NEW PLANT BREEDING TECHNIQUES

(1)

NEW PLANT BREEDING TECHNIQUES

CLAUDIA PARISI

(2)

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

AUTOR: CLAUDIA PARISI

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

www.uco.es/publicaciones [email protected]

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i

Acknowledgments

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iii

Resumen

New Plant Breeding Techniques

Freedom to Operate

(11)

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v

Summary

(13)

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vii

xi

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

(19)

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

(20)

Chapter 1. Technical

innovation in plant breeding

(21)

1.1 Introduction

et al.

(22)

et al.

1.2 Plant Breeding Techniques not considered as GM in EU legislation

(Directive 2001/18/EC)

(23)

1.2.1 Breeding by crossing

et al.

1.2.2 Overcoming barriers for crossing

(24)

et al.

et al. In planta

In vitro

in vitro in vitro

1.2.3 Change of ploidy level

Triticum durum

Triticum aestivum

et al.

(25)

1.2.4 Increasing genetic variation

et al.

in vitro

in vitro et al.

(26)

1.2.5 Selection of desirable traits through molecular tools

et al.

et al.

et al.

(27)

et al.

1.3 Plant Breeding Techniques considered as GM in EU legislation (Directive 2001/18/EC)

Agrobacterium

(28)

et al.

in vitro

1.3.1 Agrobacterium mediated transformation

Agrobacterium tumefaciens

et al.

Agrobacterium

et al.

Agrobacterium

et al.

Agrobacterium

et al.

vir Agrobacterium

Vir

(29)

Figure 1.1 Agrobacterium tumefaciens

Agrobacterium

Vir

vir et al.

Agrobacterium

Agrobacterium Ti plasmid

VIRULENCE REGION (vir genes)

T-REGION (T-DNA) left T-DNA border

ORI

(origin of replication)

right T-DNA border

(30)

Agrobacterium et al.

1.3.2 Particle bombardment

et al.

Agrobacterium

Agrobacterium Agrobacterium

1.3.3 Extra sequences: not just the transgene.

(31)

Promoters

et al.

nos

Agrobacterium

Ubi

Marker genes

(32)

nptII hpt

pmi E.Coli

nptII hpt

et al. et al.

(33)

1.3.4 Current commercial applications of GM plants

cp4 epsps

Agrobacterium bar Streptomyces

hygroscopicus et al.

Bacillus thuringensis et al.

Ostrinia nubilalis

(34)

1.4 New Plant Breeding Techniques (NPBTs) based on biotechnology

1.4.1 Targeted mutagenesis: ODM, ZFN, MGN, TALEN

(35)

et al. et al. et

al. et al.

Oligonucleotide directed mutagenesis (ODM)

et al.

et al. et al.

(36)

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

Flavobacterium okeanokoites

et al. et al.

Agrobacterium

et al. et al.

(37)

et al.

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

et al. et al. et al.

(38)

Transcription Activator Like Effector Nuclease (TALEN) techniques

Xanthomonas

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

et al.

(39)

RNA dependent DNA methylation (RdDM)

Reverse Breeding

(40)

Accelerated breeding through induction of early flowering

Arabidopsis et al.

Arabidopsis

et al. et al.

(41)

1.4.3 Variants of plant transformation techniques: Cisgenesis, Intragenesis and Grafting on GM rootstock

Agrobacterium

Cisgenesis and Intragenesis

et al.

(42)

Agrobacterium

et al.

Grafting non GM varieties onto GM rootstocks

et al. et al.

Agrobacterium

et al. et al.

