Woody tree species

The forestry industry –which relies on woody tree species– is a major economical factor for several countries. Tree species like Eucalyptusand Pinus are very important in the paper and cellulose industry. Because of their long generation times, novel breeding methodologies are sought to introduce disease resistance genes, influence lignin production or modulate flowering time for example. Patents covering transformation technologies could be of commercially strategic relevance.

Summary

  • The European application assigned to the Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA) and theInstituto Valenciano de Investigaciones Agrarias (IVIA), both in Spain, is related to a United States patent analyzed in the ‘Citrus’ section. The present application discloses a procedure for the transformation of any woody species using adult plant material as primary explant. The explant is grafted on a stock to generate shoots which are then used as target tissue for Agrobacterium-mediated transformation. Treated shoots are then re-grafted in vitro to select for transformed shoots, which are then regenerated into full transgenic plants. This method addresses the problem of how to obtain improved clones from elite woody trees from which it is usually hard to obtain totipotent, regenerable material.
  • The PCT application assigned to the Companhia Suzano de Papel e Celulose and the University of Sao Paulo in Brazil discloses a method to transform woody tree species starting from pre-germinated seed material. The method disclosed was developed for transformation of Eucalyptus spp but the claims have been worded to encompass any woody tree species, which could include conifers. The method disclosed in the independent claims is rather detailed in terms of timing of the multiple steps. This timing could be very different for other tree species than Eucalyptus, which would allow ways to design around the claim in this patent (if granted) and thus avoid infringement.

Specific Patent Information

Patent Number Tite, Independent Claims and Summary of Claims Assignee
WO 2002/14463 A2

  • Earliest priority – 18 August 2000
  • Filed – 16 August 2001
  • OPI – 21 February 2002
  • International search report (A3) – 6 May 2004
  • Modified first page (C1) – 1 July 2004
Title – Method for genetic transformation of woody trees

Claim 1METHOD FOR GENETIC TRANSFORMATION OF WOODY TREES, which comprises the following steps:
-sterilization and washing of seeds of woody trees;
-transference of said seeds to an appropriate medium of culture;
-germination for a period between about 2 and 17 days;
-collection of germinated material;
-inoculation with Agrobacterium, containing one or more genes of interest and optionally one or more marker genes, under concentration between about 10E+7 and 10E+9 cells per ml;
-await inoculation between about 20 and 30 hours in a liquid medium;
-transference of the material to a solid medium for a period between about 38 and 50 hours in the dark, under temperature between about 25 and 31 C and ambient humidity;
-transference of the material to start plantlet growth in the light;
-withdrawal of a plantlet leaf obtained between about 5 and 17 days during growth in the light;
-transference of the plantlet leaf to the MS medium containing auxins derived from urea;
-await germination of the plant tissue for approximately 20 days; and
-identification and selection of the transformed plantlets.
Claim 2METHOD FOR GENETIC TRANSFORMATION OF WOODY TREES, which comprises the following steps:
-sterilization and washing of seeds of woody trees;
-transference of said seeds to an appropriate medium of culture;
-germination for a period between about 2 and 17 days;
-collection of germinated material;
-sonication of the material as collected ; inoculation with Agrobacterium, containing one or more genes of interest and optionally one or various marker genes, under concentration between about 107 and 109 cells per ml;
-await inoculation between about 20 and 30 hours in a liquid medium;
-transference of the material to a solid medium for a period between about 38 and 50 hours in the dark, under temperature between about 25 and 31 C and ambient humidity;
-transference of the material to start the plantlet growth in the light;
-withdrawal of a plantlet leaf obtained between about 5 and 17 days during growth in the light;
-transference of the plantlet leaf to the MS medium containing auxins derived from urea;
-await germination of the plant tissue for approximately 20 days and
-identification and selection of transformed plantlets.
Claim 9METHOD TO OBTAIN TRANSGENIC WOODY TREE PLANTS, which comprises the following steps:
-sterilization and washing of seeds of woody trees;
-transference of said seeds to an appropriate medium of culture;
-germination for a period between about 2 and 17 days;
-collection of germinated material;
-inoculation with Agrobacterium, containing one or more genes of interest and optionally one or more marker genes, under concentration between about 107 and 109 cells per ml;
-await inoculation between about 20 and 30 hours in a liquid medium ;
-transference of the material to a solid medium for a period between about 38 and 50 hours in the dark, under temperature between about 25 and 31 C and ambient humidity;
-transference of the material to start the plantlet growth step in the light;
-withdrawal of a plantlet leaf obtained between about 5 and 17 days during growth in the light;
-transference of plantlet leaf to the MS medium containing auxins derived from urea;
-await germination of the plant tissue for approximately 20 days;
-identification and selection of transformed plantlets;
-maintaining transforming plantlets in a multiplication and elongation medium for about 20 days;
-regeneration of the final transformed plant from one of the following regions hypocotyl; cotyledon; primary leaves and col.

A method for transformation of woody trees using pre-germinated seed as target tissue for Agrobacterium-mediated transformation.

Companhia Suzano de Papel e Celulose and Universidade De Sao Paulo
Remarks
  1. National phase entry of WO 2002/14463 in Australia (AU 2001/279510) is pending.
  2. National phase entry of WO 2002/14463 in China (CN 1630723) is pending.
  3. National phase entry of WO 2002/14463 in Europe (EP 1448777) is pending.
  4. National phase entry of WO 2002/14463 in the United States (US 2004/055041) is pending.
  5. Other national phase entries of WO 2002/14463 include Brazil (BR 200113455), South Africa (ZA 200301299).
EP 0870838 A2

  • Earliest priority – 5 March 1997
  • Filed – 4 March 1998
  • Application pending
Title – Procedure for the genetic transformation of adult plants of woody species

Claim 1A genetic transformation procedure for adult plants of woody species which consists of:

  • inoculating explants of adult tissue of woody species, from the first shoots of the grafts of buds of adult plants of woody species on stocks, with appropriate vectors which carry the genes which encode the characteristics of interest, under conditions which permit the development of transgenic shoots; and
  • micrografting in vitro said transgenic shoots, their buds or apices, on stock cultivated in vitro and,
  • subsequently, grafting the resulting micrografted plants onto other stocks which give vigor and let the taken shoots grow to generate complete adult transgenic plants, or directly transplanting the in vitro micrografted plants into the soil, to give complete adult transgenic plants.

A procedure for transformation of adult woody species with appropriate vectors, which is not limited to Agrobacterium. Adult tissue is grafted on stocks to generate new buds or apices that are used as target tissue for transformation followed by in vitro micrografting of transgenic shoots.

Remarks
  1. Related United States patent US 6103955 recites an Agrobacterium-mediated transformation method of citrus (see section on citrus).
  2. Other related patent application filed in Spain (ES 2151338).

Note: Patent information on this page was last updated on 28 March 2006.