NEW PLANT BREEDING TECHNIQUES

CLAUDIA PARISI
TITULO: New plant breeding techniques: State of the art, potential and
challenges.

AUTOR: CLAUDIA PARISI

© Edita: Servicio de Publicaciones de la Universidad de Córdoba.

Campus de Rabanales
Ctra. Nacional IV, Km. 396 A
14071 Córdoba

www.uco.es/publicaciones
publicaciones@uco.es
Acknowledgments

i
Resumen

New Plant Breeding Techniques

Freedom to Operate

iii
Summary

v
Table of Contents

ACKNOWLEDGMENTS I

RESUMEN III

SUMMARY V

LIST OF ABBREVIATIONS XI

CHAPTER 1. TECHNICAL INNOVATION IN PLANT BREEDING 1

1.1 INTRODUCTION 2
1.2 PLANT BREEDING TECHNIQUES NOT CONSIDERED AS GM IN EU LEGISLATION (DIRECTIVE
2001/18/EC) 3

1.3 PLANT BREEDING TECHNIQUES CONSIDERED AS GM IN EU LEGISLATION (DIRECTIVE

2001/18/EC) 8
AGROBACTERIUM

1.4 NEW PLANT BREEDING TECHNIQUES (NPBTS) BASED ON BIOTECHNOLOGY 15

CHAPTER 2. THE REGULATORY ISSUES IN PLANT BREEDING 25

2.1 INTRODUCTION 26
2.2 GMO LEGISLATION 26

2.3 REGULATORY STATUS OF NPBTS 35

CHAPTER 3. INTELLECTUAL PROPERTY IN PLANT BREEDING 41

3.1 INTRODUCTION 42
3.2 HISTORICAL BACKGROUND OF IP IN PLANT BREEDING 43

vii
3.3 PLANT VARIETY REGISTRATION 48

3.4 PATENTS IN PLANT BREEDING 50

CHAPTER 4. SCOPE AND OBJECTIVES OF THE THESIS 59

4.1 SCOPE 60
4.2 OBJECTIVES 61

CHAPTER 5. METHODOLOGICAL APPROACH 63

5.1 LITERATURE SEARCH ON NPBTS 64

5.2 FIELD TRIALS ANALYSIS FOR CISGENESIS, INTRAGENESIS AND GRAFTING ON GM ROOTSTOCK 66
5.3 PATENT SEARCH ON NPBTS 67

5.4 COMPARATIVE ANALYSIS OF PLANT BREEDING TECHNIQUES 71

5.5 ANALYSIS OF FREEDOM TO OPERATE IN NPBTS 73

5.6 EXPERTS' OPINION 74

CHAPTER 6. RESULTS: RESEARCH LANDSCAPE ON NPBTS 75

6.1 INTRODUCTION 76
6.2 RESEARCH IN NPBTS 76

6.3 SAFETY AND REGULATORY ISSUES ON NEW TECHNIQUES IDENTIFIED IN LITERATURE 103

6.4 CONCLUSIONS 118

CHAPTER 7. RESULTS: PATENT ANALYSIS OF NPBTS 121

7.1 INTRODUCTION 122

7.2 PATENTS IN NPBTS 122
7.3 CONCLUSIONS 143

CHAPTER 8. RESULTS: COMPARATIVE ANALYSIS OF NPBTS THROUGH CASE STUDIES 145

8.1 INTRODUCTION 146

8.2 CISGENESIS IN WHEAT 149

8.3 GRAFTING ON GM ROOTSTOCK IN CITRUS 165

8.4 ANALYSIS OF FTO (FREEDOM TO OPERATE) 180

CHAPTER 9. OVERALL DISCUSSION AND CONCLUSIONS 199

9.1 DISCUSSION 200

9.2 CONCLUSIONS 215
9.3 CONCLUSIONES 217

LIST OF REFERENCES 219

ANNEX I. LITERATURE SEARCH RESULTS ON NEW PLANT BREEDING TECHNIQUES 239

ANNEX II. PATENT SEARCH RESULTS ON NEW PLANT BREEDING TECHNIQUES 259
List of Abbreviations

AHAS
ALS
ARMG
BAC
bar
Bt
CA
CaMV
CIMMYT
COGEM
CPVO
CSIC
CTV
DNA
DSB
dsRNA
EC
ECLA
EFSA
EMS
EPA
EPO
EU
FAO
FDA
FISH
FTO
GA
GFP
GM
GMO
GUS
HMW GS
IAEA
IAS
IHCP
INIA
INPADOC
IP
IPK
IPR
IPTS
ISAAA
IVIA
JPO
JRC
MAS
MGN
NHEJ

xi
nos
NPBT
npt
NTTF
NTWG
ODM
OECD
ORF
PAT
PCR
PCT
P DNA
PEG
PIPRA
PPA
PPT
PVPA
qRT PCR
RdDM
RNA
RNAi
SCoFCAH
SDS PAGE
TALE
TALEN
T DNA
TGS
Ti
TILLING
TRIPS
UPOV
USDA
WIPO
WTO
ZF
ZFN
 Chapter 1. Technical
innovation in plant breeding
1.1 Introduction

et al.
 et al.

1.2 Plant Breeding Techniques not considered as GM in EU legislation

(Directive 2001/18/EC)
1.2.1 Breeding by crossing

et al.

et al.

1.2.2 Overcoming barriers for crossing

 et al.

et al. In planta

In vitro
in vitro
in vitro
in vitro
in vitro

1.2.3 Change of ploidy level

Triticum durum
Triticum aestivum

et al.

et al.
1.2.4 Increasing genetic variation

et al.
et al.

et al.

in vitro

in vitro
et al.
1.2.5 Selection of desirable traits through molecular tools

et al.

et
al.

et al.

et al.
 et al.

et al.

et al.
et al.

1.3 Plant Breeding Techniques considered as GM in EU legislation

(Directive 2001/18/EC)

Agrobacterium
 et al.
in vitro

1.3.1 Agrobacterium mediated transformation

Agrobacterium tumefaciens

et al.
et al.

Agrobacterium

et al.

Agrobacterium
et al.

