Duckweed

Summary

Duckweeds are small, fresh-water plants with a world-wide distribution. They are exploited for protein production due to two unusual aspects of their growth: the plants reproduce vegetatively by budding and under intensive culture they accumulate a very high rate of biomass. The level of protein production can achieve that obtained with yeast gene expression systems.

Two entities have patents and patent applications directed to Agrobacterium-mediated transformation of duckweed:

  • North Carolina State University has been granted a United States patent directed to transformation of duckweed tissue with Agrobacterium having a gene of interest. The invention further comprises a method for mass production of recombinant proteins or peptides from duckweed cultures.
  • Yeda Research & Development Co. has filed patent applications in Europe, Australia and Canada directed to a transformed Lemnaceae plant with Agrobacterium. Lemnaceae is the name of the botanical family to which duckweed belongs. Duckweeds from the genera SpirodelaLemna and Wolffia are used for the production of various chemical and biological products. The claims as filed recite different methods for the transformation of Lemnaceae such as in planta transformation, microinjection of Agrobacterium cells into meristematic plant cells and incubation of meristematic plant cells with Agrobacterium.

Although the inventions disclosed by both institutes may overlap in terms of the subject matter, they have been filed in different countries. The claims as filed by Yeda Research & Development Co. in the European patent application are broad and may have a different scope if granted.

Duckweed – Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 6040498

  • Earliest priority – 11 August 1998
  • Filed – 11 August 1998
  • Granted – 21 March 2000
  • Expected expiry – 10 August 2018
Title – Genetically Engineered Duckweed

Claim 1A method for stably transforming duckweed tissue with a nucleotide sequence of interest, the method comprising the steps of:

A) inoculating a duckweed plant tissue with Agrobacterium comprising a vector which comprises a nucleotide sequence of interest; and
B) co-cultivating the tissue with the Agrobacterium to produce stably transformed tissue.

Claim 20A stably transformed duckweed plant comprising a heterologous nucleic acid of interest incorporated in its genome wherein said plant is produced via an Agrobacterium-mediated method.
Claim 30A method of producing recombinant proteins or peptides, comprising the steps of:

A) culturing a stably transformed duckweed plant that expresses at least one heterologous protein or peptide; and
B) collecting the at least one heterologous protein or peptide from the duckweed cultures.

Claim 48A method for stably transforming duckweed tissue from the genus Lemna with a nucleotide sequence of interest, the method comprising the steps of:

A) inoculating a duckweed plant tissue with Agrobacterium comprising a vector which comprises a nucleotide sequence of interest, wherein the duckweed plant tissue is from the genus Lemna; and
B) co-cultivating the tissue with the Agrobacterium to produce stably transformed tissue.

Claim 59A stably transformed duckweed plant from the genus Lemna comprising a heterologous nucleic acid sequence incorporated in its genome wherein said plant is produced via an Agrobacterium -mediated method.
Claim 62A method of producing recombinant proteins or peptides, comprising:

A) culturing a stably transformed duckweed plant from the genus Lemna that expresses at least one heterologous protein or peptide; and
B) collecting the at least one protein or peptide from the duckweed cultures.

A method for transforming a duckweed tissue by co-cultivating the tissue with Agrobacterium having a sequence of interest. A method for producing recombinant proteins or peptides by culturing transformed duckweed plants expressing the proteins or peptides and collecting them form the cultured plants.
The same methods are used to transform a duckweed from the genus Lemna.

North Carolina State University

AU 775632 B2

  • Earliest priority – 12 August 1997
  • Filed – 11 August 1998
  • Granted – 19 December 2002
  • Expected expiry – 10 August 2018
Title – Genetically engineered duckweed

Claim 1
A method for producing a stably transformed duckweed plant comprising a heterologous nucleotide sequence of interest, the method comprising the steps of:(a) inoculating a duckweed callus tissue with an Agrobacterium comprising a vector which is not a super-virulent vector and which comprises a heterologous nucleotide sequence of interest, wherein the nucleotide sequence comprises at least one expression cassette comprising a gene which confers resistance to a selection agent;
(b) co-cultivating the callus tissue with the Agrobacterium to produce stably transformed callus tissue; and
(c) regenerating a stably transformed duckweed plant from the stably transformed callus tissue.
Claim 40
A method for producing a stably transformed duckweed plant comprising a chimeric nucleotide sequence of interest, the method comprising the steps of:(a) inoculating a duckweed callus tissue with an Agrobacterium comprising a vector which is not a super-virulent vector and which comprises a chimeric nucleotide sequence of interest, said chimeric nucleotide sequence comprising a coding sequence operably linked to a transcription initiation region that is heterologous to said coding sequence;
(b) co-cultivating the callus tissue with the Agrobacterium to produce stably transformed callus tissue; and
(c) regenerating a stably transformed duckweed plant from the stably transformed callus tissue.

