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CRISPR is a crucial technology in plant physiology and molecular biology, resulting in more sustainable agricultural practices, including outcomes of better plant stress tolerance and crop improvement. CRISPR and Plant Functional Genomics explores ways to release the potential of plant functional genomics, one of the prevailing topics in plant biology and a critical technology for speed and precision crop breeding. This book presents achievements in plant functional genom ics and features information on diverse applications using emerging CRISPR‑based genome edit ing technologies producing high‑yield, disease‑resistant, and climate‑smart crops. It also includes theories on organizing strategies for upgrading the CRISPR system to increase efficiency, avoid off‑target effects, and produce transgene‑free edited crops.
Features:
• Presents CRISPR‑based technologies, releasing the potential of plant functional genomics
• Provides methods and applications of CRISPR/Cas‑based plant genome editing technologies
• Summarizes achievements of speed and precision crop breeding using CRISPR‑based technologies
• Il lustrates strategies to upgrade the CRISPR system
• Supports the UN’s sustainable development goals to develop future climate‑resilient crops
CRISPR and Plant Functional Genomics provides extensive knowledge of CRISPR‑based tech nologies and plant functional genomics and is an ideal reference for researchers, graduate students, and practitioners in the field of plant sciences as well as agronomy and agriculture.
CRISPR and Plant Functional Genomics
Edited by Jen‑Tsung Chen
First edition published 2024 by CRC Press
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Chapter 1 Advances in CRISPR/Cas‑based Genome Editing: Break New Ground for Plant Functional Genomics .......................................................................................... 1
Hao Hu and Fengqun Yu
Chapter 2 Functional Genome Analysis and Genome Editing in Plants: An Updated Overview 20
Chapter 3 Recent Tools of Genome Editing and Functional Genomics in Plants: Present and Future Applications on Crop Improvement ............................................ 40
Johni Debbarma, Mamta Bhattacharjee, Trishna Konwar, Channakeshavaiah Chikkaputtaiah, and Dhanawantari L. Singha
Chapter 4 St rategies and Applications of Genomic Editing in Plants with CRISPR/Cas9 59
Chapter 5 Vi rus‑Induced Genome Editing: Methods and Applications in Plant Breeding 81 Mireia Uranga
Chapter 6 CR ISPR/Cas9: The Revolutionary Genome Editing Tools for Crop Genetic Tweak to Render It ‘Climate Smart’ ........................................................................ 108
Abira Chaudhuri, Asis Datta, Koushik Halder, and Malik Z. Abdin
Chapter 7 Functional Genomics for Abiotic Stress Tolerance in Crops ................................... 12 4
Muhammad Khuram Razzaq, Guangnan Xing, Muhammad Basit Shehzad, Ghulam Raza, and Reena Rani
Chapter 8 CR ISPR/Cas9 for Enhancing Resilience of Crops to Abiotic Stresses 133
Chapter 9 CR ISPR‑Based Genome Editing for Improving Nutrient Use Efficiency and Functional Genomics of Nutrient Stress Adaptation in Plants................................. 144
Lekshmy Sathee, Sinto Antoo, Gadpayale Durgeshwari Prabhakar, Arpitha S R, Sudhir Kumar, Archana Watts, Viswanathan Chinnusamy, Balaji Balamurugan, and Asif Ali Vadakkethil
Chapter 10
CR ISPR‑Cas Genome Modification for Non‑Transgenic Disease‑Resistant, High Yielding and High‑Nutritional Quality Wheat 175
S. M. Hisam Al Rabbi, Israt Nadia, and Tofazzal Islam
Chapter 11 Using CRISPR to Unlock the Full Potential of Plant Immunity
Mumin Ibrahim Tek, Abdul Razak Ahmed, Ozer Calis, and Hakan Fidan
Chapter 12 Advancement in Gene Editing for Crop Improvement Highlighting the Application of Pangenomes ......................................................................................
Biswajit Pramanik , Sandip Debnath , and Anamika Das
189
Chapter 13 Characterization of CRISPR‑Associated Protein for Epigenetic Manipulation in Plants 223
Chapter 15 Gene Editing Using CRISPR/Cas9 System: Methods and Applications 258 G opika, Boro Arthi, Arumugam Vijaya Anand, Natchiappan Senthilkumar, Senthil Kalaiselvi, and Santhanu Krishnapriya
Chapter 16 CR ISPR/Cas13 for the Control of Plant Viruses......................................................
