Solanum tuberosum 1st Edition David

Dobnik Kristina Gruden Živa Ramšak

Anna Coll
Visit to download the full and correct content document:
https://ebookmeta.com/product/solanum-tuberosum-1st-edition-david-dobnik-kristina-
gruden-ziva-ramsak-anna-coll/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...

Chess Explained The c3 Sicilian 1st Edition Sam Collins

https://ebookmeta.com/product/chess-explained-
the-c3-sicilian-1st-edition-sam-collins/

Starting Out The c3 Sicilian 1st Edition John Emms

https://ebookmeta.com/product/starting-out-the-c3-sicilian-1st-
edition-john-emms/

Harmony of Colour 85 Big Cats 1st Edition Various

https://ebookmeta.com/product/harmony-of-colour-85-big-cats-1st-
edition-various/

English Grammar Exercises with answers Part 4 Your

quest towards C2 1st Edition Daniel B. Smith

https://ebookmeta.com/product/english-grammar-exercises-with-
answers-part-4-your-quest-towards-c2-1st-edition-daniel-b-smith/
Studies in Law, Politics, and Society (Volume 85) 1st
Edition Austin Sarat (Editor)

https://ebookmeta.com/product/studies-in-law-politics-and-
society-volume-85-1st-edition-austin-sarat-editor/

85 Years IFLA A History and Chronology of Sessions 1927

2012 1st Edition Jeffrey M Wilhite

https://ebookmeta.com/product/85-years-ifla-a-history-and-
chronology-of-sessions-1927-2012-1st-edition-jeffrey-m-wilhite/

85 Years of the Oscar The Official History of the

Academy Awards 6th Edition Robert Osborne

https://ebookmeta.com/product/85-years-of-the-oscar-the-official-
history-of-the-academy-awards-6th-edition-robert-osborne/

Thirty Years Hundred Stories Engineering

Accomplishments in Singapore as Told by the NTI Pioneer
Engineering Class of 85 1st Edition Liu Fook Thim

https://ebookmeta.com/product/thirty-years-hundred-stories-
engineering-accomplishments-in-singapore-as-told-by-the-nti-
pioneer-engineering-class-of-85-1st-edition-liu-fook-thim/

The Unofficial Sims Cookbook : From Baked Alaska to

Silly Gummy Bear Pancakes, 85+ Recipes to Satisfy the
Hunger Need 1st Edition Taylor O’Halloran

https://ebookmeta.com/product/the-unofficial-sims-cookbook-from-
baked-alaska-to-silly-gummy-bear-pancakes-85-recipes-to-satisfy-
the-hunger-need-1st-edition-taylor-ohalloran/
Methods in
Molecular Biology 2354

David Dobnik
Kristina Gruden
Živa Ramšak
Anna Coll Editors

Solanum
tuberosum
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series
was the first to introduce the step-by-step protocols approach that has become the standard
in all biomedical protocol publishing. Each protocol is provided in readily-reproducible
step-by step fashion, opening with an introductory overview, a list of the materials and
reagents needed to complete the experiment, and followed by a detailed procedure that is
supported with a helpful notes section offering tips and tricks of the trade as well as
troubleshooting advice. These hallmark features were introduced by series editor Dr. John
Walker and constitute the key ingredient in each and every volume of the Methods in
Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols
from the series are indexed in PubMed.
 Solanum tuberosum

Methods and Protocols

Edited by

David Dobnik, Kristina Gruden, Živa Ramšak and Anna Coll

Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia
Editors
David Dobnik Kristina Gruden
Department of Biotechnology Department of Biotechnology and Systems Biology
and Systems Biology National Institute of Biology
National Institute of Biology Ljubljana, Slovenia
Ljubljana, Slovenia

Živa Ramšak Anna Coll

Department of Biotechnology Department of Biotechnology and Systems Biology
and Systems Biology National Institute of Biology
National Institute of Biology Ljubljana, Slovenia
Ljubljana, Slovenia

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1608-6 ISBN 978-1-0716-1609-3 (eBook)
https://doi.org/10.1007/978-1-0716-1609-3

© Springer Science+Business Media, LLC, part of Springer Nature 2021

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface

Solanum tuberosum, commonly known as potato, has its origins way back in the past.
Evidences of potato cultivation date back to at least 7000 years. Today, potato, as we
know it, is the fourth most important crop in the world, and oftentimes the potato project
applications usually begin with this statement, stressing the importance of conducting
research on potato. The crop is very versatile and at the same time very environmentally
friendly, easy to grow, and does not require excessive amounts of fertilizer and chemical
additives. Some scientists also claim that one could live almost entirely off potatoes—if
paired with milk or butter. Due to its essential vitamin C content, it was worth its weight in
gold during the Alaskan Klondike Gold Rush. A bouquet of potato flowers given to Marie
Antoinette inspired first the use of it in fashion, and to be later included in French cuisine.
And prominent artists, such as Shakespeare, Van Gogh, and James Joyce, have directly or
indirectly used this crop in their esteemed works. And lastly, in 1995, it became the first
vegetable grown in space on the Columbia space shuttle.
With the above quick collection of whimsical facts about potato, we, the editors, would
like to succinctly introduce this potato protocols book, which brings to a reader a collection
of protocols to address potato research from several different perspectives. The book is
divided into five parts and begins with chapters on the history of potato, introduction to
potato breeding, and its long-term storage. Further three parts are dedicated to different
research fields from molecular biology to omics approaches and bioinformatics. High-
throughput omics techniques aim at a wider understanding of potato-related processes,
and the bioinformatics in silico approaches are useful to make sense of the big data, to allow
proper biological interpretation. Within molecular biology section, we get information on
how to study selected genes, proteins, or processes of interest, which were identified as
potentially important by systems studies. Finally, the generated knowledge can lead to
applications in a form of actual crop improvement and development of diagnostic assays,
which are covered in the last part of the volume. In such a way, we tried to prepare a
comprehensive collection of research that is currently being conducted on potato.
We hope that this collection of knowledge from the potato research community will
enable the reader to get a better insight into the interesting world of potato and also give
them direct practical advices in forms of protocols on how to address potato-related research
challenges. With this in mind, we wish for this book to become a valuable resource for the
everyday researcher already working or only starting to work with potato.

Ljubljana, Slovenia David Dobnik

Kristina Gruden
Živa Ramšak
Anna Coll

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I HISTORY, BREEDING AND LONG-TERM STORAGE

1 Importance of Potato as a Crop and Practical Approaches to

Potato Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Peter Dolničar
2 Cryopreservation of Potato Shoot Tips for Long-Term Storage. . . . . . . . . . . . . . . 21
Rainer Vollmer, Janeth Espirilla, Rosalva Villagaray, José Cárdenas,
Mario Castro, Juan Carlos Sánchez, Norma Manrique-Carpintero,
David Ellis, and Noelle Lynette Anglin

PART II HIGH-THROUGHPUT OMICS TECHNIQUES

3 RNA Sequencing Analyses for Deciphering Potato Molecular Responses. . . . . . . 57

Živa Ramšak, Marko Petek, and Špela Baebler
4 Yeast Two-Hybrid Screening for Identification of Protein-Protein
Interactions in Solanum tuberosum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Hazel McLellan, Miles R. Armstrong, and Paul R. J. Birch
5 Potato as a Model for Field Trials with Modified Gene Functions
in Research and Translational Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Nam Phuong Kieu, Marit Lenman, and Erik Andreasson
6 DAP-Seq Identification of Transcription Factor-Binding Sites in Potato . . . . . . . 123
José M. Franco-Zorrilla and Salomé Prat
7 Mass Spectrometric Monitoring of Plant Hormone Cross Talk
During Biotic Stress Responses in Potato (Solanum tuberosum L.) . . . . . . . . . . . . 143
Marta-Marina Pérez-Alonso, Paloma Ortiz-Garcı́a,
José Moya-Cuevas, and Stephan Pollmann
8 A Comprehensive Guide to Potato Transcriptome Assembly . . . . . . . . . . . . . . . . . 155
Maja Zagorščak and Marko Petek

PART III BIOINFORMATICS AND IN SILICO APPROACHES

9 MapMan Visualization of RNA-Seq Data Using Mercator4

Functional Annotations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Marie Bolger, Rainer Schwacke, and Björn Usadel
10 Identification of Resistance Genes Using Diagnostic R-Gene
Enrichment Sequencing (dRenSeq) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Miles Armstrong and Ingo Hein

vii
viii Contents

11 Methodologies for Discovery and Quantitative Profiling of

sRNAs in Potato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Maja Križnik, Maja Zagorščak, and Kristina Gruden
12 Co-expression for Genotype-Phenotype Function Annotation
in Potato Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Oren Tzfadia
13 Computer Vision and Less Complex Image Analyses to Monitor
Potato Traits in Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Junfeng Gao, Jesper Cairo Westergaard, and Erik Alexandersson

PART IV MOLECULAR BIOLOGY APPROACHES

14 Quantifying the Contribution to Virulence of Phytophthora

infestans Effectors in Potato. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Aviv Andriani, Doret Wouters, Pieter J. Wolters, and Vivianne G. A. A.
Vleeshouwers
15 Identification of Solanum Immune Receptors by Bulked Segregant
RNA-Seq and High-Throughput Recombinant Screening . . . . . . . . . . . . . . . . . . . 315
Yerisf Torres Ascurra, Xiao Lin, Pieter J. Wolters, and Vivianne G. A. A.
Vleeshouwers
16 Gene Editing in Potato Using CRISPR-Cas9 Technology . . . . . . . . . . . . . . . . . . . 331
Laura Chauvin, François Sevestre, Tjaša Lukan, Fabien Nogué,
Jean-Luc Gallois, Jean-Eric Chauvin, and Florian Veillet
17 Gene Downregulation in Potato Roots Using Agrobacterium rhizogenes-
Mediated Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Sandra Fernández-Piñán, Carla Sànchez-Guirado,
Mercè Figueras, and Olga Serra

PART V CROP IMPROVEMENT AND DIAGNOSTIC ASSAYS

18 Molecular Detection of Ralstonia solanacearum to Facilitate
Breeding for Resistance to Bacterial Wilt in Potato . . . . . . . . . . . . . . . . . . . . . . . . . . 375
Virginia Ferreira, Matı́as González, Marı́a Julia Pianzzola,
Núria S. Coll, Marı́a Inés Siri, and Marc Valls
19 Toward the Design of Potato Tolerant to Abiotic Stress . . . . . . . . . . . . . . . . . . . . . 387
Raymond Campbell, Laurence J. M. Ducreux, Elena Mellado-Ortega,
Robert D. Hancock, and Mark A. Taylor
20 Rapid Loop-Mediated Isothermal Amplification for Detection
of the Ralstonia solanacearum Species Complex Bacteria
in Symptomatic Potato Tubers and Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
Manca Pirc, Špela Alič, and Tanja Dreo

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
Contributors

ERIK ALEXANDERSSON • Department of Plant Protection Biology, Swedish University of