Agrobacterium
et al.

vir
Agrobacterium
Vir
 T-REGION
(T-DNA)
left T-DNA border right T-DNA border

VIRULENCE Agrobacterium
REGION
(vir genes) Ti plasmid

ORI
(origin of replication)

Figure 1.1 Agrobacterium tumefaciens

Agrobacterium

Vir

vir et al.

Agrobacterium
 Agrobacterium

et al.

1.3.2 Particle bombardment

et al.

Agrobacterium

Agrobacterium
Agrobacterium

1.3.3 Extra sequences: not just the transgene.

Promoters

et al.

nos
Agrobacterium

Ubi

Marker genes
 nptII

hpt

pmi E.Coli

nptII hpt

et al. et al.
1.3.4 Current commercial applications of GM plants

cp4 epsps
Agrobacterium bar Streptomyces
hygroscopicus et al.

Bacillus thuringensis
et al.

Ostrinia nubilalis
1.4 New Plant Breeding Techniques (NPBTs) based on biotechnology

1.4.1 Targeted mutagenesis: ODM, ZFN, MGN, TALEN

 et al. et al. et
al. et al.

Oligonucleotide directed mutagenesis (ODM)

et al.
et al. et al.
Zinc finger nuclease (ZFN) techniques (ZFN 1,2,3)

Flavobacterium okeanokoites

et al. et al.
et al. et al.

Agrobacterium

et al. et al.
 et al.

et al.

et al.

Meganuclease (MGN) techniques (MGN 1,2,3)

et al. et al. et al.

Transcription Activator Like Effector Nuclease (TALEN) techniques

Xanthomonas

et al. et al. et al.

1.4.2 Techniques resulting in "Negative segregants": Reverse breeding, RdDM, Early

flowering

et al.
RNA dependent DNA methylation (RdDM)

Reverse Breeding
Accelerated breeding through induction of early flowering

Arabidopsis et al.

Arabidopsis
et al. et al.
1.4.3 Variants of plant transformation techniques: Cisgenesis, Intragenesis and Grafting
on GM rootstock

Agrobacterium

Cisgenesis and Intragenesis

et al.
Agrobacterium

Agrobacterium

et al.

Grafting non GM varieties onto GM rootstocks

et al. et al.

Agrobacterium

et al. et al.
Chapter 2. The regulatory
issues in plant breeding
2.1 Introduction

2.2 GMO legislation

2.2.1 The EU GMO legal framework

E. coli
"An organism, with the exception of human beings, in which the genetic material has been altered
in a way that does not occur naturally by mating and/or natural recombination"

Techniques of genetic modification

Techniques which are not considered to result in GMOs

Techniques of genetic modification which are excluded from the scope of the Directive
BOX 1: Legislative instruments of EU legislation on GMOs.
Regulation (EC) No 258/97

Official Journal L 043 , 14/02/1997 P. 0001 0006

Directive 2000/13/EC

Official Journal L 109 , 06/05/2000 P. 0029 0042

Directive 2001/18/EC

Official Journal L 106 , 17/04/2001 P. 0001 0039

Regulation (EC) No 1829/2003

Official Journal L 268 , 18/10/2003 P. 0001 0023

Regulation (EC) No 1830/2003

Official Journal L 268 , 18/10/2003 P. 0024 0028

Regulation (EC) No 1946/2003

Official Journal L 287 , 05/11/2003 P. 0001 0010

Commission Regulation (EC) No 65/2004

Official Journal L 010 , 16/01/2004 P. 0005 0010

2004/204/EC: Commission Decision

Official Journal L 065 , 03/03/2004 P. 0020 0022

Commission Regulation (EC) No 641/2004

Official Journal L 102 , 07/04/2004 P. 0014 0025

Commission Recommendation 2004/787/EC

Official Journal L 348 , 24/11/2004 P. 0018 0026

Regulation (EC) No 882/2004 of the European Parliament and of the Council

OJ L 165, 30.4.2004, p. 1–141

Regulation (EC) No 1981/2006 of the European Parliament and of the Council

Official Journal L 97 , 09/04/2008 P. 0064 0066

Directive 2009/41/EC

Official Journal L 125 , 21/05/2009 P. 0075 0097

Regulation (EC) No 767/2009

Official Journal L 229 , 01/09/2009 P. 0001 0028

2009/770/EC: Commission Decision

Official Journal L 275 , 21/10/2009 P. 0009 – 0027

2009/866/EC: Commission Decision

Official Journal L 314 , 01/12/2009 P. 0102 0105

Commission Recommendation 2010/C200/01

Official Journal C 200 , 22/07/2010 P. 0001 0005

Commission Regulation (EU) No 619/2011

Official Journal L 166 , 25/06/2011 P. 0009 – 0015

experimental release

commercial release
 COMPANY

DOSSIER

NATIONAL
AUTHORITY of the
Member State

MEMBER
EFSA
STATES

EFSA
OPINION

FOR FINAL EUROPEAN FOR

DECISION COMMISSION ADOPTION

DRAFT PROPOSAL

ACCEPT
SCoFCAH QUALIFIED
MAJORITY
REJECT
EUROPEAN NO QUALIFIED
PARLIMENT MAJORITY

REJECT
NO COUNCIL OF QUALIFIED
REACTION MINISTERS MAJORITY
ACCEPT

NO QUALIFIED
MAJORITY

Figure 2.1

2.2.2 European Food Safety Authority

2.2.3 GMO legislation in other countries
2.2.4 The regulatory costs for breeding GM plants
2.3 Regulatory status of NPBTs

2.3.1 The new techniques working group (NTWG) in the EU

 sensu stricto

et al.
2.3.2 Regulatory developments for NPBTs in other countries

Agrobacterium
Table 2.1
COMPANY TECHNIQUE PRODUCT USDA response to inquiry

Agrobacterium
Agrobacterium

2.3.3 Detection of products of NPBTs

et al.
 Chapter 3. Intellectual
Property in plant breeding
3.1 Introduction

sui
generis

et al. Union Internationale

pour la Protection des Obtentions Végétales
3.2 Historical background of IP in plant breeding

3.2.1 From plant domestication to IP in plant breeding

 sui generis

3.2.2 Townsend Purnell Plant Patent Act (PPA)

et al.