Joana A. Ribeiro, Patrick Materatski, Carla M. R. Varanda, Maria Doroteia Campos, Mariana Patanita, André Albuquerque, Nicolás Garrido, Tomás Monteiro, Filipa Santos, and Maria do Rosário Félix
Chapter 17 Genome Editing in Ornamental Plants: Current Findings and Future Perspectives 289
Chapter 18 A Convenient CRISPR/Cas9 Mediated Plant Multiple Gene Editing Protocol by In‑Fusion Technology 301
Jun‑Li Wang, Luo‑Yu Liang, and Lei Wu
Chapter 19 The Application of CRISPR Technology for Functional Genomics in Oil Palm and Coconut .............................................................................................. 313
Siti Nor Akmar Abdullah and Muhammad Asyraf Md Hatta
Chapter 20 Advances and Perspectives in Genetic Engineering and the CRISPR/Cas‑Based Technology for Oil Crop Enhancement 332
Chapter 21 CR ISPR/Cas9‑Based Technology for Functional Genomics and Crop Improvement in Soybean
Cuong Xuan Nguyen and Phat Tien Do
Preface
For decades, functional genomics has been conducted intensively to explore plant biological net works and continues to identify critical components of regulatory and controlling machinery as well as their functions in sustaining growth and development. In the meantime, the resulting knowledge and technologies keep benefiting agricultural production through the creation of stress‑tolerant, high‑yield, and high‑quality crops using multi‑omics‑assisted breeding strategies. In recent years, a goal that fits SDGs (Sustainable Development Goals) by the United Nations is to precisely breed climate‑smart crops against global climate change, and therefore, definitely, functional genomics is the major technology for future sustainable agriculture.
The system of functional genomics evolves rapidly, and one of the game‑changing tools is genome editing technology. Among them, an emerging one is based on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the associated Cas proteins. CRISPR‑based genome editing technology can specifically introduce targeted sequence alterations, and thus, it leads to a new era of plant functional genomics and, meanwhile, a practical system of precision molecular plant breeding. This book summarizes and comprehensively discusses the introduction and integra tion of CRISPR‑based technology into plant functional genomics and precision breeding. It consists of 21 chapters to refine current knowledge from the literature and is organized by scientists and experts in this field.
Since the post‑genomics era, bioinformatics has become an essential tool for dealing with huge amounts of genomic datasets and for the research field of systems biology, contributing to an array of research fields, including the construction of CRISPR systems. In this book, a part of the content pays attention to the design of guide RNA for CRISPR technology, which is critical to ensure efficient and precisely targeted modification of interest genes, and it can be customized by some tools belonging to bioinformatics. Another essential component of the CRISPR system is the association of Cas proteins, and a chapter was organized to illustrate the structures and functions of their va rieties, which provide available types for developing appropriate systems in epigenetic manipulation.
To deal with the growing human population, the UN sets SDG goals to achieve zero hunger around the world. The issue of food security majorly depends on agricultural production, and it can be enhanced by crop improvement. Without a doubt, in the future, CRISPR‑assisted precision plant breeding will be a promising tool to edit bases with the aid of advanced genomics tools, such as NGS (next‑generation sequencing), single‑cell omics, spatial transcriptomics, and so on. In mod ern times, another global threat is climate change, and therefore, scientists attempt to mitigate the effect by gaining future crops with the capacity of “climate‑smart” or “climate‑resilient” through cutting‑edge breeding strategies. One of the effective ways is to apply the emerging CRISPR/Cas technology, particularly using Cas9, and some achievements have been reported, chiefly focusing on environmental/abiotic stress tolerance under heat, drought, flood, and so on, and disease resistance when faced with a changing climate that probably leads to severe damage on yield and quality of crops. For developing future crops, a revolutionary system must be taken into consideration, namely pan‑genomics which explores a collection of genomic sequences for the entire species or population and can provide a rich resource for identifying critical genetic variations to advance plant breeding. In the future, crop breeders can perform CRISPR more efficiently based on the information and knowledge of the research in pan genomes to optimize their programs for “speed breeding”. In agricultural production, a serious negative factor that impacts food security is the huge loss of crop yield and quality caused by pathogens and pests. Fortunately, plants have evolved an innate system of defense against viruses, bacteria, fungi, herbivores, nematodes, and so on. Theoretically, plant immunity has the potential to be released or improved, and it can be achieved by CRISPR‑based technology through the understanding of the interactions between plants and microbes or pests and
subsequently ensuring the editing of targeted genes for resistance. For plant pathologists, itʼs often challenging to monitor and control plant viral diseases, and some efforts have been made to use CRISPR‑based technology against plant viruses. In this book, the current achievements using the emerging CRISPR/Cas13 system to successfully produce virus‑resistant plants are systematically summarized.