Agricultural Sciences, Alnarp, Sweden
ŠPELA ALIČ • Department of Biotechnology and Systems Biology, National Institute of Biology,
Ljubljana, Slovenia
ERIK ANDREASSON • Department of Plant Protection Biology, Swedish University of
Agricultural Sciences, Alnarp, Sweden
AVIV ANDRIANI • Wageningen UR Plant Breeding, Wageningen University and Research,
Wageningen, The Netherlands
NOELLE LYNETTE ANGLIN • International Potato Center (CIP), Cryobank, Lima, Peru
MILES R. ARMSTRONG • Division of Plant Sciences, School of Life Science, University of
Dundee (at the James Hutton Institute), Dundee, UK; Cell and Molecular Sciences,
The James Hutton Institute, Dundee, UK
ŠPELA BAEBLER • Department of Biotechnology and Systems Biology, National Institute of
Biology, Ljubljana, Slovenia
PAUL R. J. BIRCH • Division of Plant Sciences, School of Life Science, University of Dundee
(at the James Hutton Institute), Dundee, UK; Cell and Molecular Sciences, The James
Hutton Institute, Dundee, UK
MARIE BOLGER • Institute of Bio- and Geosciences (IBG-4: Bioinformatics),
Forschungszentrum Jülich, Jülich, Germany
RAYMOND CAMPBELL • Cell and Molecular Sciences, The James Hutton Institute, Dundee,
UK
JOSÉ CÁRDENAS • International Potato Center (CIP), Cryobank, Lima, Peru
MARIO CASTRO • International Potato Center (CIP), Cryobank, Lima, Peru
JEAN-ERIC CHAUVIN • IGEPP, INRAE, Institut Agro, Univ Rennes, Ploudaniel, France
LAURA CHAUVIN • IGEPP, INRAE, Institut Agro, Univ Rennes, Ploudaniel, France
NÚRIA S. COLL • Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB),
Bellaterra, Catalonia, Spain
PETER DOLNIČAR • Agricultural Institute of Slovenia, Ljubljana, Slovenia
TANJA DREO • Department of Biotechnology and Systems Biology, National Institute of
Biology, Ljubljana, Slovenia
LAURENCE J. M. DUCREUX • Cell and Molecular Sciences, The James Hutton Institute,
Dundee, UK
DAVID ELLIS • International Potato Center (CIP), Cryobank, Lima, Peru
JANETH ESPIRILLA • International Potato Center (CIP), Cryobank, Lima, Peru
SANDRA FERNÁNDEZ-PIÑÁN • Laboratori del Suro, Biology Department, Universitat de
Girona, Girona, Spain
VIRGINIA FERREIRA • Área Microbiologı́a, Departamento de Biociencias (DEPBIO),
Facultad de Quı́mica, Universidad de la República, Montevideo, Uruguay
MERCÈ FIGUERAS • Laboratori del Suro, Biology Department, Universitat de Girona, Girona,
Spain
JOSÉ M. FRANCO-ZORRILLA • Department of Plant Molecular Genetics, Centro Nacional de
Biotecnologı́a-CSIC, Madrid, Spain
JEAN-LUC GALLOIS • INRAE, GAFL, Montfavet, France

ix
x Contributors

JUNFENG GAO • Lincoln Agri-Robotics, Lincoln Institute for Agri-Food Technology,

University of Lincoln, Lincoln, UK
MATÍAS GONZÁLEZ • Instituto Nacional de Investigaciones Agropecuarias (INIA), Estacion
Experimental Salto Grande, Salto, Uruguay
KRISTINA GRUDEN • Department of Biotechnology and Systems Biology, National Institute of
Biology, Ljubljana, Slovenia
ROBERT D. HANCOCK • Cell and Molecular Sciences, The James Hutton Institute, Dundee,
UK
INGO HEIN • Division of Plant Sciences, School of Life Science, University of Dundee (at the
James Hutton Institute), Dundee, UK; The James Hutton Institute. CMS, Dundee, UK
NAM PHUONG KIEU • Department of Plant Protection Biology, Swedish University of
Agricultural Sciences, Alnarp, Sweden
MAJA KRIŽNIK • Department of Biotechnology and Systems Biology, National Institute of
Biology, Ljubljana, Slovenia
MARIT LENMAN • Department of Plant Protection Biology, Swedish University of
Agricultural Sciences, Alnarp, Sweden
XIAO LIN • Wageningen UR Plant Breeding, Wageningen University and Research,
Wageningen, The Netherlands
TJAŠA LUKAN • Department of Biotechnology and Systems Biology, National Institute of
Biology, Ljubljana, Slovenia
NORMA MANRIQUE-CARPINTERO • International Potato Center (CIP), Cryobank, Lima,
Peru
HAZEL MCLELLAN • Division of Plant Sciences, School of Life Science, University of Dundee
(at the James Hutton Institute), Dundee, UK
ELENA MELLADO-ORTEGA • Cell and Molecular Sciences, The James Hutton Institute,
Dundee, UK
JOSÉ MOYA-CUEVAS • Centro de Biotecnologı́a y Genomica de Plantas, Universidad Polité
cnica de Madrid (UPM)—Instituto Nacional de Investigacion y Tecnologı́a Agraria y
Alimentacion (INIA), Madrid, Spain
FABIEN NOGUÉ • Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université Paris-
Saclay, Versailles, France
PALOMA ORTIZ-GARCÍA • Centro de Biotecnologı́a y Genomica de Plantas, Universidad Polité
cnica de Madrid (UPM)—Instituto Nacional de Investigacion y Tecnologı́a Agraria y
Alimentacion (INIA), Madrid, Spain
MARTA-MARINA PÉREZ-ALONSO • Centro de Biotecnologı́a y Genomica de Plantas,
Universidad Politécnica de Madrid (UPM)—Instituto Nacional de Investigacion y
Tecnologı́a Agraria y Alimentacion (INIA), Madrid, Spain
MARKO PETEK • Department of Biotechnology and Systems Biology, National Institute of
Biology, Ljubljana, Slovenia
MARÍA JULIA PIANZZOLA • Área Microbiologı́a, Departamento de Biociencias (DEPBIO)—
Facultad de Quı́mica, Universidad de la República, Montevideo, Uruguay
MANCA PIRC • Department of Biotechnology and Systems Biology, National Institute of
Biology, Ljubljana, Slovenia
STEPHAN POLLMANN • Centro de Biotecnologı́a y Genomica de Plantas, Universidad Polité
cnica de Madrid (UPM)—Instituto Nacional de Investigacion y Tecnologı́a Agraria y
Alimentacion (INIA), Madrid, Spain; Departamento de Biotecnologı́a-Biologı́a Vegetal,
Escuela Técnica Superior de Ingenierı́a Agronomica, Alimentaria y de Biosistemas,
Universidad Politécnica de Madrid (UPM), Madrid, Spain
 Contributors xi

SALOMÉ PRAT • Department of Plant Molecular Genetics, Centro Nacional de Biotecnologı́a-

CSIC, Madrid, Spain
ŽIVA RAMŠAK • Department of Biotechnology and Systems Biology, National Institute of
Biology, Ljubljana, Slovenia
CARLA SÀNCHEZ-GUIRADO • Laboratori del Suro, Biology Department, Universitat de
Girona, Girona, Spain
JUAN CARLOS SÁNCHEZ • International Potato Center (CIP), Cryobank, Lima, Peru
RAINER SCHWACKE • Institute of Bio- and Geosciences (IBG-4: Bioinformatics),
Forschungszentrum Jülich, Jülich, Germany
OLGA SERRA • Laboratori del Suro, Biology Department, Universitat de Girona, Girona,
Spain
FRANÇOIS SEVESTRE • Unité de Glycobiologie Structurale et Fonctionnelle, Univ. Lille, CNRS,
UMR8576, UGSF, Lille, France; Univ. Lille, CNRS, USR 3290, MSAP, Miniaturisation
pour la Synthèse, l’Analyse et la Protéomique, Lille, France
MARÍA INÉS SIRI • Área Microbiologı́a, Departamento de Biociencias (DEPBIO)—Facultad
de Quı́mica, Universidad de la República, Montevideo, Uruguay
MARK A. TAYLOR • Cell and Molecular Sciences, The James Hutton Institute, Dundee, UK
YERISF TORRES ASCURRA • Wageningen UR Plant Breeding, Wageningen University and
Research, Wageningen, The Netherlands
OREN TZFADIA • Faculty of Pharmaceutical, Biomedical and Veterinary Sciences,
Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
BJÖRN USADEL • Institute of Bio- and Geosciences (IBG-4: Bioinformatics),
Forschungszentrum Jülich, Jülich, Germany
MARC VALLS • Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB),
Bellaterra, Catalonia, Spain; Department of Genetics, University of Barcelona, Barcelona,
Catalonia, Spain
FLORIAN VEILLET • IGEPP, INRAE, Institut Agro, Univ Rennes, Ploudaniel, France
ROSALVA VILLAGARAY • International Potato Center (CIP), Cryobank, Lima, Peru
VIVIANNE G. A. A. VLEESHOUWERS • Wageningen UR Plant Breeding, Wageningen
University and Research, Wageningen, The Netherlands
RAINER VOLLMER • International Potato Center (CIP), Cryobank, Lima, Peru
JESPER CAIRO WESTERGAARD • Department of Plant and Environmental Sciences, University
of Copenhagen, Taastrup, Denmark
PIETER J. WOLTERS • Wageningen UR Plant Breeding, Wageningen University and
Research, Wageningen, The Netherlands
DORET WOUTERS • Wageningen UR Plant Breeding, Wageningen University and Research,
Wageningen, The Netherlands
MAJA ZAGORŠČAK • Department of Biotechnology and Systems Biology, National Institute of
Biology, Ljubljana, Slovenia
 Part I

History, Breeding and Long-term Storage

 Chapter 1

Importance of Potato as a Crop and Practical Approaches

to Potato Breeding
Peter Dolničar

Abstract
Potatoes (Solanum tuberosum L. subsp. tuberosum and andigena) and seven other related species, which are
cultivated today, have become the most important non-cereal crop in the world. It is grown on a significant
scale in 130 countries, with a gross production value of 63.6 billion US dollars in 2016, with the yearly
potato production of 368 million tons in 2018. Today potato is grown for food, animal feed, industrial
uses, and seed tuber production, depending on the region, country development, and historical reasons.
The food production is both for fresh ware markets and for processing into crisps, french fries, canned
potatoes, flakes, etc. More than 10,000 potato varieties have been grown worldwide to date, many of which
are still grown. Despite such a large number of varieties, there is still a need for new varieties. Classical
breeding of new potato varieties in many programs around the world has changed little in decades and
differs mainly in terms of scope and technologies used. Until the turn of the millennium, it was based
primarily on empirical experience and selection of individual phenotypic traits. The great genetic diversity
that exists in potato and its wild relatives is both an opportunity and a challenge to introduce traits that do
not currently exist in the potato gene pool into modern potato varieties. Molecular marker technology
development has reached the point where published markers for use in commercial breeding are available.
Markers can be used during the whole selection process, with an even more important role of molecular
breeding in pre-breeding programs and creation of the most appropriate parental lines.

Key words Potato, Solanum tuberosum, Origin and history, Market and utilization, Breeding pota-
toes, Resistance to pests and diseases, Pyramiding R genes, Molecular markers

1 Origin and History of Cultivated Potatoes

Cultivated potato as we know it, often called Irish potato (Solanum

tuberosum L. subsp. tuberosum), is a tetraploid species with a num-
ber of chromosomes (n ¼ 12), i.e., a total of 48 chromosomes,
adapted to the long day in the temperate zone. It belongs to the
genus Solanum of the Solanaceae family, which according to the
classical Hawkes taxonomy [1] comprises 7 cultivated potato spe-
cies (among them Solanum tuberosum with subspecies tuberosum
and andigena) and 228 wild potato species of different ploidy, from
diploid to hexaploid. Many species of the genus Solanum do not

David Dobnik et al. (eds.), Solanum tuberosum: Methods and Protocols, Methods in Molecular Biology, vol. 2354,
https://doi.org/10.1007/978-1-0716-1609-3_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021

3
4 Peter Dolničar

even form tubers, but they are nevertheless extremely important as

a source of various traits (genes) for modern potato breeding
programs. Newer molecular approaches to the basic taxonomy of
the genus Solanum have not changed it significantly, only that they
have identified a new cultivated potato species, so today we distin-
guish 8 cultivated potato species [2, 3]. The cultivated potatoes
(Solanum tuberosum L. subsp. tuberosum and andigena) and the
seven related cultivated species (Solanum ajanhuiri, Solanum chau-
cha, Solanum curtilobum, Solanum goniocalyx, Solanum juzepczu-
kii, Solanum phureja, and Solanum stenotomum) have become the
most important non-cereal crop in the world. Potato is grown as a
major crop in countries with very large populations, in different
climatological zones, including temperate regions, subtropics, and
tropics, under very different agroecological conditions, lowlands,
as well as highlands and in very different socioeconomic environ-
ments, by large-scale farmers in high-external-input agriculture as
well as smallholders in most of the developing countries.
The center of origin of potato cultivation may well have been
the Andes of southern Peru and northern Bolivia. Potato archaeo-
logical remains have been radiocarbon-dated to 7000 years ago
[4]. It is highly likely that the potato was domesticated even before
that date, but there is no information to support that assumption.
There is much later evidence from rubbish heaps, graves, and food
stores of potato cultivation 4500–3500 years ago [5]. There are
also ceramics dating from the Moche culture in northern Peru
(C. AD 1–600), and the Chimú peoples (C. AD 900–1450), as
well as Huari of Pacheco urns from Nazca valley in southern Peru
(C. AD 650–700). It is assumed that all this ceramics from coastal
areas was obtained from farmers in the highlands, where potatoes
were cultivated. Consistent with archaeological evidence that sug-
gests that potato was first grown in South America, Vavilov identi-
fied the New World region of Mexico, Peru, and adjoining
countries as a center of origin for potato. A more exact geographic
origin for potato has been the topic of scientific study in recent
decades, suggesting different regions and multiple domestication
events, sometimes involving complex interspecific hybridization of
various Solanum species [2, 6–8]. Spooner et al. [9] however
provided molecular evidence refuting these claims. Applying
AFLP marker technology to collection of 98 potato landraces and
261 wild potato accessions, these authors demonstrated a clear
distinction between “Northern” (Peru) and “Southern” (Bolivia
and Argentina) wild potato species. They clustered together all
cultivated types suggesting a single domestication event. Cultivated
potatoes were more closely related to “Northern” species and
Spooner et al. [9] concluded that potato was domesticated in
what is now southern Peru [10].
The first historical record of the first European to see the
potatoes was in 1537, when a band of Spaniards led by Jimenez
Importance of Potato as a Crop and Practical Approaches to Potato Breeding 5