(43)

(44)

Chapter 2. The regulatory

issues in plant breeding

(45)

2.1 Introduction

2.2 GMO legislation

2.2.1 The EU GMO legal framework

E. coli

(46)

"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

(47)

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 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

(48)

experimental release

commercial release

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 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

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Figure 2.1

2.2.2 European Food Safety Authority

COMPANY

NATIONAL AUTHORITY of the

Member State

EFSA

EUROPEAN COMMISSION

SCoFCAH

MEMBER STATES

COUNCIL OF MINISTERS DOSSIER

EFSA OPINION

DRAFT PROPOSAL

ACCEPT QUALIFIED

MAJORITY NO QUALIFIED

MAJORITY EUROPEAN

PARLIMENT

REJECT

NO QUALIFIED MAJORITY

QUALIFIED MAJORITY

ACCEPT REJECT FOR

ADOPTION FOR FINAL

DECISION

NO REACTION

(52)

2.2.3 GMO legislation in other countries

(53)

2.2.4 The regulatory costs for breeding GM plants

(54)

2.3 Regulatory status of NPBTs

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

(55)

sensu stricto

et al.

(56)

2.3.2 Regulatory developments for NPBTs in other countries

Agrobacterium

(57)

Table 2.1

COMPANY TECHNIQUE PRODUCT USDA response to inquiry

Agrobacterium Agrobacterium

2.3.3 Detection of products of NPBTs

et al.

(58)

(59)

(60)

Chapter 3. Intellectual

Property in plant breeding

(61)

3.1 Introduction

sui generis

et al. Union Internationale

pour la Protection des Obtentions Végétales

(62)

3.2 Historical background of IP in plant breeding

3.2.1 From plant domestication to IP in plant breeding

(63)

(64)

sui generis

3.2.2 Townsend Purnell Plant Patent Act (PPA)

et al.

3.2.3 UPOV convention

sui generis

et al.

(65)

et al.

(66)

3.2.4 The advent of biotechnology in plant breeding

sui generis

(67)

3.3 Plant variety registration

3.3.1 Plant Variety definition

(68)

3.3.2 Application for Plant Variety Rights

(69)

3.4 Patents in plant breeding

3.4.1 Patents in general

(70)

et al.

(71)

et al.

3.4.2 Patents in plant breeding

(72)

Table 3.1

Agrobacterium

et al.

(73)

3.4.3 Freedom to operate (FTO)

et al.

(74)

et al.

(75)

3.4.4 Patent licensing

(76)

(77)

(78)

Chapter 4. Scope and

objectives of the thesis

(79)

4.1 Scope

(80)

4.2 Objectives

4.2.1 State of the art of NPBTs

4.2.2 Patent landscape analysis

4.2.3 Comparative analysis

(81)

(82)

Chapter 5. Methodological

approach

(83)

5.1 Literature search on NPBTs

5.1.1 Literature database and search method

(84)

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

(85)

GRAFTING ON GM ROOTSTOCK

5.1.2 Literature data analysis

5.2 Field trials analysis for cisgenesis, intragenesis and grafting on

GM rootstock

(86)

5.3 Patent search on NPBTs

5.3.1 Patent databases and searches methods

(87)

et al et al.

(88)

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

(89)

5.3.3 Analysis of patent citations and claims

(90)

5.4 Comparative analysis of plant breeding techniques

5.4.1 Case studies selection

5.4.2 Data collection

(91)

et al.

opaque2,

(92)

et al.

5.5 Analysis of Freedom to Operate in NPBTs

(93)

5.6 Experts' opinion

(94)

Chapter 6. Results: Research

landscape on NPBTs

(95)

6.1 Introduction

6.2 Research in NPBTs

(96)

et al.

Arabidopsis

(97)

SuRA

gfp Arabidopsis

Figure 6.1

(98)

Table 6.1

Authors country N. publications

EU 27 9

North America 13

Asia 2

Other countries 2

Table 6.2

Research paper Journal 5 year impact

factor Institution

et al.

Table 6.3

Plant Traits N. publications

Arabidopsis

(99)

Arabidopsis

(100)

Figure 6.2

Table 6.4

EU 27 4

Asia 4

(101)

Table 6.5

et al.