Altogether, this book presents the current knowledge of the CRISPR technology based on refining literature with an emphasis on the fundamental technologies as well as upgraded tools for the exploration of functional genomics and then toward accelerated and precision plant breeding. The applications have been reported in some significant crops, such as rice, wheat, maize, soybean, oil palms, coconut, and ornamental plants. This book provides sufficient and updated knowledge of CRISPR technology and plant functional genomics and thus is an ideal reference for graduate students, teachers, researchers, and experts in the field of plant biology as well as agronomy and agriculture.
As the book editor, I’d like to thank all the authors of this book for their expertise and for their time and effort in organizing chapters. The friendly assistance and instructions from the staff of CRC Press/Taylor & Francis Group are very much appreciated.
Editor
Jen‑Tsung Chen is a professor of cell biology at the National University of Kaohsiung in Taiwan. He also teaches genomics, proteomics, plant physiology, and plant biotechnology. His areas of research interest include bioactive compounds, chromatography techniques, plant molecular biol ogy, plant biotechnology, bioinformatics, and systems pharmacology. He is an active editor of academic books and journals to advance the exploration of multidisciplinary knowledge involving plant physiology, plant biotechnology, nanotechnology, ethnopharmacology, and systems biology. He serves as an editorial board member in reputed journals, including Plant Methods, GM Crops & Food, Plant Nano Biology, Biomolecules, International Journal of Molecular Sciences, and a guest editor in Frontiers in Plant Science, Frontiers in Pharmacology, Journal of Fungi, and Current Pharmaceutical Design. Dr. Chen published books in collaboration with Springer Nature, CRC Press/Taylor & Francis Group, and In‑TechOpen, and he is handling book projects for more international publishers on diverse topics such as drug discovery, herbal medicine, medicinal bio technology, nanotechnology, bioengineering, plant functional genomics, plant speed breeding, and CRISPR‑based plant genome editing.
Contributors
Malik Z. Abdin
Centre for Transgenic Plant Development, Department of Biotechnology
School of Chemical and Life Sciences
New Delhi, India
Siti Nor Akmar Abdullah
Department of Agriculture Technology, Faculty of Agriculture
Universiti Putra Malaysia Selangor, Malaysia and Laboratory of Agronomy and Sustainable Crop Protection, Institute of Plantation Studies
Universiti Putra Malaysia Selangor, Malaysia
Mahtab Ahmad
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur), India
Abdul Razak Ahmed
Molecular Mycology Laboratory, Plant Protection Department, Faculty of Agriculture
Akdeniz University Antalya, Turkey
André Albuquerque
MED ‑ Mediterranean Institute for Agriculture, Environment and Development & CHANGE ‑ Global Change and Sustainability Institute, Institute for Advanced Studies and Research
Universidade de Évora, Pólo da Mitra Évora, Portugal
Abdel Rahman Al‑Tawaha
Department of Biological Sciences
Al‑Hussein Bin Talal University Maan, Jordon
Arumugam Vijaya Anand
Department of Human Genetics and Molecular Genetics
Bharathiar University Coimbatore, India
Kumari Anjani
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur), India
Sinto Antoo
Division of Plant Physiology
ICAR‑Indian Agricultural Research Institute
New Delhi, India
Anuradha
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur), India
Arpitha S R
Division of Biochemistry
ICAR‑Indian Agricultural Research Institute
New Delhi, India
Boro Arthi
Department of Human Genetics and Molecular Genetics
Bharathiar University Coimbatore, India
Muthukrishnan Arun
Department of Biotechnology
Bharathiar University Coimbatore, India
Balaji Balamurugan
ICAR‑National Institute for Plant Biotechnology
New Delhi, India
Mamta Bhattacharjee
Department of Agricultural Biotechnology
Assam Agriculture University
Jorhat, India
Sayan Bhattacharya
School of Ecology and Environment Studies
Nalanda University
Rajgir, India
Karma Landup Bhutia
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur), India