de Quesada penetrated into what is now Colombia. This was fol-

lowed by López de Gómara (1552) who found potatoes in south-
ern Peru and Pedro Cieza de León (1553) in the area of today’s
southern Colombia and northern Ecuador. Potatoes in Chile
received first mention by Sir Francis Drake in 1578 [1].
How the potato came to Europe after the discovery of America
and spread around the world is known. There were two introduc-
tions into Europe, one into Spain in 1570 and the second into
England in 1590 [4]. European herbalists obtained their potatoes
from the Flemish herbalist Carolus Clusius (1601), his specimens
being derived from Spain, via Italy. It is interesting that these first
European potatoes’ early entries were adapted to develop tubers
under a 12-h day in the Andes and not as our present potatoes do,
under a 16–18-h day of Europe. Evidence from Spanish archives
clearly shows that the first European potatoes were short-day-
adopted, tuberizing in December to January in the frost-free
zones of southern Spain and Italy [1, 11]. These potatoes were
the Andean form of the tetraploid potato species (Solanum tuber-
osum L. subsp. andigena Hawkes) which was initially widespread in
Europe. They needed several centuries to adapt to a long day in
northern Europe, also with inputs of new genetic resources from
Chile [4]. Potatoes in North American colonies were first received
from Bermuda in 1691, where they had been grown from an earlier
importation from England in 1613. British missionaries took pota-
toes to India and China in the seventeenth century and they were
introduced into Japan and parts of Africa at about the same time.
They appeared in New Zealand in 1769, thus turning the potato
into one of the most important world crops in less than
300 years [4].
Potatoes soon became a very important staple food across
Europe, saving most of its population from starvation in the fol-
lowing centuries. On the other hand, the potato crop failure was
the reason for the Irish Potato Famine, also known as the Great
Hunger, which began in 1845, when an oomycete called late blight
(Phytophthora infestans (Mont.) de Bary) spread rapidly throughout
Ireland. The worst year of the period was 1847, known as “Black
47,” when the infestation ruined up to one-half of the potato crop
that year, and about three-quarters of the crop over the next
7 years. Because the tenant farmers of Ireland, then ruled as a
colony of Great Britain, relied heavily on the potato as a source of
food, the infestation had a catastrophic impact on Ireland and its
population. Before it ended in 1852, the Potato Famine resulted in
the death of roughly 1 million Irish from starvation and related
causes, with at least another million forced to leave their homeland
as refugees, mainly to North America and Great Britain, causing
Ireland’s population to fall by between 20% and 25% [12].
Late blight probably came to Europe with potatoes imported
from North America, on the Cureghem estate in West Flanders in
6 Peter Dolničar

1844. The Belgian Government decided to import them after an

epidemic of dry rot and curliness a few years earlier. Already in the
first year, “unusual brown spots” were noticed, and some tubers
collapsed (rotted) in the warehouse. The late blight spread rapidly
from Europe across Belgium. As early as in 1844, it was spotted in
northern France and south-east England. In 1845, the late blight
also reached the northern provinces of the Austrian Empire, Slesia
and Galicia (now Poland), Bohemia and Moravia, and Bukovina
(now Ukraine and Romania). Yield losses were also up to 75%.
Unger also reported late blight in Styria in 1847 [13].
The potato blight returned to Europe and Ireland in 1879, but
by that point the Land War, one of the largest agrarian movements
to take place in nineteenth-century Europe, had begun in Ireland,
and the consequent reduction in homelessness and house demoli-
tion resulted in a drastic reduction in the number of deaths.

2 The Importance of Potato Today

Potato is one of the world’s major staple food crops and produces
more dry matter and protein per hectare than the other major cereal
crops [14]. The crop is grown on a significant scale in 130 countries.
Today, potatoes are the fourth most important crop in the world
after rice, maize, and wheat, looking at a gross production value of
63.6 billion US dollars in 2016. With the yearly potato production
of 368 million tons in 2018 and more than a half of gross produc-
tion value of root crops, potato is the most important root crop in
the world, followed by cassava, yams, sweet potatoes, and taro
(Table 1) [15].

Table 1
The area harvested and total production in 2018 and gross production value in 2016 of some major
cereal and root crops [15]

Crop Area harvested (ha) Production (tons) Gross production value 2016 (in 1000 $)
Rice, paddy 167,132,623 782,000,147 206,476,316
Maize 193,733,568 1,147,621,938 150,180,124
Wheat 214,291,888 734,045,174 118,251,309
Potato 17,578,672 368,168,914 63,601,232
Cassava 24,590,181 277,808,759 28,946,965
Yams 8,690,716 72,580,851 16,816,988
Sweet potatoes 8,062,737 91,945,358 7,944,828
Taro 1,660,170 10,639,850 2,148,219
 Importance of Potato as a Crop and Practical Approaches to Potato Breeding 7

Table 2
The area harvested, average yield and total production of potato in the world, regions, and some
major potato-producing countries in 2018 [16]

Area harvested (ha) Yield (tons/ha) Production (tons)

World 17,578,672 20.9 368,168,914
Asia 9,601,798 20.3 188,644,711
China 4,810,888 18.8 90,259,155
India 2,151,000 22.5 48,529,000
Europe 4,754,037 22.1 105,181,286
Germany 252,200 35.4 8,920,800
France 199,886 39.4 7,870,973
Poland 297,484 25.1 7,478,184
The Netherlands 164,689 36.6 6,029,734
The United Kingdom 140,000 35.9 5,028,000
Russian Federation 1,313,495 17.0 22,394,960
Ukraine 1,319,900 17.0 22,503,970
North America 548,255 48.2 26,399,105
The United States 414,115 49.8 20,607,342
Canada 134,102 43.2 5,790,838
Africa 1,904,121 13.6 26,041,720
South and Central America 1,029,994 19.6 20,197,255
Oceania 40,467 42.1 1,704,835

According to FAO data for 2018, 17,578,672 ha of potatoes

were planted in the world with a moderate average yield of
20.9 tons/ha and a total yield of 368,168,914 tons of potatoes
(Table 2). There are large differences between regions and
countries in terms of area, average yield, as well as growth dynamics
or declining potato production. In the last decades, the potato
acreage in developed countries decreased with an increase of aver-
age yields. It decreased significantly also in the eastern Europe (e.g.,
acreage in Poland dropped by sixfold, from 1.8 million to
297,000 ha) and in the former Soviet Union countries. Despite
that, the Russian Federation and Ukraine remain among the largest
potato-producing countries, with more than 1.3 million ha each,
but with rather low average yields of 17 tons/ha in 2018. Since the
early 1960s, the increase of area planted with potato in the devel-
oping countries (especially Asia and Africa) has been greater than
that for any other major crop. The potato has become an important
8 Peter Dolničar

staple food in parts of the world where there is a limited (but

increasing) purchasing power, an increasing demand for food, and
increasing pressure on scarce land. Currently 54% of the
potato-cropped world’s area is in Asia, of that 40% in China and
India, the two most populated countries worldwide [16].
Statistically Europe remained the second largest potato produc-
tion region in the world, with Russian Federation and Ukraine
attributing more than a half. Other large potato-producing
countries in the European Union in 2018 were Germany, France,
Poland, the Netherlands, and the United Kingdom, with average
yields between 35 and 40 tons/ha, except for Poland with lower
yields. In some other years average yields in these countries came
close to 45 tons/ha, coming closer to the United States, where the
highest average yield was 49.8 tons/ha, followed by Canada and
Oceania (of the latter mainly Australia) (Table 2). In the Nether-
lands the ware potato yields usually exceed 45 tons/ha, but
national average is lower due to a large proportion of seed potato
with lower yields [16].

3 The Potato Markets and Utilization

Potato is grown for food, animal feed, industrial uses, and seed
tuber production, depending on the region, country development,
historical reasons, etc. The food production is both for fresh ware
markets and for processing into crisps, french fries, canned pota-
toes, flakes, etc.
The main use of potato is still as a direct fresh food, but
increased proportion is processed as a snack. The fresh ware markets
differ a lot in consumer demands. In developed countries consu-
mers demand high-quality uniform tubers with a nice skin finish,
with additional specific requirements for certain purposes and uses.
The purchased potato type or variety varies even according to the
meal occasion, which influences expected packaging or presenta-
tion. Some practical examples include jacket potatoes, a popular
choice for those looking for a healthy meal, and roast potatoes as a
firm favorite for family meals, whereas french fries are rated highly
as a quick and simple kids’ meal. High proportion of fresh potato
sales is done in supermarkets, with tubers washed and prepacked in
small packages. This gives the retailers massive purchasing power
and influence over the supply chain. The situation is completely
opposite in the developing countries. There, potato tubers are sold
mostly in local markets by small producers, consumers’ quality
standards are much lower, and tubers are less uniform and in poorer
shape. In some parts of the developing world, potato is produced
from old varieties and landraces or in Latin America from other
cultivated Solanum species, resulting in very ununiform, but some-
times very tasty, tubers. In some tropical countries, true potato seed
Importance of Potato as a Crop and Practical Approaches to Potato Breeding 9

is often used for production of ware potatoes, but the crop quality
standards fall far from those in developed countries.
Potato production for processing occurs mainly in developed
countries and increasingly in some countries of Asia and Latin
America. It is based on special-purpose varieties grown by large
specialized producers, with up-to-date production and storage
technologies and facilities. Most of the production is contracted.
Processing facilities for french fries and chips are concentrated in
the main potato production areas. During the last 40 years signifi-
cant expansion occurred not only in the global market of chips, but
also for french fries and other processed potato products [17].
Production for animal feed is still important in some countries
in Eastern Europe and Russian Federation, and other countries of
former Soviet Union region. Potato domestic production for
industrial uses is also of local importance, e.g., starch and its deri-
vates (the Netherlands and Japan), industrial alcohol (Poland and
Denmark), or vodka (countries of the former Soviet Union region).
Commercial production of seed potato is organized in many
countries across the world, but it is of major importance in specific
regions, where disease and pest pressure are low [18].
Potato provides significant amounts of carbohydrates, proteins,
vitamin C, and iron. Their content varies among cultivars. Carbo-
hydrates, which constitute about 75% of the total dry matter, are
the main energy source. Average consumption of 240 g of potato
per day provides around 5% of daily energy requirements. It is also
an important source of dietary fibre, contributing up to 15% of
daily required intake. Protein content estimates range between 1.6
to 2.1 g per 100 g of fresh weight. Potatoes are a very good source
of lysine, but with lower concentrations of the sulfur-containing
amino acids (methionine, cysteine). Although potatoes are not
usually regarded as a protein source because of low protein content
on a fresh weight basis compared to other foods, they can make a
significant nutritional contribution to the diet in countries with
high potato consumption per capita. In mixed diets, an important
role of potatoes is supplementing foods low in lysine, e.g., rice and
pasta [19].
Vitamin C is the main vitamin found in potatoes, its content
ranging between 15 and 25 mg per 100 g of fresh weight, con-
tributing up to 30% of daily required intake. It is present in both the
reduced state (ascorbic acid) which predominates in the tuber
(85–100%) and the oxidized state (dehydroascorbic acid); the two
forms are readily interchangeable. Vitamin C losses which occur
during cooking and processing can be considerable: unpeeled-
steamed 10–15%, boiled and baked 20%, peeled-steamed 10–30%,
pressure cooked 25%, microwaved 25%, roasted 20–45%, chipped
35–50%, and instant powder or flake 70%. Although the losses from
processing can be large, addition of ascorbic acid by manufacturers
can give high vitamin C content in the final product [19].
10 Peter Dolničar

4 Potato Varieties

More than 10,000 potato varieties have been grown worldwide to

date, many of which are still grown. In Latin America alone, the
International Potato Centre (CIP [20]) collected 17,326 acces-
sions of local varieties in the last 45 years. After the elimination of
duplicates, the active cultivated potato collection now totals 4727
accessions including 4354 traditional landrace cultivars from
17 countries (mainly from the Andean region) and improved vari-
eties. These varieties were bred over centuries of cultivation by
selecting natural diversity. The European Cultivated Potato Data-
base [21] consists of 4174 varieties and 1513 potato breeding lines
collected in gene banks and collections from 13 participating
countries, while the World Potato Atlas [22] describes more than
4000 varieties from more than a hundred countries around the
world. The common catalog of crop varieties in the EU in 2020,
which is the basis for cultivation in the EU, includes 1774 varieties
of potatoes [23].
Despite such a large number of varieties, there is still a need for
new varieties. In developing countries of Asia and Africa, where
potato production is on the rise, new varieties must provide higher
and stable yields with low inputs, be resistant to diseases and pests,
and be tolerant to stresses such as heat, frost, drought, and salinity.
They should also have improved nutritional properties. On the
other hand, the developed markets of the Western world, such as
the European Union, want economically efficient and environmen-
tally friendly production, with better market efficiency and more
efficient use of water and nutrients. They must also meet customer
requirements for improved nutritional value, taste, as well as devel-
opment of new processed products [24].