3.2.3 UPOV convention

sui generis

et al.
 et al.

et al.
3.2.4 The advent of biotechnology in plant breeding

sui generis
3.3 Plant variety registration

3.3.1 Plant Variety definition

3.3.2 Application for Plant Variety Rights
3.4 Patents in plant breeding

3.4.1 Patents in general

et al.
 et al.

3.4.2 Patents in plant breeding

Table 3.1

Agrobacterium

et al.
3.4.3 Freedom to operate (FTO)

et al.

et al.

et al.
 et al.

et al.

et al.
3.4.4 Patent licensing
 Chapter 4. Scope and
objectives of the thesis
4.1 Scope
4.2 Objectives

4.2.1 State of the art of NPBTs

4.2.2 Patent landscape analysis

4.2.3 Comparative analysis

Chapter 5. Methodological
approach
5.1 Literature search on NPBTs

5.1.1 Literature database and search method

Table 5.1

Technique Keywords
TARGETED MUTAGENESIS

ZFN

ODM

MGN

TALEN

targeted
mutagenesis in
general

NEGATIVE SEGREGANTS

RdDM

REVERSE BREEDING

EARLY FLOWERING

VARIANTS OF PLANT TRANSFORMATION

CISGENESIS
INTRAGENESIS
 GRAFTING ON GM
ROOTSTOCK

5.1.2 Literature data analysis

5.2 Field trials analysis for cisgenesis, intragenesis and grafting on

GM rootstock
5.3 Patent search on NPBTs

5.3.1 Patent databases and searches methods

et al et al.
Tab 5.2

Technique Keywords
TARGETED MUTAGENESIS
ZFN

ODM

MGN

TALEN

NEGATIVE SEGREGANTS
RdDM

REVERSE BREEDING

VARIANTS OF PLANT TRANSFORMATION

CISGENESIS
INTRAGENESIS

GRAFTING ON GM
ROOTSTOCK

5.3.2 Patent data analysis

5.3.3 Analysis of patent citations and claims
5.4 Comparative analysis of plant breeding techniques

5.4.1 Case studies selection

5.4.2 Data collection

5.4.3 Comparative analysis

et al.

opaque2,
 et al.

5.5 Analysis of Freedom to Operate in NPBTs

5.6 Experts' opinion
Chapter 6. Results: Research
landscape on NPBTs
6.1 Introduction

6.2 Research in NPBTs

6.2.1 Targeted mutagenesis: ODM, ZFN, MGN, TALEN

 et al. et al. et al.

et al. et al. et al.

et al.

Arabidopsis

Oligonucleotide directed mutagenesis (ODM)

 SuRA

SuRA

gfp
Arabidopsis

Figure 6.1
Table 6.1

Authors country N. publications

EU 27 9

North America 13

Asia 2

Other countries 2

Table 6.2

5 year impact
Research paper Journal Institution
factor
et al.
et al.
et al.

Table 6.3

Plant Traits N. publications

Arabidopsis
Zinc finger nuclease (ZFN) techniques (ZFN 1,2,3)

Arabidopsis

Arabidopsis

Arabidopsis
Figure 6.2

Table 6.4

Authors country N. publications

EU 27 4

North America 24

Asia 4

Other countries 5
Table 6.5

5 year impact
Research paper Journal Institution
factor
et al.

et al.

et al.

Table 6.6
Plant Traits Techniques N. publications

Arabidopsis

Meganuclease (MGN) techniques (MGN1,2,3)

et al.
 N. scientific

EU 27
publications

Table 6.7
Figure 6.3
0
1
2
19

North America
9
19 8

Authors country
9
20 9
0
20 0
0
20 1
0
20 2
0
20 3
0
20 4
0
20 5

Year
0
20 6
0
20 7
0
20 8
0
20 9
1

N. publications

5
6
20 0
11
Arabidopsis
 Table 6.8

5 year impact
Research paper Journal Institution
factor
et al.
et al.

Table 6.9
Plant Traits Techniques N. publications

Arabidopsis

Transcription Activator Like Effector Nuclease (TALEN) techniques

Arabidopsis
Arabidopsis

Arabidopsis
et al.

Os11N3

8
publications
N. scientific

6
4
2
0
2010 2011

Year

Figure 6.4

Table 6.10

Authors country N. publications

EU 27 3

North America 2

Asia 3
 Table 6.11

5 year impact
Research paper Journal Institution
factor
et al.

et al.

et al.

et al.

Table 6.12
Plant Traits Techniques N. publications
Arabidopsis

Arabidopsis

Targeted mutagenesis – aggregated data

 14

12

10

0
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

ODM ZFN MEGA TALEN

Figure 6.5

Table 6.13

Authors country N. publications

EU 27 23

North America 44

Asia 9

Other countries 8
6.2.2 Techniques resulting in "Negative segregants": Reverse breeding, RdDM, Early
flowering

RNA dependent DNA methylation (RdDM)

Arabidopsis
nptII gfp

carrot leafy cotyledon 1, C LEC1

OsRac
Figure 6.6

Table 6.14

Authors country N. publications

EU 27 29

North America 4

Asia 9

South America 1

Other countries 4
 Table 6.15

5 year impact
Research paper Journal Institution
factor

et al.

et al.
et al.

Table 6.16
Plant Traits N. publications
Arabidopsis

Reverse Breeding

Arabidopsis et al.
Table 6.17
Publication Year Country Sector Institution

Accelerated breeding through induction of early flowering

et al. et al. Arabidopsis

FPF1 Arabidopsis OsMADS7/8
 12

10
N. scientific publications

0
1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

Year

Figure 6.7

6.2.3 Variants of plant transformation techniques: Cisgenesis, Intragenesis and Grafting

non GM varieties onto GM rootstock

Cisgenesis and Intragenesis

 et al.

Agrobacterium
 10

8
N. scientific publications

0
2003 2004 2005 2006 2007 2008 2009 2010 2011

Year

Figure 6.8

Table 6.18

Authors country N. publications

EU 27 26

North America 15

Asia 3

South America 1

Other countries 2
Table 6.19

5 year impact
Research paper Journal Institution
factor
et al.
et al.
et al.