Nangsol Dolma Bhutia
Department of Vegetable Sciences, College of Horticulture and Forestry
Central Agricultural University (Imphal) Pasighat, India
Bhavna Borah
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur), India
Ercan Bursal
Department of Biochemistry
Mus Alparslan University
Merkez/Muş, Turkey
Ozer Calis
Molecular Virology Laboratory, Plant Protection Department, Faculty of Agriculture
Akdeniz University
Antalya, Turkey
Maria Doroteia Campos
MED ‑ Mediterranean Institute for Agriculture, Environment and Development & CHANGE ‑ Global Change and Sustainability Institute, Institute for Advanced Studies and Research
Universidade de Évora, Pólo da Mitra
Évora, Portugal
Abira Chaudhuri
National Institute of Plant Genome Research
New Delhi, India
Channakeshavaiah Chikkaputtaiah
Biological Sciences and Technology Division
CSIR‑North East Institute of Science and Technology (CSIR‑NEIST) Jorhat, India
Viswanathan Chinnusamy Division of Plant Physiology
ICAR‑Indian Agricultural Research Institute New Delhi, India
Anamika Das
Department of Genetics and Plant Breeding
Bidhan Chandra Krishi Viswavidyalaya Nadia, India and
Department of Agriculture
Netaji Subhas University Jamshedpur, India
Asis Datta
National Institute of Plant Genome Research New Delhi, India
Johni Debbarma
Department of Botany
Assam Don Bosco University Sonapur, India and
Biological Sciences and Technology Division
CSIR‑North East Institute of Science and Technology (CSIR‑NEIST) Jorhat, India
Sandip Debnath
Department of Genetics and Plant Breeding
Palli Siksha Bhavana (Institute of Agriculture), Visva‑Bharati Sriniketan, India
Barasha Rani Deka
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur), India
Abhijit Dey
Department of Life Sciences
Presidency University
Kolkata, India
Phat Tien Do
Institute of Biotechnology
Vietnam Academy of Science and Technology
Hanoi, Vietnam and
Graduate University of Science and Technology
VietnamAcademy of Science and Technology
Hanoi, Vietnam
Maria do Rosário Félix
MED ‑ Mediterranean Institute for Agriculture, Environment and Development & CHANGE ‑ Global Change and Sustainability Institute, Departamento de Fitotecnia, Escola de Ciências e Tecnologia
Universidade de Évora, Pólo da Mitra
Évora, Portugal
Hakan Fidan
Molecular Mycology Laboratory, Plant Protection Department, Faculty of Agriculture
Akdeniz University
Antalya, Turkey
Nicolás Garrido
MED ‑ Mediterranean Institute for Agriculture, Environment and Development & CHANGE ‑ Global Change and Sustainability Institute, Institute for Advanced Studies and Research
Universidade de Évora, Pólo da Mitra
Évora, Portugal
Mimosa Ghorai
Department of Life Sciences
Presidency University
Kolkata, India
Gopika
Department of Human Genetics and Molecular Genetics
Bharathiar University
Coimbatore, India
Packiaraj Gurusaravanan
Plant Biotechnology Laboratory, Department of Botany
Bharathiar University
Coimbatore, India
Koushik Halder
National Institute of Plant Genome Research
New Delhi, India and
Centre for Transgenic Plant Development, Department of Biotechnology
School of Chemical and Life Sciences
New Delhi, India
Muhammad Asyraf Md Hatta
Department of Agriculture Technology, Faculty of Agriculture
Universiti Putra Malaysia
Selangor, Malaysia
Akbar Hossain
Division of Soil Science
Bangladesh Wheat and Maize Research Institute
Dinajpur, Bangladesh
Hao Hu
Agriculture and Agri‑Food Canada
Saskatoon Research and Development Centre
Saskatoon, SK, Canada
Ishfaq Majid Hurrah
Plant Biotechnology Division
CSIR‑Indian Institute of Integrative Medicine
Srinagar, India
Tofazzal Islam
Institute of Biotechnology and Genetic Engineering (IBGE)
Bangabandhu Sheikh Mujibur Rahman
Agricultural University
Gazipur, Bangladesh
M. James
School of Crop Improvement, College of Post Graduate Studies in Agricultural Sciences
Central Agricultural University (Imphal)
Imphal, India
Gayatri Jamwal
School of Biotechnology
Sher‑e‑Kashmir University of Agricultural Sciences and Technology of Jammu Chatha, India
Senthil Kalaiselvi
Department of Biochemistry Biotechnology and Bioinformatics
Avinashilingam Institute for Home Science & Higher Education for Women Coimbatore, India