5 Practical Approaches to Potato Breeding

5.1 Classical Potato Principles of classical breeding of new potato varieties have changed
Breeding Programs little in many programs around the world in recent decades and
differ mainly in terms of scope and technologies used. Until the
turn of the millennium, classical breeding was based primarily on
empirical experience and selection of individual phenotypic traits.
This was shown in a number of similarly designed breeding pro-
grams in Canada [25], England [26], Germany [27], Ireland [28],
and the Netherlands [29]. Breeding programs are based on the
selection of parents with appropriate highly heritable phenotypic
traits, so they can be effectively transmitted to offspring. Over time,
breeders determine the individual parental clones or varieties with
good combining ability and use them in crossings. Desired traits
can range from morphological properties, economic value, as well
 Importance of Potato as a Crop and Practical Approaches to Potato Breeding 11

as resistance to diseases and pests. Not a simple task, particularly

when considering the influence of the tetraploidy of potato.
In a classical breeding program crossings usually take place in a
greenhouse, with different programs using different techniques to
promote flowering and berry formation. In Europe, the most
common method is planting on a brick, but there are other meth-
ods such as grafting on tomatoes, cultivation, and crossbreeding on
stems grown in aqueous solution. The key in all methods is to
prevent the outflow of assimilates in the absence of tubers, which
are therefore only available for the growth of stems, flowers, and
berries. The strong sink of assimilates in the tubers is the main cause
of flower loss, which is not what we want when breeding. All
breeders also strive to ensure optimal conditions for flowering and
fertilization, i.e., temperatures below 22  C with sufficiently high
humidity. The year after the crossings is followed by sowing approx.
100,000 or more plants (seedlings) in pots in a greenhouse, and the
cultivation of tubers for further selection in the field [30]. The
number of plants varies between programs, and most major com-
mercial programs still sow about the same number of plants today,
which realistically allows them to grow one new variety per year.
Programs with 5000 to 20,000 plants per year are considered small;
for these we can expect the cultivation of a new variety every few
years.
Up to 10 years after the selection process started, progenies are
grown on the field, with application of different selection strategies
and methods depending on the goals of the breeding program. In
general, progeny number decreases over the years, until the num-
ber of plants per progeny increases to a certain point, when the
most advanced genotypes are sent to the registration trials.

5.2 Combining Although we often do not know individual genes and instead infer
Ability the genotype of parental clones on the basis of phenotype, we can
estimate the probability that those clones will pass on their traits to
offspring, which is reflected in their general and specific combina-
tion ability. The general combining ability (GCA) represents the
average of a clone (line, variety) when crossed with various other
clones (lines, varieties) and is calculated as the average of all
offsprings that have this clone as one of the parental components
in the cross. It is usually expressed as the deviation from the average
of all crosses in testing, with clones of interest to be used for testing,
or they can be randomly selected [31]. Two types of crossover
factor schemes are usually used to calculate it: in the first one the
set of clones is crossed with another for a certain property of
complementary sets, and in the second with a diallel set of crosses
between clones to get the whole spectrum of values for each prop-
erty. Where it is possible, reciprocal crossings make sense [32].
The assessment of specific combining ability (SCA) is obtained
by determining the deviation of the actual mean offspring values
12 Peter Dolničar

from the expected GCA value. Where we find that the overall
variability between progeny of different crosses is due to SCA,
progeny performance cannot be predicted for any combination of
crosses [31]. GCA and SCA typically contribute different percen-
tages of variability. Neele et al. [33] found that in breeding schemes
where parental clones are closely related SCA appears to be more
important than GCA. Due to the presence of male sterility, e.g., in
extremely resistant varieties to potato virus Y, combining ability
tests (GCA and SCA) cannot be performed as previously described.

5.3 Selection When growing seedlings in the greenhouse in the first clonal year,
in Breeding Programs one tuber per clone is usually selected for planting the following
year in the field. In all breeding programs, the first year of clonal
selection in the field is followed by selection in subsequent years,
with the number of plants planted per clone increasing over the
years and the number of selected clones decreasing. In the second
year of clonal selection, 3–6 plants per clone are usually selected
(depending on the program). In some programs they already start
planting clones in replicates in the second year, and also separate
seed production. In subsequent years, the number of plants per
clone increases to 6–10, and then 20, 100, 300, 700, and 2000 or
more in the tenth year. The relatively small multiplication factor
(approx. 8) of potatoes in clonal propagation prevents the simulta-
neous evaluation of many different traits. Therefore, we need at
least five or more additional years until registration attempts
begin [31].
In the program of the former Scottish Crop Research Institute
(SCRI)—today the James Hutton Institute (JHI)—they reduced
from 100,000 in the seedling stage to 40,000 plants in the first
clonal field year, to 4000 plants selected and planted for the second
clonal year (thus eliminating 96% of the original genetic variability)
[30]. In the previously listed programs of other countries, the
percentage of plants isolated in the first clonal generation was
between 85% and 98%. No greenhouse selections were performed
in the Canadian program [25]. Up to 99% of genetic variability was
eliminated in the field at SCRI by the second clonal year. Research
has shown that this type of selection is very inefficient [25, 30].

5.4 Efficiency The greatest genetic variability in breeding programs is available in

of Selection the early generations, at the seedling stage in the greenhouse and in
of Agronomic Traits the first and second clonal years in the field. Increasing the effi-
at an Early Stage ciency of selection at this stage would mean a significant improve-
ment in the efficiency of the breeding program in general. Brown
et al. [34] compared the offspring of randomly selected 571 geno-
types from eight crosses by simulating greenhouse seedling selec-
tion and the first clonal year in the field and compared them to
selection in the second year in the field, noting the loss of signifi-
cantly better genotypes. Compared to the selection in the second
 Importance of Potato as a Crop and Practical Approaches to Potato Breeding 13

clonal year, only 27% of the better genotypes would be successfully

selected in the greenhouse, and if we take into account the first
clonal year, 56% of the genotypes were selected. It was shown that
selection in the first 2 years in the commercial SCRI program was
not more effective than random selection. On the other hand,
research has shown that average estimated values of individual
crossings do not change significantly over the years or due to
other influences [34]. Therefore, they suggested that combinations
with a higher proportion of potential new varieties could be identi-
fied with the help of the breeder’s average visual assessment of the
offspring. This theory was confirmed by research on a further
52 offspring of crosses [35]. Theoretically, it would be optimal if
the selection could equally take into account the average breeder’s
assessment of the offspring and individual visual phenotypic assess-
ment of individual genotypes, but this would be too complicated
for a rapid visual breeder’s assessment. The average breeder’s esti-
mate of the offspring for the elimination of less successful crosses
could be used as early as in the first year when growing seedlings in
a greenhouse. In this case, a mild selection of clones could be
performed in the first clonal year, and the real selection due to the
strong interaction between genotype and early maturity at physio-
logical maturity in a food experiment in the second clone year in the
field [36]. Therefore medium-strict selection over several years is
recommended. Neele and co-workers [37] confirmed this on ran-
domly selected 600 clones from 20 different combinations. They
found that the most economical selection would be about
one-third (20–50%) of clones in the first clonal generation, fol-
lowed by selection of about 20% of genotypes in the second clonal
year, which would lead to a higher share of late-maturing clones
with more dryness in a mild selection in Dutch conditions
[29]. They also confirmed that the selection of individual proper-
ties that make up the so-called breeder’s visual preference is no
more effective than common breeder’s visual preferences (read
their common general assessments—plant appearance) [33, 38].

5.5 Efficacy An early-stage selection can be effective against some diseases and
Selections Against pests, especially if the resistance is caused by the major dominant
Diseases and Pests genes [39, 40]. Thus, according to de Bokx [41], in the case of
at an Early Stage potato virus Y (PVY), with the introduction of extreme resistance, a
mass method of early elimination of susceptible genotypes can be
introduced simply by spraying offspring with PVY in the cotyledon
phase and then eliminating genotypes with mosaic signs within
2 weeks. In this case, when selecting only resistant genotypes, we
consciously exclude a large proportion of genetic variability.

5.6 Progeny Testing Based on the above facts, in the late 1980s SCRI introduced
in Breeding Programs prognostic testing for quantitative resistance to diseases and pests
(potato cyst nematodes, common scab, powdery scab, and dry rot)
14 Peter Dolničar

in the breeding program. In practice, it is sufficient to estimate

80 genotypes per cross-combination in 4 replicates, and even
40 or 20 for a larger number of combinations [30]. For example
in 2014 the James Hutton Institute used progeny testing of com-
binations of crosses with an assessment of 30 genotypes in two
replicates. The average breeder’s score of offspring was calculated
by evaluating all genotypes of the combination with a total score of
overall impression and appearance, uniformity, and yield from 1 to
5, and then calculating the average score. In addition, the number
of above-average genotypes that the breeder would select and the
number of genotypes with a long shape (if the crosses were
intended for the preparation of fried potatoes) were monitored.

5.7 Selection When designing a potato breeding program, we need to decide

of Qualitative what the breeding goals will be. If we wanted to take into account
and Quantitative Traits all 50 traits that are important in potatoes and thus combine them
in the Breeding into one optimal genotype—a new variety, according to some
Program calculations—we would need 1,000,000 seedlings [18]. If we take
into account that larger programs usually reach up to 100,000
seedlings per year, it is clear that we also have to accept many
compromises and focus only on the most important traits for
us. This is even more important for smaller breeding programs.
The most important characteristics and traits that a modern breed-
ing program can take into account are [31, 42]:
l Yield
l Quality of tubers for cooking and processing:
– Tuber defects: cracking of tubers, hollow heart, brown heart,
brown spotting, regrowth
– Damage resistance
– Glycoalkaloid content
– Sensitivity to greening
l Edible quality and dietary value for cooking, baking, frying,
starch, and other potato products: dry matter content, texture,
taste, protein content, vitamins, after-cooking darkening, enzy-
matic darkening, sugar content
l Biotic stress: resistance to nematodes, virus diseases, late blight
and other fungal diseases, bacterial diseases, resistances to other
pests and diseases
l Properties associated with abiotic stress: resistance to high tem-
peratures or heat stress, resistance to water stress or resistance to
drought conditions, resistance to cold stress (frost tolerance,
sweetening of tubers during cold storage)
 Importance of Potato as a Crop and Practical Approaches to Potato Breeding 15

5.8 Selection Selection procedures include several parallel methods that depend
Schemes in Potato on the breeder’s objectives:
Breeding Programs l Selection of qualitatively inherited traits (tuber shape, eye depth,
skin and flesh color, stolon length, habitus, . . .) in the first and
the second clonal years in the field
l Selection of quantitatively inherited traits (yield, number of
tubers, quality, . . .), in later years of the selection in the field,
very often in replicated trials
l Determination of resistance to various defects and diseases and
pests depends on the studied properties and methods used and
takes place from the first year in the greenhouse and in the field
until final selection [31]
Finally, advanced clones which pass the selection during several
years must pass the trials for value of cultivation and use (VCU) and
distinctness, uniformity, and stability (DUS) testing, in order to be
registered as a new variety in the EU database of registered plant
varieties.
The success of the potato breeding program depends on the
successful introduction of resistant genes, the expression of other
agronomic and morphological characteristics of different sources of
resistance in the offspring in local growing conditions, their suit-
ability for genetic recombination or interspecific crosses (male ste-
rility, endosperm balance number ¼ EBN), their general and
specific combination ability, and many other factors.
Many agronomic and morphological traits are less pronounced
in resistant genotypes due to the introduction of resistant genes
from potato-related species. Despite repeated backcrosses and
selections, many resistant genotypes still carry many undesirable
traits such as poorer morphological properties (regularity of shape,
eye depth, stolon length, . . .), poorer edible quality, lower yield,
and often also late maturity when carrying R genes responsible for
late blight resistance. This is expressed both in genes from the
species Solanum demissum [43] and in R8 genes originating from
the Solanum tuberosum Sarpo Mira variety. Therefore, the path to a
successful introduction of resistance is often not easy, as it is neces-
sary to simultaneously remove the unwanted properties of the
parent plants. The effectiveness of the selection depends on the
combination ability of the parents. Both specific and general com-
bination abilities of the selected parents can be determined in the
first years of selection from the seedling phase to the third clonal
generation (second clonal year in the field), with the exception of
the yield and its characteristics, where this is possible only from the
second clonal generation [44].

