Method of plant tissue culture and regeneration

Details Method of plant tissue culture and regeneration Method of plant tissue culture and regeneration

1996-03-14

1999-10-19

Campell, Bruce R.

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C12N 1500

Patent

active

059692154

DESCRIPTION:

BRIEF SUMMARY
This application claims benefit of international application PCT/GB94/02252, filed Oct. 14, 1994.


BACKGROUND OF THE INVENTION

This invention relates to a method of plant tissue culture and regeneration. More particularly the invention relates to a method for the genetic transformation of plant cells, the regeneration of whole plants from such cells and the plants so produced. Particularly, but not exclusively, the invention relates to a method for the transformation of Beta vulgaris, which includes sugar beet, fodder beet, table beet and Swiss chard.
Although genetic transformation and subsequent regeneration is largely a matter of routine nowadays for many plants species, some species have remained recalcitrant to transformation by most of the numerous methods which are available. Beta vulgaris is one such example where, despite transient expression in some cells and occasional success with specific genotypes, no simple routine method is available for the production of transgenic plants (International Patent Application No. WO 91/13159; D'Halluin, K. et.al., Biotechnology 10 309-314 (1992)). More particularly, no method for transformation via direct gene transfer and subsequent regeneration has yet been published. The recalcitrance of sugar beet protoplasts is well documented (Lindsey et.al. Transformation in Sugar Beet (Beta vulgaris L.) Biotechnology in Agriculture and Forestry, Vol 23, Plant protoplasts and Genetic Engineering IV" Y. P. S. Bajaj, Ed., Springer-Velag, Berlin, 1993). Cell division in vitro is restricted and totipotent colonies are generally obtained only at low frequency (0.1% or less). Protoplasts isolated from sugar beet leaves vary in size and morphology, reflecting the high degree of cellular heterogeneity present within the source tissue both at physiological (resulting from the relative location of the cells in vivo) and cytogenetic (ploidy, cell cycle phase) levels.
There is, therefore, a continuing need for a simple, high frequency transformation method which may be applicable to beet.
In order that a cell may be efficiently transformed, certain requirements must be satisfied. First the gene to be inserted must be assembled within a construct which contains effective regulatory elements which will drive transcription of the gene. Next, there must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. The probability of integration may be improved by certain means but, in general, integration is simply a matter of uncontrolled chance. Finally, as far as plants are concerned the target cell type must be such that cells can be regenerated into whole plants.
Plant cells are more difficult to transform than bacterial or animal cells because the presence of hard cell walls presents a barrier to insertion of the construct through that wall.
Thus the choice of method for the transformation of plant cells tends to be limited to those which are convenient for the target plant type (Potrykus, I, in Plant Breeding: Principles and Prospects, Ed. Hayward et.al., Publ. Chapman & Hall, London (1993)). As a generalisation, dicotyledonous plants are relatively easy to transform whereas monocotyledonous plants are very difficult, there being only a few techniques available in respect of which success has been reported, and that with very low success rate.
One method which is claimed to transform plant cells is the procedure known as "microinjection" where, under the microscope, a DNA construct is injected from a hollow needle into a target cell. A variant of that procedure is the rupturing of the cell wall with a needle, the DNA being added to the surrounding medium and allowed to diffuse into the cell through the break in the cell wall. This variant is known as "micropricking". Both of these procedures require a high degree of manipulative skill by the operator and are very time consuming. Japanese Published Patent Application Number 03103183 of 1991 pr

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