GMO. Another Perspective. The dark side of Patents

Tito Schiva

• Publisher: Youcanprint
• Year: 2017
• ISBN:
  9788892645141
• Topic: Biological Sciences
• Subtopic: Genetics & Genomics
• Index: Biological Sciences

Tito Schiva, geneticist and past Director of the Experimental Institute for Floriculture Sanremo I for 30 years, attended the UPOV Workshop (International Convention for the New Varieties of Plants Protection) as Italian delegate. In the pre-DNA period, together with A. Mercuri, he developed a method for genotype identification based on the isoenzymatic fingerprinting for plant varieties with a view to protecting intellectual property. At the advent of genetic transformation techniques, again working with A. Mercuri, he created dwarf compact plants on Limonium sp. using the ROL genes, and fluorescent flowers on Lisianthus and Rinchospermum using. GFP genes (Green Fluorescent Protein).
So far the controversy on GMO has concerned essentially the wealthy and the environment not highlighting the consequences of the Patent on living matter.
To apply a Patent on a gene provokes unique biological/economical synergy and has a great impact on our lives.
Gunter Reimann, in "Patent for Hitler" (1942), showed how the Patent was stifling the development of technology. In this reality the food step crops appear to be the most vulnerable.
Slowing down innovation is the most negative aspect of the Patent system, but the greatest tragedy lies in the political mistake of not pointing out the guidelines or worse forbidding the development of these bio-technologies, and then leaving this know-how as a privilege of the few.

ANNEX 1

Patent application for Carnation GM. (Down loud from www.espacenet.com) showing in particular the claims on several methodological step referred to the protocol of invention.

1) Genetically transformed Carnation plants and methods for their propagation

Nov 10, 1993 - Florigene Europe B.V.

• Carnation plant material is transformed by co-cultivation with Agrobacterium cells carrying an exogenous DNA sequence. In particular, by initiating callus formation on the plant material, transformed shoots may be produced in a suitable medium. Plantlets may be produced from the shoots by initiating root formation and subsequently transferring the rooted shoots to soil.
• Genetically transformed Carntion plants and methods for their production

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Florigene Europe B.V. Patents:

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods for culturing and genetically altering higher plants. More particularly, the present invention relates to methods for culturing and genetically transforming tissue material from carnation plants.

The carnation, Dianthus caryophyllus, is a popular ornamental plant and highly valued for its cut flowers. As with many ornamental plant species, breeders have long sought to improve existing varieties and to create new cultivars using conventional techniques, such as cross-breeding and somatic clonal variation. Phenotypic variations of particular interest include color, fragrance, morphology, herbicide resistance, pesticide resistance, environmental tolerance, vase life of the cut flower, and the like. While improvements and variations in many or all of these characteristics have been achieved, progress is slow because of the inherently random nature of such breeding approaches. Indeed, the introduction of any particular characteristic requires a substantial effort if it can be achieved at all.

The propagation of carnation plants through the regeneration of plant parts and callus, as well as through micropropagation of shoot cultures, can be difficult even when it is not desired to introduce genetic alterations. In particular, methods proposed for the regeneration of carnation tissue often result in “vitrified” shoots having a glassy or translucent appearance and an abnormal morphology. Moreover, conventional regeneration methods often have regeneration frequencies below about 10% Micropropagation methods often are slow and produce few shoots per original cultured shoot. Those methods involving liquid medium cultures can be laborious and care subject to contamination.

For these reasons, it would be desirable to provide improved regeneration and micropropagation procedures for the efficient in vitro reproduction of carnation plant material. It would be particularly desirable to combine recombinant DNA technology with the regeneration and micropropagation techniques in order to produce new carnation cultivars in a controlled and predictable manner. Such recombinant DNA methods should provide for transformation, should be capable of introducing preselected exogenous gene(s) to the carnation plant, and should permit selection of transformed plant materials which are capable of expressing the exogenous gene(s). The method should also produce regenerated carnation plants which have stably incorporated the desired exogenous DNA sequences.

2. Description of the Background Art

Woodson (1989) Hort. Science 24:80 (Abstract No. 172) briefly describes the use of Agrobacterium tumefaciens to transform carnation petal explants. While the regeneration of roots in putative transformants is asserted, no regeneration of whole plants is described. The regeneration of carnation plants (without transformation) from tissue has been described. See, e.g., Petru et al. (1974) Biologia Plantarum 16:450-453 (stem formation in callus from hypocotyl and apical meristems); Lesham (1986) Hort. Science 21:320-321 (regeneration from callus and petals); and Frey et al. (1989) Hort. Science 24:74 (Abstract No. 124) (regeneration from petals and other explants with a callus stage; 1.5-3% of shoots survived transfer to soil). The micropropagation (shoot multiplication) of cultured shoots and meristem material has been described. See, e.g., Hackett et al. (1987) Amer. Soc. Hort. Sci. 90:365-369 (shoot tip propagation); Earle et al. (1975) Hort. Science 10:608-610 (shoot tip propagation); Davis et al. (1977) J. Amer. Soc. Hort. Sci. 102:48-53 (shoot tip propagation); and Ziv et al. (1983) Plant Cell Tissue Organ Culture 2:55-65 (describes vitrification in shoot tip culture).

SUMMARY OF THE INVENTION

The present invention comprises methods for genetically transforming carnation plant material, particularly leaf and petal explants, by cocultivating (or otherwise inoculating) the plant material with Agrobacterium cells carrying an exogenous DNA sequence. After cocultivation, callus formation is preferably initiated in the plant material. Transformed plant material (calli or shoots) is selected, typically by exposure to a plant selection agent which inhibits formation of plant material which does not express the exogenous DNA sequence. In the preferred embodiments, plantlets are produced from the selected (transformed) plant material by first regenerating transformed shoots and then inducing root formation in the regenerated transformed shoots.