Table 6.20

Plant Traits N. publications

Table 6.21

INSTITUTE/ NOTIFICATION GENE

TECH. MS YEARS PLANT TRAIT
COMPANY NUMBER INSERTED

Rpi blb1

Rpi genes

HcrVf2

Rpi vnt1

Rpi vnt1.1

HvPAPhy_a

GBSS
Grafting non GM varieties onto GM rootstocks

rol Agrobacterium
rhizogenes

Phytophtora
 N. scientific publications

Asia
EU 27
0
1
2
3
4
19
91

Figure 6.9

Table 6.22
19
92

North America
19

Other countries
93

Authors country
19
94
19
95
19
96
19
97
19
98
19
99
20
00
20
01
20
Year

02
20
03
20
04
20
05
20

N. publications

3
6
8
13
06
20
07
20
08
20
09
20
10
20
11
Table 6.23

5 year impact
Research paper Journal Institution
factor

et al.
et al.

et al.

Table 6.24

Plant Traits N. publications

Table 6.25

INSTITUTE/ NOTIFICATION GENE

MS YEARS PLANT TRAIT
COMPANY NUMBER INSERTED

Phytophtora PR P23
citrophtora

rolB
6.2.4 Aggregated results
 30

25

20

15

10

0
1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

ODM ZFN MEGA TALEN CISG-INTRA RdDM REV BREED GRAFT

Figure 6.10

Other countries
12.1%

Asia
12.6% EU-27
42.6%

North America
32.7%

Figure 6.11
Table 6.26

N. publications
Authors country CISG REV % IN
ODM ZFN MGN TALEN RdDM GRAFT TOTAL
INTRA BREED TOTAL
EU 27 9 4 7 3 26 29 4 13 95 42,6

North America 13 24 5 2 15 4 2 8 73 32,7

Asia 2 4 0 3 3 9 1 6 28 12,6

Other countries 3 5 0 0 11 5 0 3 27 12,1

Table 6.27

Institution Sector Country City N. publication Techniques

Industry

Industry

Table 6.28
5 year
NPBT Research paper Journal impact Institution
factor
et al.
et al.

et al.

et al.

et al.

et al.

et al.

et al.

et al.
6.3 Safety and Regulatory issues on new techniques identified in
literature
6.3.1 Targeted mutagenesis: ODM, ZFN, MGN, TALEN

Reasons for excluding the techniques from the scope of Directive 2001/18

Reasons for including the techniques in the scope of Directive 2001/18

in vitro
6.3.2 Negative segregants: RdDM, Reverse breeding and Early flowering

Reasons for excluding the techniques from the scope of Directive 2001/18

Reasons for including the techniques in the scope of Directive 2001/18

6.3.3 Variants of plant transformation techniques: Cisgenesis, Intragenesis and Grafting

non GM varieties onto GM rootstock
Cisgenesis and Intragenesis

Reasons for excluding the techniques from the scope of Directive 2001/18

Reasons for including the techniques in the scope of Directive 2001/18

Agrobacterium
Grafting non GM varieties onto GM rootstocks

Reasons for excluding the techniques from the scope of Directive 2001/18

Reasons for including the techniques in the scope of Directive 2001/18

et al. et al.
Table 6.29

Issues covered
NPBTs Food and Safety for the Regulatory Main conclusions, discussions or remarks
Feed Safety environment issues
Table 6.29

Issues covered
ODM Food and Safety for Main conclusions, discussions or remarks
Regulator
Feed the
y issues
Safety environment

Issues covered
ZFN Food and Safety for Main conclusions, discussions or remarks
Regulator
Feed the
y issues
Safety environment
Table 6.29

Issues covered
MGN Food and Safety for Main conclusions, discussions or remarks
Regulator
Feed the
y issues
Safety environment

Issues covered
TALEN Food and Safety for Main conclusions, discussions or remarks
Regulator
Feed the
y issues
Safety environment
Table 6.29

Issues covered
RdDM Food and Safety for Main conclusions, discussions or remarks
Regulator
Feed the
y issues
Safety environment

Issues covered
REVERSE BREEDING Food and Safety for Main conclusions, discussions or remarks
Regulator
Feed the
y issues
Safety environment
Table 6.29

Issues covered
CISGENESIS, Food and Safety for
Regulator Main conclusions, discussions or remarks
INTRAGENESIS Feed the
y issues
Safety environment
Table 6.29

Agrobacterium
Table 6.29
Table 6.29

Issues covered
GRAFTING Food and Safety for Main conclusions, discussions or remarks
Regulator
Feed the
y issues
Safety environment
Table 6.29
6.4 Conclusions

Agrobacterium
et al.

et al.
Chapter 7. Results: patent
analysis of NPBTs
7.1 Introduction

et al.

7.2 Patents in NPBTs

7.2.1 Targeted mutagenesis: ODM, ZFN, MGN, TALEN

Oligonucleotide directed mutagenesis (ODM)

et al.

et al.

Thomas Jefferson University (US), University of Miami (US)

 University of Delaware
(US)

University of Delaware (US)

Thomas Jefferson University (US)

et al.
et al. et al.

et al.

et al. et al.
 N. of patents

0
1
2
3
4
5
6
7
19
91

Figure 7.1
19
92
19
93
19
94
19
95
19
96
19
97
19
98
19
99
20
00
20
01

Year
20
02
20
03
20
04
20
05
20
06
20
07
20
08
20
09
20
10
 Table 7.1

Authors country N. patents

EU 27 10

North America 25

Asia 1

Zinc finger nuclease (ZFN) techniques (ZFN 1,2,3)

Sangamo Bioscience Inc (US)

Sangamo Biosciences Inc (US)

University of Utah
Research Foundation (US)
Sangamo Biosciences Inc (US)

et al.
et al.
 Arabidopsis
Agyranthemum Populus tremula
Arabidopsis

12

10

8
N. of patents

0
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011
Year

Figure 7.2

Table 7.2

Authors country N. patents

EU 27 6

North America 27

Asia 5

Other countries 4
Meganuclease (MGN) techniques (MGN1,2,3)

Cellectis (FR)

Cellectis (FR)

Cellectis (FR)

Precision
Biosciences (US)

Fred Hutchinson Cancer Research Center (US)

 N. of patents

0
1
2
3
4
5
6
7
8

Figure 7.3
1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

Year
2004

2005

2006

2007

2008

Arabidopsis
2009

2010

2011
et al.
 Table 7.3

Authors country N. patents

EU 27 27

North America 4

Asia 1

Transcription Activator Like Effector Nuclease (TALEN) techniques

et al.
 Table 7.4
Patent Year Country Sector Institution
et al.

et al.

et al.

et al.