Danish Mushtaq Khanday
Division of Plant Breeding and Genetics
Sher‑e‑Kashmir University of Agricultural Sciences and Technology of Jammu Chatha, India
Anima Kisku
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur), India
Trishna Konwar
Department of Agricultural Biotechnology
Assam Agriculture University Jorhat, India
Kumaresan Kowsalya
Department of Biotechnology Bharathiar University Coimbatore, India
Santhanu Krishnapriya
Department of Biochemistry Biotechnology and Bioinformatics
Avinashilingam Institute for Home Science & Higher Education for Women Coimbatore, India
Sudhir Kumar
Division of Plant Physiology
ICAR‑Indian Agricultural Research Institute New Delhi, India
Sarita Kumari
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur), India
Bharti Lap
School of Crop Improvement, College of Post Graduate Studies in Agricultural Sciences Central Agricultural University (Imphal) Imphal, India
Luo‑Yu Liang
Department of MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences
Lanzhou University Lanzhou, China and
Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences
Lanzhou University Lanzhou, China
Limasunep Longkumer
Department of Agricultural Biotechnology
Assam Agricultural University Jorhat, India
Sayanti Mandal
Department of Biotechnology, Dr. D. Y. Patil Arts
Commerce & Science College (affiliated to Savitribai Phule Pune University) Pune, India and
Institute of Bioinformatics and Biotechnology
Savitribai Phule Pune University Pune, India
Patrick Materatski
MED ‑ Mediterranean Institute for Agriculture, Environment and Development & CHANGE ‑ Global Change and Sustainability Institute, Institute for Advanced Studies and Research Universidade de Évora, Pólo da Mitra Évora, Portugal
Kanshouwa Modunshim
Department of Agricultural Biotechnology Assam Agricultural University Jorhat, India
Tabasum Mohiuddin Govt. Degree College for Women Baramulla, India
Tomás Monteiro
MED ‑ Mediterranean Institute for Agriculture, Environment and Development & CHANGE ‑ Global Change and Sustainability Institute, Institute for Advanced Studies and Research Universidade de Évora, Pólo da Mitra Évora, Portugal
Ponnuchamy Mugudeshwari Indian Institute of Rice Research Hyderabad, India
Israt Nadia Mawlana Bhashani Science and Technology University Tangail, Bangladesh
Cuong Xuan Nguyen
Institute of Biotechnology
Vietnam Academy of Science and Technology Hanoi, Vietnam
Prabhakar Nishi
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University Pusa (Samastipur), India
Potshangbam Nongdam Department of Biotechnology
Manipur University Imphal, India
Devendra Kumar Pandey Department of Biotechnology Lovely Professional University Punjab, India
Mariana Patanita
MED ‑ Mediterranean Institute for Agriculture, Environment and Development & CHANGE ‑ Global Change and Sustainability Institute, Institute for Advanced Studies and Research Universidade de Évora, Pólo da Mitra Évora, Portugal
Ayyadurai Pavithra Department of Human Genetics and Molecular Genetics
Bharathiar University Coimbatore, India
Gadpayale Durgeshwari Prabhakar Division of Biochemistry
ICAR‑Indian Agricultural Research Institute New Delhi, India
Biswajit Pramanik Department of Genetics and Plant Breeding Palli Siksha Bhavana (Institute of Agriculture), Visva‑Bharati Sriniketan, India
S. M. Hisam Al Rabbi Biotechnology Division Bangladesh Rice Research Institute Gazipur, Bangladesh
Gyanendra Kumar Rai School of Biotechnology
Sher‑e‑Kashmir University of Agricultural Sciences and Technology of Jammu Chatha, India
Bashyam Ramya Department of Biochemistry Holy Cross College (Autonomous) Tiruchirappalli, India
Reena Rani
National Institute for Biotechnology and Genetic Engineering
Faisalabad, Pakistan and
Constituent College Pakistan Institute of Engineering and Applied Sciences Faisalabad, Pakistan
Ghulam Raza
National Institute for Biotechnology and Genetic Engineering
Faisalabad, Pakistan and
Constituent College Pakistan Institute of Engineering and Applied Sciences
Faisalabad, Pakistan
Muhammad Khuram Razzaq
Soybean Research Institute & MARA National Center for Soybean Improvement & MARA
Key Laboratory of Biology and Genetic Improvement of Soybean & National Key Laboratory for Crop Genetics and Germplasm Enhancement & Jiangsu
