Progress in genetic improvement of plum

Modern Agricultural Biotechnology:

Progress in genetic improvement of plum

Ralph Scorza

USDA
-
ARS

Appalachian Fruit Research Station

Kearneysville, West Virginia

ralph.scorza@ars.usda.gov

World
-
Wide Adoption of Genetically Engineered Crops

http://chartsbin.com/view/578

Soybean, Maize, Canola, Cotton………

Sugarbeet


Biotech herbicide tolerant sugarbeet was grown on 0.5 million


hectares in the USA and Canada at 97% adoption rate.


Alfalfa

425,000 hectares of biotech RR®alfalfa was planted in the US in 2012.


Poplar

Biotech Bt poplar was grown on ~500 hectares in China.


Papaya

Biotech PRSV resistant papaya was planted in Hawaii (2,000 hectares


with a 60% adoption) and in China at 6,275 hectares.


Squash

2,000 hectares of biotech virus resistant squash was grown in the US.

http://www.isaaa.org/resources/publications/biotech_crop_annual_update/download/05%20Alfalfa%202013.pdf

-

16.7 million
-

F
armers planting biotech crops


-

29 countries
-

planting biotech crops


-

18

Years

-

Farmers have planted biotech crops


-

160 M hectares

-

Global biotech crop area


-

$44 billion

-

Increase in net income for farmers


-

1 trillion

-

Estimated number of meals consumed with biotech


ingredients


-

0

-

Reliably documented human or animal safety issues


Biotech crop Facts

ISAAA report 2011

We all can benefit from the use of biotech crops:


Farmer benefits

increasing yield

improving protection from insects and disease

increasing their crops’ tolerance to heat, drought, and other environmental
stress


Processor benefits


improve the quality of animal feed, food and energy sources.


Consumer benefits

Increased protein

Healthier oils

Reduced exposure to agricultural chemicals


Environmental benefits

Conserve soil and energy

Reduce greenhouse gases

Minimize use of toxic chemicals

Conserve soil fertility and natural resources

Reduce need for new agricultural land



Biotech crop benefits








-
1.9 billion

kilograms of reduced carbon dioxide emissions



-
21.1 billion

kilograms of carbon sequestered in the soil



-
473 million

kilogram reduction in pesticides applied from 1996 to 2011



-
108.7 million

hectares of land preserved between 1996 and 2011



-
328 million tons

of additional food, feed and fiber produced from 1996 to 2011



(ISAAA report 2013)

-

pesticide use would decrease by 14.5 million kg /year


-

reduction of 7.5 million ha sprayed


-

20.5 million litres of diesel saved


-

reduction of 73,000 tons of carbon dioxide being released into the atmosphere.

Some potential environmental

benefits for 50% adoption of the

currently available biotech crops

in the EU

Park and Phipps (2002) Journal of Animal and Feed Sciences, 11:1
-
18.


HoneySweet Plum Resistant to Sharka:

A Potential Benefit to European agriculture


-

Co
-
developed by U.S. and European partners


-

F
ield tested in Europe for over 15 years


-

Solely the work of publicly
-
supported scientists, at public research institutions


–

For the benefit of growers and consumers






PPV Symptoms

-
Fruit deformation


and reduced quality

-
Premature fruit drop

-
Leaf chlorosis

-
Tree decline in severe


infections

The need for PPV resistance

PPV infection in selected European countries
:


Serbia
-


42 million bearing plum trees; 58% are infected with PPV



Croatia
-

2004 survey found 51% of sampled plum trees infected


Bosnia
-
Herzegovina
-

up to 41% of plum trees infected.


Bulgaria

-

infection in plums 62%


Romania

–

infection in plums 70%



Moldova

–

yield loses in plum 16
-
48%


Czech Republic
–

30 years average fruit yield
dropped by 80%

and
the number of


plum trees has been reduced from 18 million to

4 million
.


Greece

–

apricot production decreased from 35% to 13% of world production due to PPV


Spain

-

2.3 million PPV
-
infected trees removed between 1989 and 2006


at a cost of over 63 M Euros


2006 OEPP/EPPO 36 (2)

Zagrai et al UASMV 67 2010

Development of ‘HoneySweet’ PPV resistant plum variety

Gene discovery, Vector construction, Transformation 1990
-

1992


Plant establishment, Propagation, Greenhouse testing 1992
-

1995


Field testing U.S. 1995
–

2005


Field testing Europe 1996
–

2005 and continues today


Research and regulatory data accumulation 1990
–

2005

(Over 40 publications from this work)








14 Years

Development of ‘HoneySweet’ PPV resistant plum variety

14 Years

7 Years

Full Regulatory

Approval in U.S.

2011

R&D

Regulatory approval for ‘HoneySweet’

U.S. Regulatory Approval

Three agencies: Jurisdiction
__________________



Safety for Agriculture








Safety for Food









Safety for Environment



Examples of some of the information provided to U.S. regulators
:


Transformation system


Donor genes and sequences


Molecular characterization (DNA, RNA, protein)


Mechanism of resistance


Stability of resistance


Inheritance of resistance


Gene flow


Fruit compositional analyses


Allergenicity potential


Environmental consequences



European colleagues have worked together with

The U.S. team since 1990. In 1996 field tests were initiated

In Poland, Romania, Spain, and in 2002 in the Czech Republic.

