A series of check points were drawn up to deal with the situation would the clone prove to be the wrong one and a new start have to be made.

It is presumed that the best approach to ensure minimum effect on the rest of the phenotype of the final transformed plants is to have the chosen genes expressed specifically in the tubers under cold storage conditions. Progress to obtaining controls that would allow this pattern of expression is continuing.

Genomic clones containing the promoter region of three different cold inducible genes have been isolated (C17, Cl9, C115/C121). One promoter was sequence characterised and is now ready for expression studies (C17). One of the cold induced cDNAs (Cl 13) is coding for a glycerinaldeyde3phosphate dehydrogenase (GAPDH) of the glycolytic pathway.

SCOTTISH CROP RESEARCH INSTITIUTE

At the Scottish Crop Research Institute progress has been made in identifying and classifying invertases:

Invertase I is a gene encoding a protein similar to the carrot cell wall invertase. Using the carrot gene as a probe a genomic clone has been isolated and nearly completely characterised. Its predicted amino acid sequence is 50% homologous to the carrot enzyme and also to a tomato vacuolar invertase. This gene does not appear to be expressed in tubers under normal temperatures.

Invertase 2 is the major soluble invertase from tubers. A cDNA clone has been isolated which has a high probability of being the corresponding cDNA sequence to the protein. It is expressed in tubers.

Invertase 3 is a soluble alkaline invertase purified from leaves. It is related to the tuber soluble invertase on the basis of its reaction with antibodies.

Invertase 4 is a tenaciously bound (probably cell wall) invertase found in leaves, stems and roots but not tubers. It may be equivalent to the protein encoded by the gene for invertase 1.

Constructs in the sense and antisense directions have been made for both Invertases I and 2 to transform potatoes. The first antisense constructions have been put into plants to attempt to turn this enzyme off. Probably the antisense constructs for INV I will not affect invertase levels in tubers but this is not certain. However, the results with INV 2, which apparently codes it for a cytoplasmic broadspecificity enzyme may give a more interesting phenotype.