Collaborative Innovation Center for Modern Crop Production
Nanjing Agricultural University
Nanjing, China
Joana A. Ribeiro
MED ‑ Mediterranean Institute for Agriculture, Environment and Development & CHANGE ‑ Global Change and Sustainability Institute, Institute for Advanced Studies and Research Universidade de Évora, Pólo da Mitra
Évora, Portugal
Kattilaparambil Roshna
Plant Biotechnology Laboratory, Department of Botany
Bharathiar University Coimbatore, India
Filipa Santos
MED ‑ Mediterranean Institute for Agriculture, Environment and Development & CHANGE ‑ Global Change and Sustainability Institute, Departamento de Fitotecnia, Escola de Ciências e Tecnologia
Universidade de Évora, Pólo da Mitra Évora, Portugal
Chinnasamy Sashtika
Department of Biochemistry Biotechnology and Bioinformatics
Avinashilingam Institute for Home Science & Higher Education for Women
Coimbatore, India
Lekshmy Sathee
Division of Plant Physiology
ICAR‑Indian Agricultural Research Institute
New Delhi, India
Natchiappan Senthilkumar
Division of Bioprospecting Institute of Forest Genetics and Tree Breeding (IFGTB)
Coimbatore, India
Vinay Kumar Sharma
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur), India
Muhammad Basit Shehzad
College of Plant Protection
Nanjing Agricultural University
Nanjing, China
Mahipal S Shekhawat
Plant Biotechnology Unit
KM Government Institute for Postgraduate Studies and Research
Puducherry, India
Monika Singh
Department of Applied Sciences & Humanities
GL Bajaj Institute of Technology and Management
Greater Noida, India
Dhanawantari L. Singha Department of Botany
Rabindranath Tagore University Hojai, Assam
Annamalai Sivaranjini Department of Biotechnology
Dwaraka Doss Goverdhan Doss Vaishnav College
Chennai, India
Rajalingam Amutha Sudhan
Department of Agricultural Biotechnology and Molecular Biology, College of Basic Sciences & Humanities
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur), India
Mumin Ibrahim Tek
Molecular Virology Laboratory, Plant Protection Department, Faculty of Agriculture
Akdeniz University Antalya, Turkey
Mireia Uranga
Department of Plant Biotechnology and Bioinformatics
Ghent University
Ghent, Belgium and
VIB Center for Plant Systems Biology Ghent, Belgium and
KU Leuven Plant Institute (LPI) Heverlee, Belgium
Asif Ali Vadakkethil
Centre for Agricultural Bioinformatics
ICAR‑Indian Agricultural Statistics Research Institute
New Delhi, India
Carla M. R. Varanda
ESAS, Instituto Politécnico de Santarém Santarém, Portugal and
MED ‑ Mediterranean Institute for Agriculture, Environment and Development & CHANGE ‑ Global Change and Sustainability Institute, Institute for Advanced Studies and Research Universidade de Évora, Pólo da Mitra Évora, Portugal
Nandakumar Vidya
Department of Botany Bharathiar University Coimbatore, India
Sathasivam Vinoth
Department of Biotechnology
Sona College of Arts and Science
Salem, India
Jun‑Li Wang
Department of MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences
Lanzhou University
Lanzhou, China and
Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences
Lanzhou University
Lanzhou, China
Archana Watts
Division of Plant Physiology
ICAR‑Indian Agricultural Research Institute
New Delhi, India
Lei Wu
Department of MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences
Lanzhou University
Lanzhou, China and Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences
Lanzhou University
Lanzhou, China
Guangnan Xing
Soybean Research Institute & MARA National Center for Soybean Improvement & MARA Key Laboratory of Biology and Genetic Improvement of Soybean & National Key Laboratory for Crop Genetics and Germplasm Enhancement & Jiangsu Collaborative Innovation Center for Modern Crop Production
Nanjing Agricultural University
Nanjing, China
Fengqun Yu
Agriculture and Agri‑Food Canada, Saskatoon Research and Development Centre
Saskatoon, SK, Canada
1 Advances in CRISPR/ Cas-ba sed Genome Editing
Break New Ground for Plant Functional Genomics
Hao Hu and Fengqun Yu
1.1 INTRODUCTION