Targeted mutagenesis – aggregated data

 18

16
14

12

10
8

2
0
91

92

93

94

95

96

97

98

99

00

01

02

03

04

05

06

07

08

09

10

11
19

19

19

19

19

19

19

19

19

20

20

20

20

20

20

20

20

20

20

20

20
ODM ZFN MEGA TALEN

Figure 7.4

Table 7.5

Authors country N. patents

EU 27 45

North America 59

Asia 9

Other countries 4
7.2.2 Techniques resulting in "Negative segregants": Reverse breeding, RdDM, Early
flowering

RNA dependent DNA methylation (RdDM)

Table 7.6
Patent Year Country Sector Institution
et al.

Reverse Breeding

et al.
 Table 7.7
Patent Year Country Sector Institution
et al.

Accelerated breeding through induction of early flowering

7.2.3 Variants of plant transformation techniques: Cisgenesis, Intragenesis and Grafting

non GM varieties onto GM rootstock

Cisgenesis and Intragenesis

 J.R. Simplot Company
(US)

P.infestans

et al.
et al. et al.
et al.
 7

5
N. of patents

0
98

99

00

01

02

03

04

05

06

07

08

09

10
19

19

20

20

20

20

20

20

20

20

20

20

20
Year

Figure 7.5

Table 7.8

Authors country N. patents

EU 27 10

North America 10

Other countries 2

Grafting non GM varieties onto GM rootstocks

 et al. et al.
et al.

et al.
et al.

3
N. of patents

0
1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

Year

Figure 7.6
 Table 7.9

Authors country N. patents

EU 27 1

North America 10

Asia 2

Other countries 2

7.2.4 Aggregated results

 25

20

15

10

0
1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

ODM ZFN MEGA TALEN CISG-INTRA RdDM REV BREED GRAFT

Figure 7.7
 Table 7.10

Authors country N. patents

EU 27 59

North America 79

Asia 11

Other countries 8

Other countries
5.1%
Asia
7.0%
EU-27
37.6%

North America
50.3%

Figure 7.8
 Table 7.11

N.
Institution Sector Country Techniques
patents
Industry
Industry
Industry
Industry
Industry

Industry
Industry
Industry
Industry

150
140
130
120
110
Number of patents

100
90
80
70
60
50
40
30
20
10
0
G

NG

L
N

N
N
M

M
A

TA
N
ZF

LE
G
D

dD
TR

DI

TI
M
O

TO
TA

AF
R

EE
/IN

R
BR
G

G
IS

SE
C

ER
EV
R

Total N. of patents WIPO USPTO EPO CA AU JP CN

Figure 7.9
7.3 Conclusions
 Chapter 8. Results:
Comparative analysis of
NPBTs through case studies
8.1 Introduction

et al. et al. et al.

et al. et al. et al.

et al.

et al.
Table 8.1
8.2 Cisgenesis in wheat

8.2.1 Bread making quality

et al.
Triticum durum
Triticum aestivum

et al.

et al.
 et al.

et al.
et al.

et al. et al.

8.2.2 Introgression breeding

et al. et al.

HorD et al.
H. chilense
Triticum turgidum conv. durum
 tritordeum
Triticum aestivum.

Gli 1 Gli 3

tritordeum

D Hor
Figure 8.1
Box 8.1. Introgression breeding protocol

1) PRELIMINARY WORK
Hordeum chilense Tritordeum… Triticum
aestivum

2) CROSS OF DONOR AND RECIPIENT LINE (Time = 0, duration 6 months)

F1 generation

3) BACK CROSSING AND SELECTION (Time = 6, duration 18 months)

BC1F1 generation

o
o
o
generations BC2F1 and BC3F1

o
o
o

4) SELF POLLINATION (Time = 24, duration 6 months)

generation BC2F2

Total time = 30 months

8.2.3 Transgenesis

et al.
et al. et al. et al.

et al. et al. bar

bar

bar

8.2.4 Cisgenesis

et al. et al. et al.

 et al.

1Dx5,
1Ax1 1Dy10 bar

bar

bar

bar

bar
Figure 8.2
Figure 8.3
8.2.5 Comparative analysis

Time requirements

Technical and labour costs

bar
Table 8.1

CISGENESIS INTROGRESSION
TRANSGENESIS BREEDING
costs costs

TRANSFORMATION

PLANT SELECTION

PLANT CULTIVATION

EXTRA MATERIAL

TOTAL TECHNICAL COSTS €10,149.89 €4,426.25

LABOUR

OVERALL TOTAL COSTS €16,139.89 €9,766.25

Quality of the final product

bar
Figure 8.4

Patent licensing

bar
et al.

HorD
 bar

HorD

Regulatory costs
8.2.6 Conclusions
8.3 Grafting on GM rootstock in citrus
8.3.1 The importance of grafting in citrus

Citrus sinensis Citrus

aurantium

Citrus sinensis Poncirus trifoliata et al.

8.3.2 Dwarfing citrus

 et al.

et al. et al.

et al.
CcGA20ox1
Arabidopsis et al. et al.
et al. et al.

Arabidopsis et al.
et al.
Figure 8.5
et al.

8.3.3 Conventional breeding

Poncirus
trifoliata Mandarino King

Poncirus trifoliata
Poncirus trifoliata
 Poncirus trifoliata Mandarino King
(male parent) (female parent)

Anthers collection Flowers emasculation

and storage

POLLINATION

Fruit setting and ripening

Seeds extraction

Sowing in
nutrient medium

Hybrids
identification and
transfer into soil

Hybrids
multiplication
through seeds

HYBRID SELECTION

In the In the
greenhouse field

• Citrus Tristeza Virus (CTV) • Phytophtora

• Nematodes • Iron chlorosis
• Salinity, drought, iron chlorosis • Productivity and Fruits quality
• Junction scion and rootstock

Figure 8.6
Box 8.3. Conventional breeding protocol (Forner Alcaide 517)

1) PRELIMINARY WORK

2) CROSS OF PARENT LINES TO OBTAIN SEEDS (Time = 0, duration 6 months)

Poncirus trifoliata

3) HYBRIDS GROWTH AND MULTIPLICATION (Time = 6 months, duration 2.5 years)

Poncirus trifoliata

4) HYBRIDS SELECTION (Time = 3 years, duration 10 years)

Total time = 13 years

8.3.4 Transgenic rootstock

et al.