Table 6.6

Plant Traits Techniques N. publications

Arabidopsis

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

et al.

(102)

Arabidopsis

0 1 2

1998 1999

2000 2001

2002 2003

2004 2005

2006 2007

2008 200

9 2010

2011 Year

N. scientific publications

Figure 6.3

Table 6.7

EU 27 6

North America 5

(103)

Table 6.8

et al.

Table 6.9

Arabidopsis

(104)

Arabidopsis

et al.

Os11N3

0 2 4 6 8

2010 2011

Year N. scientific publications

Figure 6.4

Table 6.10

EU 27 3

North America 2

Asia 3

(105)

Table 6.11

et al.

Table 6.12

Arabidopsis

Targeted mutagenesis – aggregated data

(106)

0 2 4 6 8 10 12 14

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 ODM ZFN MEGA TALEN

Figure 6.5

Table 6.13

EU 27 23

Asia 9

(107)

Arabidopsis

nptII gfp

carrot leafy cotyledon 1, C LEC1 OsRac

(108)

Figure 6.6

Table 6.14

EU 27 29

North America 4

Asia 9

South America 1

(109)

Table 6.15

factor Institution et al.

et al.

Table 6.16

Arabidopsis

(110)

Table 6.17

Publication Year Country Sector Institution

et al. et al. Arabidopsis

FPF1 Arabidopsis OsMADS7/8

(111)

0 2 4 6 8 10 12

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

N. scientific publications

Figure 6.7

6.2.3 Variants of plant transformation techniques: Cisgenesis, Intragenesis and Grafting non GM varieties onto GM rootstock

(112)

et al.

Agrobacterium

(113)

0 1 2 3 4 5 6 7 8 9 10

2003 2004 2005 2006 2007 2008 2009 2010 2011

Year

Figure 6.8

Table 6.18

EU 27 26

Asia 3

South America 1

(114)

Table 6.19

factor Institution et al.

et al.

Table 6.20

Table 6.21

TECH. INSTITUTE/

COMPANY MS NOTIFICATION

NUMBER YEARS PLANT TRAIT GENE

INSERTED Rpi blb1 Rpi genes HcrVf2 Rpi vnt1 Rpi vnt1.1

HvPAPhy_a

GBSS

(115)

rol Agrobacterium

rhizogenes

Phytophtora

(116)

0 1 2 3 4

1991 1992 1993 1994 1995 1996

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

2008 2009 2010 2011 Year

Figure 6.9

Table 6.22

EU 27 13

North America 8

Asia 6

(117)

Table 6.23

et al.

Table 6.24

Table 6.25

INSTITUTE/

COMPANY MS NOTIFICATION

NUMBER YEARS PLANT TRAIT GENE

INSERTED

Phytophtora

citrophtora PR P23

rolB

(118)

6.2.4 Aggregated results

(119)

0 5 10 15 20 25 30

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

EU-27 42.6%

North America 32.7%

Asia 12.6%

Other countries 12.1%

Figure 6.11

(120)

Table 6.26

N. publications Authors country

ODM ZFN MGN TALEN CISG

INTRA RdDM REV

BREED GRAFT TOTAL % IN 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

(121)

Table 6.27

Institution Sector Country City N. publication Techniques

Industry

Table 6.28

NPBT Research paper Journal 5 year impact factor

Institution

et al.

(122)

6.3 Safety and Regulatory issues on new techniques identified in

literature

(123)

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

(124)

6.3.2 Negative segregants: RdDM, Reverse breeding and Early flowering

(125)

Agrobacterium

(126)

(127)

et al. et al.