In potatoes, several cases are known where resistance to important

diseases and pests is based on dominant genes with a strong effect,
16 Peter Dolničar

5.9 Dominant Genes which are inherited according to Mendel’s rules. We call them
with Strong Effect R genes. Among them are, e.g., H1 for resistance to Ro1 to Ro4
(R Genes) pathotypes of yellow potato cyst nematode (Globodera rostochien-
sis), many R genes acting against different races of late blight
(Phytophthora infestans (Mont.) de Bary), and of course genes
carrying race-specific and general virus resistance against PVY and
PVX viruses, e.g., Nx, Ny and Rxadg, Rysto [30].
Due to the nature of the host/pathogen relationship, R genes
for resistance to viral diseases are very efficient and stable, as there
have been no cases of breakdown of their resistance in potatoes, and
at least in the near future this is not expected. The likelihood of
such an aggressive variation occurring is also small, because there is
no selection pressure in susceptible hosts toward the emergence of
resistant mutants [31].
In contrast, late blight and its ability to adapt and develop
R gene-resistant pathotypes have discouraged many breeders
from using R genes in the past [45]. The use of a single R gene
alone cannot provide long-term resistance to such a rapidly chang-
ing pathogen as late blight [46]. Only the accumulation of R genes
resistant to late blight, possibly even in combination with QTL, can
yield a more lasting resistance.

5.10 Efficiency The probability of inheriting monogenic dominant resistance

of Selection (R genes) if one parent has a single copy (simplex) of the dominant
and Pyramiding allele in tetraploid potatoes is 0.5. If both parents have a single copy
of R Genes of the dominant allele, the probability of inheritance is already 0.75.
If one of the parents has a double copy of the same allele (duplex)
and the other is sensitive, the probability of inheriting resistance is
0.83, and with a triple copy of the same allele (triplex), all offspring
are resistant. The same ratio applies to two R genes at different loci
[32]. In practice, these relationships deviate from theoretical values
due to chromatid segregation for various reasons [47]. Therefore,
for effective selection, it would be best for the selected parents to
possess resistant alleles in as many copies as possible (two—duplex,
three—triplex, four—quadruplex). When selecting resistant geno-
types, we exclude about half of the genotypes from the outset,
regardless of other possible good traits they have; thus the efficiency
of selection is automatically 50% lower. Therefore, a selection pro-
gram must be adapted to the accumulation of genes for a particular
trait, which must distinguish not only between susceptible and
resistant genotypes, but also between genotypes with one resistant
dominant gene and genotypes with several resistant genes [48]. To
avoid time-consuming further crosses to determine the number of
copies of genes in the offspring, the use of molecular markers is
most optimal.
A program to breed breeding lines with multiplex resistance in
the Ryadg gene was carried out at the International Potato Center
(CIP) in Peru. Two duplex genotypes (YYyy  YYyy) were used for
Importance of Potato as a Crop and Practical Approaches to Potato Breeding 17

crosses and the resulting 182 clones were bred by crossing with a
sensitive tester (yyyy). Analysis of the ratio of resistant and suscep-
tible genotypes of crossbreeds taking into account the random
chromatid segregation model showed that from 182 crosses, two
clones possessed triplex resistance to PVY (YYYy), while no geno-
types with quadruplex resistance were detected [49].
Probably the most effective example of R gene accumulation in
potatoes took place during the early 1980s in Hungary, where
many sources of resistance and tolerance to late blight were col-
lected. Through numerous crossbreeds, they achieved the greatest
possible diversity of offspring, systematically infecting them with a
mixture of potato late blight pathotypes. Once 95% of the plants
perished, the remainder were sprayed with fungicides and allowed
to mature. In further crosses, only the tubers of the surviving plants
were used. In the early 1980s, the program was halted. Part of the
genetic material remained in Hungary at the University of
Keszthely [50], and part was transferred to Denmark after a short
period in Romania, where the Danish company Danespo developed
the Sarpo varieties, including the “Sarpo Mira,” which is now
considered as one of the most resistant varieties to late blight
carrying R8 gene [51]. The variety is also extremely resistant to
PVY (Rychc gene) [50].
In an attempt to accumulate genes for resistance to late blight
in Poland, the offspring of 83 potato clones from crosses between
the “Sarpo Mira” variety and donors of the Rpi-phu1 gene from
Solanum phureja were classified into four groups using molecular
markers and the stand-alone method: without R genes (N ¼ 26),
resistant R8 “Sarpo Mira” genes (N ¼ 19), group with Rpi-phu1
(N ¼ 28), and group of clones with “Sarpo Mira” R8 gene and Rpi-
phu1 gene (N ¼ 10). The highest average resistance of clones to late
blight was found in the last two groups, and it is expected that the
resistance of clones of the last group will be more permanent
[52]. Witworth et al. [53] successfully pooled R genes for resis-
tance to late blight from Solanum demissum and Solanum stoloni-
ferum. Up to 64% of the offspring were resistant when crossing two
resistant parents, compared to 29% of the resistant offspring when
crossing resistant and sensitive parent.
Accumulation of R resistance genes is not necessarily always
effective. Tan and colleagues [54] found that the accumulation of
two known genes for resistance to northern root-knot nematodes
(Meloidogyne hapla) did not result in greater resistance. The pres-
ence of the RMh-chcA gene from Solanum chacoense reduced the
egg number by 88% and the RMh-tar gene from Solanum tariense
gene by 55%, while the combined presence of both genes did not
cause a further reduction in the number of eggs compared to the
RMh-chcA gene.
Similarly, the accumulation of polygenic resistance by weak-
effect genes was not effective. At JKI Gross Lusewitz, Darsow
18 Peter Dolničar

(2014) successfully bred clones with resistance to potato late blight

in a diploid and tetraploid pre-breeding program. In contrast to
current research on the association of polygenic resistance to potato
blight on leaves with late maturity, as many as 6% of the clones were
very early, 20% early, and 31% medium early—altogether more than
half, due to properly targeted selection. Since 1994, as many as
69 resistant parent clones have been handed over to German bree-
ders [55]. Unfortunately, the offspring of crosses between resistant
parents and susceptible breeding populations were not as resistant
as the parents, due to the dilution effect of accumulated genes with
weak effects.