CcGA20ox1 Carrizo citrange

pBinJIT
CaMV 35S
nptII Agrobacterium
tumefaciens

Carrizo citrange

Carrizo citrange

et al.
et al.

Agrobacteria

CcGA20ox1
in vitro Troyer citrange

CcGA20ox1
 CcGA20ox1
nptII

Clemenules
Clementine
 Carrizo citrange Carrizo citrange

gene CcGA20ox1 buds grafted onto

vigorous rootstock

2-3 months
CcGA20ox1 - antisense culture

pBIN-JIT

nptII

Co-cultivation

internodes First
A. tumefaciens
sproutings

Shoot regeneration
medium with Kanamycin

PCR

Micrografting

PCR
Southern blot

Selected transformant
(3 months old)

Permission of buds grafted onto

the Competent vigorous rootstock
Authority in the field
Phenotypic
1 year assessment Grafting of
6-8 months non-GM
Green- scion
seeds
house

Figure 8.7
Box 8.4. Protocol grafting on a GM rootstock

1) PRELIMINARY WORK
Carrizo citrange

nos
Agrobacterium tumefaciens

Agrobacteria

2) TRANSFORMATION AND TRANSFORMANTS SELECTION (Time = 0, duration 7 months)

Carrizo citrange

Agrobacteria

in vitro Troyer citrange

3) GRAFTING OF A NON GM SCION ONTO THE TRANSFORMED ROOTSTOCK (Time = 10 months,

duration 20 months)

Clemenules Clementine

o
o
o
o
o

4) OBTAINMENT OF AN ADULT PLANT (Time = 30 months, duration 3 years)

Total time = 5 5 years

8.3.5 Comparative analysis

Time requirements

Technical and labour costs

Quality of the final product

Carrizo citrange
Poncirus trifoliata

Table 8.2
Patent licensing

Regulatory costs
Table 8.3

TRANSGENESIS CROSS BREEDING

costs costs

TRANSFORMATION

Agrobacterium

PLANT SELECTION

PLANT CULTIVATION

TOTAL TECHNICAL COSTS €11,555.60 €100.00

LABOUR

OVERALL TOTAL COSTS €25,785.60 €2,015.00

8.3.6 Conclusions

Poncirus trifoliata
8.4 Analysis of FTO (Freedom to operate)
et al.
8.4.1 Product deconstruction

Agrobacterium
Table 8.4

Phase of the process Components

cDNA synthesis

Transformation construct
preparation

Adult plant material Agrobacterium

transformation

Transformants selection

8.4.2 Patent review

CaMV 35s promoter

 nptII
nos
Table 8.5

Chimeric genes suitable for expression in plant cells

WO8402913 Assignee: Monsanto Co.
Priority: 17/01/1983
Family (INPADOC)
Filing date Estimated expiry
members

US5530196 02/09/1994 25/06/2013

US6174724 04/05/1995 16/01/2018

US6255560 11/01/1999 11/01/2019

BR1101069 14/05/1997 14/05/2017

JP6315381 28/03/1994 28/03/2014
JP2645217 28/03/1994 28/03/2014

Table 8.6

Plant promoters
US5097025 Assignee: Rockefeller University (US)
Priority: 01/12/1989

Family (INPADOC)
Filing date Estimated expiry
members

Selective gene expression in plants

US5110732 Assignee: Rockefeller University
Priority: 14/03/1989
Family (INPADOC)
Filing date Estimated expiry
members
 Table 8.7

DNA construct for enhancing the efficiency of

transcription
US5164316
Assignee: University of British Columbia (CA)
Priority: 17/08/1989
Family (INPADOC)
Filing date Estimated expiry
members

BR1101045 14/05/1997 14/05/2017

nptII marker gene

nptII

nptII CcGA20ox1

nptII
npt

nptII
nptII

gusA
nptII
Table 8.8
nptII
Method for selecting transformed cells
WO9905296 Assignee: Japan Tobacco Inc
Priority: 23/07/1997
Family (INPADOC)
Filing date Estimated expiry
members

EP0927765 23/07/1998 23/07/2018

CA2265570 23/07/1998 23/07/2018

CN1239513 23/07/1998 23/07/2018

Table 8.9
nptII gusA.
Bifunctional genetic markers
WO921759 Assignee: The National Research Council of Canada
Priority: 28/03/1991
Family (INPADOC)
Filing date Estimated expiry
members

US5639663 21/01/1994 17/06/2014

nos promoter

nos
nptII nptII nos

nos

nos
Table 8.10 nos

Methods and compositions for the introduction and

regulated expression of genes in plants
WO2011082318
Assignee: Pioneer Hi Bred Int.
Priority: 30/12/2009
Family (INPADOC)
Filing date Estimated expiry
members

US2011167516 30/12/2010 30/12/2030

Methods and compositions for targeted polynucleotide
modification
WO2011082310
Assignee: Pioneer Hi Bred Int.
Priority: 30/12/2009
Family (INPADOC)
Filing date Estimated expiry
members

US2011165679 30/12/2010 30/12/2030

Transgenic plants
WO2012038717 Assignee: Cambridge Advanced Tech
Priority: 22/09/2010
Family (INPADOC)
Filing date Estimated expiry
members

GA20 oxidase gene

Processes for accelerating plant growth and increasing cellulose yield

 Methods for
controlling gibberellin levels

Table 8.11

GA 20 oxidase gene sequences

WO9428141 Assignee: Long Ashton Research Station (UK)
Priority: 28/05/1993
Family (INPADOC)
Filing date Estimated expiry
members