These data were included in the U.S. regulatory dossier
.



European HoneySweet field test harvest

In over 15 years of field testing no ‘HoneySweet’ trees

have been naturally Infected with PPV by aphids

How effective is HoneySweet?

Temporal spread of Plum pox virus
-

Romania

0

10

20

30

40

50

60

1998

2000

2004

2005

2006

years

% PPV

conventional

C5

Graft inoculation of ‘HoneySweet’ with PPV


PPV infected plum

tree

HoneySweet tree

PPV infected bud

HoneySweet graft inoculum


(PPV
-
Rec)

Year 2

Year 5

Polak et al., 2008

Czech Republic

Field Test
-

Graft inoculation

0
1000
2000
3000
4000
5000
6000
7000
8000
mg GAE/Kg dry weight

Average Phenolics

Stanley
Jojo
HoneySweet
Others
0
10
20
30
40
50
60
70
% by dry weight

Average Total Sugars

Stanley
JoJo
HoneySweet
Others
0
10
20
30
40
50
60
mg per 100 dry grams

Average Vitamin C

Stanley
JoJo
HoneySweet
Others
FRUIT COMPOSITION STUDIES

HoneySweet safety studies

Molecular evaluations:



-

the entire genome of ‘HoneySweet’ was sequenced


-

no genes interrupted, no effect of insert on flanking genes


-

all RNAs were sequenced



-

no unexpected RNAs produced


-

HoneySweet produced less PPV
-
CP RNAs than


produced in PPV infected plums


-

no unexpected proteins produced







Results of Environmental Risk Studies


Zagrai et al. 2011 Transgenic plums expressing Plum pox virus coat protein gene do not assist

the development of virus recombinants under field conditions. Journal of Plant Pathology


No virus recombinants


Capote et al. 2007 Risk assessment of the field release of transgenic European plums susceptible

and resistant to Plum pox virus ITEA 2007

No effects on non
-
target insects


Capote et al. 2008 Assessment of the diversity and dynamics of Plum pox virus and aphid

populations in transgenic European Plums under Mediterranean conditions

Transgenic Research 2008

No effects on aphid populations


No effects on virus diversity


Zagrai et al. 2008 Plum pox virus silencing of C5 transgenic plums is stable under challenge

inoculation with heterologous viruses. Journal of Plant Pathology

No breakdown of resistance in presence of other
Prunus

viruses


Gene flow is low


(Scorza et al PLOS ONE in press)

‘HoneySweet’




It is highly resistant to PPV.

No trees were ever infected by aphids.


Resistance to all strains tested


Fruit quality is excellent


The transgene and resistance could be transferred to progeny through traditional
cross hybridization and the resistant seedlings could be readily identified.


HoneySweet may be useful as a PPV resistant rootstock


Approved for cultivation and consumption in the U.S.



This work is a U.S.
-

European collaboration



M. Ravelonandro, France

M. Cambra, Spain

N. Capote, Spain

T. Malinowski, Poland

I. Zagrai, Romania

J. Polak, Czech Republic

J. Kundu, Czech Republic

I. Kamenova, Bulgaria

A. Atanassov, Bulgaria

S. Paunovic, Serbia

S. Dolgov, Russia


Black Sea Biotechnology


Association



R. Scorza, USDA

A.M. Callahan, USDA

C. Dardick, USDA



and others………
.

Shouldn’t European growers

and consumers also benefit?

‘HoneySweet’ International Working Group

HoneySweet is being made freely available in the U.S.


Those outside the U.S. wishing to exploit ‘HoneySweet’ will be

responsible for obtaining the required regulatory clearances.


Qualities
-

Sweet (21% sugar), large size (60 g), productivity very good. A good
variety for the fresh market. Brandy, marmalade, drying not yet tested.


Czech Republic


2002
-

present



Spain

1996
-
present

Poland

1996
-
2007

Romania

1996
-
2006 , 2012
-


Scientists at the
Crop Research Institute
,

Czech Republic, are developing a dossier to submit to EFSA

Based on over 15 years of study in Europe and the U.S.



This technology can help solve the problems that will be

facing Europe, the U.S. and the rest of the world

including climate change, the spread of invasive species,

and population pressures for increased food production.


Wisely used, genetic engineering can be an

important tool to help meet these challenges.



‘HoneySweet’ International Working Group

Academy of Medicine, France

American Medical Association

American Society for Plant Biology

Brazilian Academy of Science

Chinese Academy of Science

Food and Agriculture Organization

Indian National Science Academy

Mexican Academy of Science

Pontifical Academy of Sciences

Romanian Academy

Romanian Academy of Agricultural and Forestry Sciences

Royal Society of London

Third World Academy of Science

U.S. Department of Agriculture

U.S. National Academy of Sciences

U.S. National Research Council

World Health Organization

and others…….




Scientific Institutions expressing positive positions on GMO Crops