5.11 The Use The use of molecular markers in potato breeding is a reality today
of Molecular Markers and is a part of any modern potato breeding program. Especially
in Potato Breeding after the “exposure” of the entire potato genome, the identification
of genes responsible for the expression of studied traits and the
allelic variability of those genes, reflected in phenotypic variability
of studied traits, is a major prerequisite for promoting a biotech-
based variety improvement. So far, there have been many successful
cases in monogenic hereditary traits (mostly in resistance to patho-
gens), while less progress has been made in identifying genes and
alleles of quantitative inherited traits [56]. Already in 2008 [57],
25 single dominant genes were localized in potatoes, of which
20 R genes were localized on all 12 chromosomes along with
more than 350 markers, evenly distributed so they covered 90%
of the potato genome. PCR markers for the Ryadg, Rychc, Rysto, Rx1,
Rx2, GroVI, Gro1, Gpa2, H1, R1, R3a, and Rpi-blb genes [57]
were available for marker-based selection. Until today, many novel
genes and corresponding molecular markers have been discovered.
In 2015, Ramakrishnan and co-workers [58] listed as many as
80 potential markers for selection, out of which 24 for resistance
against viral diseases, 24 for potato late blight, 24 for nematodes,
5 for bacterial blackleg, and 1 for potato wart disease.
Molecular research has made it possible to understand the
taxonomic links between potatoes in its wild relatives. The great
genetic diversity that exists in the potato and its wild relatives
represents both an opportunity and a challenge to introduce cur-
rently nonexistent traits into modern potato varieties. The devel-
opment of molecular marker technology has reached points where
genotyping of the entire genome is possible in many laboratories,
and already published markers for use in commercial breeding are
also available. Markers can be used during the whole selection
process, and even more important is the role of molecular breeding
in pre-breeding programs and creating the most appropriate paren-
tal lines [59].
 Importance of Potato as a Crop and Practical Approaches to Potato Breeding
References
1. Hawkes JG (1994) Origin of cultivated pota-
toes and species relationships. In: Bradshaw JE,
Mackay GR (eds) The potato genetics. CAB
International, Wallingford, pp 3–42
2. Huaman Z, Spooner D (2002) Reclassification
of landrace populations of cultivated potatoes
(Solanum sect. Petota). Am J Bot 89:947
3. van den Berg RG, Jacobs MMJ (2007) Molec-
ular taxonomy. In: Vreugdenhil D, Bradshaw J,
Gebhart C, Govers F, MacKerron DKL,
Taylor A, Ross HA (eds) Potato biology and
biotechnology, Advances and perspectives.
Elsevier, Amsterdam, pp 55–76
4. Hawkes JG (1990) The potato, evolution, bio-
diversity and genetic resources. Belhaven Press,
London
5. Urgent D et al (1982) Archeological potato
tuber remains from the Casma valley of Peru.
Econ Bot 36:181–192
6. Urgent D (1970) Solanum raphanifolium, a
Peruvian wild potato species of hybrid origin.
Botanica Gazette 131:225–233
7. Grun P (1990) The evolution of cultivated
potatoes. Econ Bot 44(Suppl. 3):39–55
8. Hosaka K (1995) Successive domestication and
evolution of the Andean potatoes as revealed
by chloroplast DNA restriction endonucleases.
Theor Appl Genet 90:356–363
9. Spooner D et al (2005) A single domestication
for potato based on multilocus amplified frag-
ment length polymorphism genotyping. Proc
Natl Acad Sci U S A 102:14694–14699
10. Braaden JM, Haynes KG (2011) Introduction
to potato. In: Braaden JM, Kole C (eds) Genet-
ics, genomics and breeding of potato. CRC
Press, Enfield, NH, pp 1–19
11. Hawkes JG, Francisco-Ortega J (1992) The
potato in Spain during the late 16th century.
Econ Bot 46:89–97
12. https://www.history.com/topics/immigra
tion/irish-potato-famine. Accessed 21 Sept
2020
13. Zadoks JC (2008) The potato murrain on the
European continent and the revolution of
1848. Potato Res 51:5–45
14. Burton WG (1989) The potato, 3rd edn.
Longman Scientific and Technical, Harlow,
p 742
15. FAO. http://www.fao.org/faostat/en/#data/
QV. Accessed 21 Sept 2020
16. FAO. http://www.fao.org/faostat/en/#data/
QC. Accessed 21 Sept 2020
17. Kirkman M (2007) Global markets for pro-
cessed potato products. In: Vreugdenhil D,
19
Bradshaw J, Gebhart C, Govers F, MacKerron
DKL, Taylor A, Ross HA (eds) Potato biology
and biotechnology, Advances and perspectives.
Elsevier, Amsterdam, pp 27–44
18. Struik PC, Wiersema SG (1999) Seed potato
technology, vol 2. Wageningen Pers, Wagenin-
gen, p 383
19. Storey RMJ, Davies HV (1992) Tuber quality.
In: Harris P (ed) The potato crop. Chapman &
Hall, London, pp 507–569
20. CIP. https://cipotato.org/genebankcip/
potato-cultivated/. Accessed 21 Sept 2020
21. European Cultivated Potato Database.
https://www.europotato.org/. Accessed
21 Sept 2020
22. Hills U, Pieterse L (2009) World catalogue of
potato varieties. Agrimedia GmbH, Bergen/
Dumme, p 326
23. https://ec.europa.eu/food/plant/plant_prop
agation_material/plant_variety_catalogues_
databases/. Accessed 21 Sept 2020
24. Bradshaw JE (2007) Potato-breeding strategy.
In: Vreugdenhil D, Bradshaw J, Gebhart C,
Govers F, MacKerron DKL, Taylor A, Ross
HA (eds) Potato biology and biotechnology,
Advances and perspectives. Elsevier, Amster-
dam, pp 157–177
25. Tarn TR et al (1992) Breeding potatoes for
long-day, temperate climates. In: Janick J
(ed) Plant breeding reviews, vol 9. John Wiley
& Sons, New York, pp 217–332
26. Howard HW (1978) The production of new
varieties. In: Harris PM (ed) The potato crop,
1st edn. Chapman & Hall, London, pp
607–646
27. Ross H (1986) Potato breeding—problems
and perspectives. Advances in plant breeding,
series no. 13, Berlin and Hamburg
28. Kehoe HW (1982) Potato breeding. An Foras
Talun research report 1982, pp 42–48
29. Neele AEF et al (1988) Effect of plot size and
selection intensity on efficacy of selection in the
first clonal generation of potato. Euphytica
Suppl 39:27–35
30. Caligari PDS (1992) Breeding new varieties.
In: Harris PM (ed) The potato crop, 2nd edn.
Chapman & Hall, London, pp 395–401
31. Bradshaw JE, MacKay GR (1994) Breeding
strategies for clonally propagated potatoes. In:
Bradshaw JE, Mackay GR (eds) The potato
genetics. CAB International, Wallingford, pp
467–497
32. Bradshaw JE (1994) Quantitative genetics the-
ory for tetrasomic inheritance. In: Bradshaw
20 Peter Dolničar
JE, Mackay GR (eds) The potato genetics.
CAB International, Wallingford, pp 71–100
33. Neele AEF et al (1991) Identification of supe-
rior parents in a potato breeding programme.
Theor Appl Genet 82:264–272
34. Brown J et al (1987) The repeatability of prog-
eny means in the early generations of potato
breeding programme. Ann Appl Biol
110:365–370
35. Brown J et al (1988) The use of cross predic-
tion methods in a practical potato breeding
programme. Theor Appl Genet 76:33–38
36. Brown J, Caligari PDS (1999) Cross prediction
in a potato breeding programme by evaluation
of parental material. Theor Appl Genet
77:246–252
37. Neele AEF et al (1989) Optimising visual selec-
tion in early clonal generation of potato based
on genetic and economic considerations.
Theor Appl Genet 78:665–671
38. Neele AEF et al (1991) Components of visual
selection in early clonal generation of a potato
breeding programme. Plant Breed 106:89–98
39. Jellis GJ (1992) Multiple resistance to diseases
and pests in potatoes. Euphytica 63:51–58
40. Plaisted RL, Thurston HD, Brodie BB,
Hoopes RW (1984) Selecting for resistance to
diseases in early generations. Am Potato J
61:395–399
41. de Bokx JA (1972) Graft and mechanical trans-
mission. In: de Bokx JA (ed) Viruses of pota-
toes and seed-potato production. PUDOC,
Wageningen, pp 26–35
42. Carputo D, Frusciante L (2011) Classical
genetics and traditional breeding. In: Bradeen
JM, Kole C (eds) Genetics, genomics and
breeding of potato. CRC Press, Science Pub-
lishers, Enfield, NH, pp 20–40
43. Simko I et al (2007) Genetics of resistance to
pests and disease. In: Vreugdenhil D,
Bradshaw J, Gebhard C, Govers F, MacKerron
DKL, Taylor MA, Ross HA (eds) Potato biol-
ogy and biotechnology, Advances and perspec-
tives. Elsevier, Amsterdam, pp 117–156
44. Kumar R, Gopal J (2006) Repeatability of
progeny mean, combining ability, heterosis
and heterobeltiosis in early generations of a
potato breeding programme. Potato Res 49
(2):131–141
45. Wastie RL (1991) Breeding for resistance. In:
Ingram DS, Williams PH (eds) Phytophthora
infestans, the cause of late blight of potato,
Advances in plant pathology, vol 7. Academic
Press, London, pp 193–224
46. Tiwari JK et al (2013) Molecular markers for
late blight resistance breeding of potato: an
update. Plant Breed 132:237–245
47. Solomon-Blackburn RM, Bradshaw JE (2007)
Resistance to potato virus Y in a multitrait
potato breeding scheme without direct selec-
tion in each generation. Potato Res 50
(1):87–95
48. Swieżyński KM (1994) Inheritance of resis-
tance to viruses. In: Bradshaw JE, Mackay GR
(eds) The potato genetics. CAB International,
Wallingford, pp 339–364
49. Mendoza HA et al (1996) Identification of
triplex (YYYy) potato virus Y (PVY) immune
progenitors derived from Solanum tuberosum
spp. andigena. Am Potato J 73:13–19
50. Polgar Z (2009) Personal communication.
University of Weszprem, Keszthely
51. VIB (Vlaams Instituut voor Biotechnologie)
(2013). http://www.vib.be/en/news/
Documents/VIBdossierPipotatoes_ENG.pdf
52. Tomczyńska I et al (2014) A locus conferring
effective late blight resistance in potato cultivar
Sárpo Mira maps to chromosome XI. Theor
Appl Genet 127:647–657
53. Witworth JL et al (2007) Multiple-site identi-
fication of potato parent clones conferring high
levels of late blight resistance with a
corresponding genetic model for resistance.
Am J Potato Res 84:313–321
54. Tan MYA et al (2009) Pyramiding of Meloido-
gyne hapla resistance genes in potato does not
result in an increase of resistance. Potato Res
52:331–340
55. Darsow U (2014) Pre-breeding and breeding
of potatoes for quantitative resistance to Phy-
tophthora infestans on foliage and tubers and
for different utilization. Problems, solutions
and results. Julius Kühn-Institut, Quedlinburg,
p 312
56. Visser RGF et al (2009) Sequencing the potato
genome: outline and first results to come from
the elucidation of the sequence of the World’s
third most important food crop. Am J Potato
Res 86:417–429
57. Song YS, Schwarzfischer A (2008) Develop-
ment of STS markers for selection of extreme
resistance (Rysto) to PVY and maternal pedi-
gree analysis of extremely resistant cultivars.
Am J Potato Res 85:159–170
58. Ramakrishnan AP et al (2015) Review of
potato molecular markers, to enhance trait
selection. Am J Potato Res 92:455–472
59. De Koeyer D et al (2011) Molecular breeding,
for potato improvement. In: Bradeen JM, Kole
C (eds) Genetics, genomics and breeding of
potato. CRC Press, Science Publishers, Enfield,
NH, pp 41–67
 Chapter 2

Cryopreservation of Potato Shoot Tips for Long-Term

Storage
Rainer Vollmer, Janeth Espirilla, Rosalva Villagaray, José Cárdenas,
Mario Castro, Juan Carlos Sánchez, Norma Manrique-Carpintero,
David Ellis, and Noelle Lynette Anglin

Abstract
Cryopreservation is currently the only method which allows long-term conservation of living clonal plant
material in the vapor or liquid phase of nitrogen (at
140 to
196  C) allowing tissue to be viable for
decades or perhaps centuries. Specifically, for species with recalcitrant seeds or requiring constant vegetative
propagation, it is the method of choice for the long-term conservation of its genetic resources. The
protocol described here is a modification of a previously developed plant vitrification solution
2 (PVS2)—droplet vitrification method of potato shoot tips, adapted from Musa species. Utilizing this
protocol, the International Potato Center (CIP) has successfully stored in the cryobank more than 3000
cultivated potato accessions, belonging to seven species and nine different taxa [16], originating principally
from ten countries in South and Central America. As part of CIP’s quality management system, all
vegetative material placed in cryo is routinely subsampled, thawed, and assessed to confirm that whole
plantlets can be produced after storage in liquid nitrogen. Complete plant recovery rates of thawed shoot
tips range from 20% to 100% (average rate: 60%). This chapter describes the complete set of steps from the
routine procedure of cryopreserving potato shoot tips for long-term conservation.

Key words Cryopreservation, Potato (Solanum species), PVS2, Droplet vitrification, Cryobank

1 Introduction

The secure and efficient conservation of genetic resources of impor-

tant staple crops such as potato, before they are lost in the natural
environment, is essential to ensure the welfare of future genera-
tions, especially in times of climate change and lack of food security
in many parts of the developing world. Conservation of genetic
resources allows the development of new varieties that can resist
environmental challenges and provide better nutrition for human-
ity, but this can only occur through preservation of diversity and
subsequent efforts in breeding and selection. Cryopreservation
offers the best available option for the secure, space-efficient, and

David Dobnik et al. (eds.), Solanum tuberosum: Methods and Protocols, Methods in Molecular Biology, vol. 2354,
https://doi.org/10.1007/978-1-0716-1609-3_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

21
22 Rainer Vollmer et al.

low-cost conservation of clonally propagated species for the long

term (decades to centuries), without the need of subsequent sub-
culturing or renewal of the plants. Although the introduction of a
new accession to the cryobank is expensive ($400–500 USD/ac-
cession), maintaining it in the cryobank is not expensive ($2 USD/
accession/year), compared to a yearly cost of $50–60 USD for
conserving an accession in the in vitro bank. After a conservation
period of ~8–10 years, cryobank storage becomes cheaper than
in vitro banking.
Different cryopreservation methods have been developed for
many plant species using desiccation, slow cooling, vitrification,
encapsulation-dehydration, encapsulation-vitrification, droplet
vitrification, and V- and D-plate [1–3]. All of these protocols strive
to avoid the formation of large intracellular ice crystals during
freezing and thawing as it is harmful to the cell membranes and
generally leads to cell death (apoptosis). Ice crystallization can only
be avoided when a glassy state is reached on the intracellular level at
freezing, mainly by increasing the viscosity to a point that the
system behaves like it is in a solid state (called glassy state or
vitrification) [4]. Plant tissue is generally treated with a mix of
cryoprotectants, e.g., glycerol, dimethyl sulfoxide (DMSO), and
ethylene glycol, to achieve vitrification. The most famous and
frequently used cryoprotectant solution for plant genetic resources
is called plant vitrification solution 2 (PVS2). It was developed
nearly three decades ago for nucellar cells of navel orange by a
Japanese research group [5]. A few years later, a German group
modified the droplet vitrification method, originally developed for
cassava, as an ultrarapid cooling method for potato [6–8]. The
droplet method uses a small strip of aluminum foil with a droplet
of cryoprotectant. The samples (generally, shoot tips of a size of
0.8–2.0 mm) are placed into a droplet of cryoprotectant and then
quickly plunged into liquid nitrogen. Initially DMSO was used as a
cryoprotectant, but later it was broadly replaced by PVS2 and its
application led to a breakthrough of successful cryopreservation of
many crops [2, 9, 10].
The International Potato Center (CIP) adapted the PVS2-
droplet vitrification method in 2004 for potato and has continu-
ously improved its efficacy and efficiency over the last 15 years
[1, 11–15]. In general, the main steps in this protocol include
in vitro propagation, pre-culture, shoot-tip excision, cryoprotec-
tant treatment of shoot tips, freezing, thawing, and plant recovery
assessment in order to determine the success of each cryo-run
(Fig. 1). One single person can cryopreserve 300–450 potato
shoot tips during an 8-h working day (including shoot-tip excision,
cryoprotection, freezing in liquid nitrogen, and transitory storage).
CIP’s cryopreservation protocol is applicable to a wide range of
potato species and genotypes (~3250) [16] and shows a high
average rate of complete plant recovery (~60%) after liquid nitrogen
 a
In vitro propagation Cold acclimatization of
cycle (3.2.) Excision of shoot tips (3.4.)
shoot tip donor plants
(3.3.)

General considerations (3.1 .) Treatment with

Preparative cryoprotectant
steps (3.5.) solutions (LS and PVS2)
(3.6.)

Freezing in liquid
Assessment of Survival
Recovery Cycle Thawing of shoot nitrogen (LN) and
and Recovery rates
(3.9.) tips (3.8.) storage in laboratory
(3.1 0.)
tank (3.7.)

Accomplishes minimum viability and quality citeria ?

Final transfer to cryobank YES Transitory storage

and safety back-up tanks (3.1 1 ).
(3.1 3.) NO

Discarding of
Monitoring of LN level
accession (3.1 2.)
and filling of cryotanks
(3.1 4.)