EP0703983 24/05/1994 24/05/2014

US5939539 27/11/1995 27/11/2015
US6198021 21/04/1999 21/04/2019
US6455675 13/12/2000 13/12/2020
AT291626 24/05/1994 24/05/2014
DE69434312 24/05/1994 24/05/2014
DK703983 24/05/1994 24/05/2014
ES2237752 24/05/1994 24/05/2014
PT703983 24/05/1994 24/05/2014
AU6929794 24/05/1994 24/05/2014
CA2163454 24/05/1994 24/05/2014
JPH08510381 24/05/1994 24/05/2014
Table 8.12

Transgenic trees exhibiting increased growth, biomass production

and xylem fibre length, and methods for their production
WO0166777
Assignee: SweTree Genomics AB (SE)
Priority: 07/03/2000
Family (INPADOC)
Filing date Estimated expiry
members

EP1261726 02/03/2001 02/03/2021

US7141422 27/01/2003 27/01/2023
US7807878 30/10/2006 30/10/2026
AT327338 02/03/2001 02/03/2021
DE60119901 02/03/2001 02/03/2021
DK1261726 02/03/2001 02/03/2021
ES2264974 02/03/2001 02/03/2021
PT1261726 02/03/2001 02/03/2021
AU4131701 02/03/2001 02/03/2021
AU2001241317 02/03/2001 02/03/2021
BR0108987 02/03/2001 02/03/2021
CA2400986 02/03/2001 02/03/2021
CN1416468 02/03/2001 02/03/2021
JP2003525618 02/03/2001 02/03/2021
NZ521066 02/03/2001 02/03/2021
ZA200206789 23/08/2002 23/08/2022

Vectors

Vectors for Cloning

Fusion gene for three sweet potato viruses and interference carrier thereof

Plant transgene expression vector containing green fluorescence protein

gene and its construction method and application
 Plant gene expression carrier, and application

Plant expression carrier for improving plant cuttage taking root and apical
dominance and its use

Nucleic acids extraction

Method for isolating and purifying nucleic acids

Method and apparatus for purifying and isolating nucleic acid

Pretreatment method for extraction of nucleic acid from biological

samples and kits therefor

Method of nucleic acid extraction

Modified surfaces as solid supports for nucleic acid purification

cDNA amplification for expression profiling

Agrobacterium

Agrobacterium
Agrobacterium

Agrobacterium

Regeneration of plants containing genetically engineered T DNA

Table 8.13

Process for the incorporation of foreign DNA into the

genome of dicotyledonous plants
US4940838
Assignee: University of Leiden
Priority: 24/02/1983
Family (INPADOC)
Filing date Estimated expiry
members

US5464763 23/12/1993 23/12/2013

JP6209779 01/12/1993 01/12/2013

Table 8.14

Process for introducing foreign DNA into the genome of plants

US5149645 Assignee: University of Leiden
Priority: 04/06/1984
Family (INPADOC)
Filing date Estimated expiry
members

Process for the site directed integration of DNA into the genome of plants
WO9102070 Assignee: Syngenta Mogen B.V.
Priority: 26/07/1989
Family (INPADOC)
Filing date Estimated expiry
members

US5501967 06/07/1993 06/07/2013

Binary BAC vector

WO9621725 Assignee: Cornell Research Foundation, Inc. (US)
Priority: 13/01/1995
Family (INPADOC)
Filing date Estimated expiry
members

EP0805851 11/01/1996 11/01/2016

US5733744 13/01/1995 31/03/2015
US5977439 22/12/1997 22/12/2017
AT267871 11/01/1996 11/01/2016
DE69632576 11/01/1996 11/01/2016
JPH11500306 11/01/1996 11/01/2016
Method of plant transformation
WO0018939 Assignee: Pioneer Hi Bred International
Priority: 01/10/1998
Family (INPADOC)
Filing date Estimated expiry
members

EP1117816 28/09/1999 28/09/2019

US6265638 28/09/1999 28/09/2019
AT313635 28/09/1999 28/09/2019
DE69929073 28/09/1999 28/09/2019
AU764100 28/09/1999 28/09/2019
AU6164899 28/09/1999 28/09/2019
CA2344700 28/09/1999 28/09/2019
 Agrobacterium

Genetic modification of plants

Transformation and foreign gene expression with woody species

Transgenic pomaceous fruit with fire blight resistance

Agrobacterium
et al.

Table 8.15 Agrobacterium

Procedure for the genetic transformation of adult plants

of woody species
US6103955
Assignee: INIA & IVIA
Priority: 05/03/1997
Family (INPADOC)
Filing date Estimated expiry
members
EP0870838 04/03/1998 04/03/2018
US6103955 05/03/1998 05/03/2018
DE69835918 04/03/1998 04/03/2018
ES2151338 05/03/1997 05/03/2017
PCR

Process for amplifying, detecting, and/or cloning nucleic acid sequences

Process for amplifying, detecting, and/or cloning nucleic acid sequences

Kit for use in amplifying and detecting nucleic acid sequences

Oligonucleotides for amplifying nucleic acid sequences and attaching a

promoter sequence

Purified thermostable and process and process for amplifying, detecting,

and/or cloning nucleic acid sequences using said enzyme

Purified thermostable enzyme

 Apparatus and method for performing automated amplification of nucleic
acid sequences and assays using heating and cooling steps

Heat exchanger for use in a temperature cycling instrument

Southern Blot

8.4.3 Product clearance

et al.
8.4.4 Final considerations
Chapter 9. Overall discussion
and conclusions
9.1 Discussion
9.2 Conclusions
9.3 Conclusiones
Oligonucleotide directed mutagenesis
Zinc Finger Nuclease
List of references
Hordeum chilense
Hordeum chilense
Annex I. Literature search
results on New Plant
Breeding Techniques
Oligonucleotide directed mutagenesis (ODM)

RESEARCH PAPERS
Paper Country Plant Trait

Proc Natl
Acad Sci U S A 96

Plant Cell Rep 25

et al.