(128)

Table 6.29

Issues covered

NPBTs _{Food and}

Feed Safety Safety for the

environment Regulatory issues

Main conclusions, discussions or remarks

(129)

Table 6.29

Issues covered

ODM Food and

Feed Safety

Safety for the environment

Regulator y issues

Issues covered

ZFN Food and

SafetyFeed

Safety for environmentthe

(130)

Table 6.29

Issues covered

MGN Food and

Feed Safety

Issues covered

TALEN Food and

Feed Safety

(131)

Table 6.29

Issues covered

RdDM Food and

Feed Safety

Issues covered REVERSE BREEDING Food and

SafetyFeed

Safety for environmentthe

(132)

Table 6.29

Issues covered CISGENESIS,

INTRAGENESIS Food and

Feed Safety

(133)

Table 6.29

Agrobacterium

(134)

Table 6.29

(135)

Table 6.29

Issues covered

GRAFTING Food and

Feed Safety

(136)

Table 6.29

(137)

6.4 Conclusions

Agrobacterium

(138)

et al.

(139)

(140)

Chapter 7. Results: patent

analysis of NPBTs

(141)

7.1 Introduction

et al.

7.2 Patents in NPBTs

(142)

et al.

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

(143)

University of Delaware (US)

University of Delaware (US)

Thomas Jefferson University (US)

et al.

et al. et al.

et al.

et al. et al.

(144)

0 1 2 3 4 5 6 7

199 1

1992 199

3 1994

1995 1996

1997 1998

199 9

2000 200

1 2002

200 3

2004 200

5 2006

2007 2008

200 9

2010 Year

N. of patents

Figure 7.1

(145)

Table 7.1

Authors country N. patents

EU 27 10

Asia 1

Sangamo Bioscience Inc (US)

Sangamo Biosciences Inc (US)

University of Utah Research Foundation (US)

(146)

Sangamo Biosciences Inc (US)

et al.

(147)

Arabidopsis Agyranthemum Populus tremula

Arabidopsis

0 2 4 6 8 10 12

2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

Year

N. of patents

Figure 7.2

Table 7.2

EU 27 6

Asia 5

(148)

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

Cellectis (FR)

Precision Biosciences (US)

Fred Hutchinson Cancer Research Center (US)

(149)

et al.

Arabidopsis

0 1 2 3 4 5 6 7 8

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

Year

N. of patents

Figure 7.3

(150)

Table 7.3

EU 27 27

North America 4

Asia 1

et al.

(151)

Table 7.4

Patent Year Country Sector Institution et al.

et al.

Targeted mutagenesis – aggregated data

(152)

0 2 4 6 8 10 12 14 16 18

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

Figure 7.4

Table 7.5

EU 27 45

Asia 9

(153)

Table 7.6

Patent Year Country Sector Institution

et al.

(154)

Table 7.7

Patent Year Country Sector Institution

et al.

(155)

J.R. Simplot Company (US)

P.infestans

et al.

et al. et al.

et al.

(156)

0 1 2 3 4 5 6 7

1998 199

9 2000

2001 2002

2003 200

4 2005

2006 2007

2008 200

9 2010 Year

N. of patents

Figure 7.5

Table 7.8

EU 27 10

(157)

et al. et al.

et al.

0 1 2 3 4

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

N. of patents

Figure 7.6

(158)

Table 7.9

EU 27 1

Asia 2

7.2.4 Aggregated results

(159)

0 5 10 15 20 25

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

(160)

Table 7.10

EU 27 59

Asia 11

Other countries 5.1%

EU-27 37.6%

North America 50.3%

Asia 7.0%

Figure 7.8

(161)

Table 7.11

Institution Sector Country N.

patents Techniques Industry

Industry Industry Industry Industry Industry Industry Industry Industry

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

ODM

ZFN

MGN

TAL EN

CISG/INTRAG RdDM REVER

SE BREED

ING

GRAFTING

TOTAL

Number of patents

Total N. of patents WIPO USPTO EPO CA AU JP CN Figure 7.9

(162)

7.3 Conclusions

(163)

(164)

Chapter 8. Results:

Comparative analysis of

NPBTs through case studies

(165)

8.1 Introduction

(166)

et al.