Fig. 1 (a) Simplified workflow of the potato cryopreservation process applied at the International Potato Center (CIP). Corresponding method sections of each step
are specified in parentheses. Principal steps are connected with a continuous arrow line and peripheric steps with dotted arrow lines. (b) Simplified scheme of the
potato cryopreservation process applied at the International Potato Center (CIP). Corresponding method sections of each step are specified in parentheses.
Cryopreservation of Potato Shoot Tips for Long-Term Storage

Transfer events between tanks are presented with dotted arrow lines
23
 24

size: 0.8-1.2 mm
80-90 plants / dish
b

Cold acclimatization of Excision of shoot tips

In vitro
shoot tip donor plants 3.4.
propagation
cycle (3.2.) (3.3.) PVS2: 50 min
(on ice)
LS: 20 min
Rainer Vollmer et al.

10 shoot ps in a droplet of

Final transfer to PVS2 (20-25 ml) on a small
cryobank and aluminum foil strip. The strip
safety back-up has a fold of 2-3 mm to
tanks (3.13.) faciliate manipulaon by Treatment with
Tranistory storage forceps cryoprotectant
(3.13.) solutions (LS and PVS2)

60 days after
thawing
30 days after
thawing RS: 20 min

Recovery Cycle (3.9.)

and Assessment of Freezing in liquid nitrogen (LN)
Survival and Recovery and storage in laboratory tank
Thawing of shoot tips in
(3.7.)
Rewarming Solution (RS)

Fig. 1 (continued)
 Cryopreservation of Potato Shoot Tips for Long-Term Storage 25

exposure and rewarming. The number of shoot tips to be cryopre-

served per accession was determined with a binomial model that
uses as input variables the number of thawed explants (sample size),
the expected recovery rate, and the confidence level [17, 18].

2 General Considerations, Supplies, Materials, Culture Media, and Solutions

Required for the Cryopreservation Process

Different types of water purification are used in specific parts of this

protocol. In the case of culture medium used for in vitro propaga-
tion, cold acclimatization, shoot-tip excision, and post-thawing
recovery, all solutions are prepared with distilled deionized water.
Loading solution (LS), plant vitrification solution 2 (PVS2), and
rewarming solution (RS) are prepared with ultrapure water to avoid
the presence of ions during the cryoprotection or thawing steps,
which can be harmful to the samples. Analytical grade reagents and
calibrated/validated equipment should be used during all protocol
steps (e.g., pH meter, autoclave, laminar flow chamber, electronic
dispensing pump [Tritech Research Inc.]). Purchased pre-prepared
full-strength Murashige and Skoog medium (MS), including
macronutrients, micronutrients, and vitamins, is used for the prep-
aration of all culture media and solutions (see Note 1). Thermola-
bile compounds (e.g., coconut water) are filter-sterilized and added
after autoclaving solutions and allowing them to cool down
(35–45  C). Store chemicals at room temperature (22  3  C),
under refrigeration (5  3  C) or in the freezer (
15  5  C),
according to what is specified in the Material Safety Data Sheet
(MSDS) of each product. Reagents are dissolved using a magnetic
stirrer at 600–1000 rpm, depending on the volume and the viscos-
ity of media/solutions that are being prepared.

2.1 Media 1. Basic medium: 4.43 g/L of commercial Murashige and Skoog
Preparation (MS) salts with vitamins (Caisson, Smithfield, UT), 25 g/L of
sucrose, and 2.8–3.0 g/L of Phytagel® (Merck Sigma-Aldrich,
St. Louis, MO) at pH 5.60  0.02 depending on which media
is being prepared (propagation or cutting). In a microwave-
resistant plastic beaker, dissolve MS salts with vitamins in
600 mL of water, add 25 g of sucrose, and mix. Bring to a
final volume of 1000 mL. Adjust pH to 5.60  0.02 with HCl
and/or NaOH as needed (see Note 2).
2. Propagation medium: Basic medium with 3.0 g of Phytagel.
Mix well and dissolve the Phytagel in a microwave (see Note 3).
Dispense 9.0  0.1 mL per 25  150 mm test tube (using an
electronic dispensing pump or by manual dispensing). Close
test tubes with caps. Autoclave for 20 min at 121  C and 15 psi.
Store at 5  3  C for maximum 14 days.
26 Rainer Vollmer et al.

3. Cold acclimatization and cutting medium: Basic medium with

2.8 g of Phytagel for standard Petri dishes (100  15 mm) and
3.0 g for deep Petri dishes (100  25 mm). Prepare in a 1 L
Pyrex® glass bottle, pour the medium in the bottle, and mix.
Autoclave for 20 min at 121  C and 15 psi. Dispense culture
medium in a warm state (~40–50  C) into sterile deep or
standard plastic Petri dishes inside a sterilized laminar flow
chamber to reduce possible contaminants. Dispense
40  2 mL per deep Petri dish (cold acclimatization medium)
and 25  1 mL per standard Petri dish (cutting medium), using
an electronic dispensing pump. Close Petri dishes with sterile
lids. Seal deep Petri dishes with parafilm. Store in sterile Petri
dish bags at 5  3  C for a maximum of 14 days.
4. Recovery medium: 4.43 g/L of commercial Murashige and
Skoog (MS) salts with vitamins, 25 g/L of sucrose, 20 mL/L
of coconut water, 0.1 mg/L of gibberellic acid, 0.4 mg/L of
kinetin, 2.8–3.0 g of Phytagel (depending on vessel type/size),
pH 5.60  0.02.
(a) In a microwave-resistant plastic beaker, dissolve MS salts
with vitamins in 600 mL of water, add 25 g of sucrose, and
mix. Add water to a volume of 980 mL. Mix well. Adjust
pH to 5.60  0.02 with HCl and/or NaOH as needed.
(b) Add 3.0 g (for test tubes) or 2.8 g of Phytagel (for Petri
dishes) in a Pyrex glass bottle and pour the solution into
the bottle. Autoclave culture medium for 20 min
(at 121  C, 15 psi).
(c) Place bottle in a laminar flow chamber and let the ster-
ilized culture medium cool down to a temperature of
~35–40  C. Using a sterile plastic pipette (10 mL), add
20 mL of coconut water stock solution (see Note 4).
Using a micropipette with sterile tip, add 100μL of gib-
berellic acid and 20μL of kinetin stock solution (see Note
5). Gently shake the bottle to get a homogeneous
solution.
(d) Dispense 25  1 mL of sterile medium per standard Petri
dish and 9.0  0.1 mL per 25  150 mm glass test tube,
using an electronic dispensing pump or by manual dis-
pensing. Close Petri dishes and/or test tubes with sterile
lids/caps. Store at 5  3  C for a maximum of 14 days
(in the dark).
5. Loading solution (LS): 4.43 g/L of commercial Murashige
and Skoog (MS) salts with vitamins, 136.8/L g sucrose, and
147.4 mL/L glycerol, pH 5.80  0.02.
(a) Dissolve the MS salts with vitamins in about 300 mL of
Milli-Q water (in a 1000 mL glass beaker). Add 136.8 g of
sucrose and mix well.
 Cryopreservation of Potato Shoot Tips for Long-Term Storage 27

(b) Using a glass measuring cylinder, measure 147 mL of

glycerol (Merck Sigma-Aldrich, St. Louis, MO) and add
it to the solution. As glycerol has a high viscosity, maintain
the cylinder on the bench for some time during pouring to
ensure an exact glycerol volume in the glass cylinder. It
will take time for the glycerol to drop down and stop
adhering to the sides of the cylinder due to its viscosity.
Mix well.
(c) Pour the solution into a glass volumetric flask (1000 mL)
and bring to a final volume of 1000 mL with Milli-Q
water. Pour the solution back into the beaker. Adjust the
pH to 5.80  0.02 with HCl and/or NaOH as needed.
(d) Under a laminar flow chamber, filter sterilize LS through a
nitrocellulose filter (pore size: 0.22μm) with a glass fiber
pre-filter (pore size: 1.0μm). Dispense ~45 mL of steri-
lized LS per black centrifuge tube (50 mL). Seal the cap
with parafilm and store at 5  3  C for maximum 14 days.
6. Plant vitrification solution 2 (PVS2): 4.43 g/L of commercial
Murashige and Skoog (MS) salts with vitamins, 136.8 g/L of
sucrose, 240.0 mL/L of glycerol, 136.4 mL/L of dimethyl
sulfoxide (DMSO), 134.8 mL/L of ethylene glycol,
pH 5.80  0.02.
(a) Dissolve MS salts with vitamins in about 200 mL of Milli-
Q water (in a 1000 mL glass beaker). Add 136.8 g of
sucrose and mix well.
(b) Using a glass measuring cylinder, measure 240.0 mL of
glycerol, 136.4 mL of DMSO, and 134.8 mL of ethylene
glycol and add them to the solution. Dissolve.
(c) Pour the solution into a glass measuring cylinder
(1000 mL) and bring to a final volume of 1000 mL with
Milli-Q water. Pour the solution back into the beaker.
Adjust the pH to 5.80  0.02 with HCl and/or NaOH
as needed.
(d) Under a laminar flow chamber, filter sterilize PVS2
through a nitrocellulose filter (pore size: 0.22μm) with
glass fiber pre-filter (pore size: 1.0μm). Dispense ~45 mL
of sterilized PVS2 per black centrifuge tube (50 mL). Seal
the cap with parafilm and store at 5  3  C for maximum
14 days.
7. Rewarming solution (RS): 4.43 g/L of commercial Murashige
and Skoog (MS) salts with vitamins, 205.2 g/L (0.6 M) of
sucrose, pH 5.80  0.02.
(a) In a 1000 mL glass beaker, dissolve the MS salts with
vitamins in about 300 mL of Milli-Q water.
(b) Add 205.2 g (¼ 0.6 M) of sucrose and dissolve.
28 Rainer Vollmer et al.

(c) Pour the solution into a glass measuring cylinder

(1000 mL) and bring to a final volume of 1000 mL with
Milli-Q water. Pour the solution back into the beaker.
Adjust pH to 5.80  0.02 with HCl and/or NaOH as
needed.
(d) Under a laminar flow chamber, filter sterilize RS through a
nitrocellulose filter (pore size: 0.22μm) with glass fiber
pre-filter (pore size: 1.0μm). Dispense ~45 mL of steri-
lized RS per sterile centrifuge tube (50 mL). Seal the cap
with parafilm and store at 5  3  C for maximum 14 days.

2.2 Supplies Needed 1. Flake ice.

for Cryopreservation 2. Liquid nitrogen (see Note 6).
Process
3. Aluminum foil (extra heavy duty, thickness: 24–30μm).
4. Sterile strips of aluminum foil (size: 5  20 mm; thickness:
24–30μm): Strips are cut with scissors and sterilized in standard
glass Petri dishes (100  15 mm).
5. Forceps for tissue culture (type 1: long straight with fine point,
type 2: short and curved [cotton forceps]).
6. Cryoboxes (capacity: 100 cryovials).
7. Sterile cryovials (1.8 mL, internal thread) (Fisher Thermo
Scientific, Branchburg, NY).
8. Freezing container (CoolBox™ 30 Systems, BioCision).
9. Ice pan suitable for liquid nitrogen.
10. Parafilm.
11. Plastic wrap.
12. Scalpel holder with blades (Nos. 10 and 11).
13. Sterile bond paper (A5).
14. Sterile filter papers (Grade 2, 10  10 mm and 25  30 mm).
15. Sterile glass Pasteur pipettes with rubber bulb (1 mL).
16. Sterile glass Petri dishes (100  15 mm).
17. Sterile glass screw-top test tubes (15 mL, 70 mm, Ø 20 mm).
18. Dewar flask for liquid nitrogen.
19. Laminar flow chamber.
20. Glass bead sterilizer.
21. Stereoscope.
22. Vortex mixer.
23. Small-, medium-, and high-capacity cryotanks (low pressure;
see Note 7).
24. High-pressure bulk tank for storage of liquid nitrogen (see
Note 8).
 Cryopreservation of Potato Shoot Tips for Long-Term Storage 29

2.3 Personal 1. Cryo-apron.

Protective 2. Cryogenic gloves.
Equipment (PPE)
3. Face shield.
4. Safety goggles and glasses.
5. Transfer hose for liquid nitrogen.