Nucleic Acids Res 28

Plant Journal 27

Plant Physiol 132

Plant Cell Rep 22

Plant Physiology 123

et al.
Plant Mol.Biol. 53

Nat Biotechnol 18

et al.
Proc Natl Acad Sci U S A 96

REVIEW PAPERS
Paper Country

Biochimie 84

et al.
Environ
Biosafety Res 8
Trends
in Plant Science 8
Proc Natl Acad Sci U S
A 96

Plant Mol Biol 59

Trends in Plant Science 6

 Curr. Opin. Biotechnol. 12

Plant Mol Biol 48

J. Plant Physiol. 160

International Review of Cytology a Survey of Cell

Biology, Vol 228 International Review of Cytology a Survey
of Cell Biology

Nature Biotechnology 19

Annals of Applied Biology 147

Biotechniques 29

Weed Sci. 50

Zinc finger nuclease (ZFN) techniques (ZFN1,2,3)

RESEARCH PAPERS
Paper Country Plant Trait ZFN 1,2,3

69

102

31
 459

57

459

44

105

REVIEW PAPERS
Paper Country

Nat. Rev. Genet. 7

et al.

Nucleic Acids Res 33

Trends in Plant Science 12

Trends Plant Sci 11

Curr Opin Plant Biol 10

Bioscience 58

Proc.
Natl. Acad. Sci. U. S. A. 102

Jarq Jpn. Agric. Res. Q. 43

 Trends in Biotechnology 23

et al.
Nat. Protoc.
1

Cellular and Molecular Life Sciences

64

Meganuclease (MGN) techniques (MGN1,2,3)

RESEARCH PAPERS
Paper Country Plant Trait MGN 1,2,3
REVIEW PAPERS
Paper Country

Transcription Activator Like Effector Nuclease (TALEN) techniques

RESEARCH PAPERS
Paper Country Plant Trait TALEN 1,2,3

REVIEW PAPERS
Paper Country
RNA dependent DNA methylation (RdDM)

RESEARCH PAPERS
Paper Country Plant Trait

Proc Natl Acad

Sci U S A 99 Suppl 4

Embo Journal 21

Plant
Journal 43
et al.

Plant Journal 60
et al.
Embo Journal
28

Plant Journal 53

Molecular and
General Genetics 263

Mol. Genet. Genomics 275

et al.

Plant Journal 43

et al.

Plant
Mol.Biol. 52

et al.

Plant Journal 54
 Embo Journal 19

Embo Journal 18

Plant Journal 56

Plant Journal 53

Gene 437

et al.
Curr Biol 11

REVIEW PAPERS
Paper Country
Plant
Journal 61

Sci. China Ser. C Life Sci. 52

Plant
Physiology 147

et al.

Biochim. Biophys. Acta Gene Struct. Expression 1769

Biochemistry Moscow 72
 et al.
Biochim. Biophys. Acta Gene Struct. Expression 1677

Science 293

Plant Mol.Biol. 43

Cellular
and Molecular Life Sciences 60

Biosci. Biotechnol.
Biochem. 71

Trends in Genetics 17

Int J Dev Biol 53

Curr. Opin. Plant Biol. 5

Plant Mol.Biol. 43

Reverse Breeding

REVIEW PAPERS
Paper Country

et al.
Plant Biotechnol J 7

NJAS
Wageningen Journal of Life Sciences 54

Trends Plant Sci 13

Accelerated breeding through induction of early flowering

RESEARCH PAPERS
Paper Country Plant
REVIEW PAPERS
Paper Country

Cisgenesis and Intragenesis

RESEARCH PAPERS
Paper Country Plant Trait
et al.
Proc. Natl. Acad. Sci. U.
S. A. 101

European Journal of Plant Pathology

125
 Journal of the
Americal Society for Horticultural Science 132

Euphytica 165

et al. Plant Physiol

139

et al.
Plant Physiology 135

Plant Biotechnology
Journal 6

J Agric Food Chem 54

et al.
Theoretical and Applied Genetics 110

et al.

Tree Genetics & Genomes 5

REVIEW PAPERS
Paper Country

Euphytica 166
 et al.
Euphytica 154
et al.

Potato Research 51

Current Science 94

Potato
Research 51

Trends Biotechnol 25

Euphytica 170

Regul Toxicol Pharmacol 50

Journal of Agricultural &

Environmental Ethics 19

Nat Biotechnol 21

Plant Breeding 128

Trends Plant Sci 9

J Agric
Food Chem 55

Trends in Plant Science 13

Plant Biotechnology Journal 8

Trends Plant Sci 12

Journal of Agricultural & Environmental Ethics 21

J. Biomed. Biotechnol.
 Trends Plant Sci 13

EMBO Rep 7

Nat Biotechnol 24

et al.
Acta Hort. 814

Nat
Biotech 24

Grafting non GM varieties onto GM rootstocks

RESEARCH PAPERS
Paper Country Plant Trait

et al.

Mol. Plant Pathol. 6

et al.

Planta 217

J. Gen. Virol. 78

et al.

Transgenic Res 14

Mol. Breed. 24

Agrobacterium rhizogenes
Bio Technology 9

Molecular Plant Microbe Interactions 4

et al.

Mol. Breed. 6

Proc. Natl. Acad. Sci. U. S. A. 91

 Sci. Hortic. 108

Hortscience 45

et al.
Plant Cell
Rep 24

et al.

J. Am. Soc. Hortic. Sci. 127

Mol. Plant Pathol. 5

REVIEW PAPERS
Paper Country

Annual Review of Plant

Biology 57

Plant Mol. Biol. Rep.

14
Annex II. Patent search
results on New Plant
Breeding Techniques
Oligonucleotide directed mutagenesis (ODM)

Patent Country Plant Trait

Et Al.
Zinc finger nuclease (ZFN) techniques (ZFN1,2,3)

ZFN
Patent Country Plant Trait
1,2,3

Et Al.
Meganuclease (MGN) techniques (MGN1,2,3)

MGN
Patent Country Plant Trait
1,2,3
Transcription Activator Like Effector Nuclease (TALEN) techniques

TALEN
Patent Country Plant Trait
1,2,3

RNA dependent DNA methylation (RdDM)

Patent Country Plant Trait

Reverse Breeding

Patent Country Plant Trait

Cisgenesis and Intragenesis

Patent Country Plant Trait

Et Al.

Et Al.

Et Al.
 Et Al.

Grafting non GM varieties onto GM rootstocks

Patent Country Plant Trait

Et Al.