The present invention further comprises carnation callus material and carnation plants which incorporate such exogenous DNA sequences. Preferably, such transformed calli and plants are obtained by the methods of the present invention.

The methods of the present invention provide a particularly convenient technique for selectively breeding new carnation cultivars in a predictable and expeditious manner. A variety of traits, such as color, fragrance, herbicide resistance, insect resistance pesticide resistance, flower vase life, environmental tolerance, disease resistance, horticultural traits, and the like, may be introduced into the carnation explant material by stably incorporating appropriate genes into the chromosomes of the selected plant cells, which may then be regenerated into carnation plants.

It has been found that use of a transformation method which involves passage of at least some of the cocultivated transformed plant material through the callus stage has certain advantages. This approach appears to reduce or eliminate chimerism, i.e., the presence of both transformed and non-transformed cells, in the regenerated shoot and rooted plant materials. Such chimerism can be a problem when the transformed explant material is directly regenerated without passage of any of the plant material through the callus stage. A preferred embodiment involves selection of initial transformants during the callus stage using a selection agent which is present at levels which inhibit (but do not kill) the non-transformed calli; this has been found to provide for high proliferation levels of transformed plant material. Such high proliferation levels greatly facilitate screening for individual calli and plants which have successfully incorporated a desired phenotype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of plasmid pSLJ1911 employed in Examples 1 and 3. LB and RB are left and right borders of T-DNA respectively. Restriction enzyme sites are indicated, e.g. as HindIII, with a number which indicates the base number distance from HIIIK. On pRK290, the number adjacent to the restriction site denotes the distance from the RIK site.

FIG. 2 is a gel showing the presence of introduced DNA amplified by PCR. The lanes are as follows.

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Lanes

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1-5 Samples from transgenic plants of Example 1.

6-7 Samples from transgenic plants of Example 2.

8 Sample from transgenic plants of Example 1.

9, 11 Samples from nontransformed control of Example 1.

12 Sample from nontransformed control of Example 2.

10 Sample from spiked-plasmid DNA in control tissues (used as positive control).

13, 14 Samples from reactions in the absence of sample DNA (negative controls).

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FIG. 3 is a map of binary plasmid pJJ3499 employed in Example 2. The construction of pJJ3499 is the same as pSLJ1911 synthase except that the nopaline synthase promoter controls the NPT gene.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

According to the present invention, genetically transformed carnation plants and calli are obtained by the selective introduction of exogenous DNA sequence(s) into the chromosomes of cultured carnation plant tissue material. The plant tissue material will be somatic tissue from any source that is capable of producing calli, such as leaf explants, petal explants, stamen filaments, stem sections, shoot tips, receptacles, sepals, and the like, with leaf explants and petals being preferred. The use of leaf explants is particularly preferred.

Any variety of carnation plant (Dianthus caryophyllus) may be used as a source of the plant tissue material. Suitable varieties include the “Sim type” which is available in a number of colors including red, white, pink, orange, and variegated forms; various Mediterranean types; and miniature types. Of particular interest are certain Sim varieties, including William Sim, Improved White Sim, Nathalie, Manon, Tibet, Cephali, Iceland, Cerise Royalette and Sandra.

Leaf explant material may be obtained from whole plant or plantlet but is preferably obtained from carnation shoots produced in tissue culture by conventional techniques. Such conventional techniques are described in the scientific literature, see, for example, Mii et al., in Handbook of Cell Culture, Vol. 5, Ammirato et al., eds., Vol. 5, McGraw-Hill Publishing Company, New York, 1990, and Besemer, Introduction to Floriculture, Larsen, ed., Chapter 2, Academic Press, Inc., 1980, the disclosures of which are incorporated herein by reference.

A suitable technique for preparing a carnation shoot culture begins with meristem tissue from the node (i.e., the slightly enlarged portion of the stem where leaves and bud arise and where branches originate) or in the shoot tip or apex (i.e., the portion of the shoot containing apical or primary meristem). The meristem material is surface sterilized, for example with 75% ethanol for two minutes followed by 20% commercial bleach (sodium hypochlorite) with 0.1% Tween.RTM.-20 for 20 minutes, followed by rinsing several times with distilled or deionized water. The sterilized meristem tissue is then divided into pieces from about 1 to 3 mm in size.

Preferably, the meristem tissue is cultured in a shoot multiplication medium containing nutrients, an energy source, an auxin, and a cytokinin, with benzyladenine (BA) being the preferred cytokinin and naphthalene acetic acid (NAA) being the preferred auxin. The BA is present at from about 0.5 to 5 mg/l preferably at about 1 mg/l and the NAA is present at from about 0,005 to 1 mg/l preferably at about 0.02 mg/l. A preferred multiplication medium is BN medium described in the Experimental section hereinafter. Using this medium, a large number of shoots, typically from about 20 to 50, may be obtained from a single meristem culture within from about 6 to 8 weeks. Additional shoots, usually from about 50 to 100, can be obtained by subculturing the first generation of shoots.

Prior to obtaining the leaf explant material, it is desirable to subculture the first or subsequent generation shoots on a medium containing nutrients and an en...

Table of contents

• GMO
• Contents
• Preface
• What is a Patent?
• How could it succeed?
• The arrival of GMO
• How did we get here?: Or better still: where are we going?
• Power of the Patent
• Collateral effect
• Fall out on scientific research
• Consequences on development
• Results of cost
• Afterword
• Bibliography
• Annex 1 - Patent Application for Carnation GM
• Annex 2 - Patent License Agreement