3 Methods

As cryopreservation is a labor-intensive, multistep process (Fig. 1)

for introducing plant material into the cryobank, it is recom-
mended to cryopreserve only pathogen-free (e.g., virus) and iden-
tity verified accessions (also known as true-to-type material).
Establishment of virus-clean plant material and identity verification
schemes will avoid conserving duplicates or non-true-to-type acces-
sions. If technically possible, all steps of the in vitro and cryopreser-
vation processes should be recorded in a real-time database using
barcode labels as input (handwriting of labels is often a potential
source of human error and may cause misidentification of acces-
sions). Furthermore, ensure that all staff are highly trained before
using and handling liquid nitrogen and associated equipment. Use
only vessels and tanks that have been especially designed for hold-
ing liquid nitrogen. Wear adequate personal protective equipment
(PPE) when using or handling liquid nitrogen (see Note 9).
Finally, it is also important to establish clear threshold values
and standards for minimum acceptability of full plant recovery
rates. The threshold varies depending on the plant species, number
of stored and thawed samples, and expected recovery rate and
probability to recover plants [15, 16]. At CIP, a minimum accept-
able plant recovery rate of 30% was established for transferring
accessions to the cryobank for long-term preservation. This recov-
ery assessment is based on a sample size of 30 shoot tips assessed
from a total of 150 cryopreserved shoot tips (per accession). Acces-
sions that show a recovery rate of 20–30% in the first cryo-run
require a second additional run, in order to increase the total
number of stored samples. Accessions that show less than 20% of
recovery rate are discarded and not transferred to the cryobank and
later cryopreserved with an alternative protocol [5]. Anything
greater than 30% is stored in the cryobank for a long term.

3.1 In Vitro 1. Always follow good laboratory practices for in vitro propaga-
Propagation Cycles tion (see Note 10).
2. Propagate stem segments of in vitro potato plants onto propa-
gation medium (see Subheading 2.1).
3. Place 5–6 stem segments per 25  150 mm test tube, with one
to two buds per segment (Fig. 2) (see Note 11).
30 Rainer Vollmer et al.

Fig. 2 In vitro propagation of potato. Planting of stem segments, with 1–2 buds,
on basic culture medium contained in 25  150 mm glass test tubes. Per test
tube place 5–6 stem segments

4. Close test tubes with sterile caps.

5. Label test tubes with an accession identifier and date of
propagation.
6. Incubate in vitro plants for 2–4 weeks at a temperature of
20  2  C with a light intensity of 96  12μmol/m2/s. As a
light source, use cold daylight fluorescent tubes (36 W), with a
photoperiod of 16-h light and 8-h darkness. The incubation
period depends on genotype and growth rate.
7. Perform a second and third propagation cycle until a total
number of 14 test tubes has been obtained (~70 in vitro
plants).

3.2 Cold 1. Place stem segments with a single axillary bud onto the cold
Acclimatization acclimatization medium. Place 80–90 stem segments per deep
of Shoot-Tip Donor Petri dish. Propagate a total number of two Petri dishes per
Plants accession (~160–180 plants) (Fig. 3).
2. Incubate Petri dishes for 7–10 days at 20  2  C and light
intensity of 96  10μmol/m2/s, followed by 10–25 days at
7  1  C and light intensity of 15  5μmol/m2/s (cold cham-
ber) (see Note 12).

3.3 Excision 1. Distribute 15 small square pieces of sterile filter paper (10  10
of Potato Shoot Tips mm) on the surface of sterile cutting medium.
 Cryopreservation of Potato Shoot Tips for Long-Term Storage 31

Fig. 3 Deep Petri dish (25  100 mm) containing ~80 potato stem segments
with a single axillary bud. Stem segments are pre-cultured for 7–10 days at
20  2  C, followed by 10–25 days at 7  1  C (cold hardening). The specific
incubation period varies depending on the genotype and physiological status of
the accession. After 17–35 days, the shoot tips are excised from these in vitro
plants, treated with cryoprotectant solutions, and fast frozen in liquid nitrogen

2. Use long tissue culture forceps and scalpel No. 11 excise shoot
tips (see Note 13) from 17- to 35-day-old in vitro plants
coming from Subheading 3.2 under the stereoscope (see
Note 14).
3. Place excised shoot tips individually onto the filter papers of the
cutting medium. First place one single shoot tip onto each of
the filter papers, then place the second shoot tip onto each of
the filter papers, then the third, and so on. At the end of this
process, each filter paper will hold 10 shoot tips. Per accession,
excise 150 shoot tips (the shoot tips were placed on a total of
15 filter papers).

3.4 Preparative 1. Using a permanent marker write accession code and freezing
Steps date on the cryotubes (temporary label).
for Cryopreservation 2. Print cryogenic barcode labels and place them onto the hand-
written identification (double identification in case the label
gets damaged) (see Note 15).
3. Label and set up glass tubes with screw-top lids (15 mL) for the
cryoprotectant treatment (see Note 16).
4. Set up two Pasteur pipettes to handle the cryoprotectant solu-
tions (LS and PVS2), shoot-tip absorption, and droplet forma-
tion (see Note 17).
5. Homogenize LS with a vortex mixer.
6. Using a Pasteur pipette, dispense ~1.0 mL of LS per sterile
screw-top test tube (15 mL).
32 Rainer Vollmer et al.

7. Fill ice pan with flake ice.

8. Place a 50 mL centrifuge tube with sterile PVS2 on ice (0  C) at
a depth of ~3/4 of the tube.

3.5 Treatment 1. Using long tissue culture forceps (23 cm), place filter paper
with Cryoprotectant with shoot tips into LS (Fig. 4). Discharge shoot tips from filter
Solutions paper. Remove filter paper from the test tube (see Note 18).
(LS and PVS2) Treat shoot tips with LS for 20 min (at room temperature).
2. Using a Pasteur pipette remove LS from the tube (see Note 19)
and replace it with ~1.0 mL of ice-cold PVS2 (see Note 20).
3. Close test tube with sterile cap and verify that all shoot tips are
submerged in PVS2 (use white absorbent paper as
background).
4. Treat the shoot tips with PVS2 for 50 min by keeping ¾ of the
tube on ice (Fig. 5).

3.6 Freezing 1. Prepare aluminum foil strips (see Note 21) and freezing con-
in Liquid Nitrogen tainer (CoolBox™ 30 Systems) (see Note 22).
2. Place labeled cryovials in the holder of freezing container (see
Note 23).
3. Using Pasteur pipette, absorb shoot tips from PVS2, place
them in a small volume of PVS2 (droplet of ~20–25μL) onto
the aluminum foil strip (see Note 24) (Figs. 6 and 7), quickly
plunge the strip into liquid nitrogen (“fast freezing”), and
transfer it to a cryovial (see Note 25) (Fig. 8).
4. Close cryovials with sterile caps (see Note 26).
5. Store cryovials in a cryotank suitable for laboratory use (see
Note 27). Cryovials are placed on aluminum canes (cryovial
holders) for temporary storage (see Note 28) (Fig. 9).

3.7 Transitory 1. Label and UV sterilize cryoboxes (capacity: 100 vials) and two
Storage liquid nitrogen pans for 15 min (see Note 29).
2. Fill pans with liquid nitrogen in a laminar flow chamber (LFC),
at a height of approximately 3–5 cm (see Note 30).
3. Place two empty cryoboxes in one of the pans (in LFC).
4. Move laboratory and transitory storage tank next to LFC (see
Note 31).
5. Place cryo-canes holding the cryovials to be transferred in the
second pan (see Note 32).
6. Using sharp-point pliers, quickly transfer cryovials from alumi-
num canes to cryoboxes (see Note 33). When the cryobox is
full, close it with its sterile lid, and transfer it to the transitory
storage tank (see Note 34).
 Cryopreservation of Potato Shoot Tips for Long-Term Storage 33

Fig. 4 A piece of filter paper (1  1 cm) with 10 potato shoot tips is introduced to
a screw-top glass tube (70  20 mm), containing ~1.0 mL of loading solution
(LS). Shoot tips are treated for 20 min with LS (at room temperature). After
20 min, LS is replaced with ~1.0 mL of ice-cooled plant vitrification solution
2 (PVS2)

Fig. 5 Shoot tips are treated with PVS2 for 50 min (at 0  C)

3.8 Thawing of Shoot 1. Set up sterile screw-top glass tubes (15 mL) for thawing of the
Tips in Rewarming control samples and set up a Pasteur pipette to handle the
Solution (RS) rewarming solution (RS) and shoot tips during the thawing
process (see Note 35).
2. Dispense ~2.0 mL of sterile RS per sterile screw-top test tube,
label tubes, and place three square pieces of filter paper on
recovery medium in sterile plastic Petri dishes (see Note 36).
3. Set up freezing container and fill it with liquid nitrogen
(as described in Note 22). Using sharp-point pliers, transfer
34 Rainer Vollmer et al.

Fig. 6 Ten potato shoot tips contained in a small volume of PVS2 in the tip of a
Pasteur pipette, before it is placed on the aluminum foil strip

Fig. 7 Shoot tips contained in a droplet of PVS2 (~20–25μL) that was placed
onto a small aluminum foil strip (5  20 mm, with a 3 mm fold). The aluminum
foil strip (with shoot tips on it) is then plunged into liquid nitrogen for quick
freezing

cryovials to be thawed from the laboratory cryotank to the

cryovial support of the freezing container.
4. Transfer aluminum foil strip with shoot tips individually to
tubes with RS. Rewarm shoot tips in RS for 20 min at room
temperature (see Note 37) (Fig. 10).
5. Place three large filter papers (~30  35 mm) onto a stack of
3–6 sterile paper bond sheets. Distribute three small filter
papers (~10  10 mm) on each of the large filter papers (see
Note 38).
 Cryopreservation of Potato Shoot Tips for Long-Term Storage 35

Fig. 8 Freezing of aluminum foil strip with ten shoot tips on it in liquid nitrogen.
The strip is plunged quickly into liquid nitrogen (“fast freezing”). A single strip is
placed per cryovial (¼ 10 shoot tips)

Fig. 9 Using sharp-point pliers or cotton forceps, capped cryovials are quickly
transferred from the freezing container to an aluminum cane that was previously
precooled in liquid nitrogen

6. Using the Pasteur pipette, absorb shoot tips from RS and place
them for approximately 1–3 min on the small sterile filter
papers (to drain excess of RS solution) (Fig. 11).
Another random document with
no related content on Scribd:
The Project Gutenberg Literary Archive Foundation is a non-profit
501(c)(3) educational corporation organized under the laws of the
state of Mississippi and granted tax exempt status by the Internal
Revenue Service. The Foundation’s EIN or federal tax identification
number is 64-6221541. Contributions to the Project Gutenberg
Literary Archive Foundation are tax deductible to the full extent
permitted by U.S. federal laws and your state’s laws.

The Foundation’s business office is located at 809 North 1500 West,

Salt Lake City, UT 84116, (801) 596-1887. Email contact links and up
to date contact information can be found at the Foundation’s website
and official page at www.gutenberg.org/contact

Section 4. Information about Donations to

the Project Gutenberg Literary Archive
Foundation
Project Gutenberg™ depends upon and cannot survive without
widespread public support and donations to carry out its mission of
increasing the number of public domain and licensed works that can
be freely distributed in machine-readable form accessible by the
widest array of equipment including outdated equipment. Many small
donations ($1 to $5,000) are particularly important to maintaining tax
exempt status with the IRS.

The Foundation is committed to complying with the laws regulating

charities and charitable donations in all 50 states of the United
States. Compliance requirements are not uniform and it takes a
considerable effort, much paperwork and many fees to meet and
keep up with these requirements. We do not solicit donations in
locations where we have not received written confirmation of
compliance. To SEND DONATIONS or determine the status of
compliance for any particular state visit www.gutenberg.org/donate.

While we cannot and do not solicit contributions from states where

we have not met the solicitation requirements, we know of no
prohibition against accepting unsolicited donations from donors in
such states who approach us with offers to donate.

International donations are gratefully accepted, but we cannot make

any statements concerning tax treatment of donations received from
outside the United States. U.S. laws alone swamp our small staff.

Please check the Project Gutenberg web pages for current donation
methods and addresses. Donations are accepted in a number of
other ways including checks, online payments and credit card
donations. To donate, please visit: www.gutenberg.org/donate.

Section 5. General Information About Project

Gutenberg™ electronic works
Professor Michael S. Hart was the originator of the Project
Gutenberg™ concept of a library of electronic works that could be
freely shared with anyone. For forty years, he produced and
distributed Project Gutenberg™ eBooks with only a loose network of
volunteer support.

Project Gutenberg™ eBooks are often created from several printed

editions, all of which are confirmed as not protected by copyright in
the U.S. unless a copyright notice is included. Thus, we do not
necessarily keep eBooks in compliance with any particular paper
edition.

Most people start at our website which has the main PG search
facility: www.gutenberg.org.

This website includes information about Project Gutenberg™,

including how to make donations to the Project Gutenberg Literary
Archive Foundation, how to help produce our new eBooks, and how
to subscribe to our email newsletter to hear about new eBooks.