Granted AU 775632 recites a method to produce stably transformed duckweed with duckweed callus tissue with a (‘heterologous’ or ‘chimeric’) nucleotide sequence of interest, which comprises

  1. inoculation of Agrobacterium,
  2. co-cultivation of Agrobacterium with the callus tissue, and
  3. regeneration of transformed callus tissue.
Remarks
  1. Continuations of the divisional patent application of US 6040498 (US 2004/73968 A1 and US 2003/115640 A1) are still pending.
  2. National phase entry of WO 1999/07210 in Canada (CA 2288895), China (CN 1272762), Europe (EP 1037523), Japan (JP 2001/513325) are pending.
  3. Other national phase entry of WO 1999/07210 includes Israel (IL 132580).
EP 1021552 A1

  • Earliest priority – 10 October 1997
  • Filed – 8 October 1998
  • Application pending
Title – Transgenic Lemnaceae

Claim 1A genetically stable, transformed Lemnaceae plant and progeny thereof.
Claim 12*A method for the stable genetic transformation of Lemnaceae plants which comprises:
incubating Lemnaceae plants and/or tissue with Agrobacterium cells containing a transforming DNA molecule, whereby cells in said plant tissue become stably transformed with said DNA.
Claim 19*A method for the genetic transformation of a plant comprising: A) cutting the plant into particles of a size such that they still contain undamaged meristematic tissue capable of developing into full plants;
B) incubating said particles with Agrobacterium cells containing transforming DNA molecules, whereby said transforming DNA is introduced into meristematic cells in said particles; and
C) producing transformed plants from the transformed meristematic tissue.
Claim 23*A method for the stable genetic transformation of a Lemnaceae plant comprising
microinjecting Agrobacterium cells containing a transforming DNA into the meristematic zone of the plant, whereby the meristemic tissue becomes stably transformed with said DNA.
Claim 25A) method for the in planta transformation of Lemnaceae plants comprising: A) exposing the plant’s meristematic zone by removal of the daughter fronds;
B) incubating the plant with Agrobacterium cells capable of targeting to the meristemic tissue.
Claim 37*A booster medium for enhancing Agrobacterium cell’s virulence comprising plant tissue culture at a pH below about 5.2.
Claim 43*A booster medium for enhancing Agrobacterium cell’s virulence comprising an extract from Lemnaceae plants.
Claim 45A method for maintaining morphogenetic Lemnaceae calli for long-periods of time comprising culturing the calli in a medium having a low level of sucrose.
Claim 47A method for the regeneration of plants from calli wherein the plant’s growth medium has sucrose levels below 1.5% and comprises: B5, minerals and organic compounds.
Claim 48A method for the production of highly regenerative calli, wherein the calli’s growth medium has sucrose levels below 1.5% and comprises B5, minerals and organic compounds.
Claim 50A method for the production of highly regenerative calli, wherein the calli’s growth medium has sucrose levels below 1.5% and comprises B5, minerals, organic compounds and selection agents.
Claim 52A method for the production of stable transformed plants, wherein the growth media has sucrose levels below 1.5% and comprises B5, minerals and organic compounds.

* Claims directly related to Agrobacterium -mediated transformation of a Lemnaceae plant.

Different methods for stable genetic transformation of a Lemnaceae plant with Agrobacterium containing a gene of interest. The methods include incubating the plants with Agrobacterium, incubating meristematic tissue with the bacterium, injecting the bacterium into the meristematic tissue and in planta transformation. The invention also includes methods for enhancing the virulence of Agrobacterium and for regenerating calli and transformed plants.

Yeda Research & Development Co.

AU 759570 B2

  • Earliest priority – 10 October 1997
  • Filed – 8 October 1998
  • Granted – 17 April 2003
  • Expected expiry – 7 October 2018
Title – Transgenic Lemnaceae

Claim 1

A stably transformed Lemnaceae plant, tissues, products and progeny thereof of the genus Spirodela, when produced by Agrobacterium-mediated transformation.

Claim 2A method for the stable genetic transformation of Lemnaceae plants, comprising the step of:
Incubating meristematic tissues of Lemnaceae with Agrobacterium cells containing a transforming DNA molecule, wherein theAgrobacterium cells are A. tumefaciens strains EHA105, EHA 101 or GVE3103, whereby cells in said tissue become stably transformed with said DNA, and regenerating Lemnaceae from the meristematic tissue.
Claim 3
A method for the stable genetic transformation of Lemnaceae plants, comprising the step of:
microinjecting into a meristematic zone of Lemnaceae plants and/or tissue Agrobacterium cells containing a transforming DNA molecule, whereby cells in said plant tissue become stably transformed with said DNA.
Claim 4
A method for the stable genetic transformation of a Lemnaceae plant, comprising the steps of:
a) cutting the plant into particles of a size such that they still contain undamaged meristematic tissue capable of developing into full plants;
b) incubating said particles with Agrobacterium cells containing transforming DNA molecules, whereby said transforming DNA is introduced into meristematic cells in said particles; and
c) producing transformed plants form the meristematic tissue.
Claim 7A method for the stable genetic transformation of a Lemnaceae plant, comprising the step of:
microinjecting Agrobacterium cells containing a transforming DNA molecule into the meristematic zone of the plant, whereby the meristematic tissue becomes stably transformed with said DNA.
Claim 8
A method for the in planta transformation of Lemnaceae plants, comprising the steps of:
a) exposing the plant’s meristematic zone by removal of daughter fronds; and
b) incubating the plant with Agrobacterium cells capable of targeting to the meristematic tissue.

This granted patent claims any Lemnaceae plant, part or whole, that is transformed using Agrobacterium.

It also claims a method of Agrobacterium-mediated transformation using the meristematic tissue of Lemnaceae plants.

Remarks
  1. National phase entry of WO 1999/19497 in Canada (CA 2312008) is still pending.
  2. Other national phase entry of WO 1999/19497 includes Israel (IL 135543).

Note: Patent information on this page was last updated on 9 March 2006.