(167)

Table 8.1

(168)

8.2 Cisgenesis in wheat

8.2.1 Bread making quality

et al.

Triticum durum

Triticum aestivum

et al.

(169)

et al.

et al. et al.

8.2.2 Introgression breeding

et al. et al.

HorD et al.

H. chilense Triticum turgidum conv. durum

(170)

tritordeum Triticum aestivum.

Gli 1 Gli 3 tritordeum

D Hor

(171)

Figure 8.1

(172)

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) F1generation

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

BC1F1generation

o o o

generations BC2F1and BC3F1

o o o

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

generation BC2F2

Total time = 30 months

(173)

8.2.3 Transgenesis

et al.

et al. et al. bar

bar

8.2.4 Cisgenesis

(174)

et al.

1Dx5,

1Ax1 1Dy10 bar

bar

(175)

Figure 8.2

(176)

Figure 8.3

(177)

Time requirements

Technical and labour costs

(178)

bar

(179)

Table 8.1

CISGENESIS TRANSGENESIS costs

INTROGRESSION BREEDING 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

(180)

Quality of the final product

bar

(181)

Figure 8.4

Patent licensing

bar

et al.

HorD

(182)

bar

HorD

Regulatory costs

(183)

8.2.6 Conclusions

(184)

8.3 Grafting on GM rootstock in citrus

(185)

8.3.1 The importance of grafting in citrus

Citrus sinensis Citrus

aurantium

Citrus sinensis Poncirus trifoliata et al.

8.3.2 Dwarfing citrus

(186)

et al.

et al. et al.

et al.

CcGA20ox1

Arabidopsis et al. et al.

et al. et al.

et al.

(187)

Figure 8.5 et al.

8.3.3 Conventional breeding

Poncirus

trifoliata Mandarino King

Poncirus trifoliata

(188)

(189)

Figure 8.6

Poncirus trifoliata (male parent)

Mandarino King (female parent) Anthers collection

and storage Flowers emasculation

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 greenhouse

In the field

•

Citrus Tristeza Virus (CTV)

•

Nematodes

•

Salinity, drought, iron chlorosis

•

Phytophtora

•

Iron chlorosis

•

Productivity and Fruits quality

•

Junction scion and rootstock

(190)

Box 8.3. Conventional breeding protocol (Forner Alcaide 517)

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

o

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

o

Total time = 13 years

(191)

8.3.4 Transgenic rootstock

et al.

CcGA20ox1 Carrizo citrange

pBinJIT

CaMV 35S

nptII Agrobacterium

tumefaciens Carrizo citrange

Carrizo citrange

et al.

Agrobacteria

CcGA20ox1 in vitro Troyer citrange

CcGA20ox1

(192)

CcGA20ox1 nptII

Clemenules Clementine

(193)

Figure 8.7

Carrizo citrange

gene CcGA20ox1

CcGA20ox1 - antisense

nptII pBIN-JIT

A. tumefaciens

Co-cultivation

Shoot regeneration medium with Kanamycin

Micrografting PCR

Southern blot

Selected transformant (3 months old)

2-3 months culture

First sproutings internodes

buds grafted onto vigorous rootstock Carrizo citrange

PCR

buds grafted onto vigorous rootstock in the field

1 year

seeds Permission of

the Competent Authority

Green- house

Grafting of non-GM scion 6-8 months

Phenotypic assessment

(194)

Box 8.4. Protocol grafting on a GM rootstock

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

(195)

8.3.5 Comparative analysis Time requirements

Technical and labour costs

(196)

Quality of the final product

Carrizo citrange

Table 8.2

(197)

Patent licensing

Regulatory costs

(198)

Table 8.3

TRANSGENESIS

costs CROSS BREEDING 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

(199)

8.4 Analysis of FTO (Freedom to operate)

(200)

et al.

NEW PLANT BREEDING TECHNIQUES