Clustered Regularly Interspaced Short Palindromic Repeats‑CRISPR associated protein (CRISPR/ Cas) technology has revolutionized the field of genetic engineering and has become a prominent tool in genome editing. It is derived from the bacterial immune system and has been adapted for precise and efficient genome editing in a wide range of organisms, including plants. The discovery of CRISPR/Cas can be traced back to the early 1990s when unusual repetitive DNA sequences were identified in the genomes of bacteria and archaea.1–4 However, it wasn’t until 2007 that researchers recognized the potential role of these repetitive sequences as part of a prokaryotic immune system.5 Further studies led to the understanding that CRISPR sequences serve as an adaptive immune defense mechanism against invading genetic elements such as viruses and plasmids.6
The CRISPR/Cas system consists of two main components: the guide RNA (gRNA) and the Cas protein. The gRNA is a synthetic RNA molecule that guides the Cas protein to the target DNA sequence of interest based on RNA‑DNA recognition and binding. The Cas protein is an endonuclease that cleaves DNA at specific sites determined by the gRNA sequence. This cleavage, i.e., double‑strand break (DSB), initiates DNA repair either by the error‑prone non‑homologous end joining (NHEJ) mechanism or by the donor‑dependent homology‑directed repair (HDR) mechanism, leading to various genetic modifications like sequence insertion/deletion (indel) and gene replacement.6 The power and versatility of CRISPR/Cas technology lie in its flexibility to be easily reprogrammed by designing a specific gRNA sequence complementary to the target DNA region, which allows researchers to precisely edit genes and introduce designed genetic modifica tions. Compared to earlier genome editing techniques, such as Zinc‑Finger Nucleases (ZFNs) and Transcription Activator‑Like Effector Nucleases (TALENs), CRISPR/Cas is simpler, faster, and more cost‑effective, making it widely adopted across various fields of research.7
Since its initial discovery, CRISPR/Cas technology has rapidly evolved and expanded, with dif ferent variants of Cas proteins, gRNA formats, and new effector proteins being discovered and engineered. Each Cas variant has unique characteristics, such as Protospacer Adjacent Motif (PAM) requirements, cleavage patterns, and target specificity. Together with other optimization strategies, especially by linking novel effectors (such as deaminase and reverse transcriptase) to Cas protein, current CRISPR/Cas genome editing in plants has been greatly improved in editing efficiency, specificity, targeting scopes, and the introduction of numerous types of precise edit ing. These advancements have deeply impacted fundamental biological research such as plant functional genomics and enabled the development of more sophisticated applications, such as mul tiplexed genome editing (simultaneously targeting multiple genes), base editing (precise nucleotide
substitutions), prime editing (precise search‑and‑replace genome editing to install indels and/or base substitutions), genome‑wide screening, and epigenomic modifications. Since its first applica tion in plants in 2013,8–10 CRISPR/Cas has been extensively utilized in agriculture to improve yield, disease resistance, and other agronomically important traits.11–13 Overall, the discovery and devel opment of CRISPR/Cas technology have transformed the field of genetic engineering, providing plant scientists with an efficient and precise tool for understanding gene function, unraveling genetic networks, and driving innovations in various plant research areas including crop improvement.
1.2 PART I. ADVANCES IN CRISPR/CAS TECHNOLOGIES
1.2.1
Cas Variants
Cas proteins, the key components of the CRISPR/Cas system, exhibit remarkable diversity across different bacterial and archaeal species. CRISPR/Cas systems can be categorized into two classes based on their mechanism of action. In Class 1 systems, a multi‑protein complex (multi‑effector) is involved in the degradation of foreign nucleic acids. On the other hand, Class 2 systems accomplish the same function using a single, large Cas protein (single effector).14–16 Due to its simplicity in application, Cas proteins from Class 2 systems are extensively studied and utilized. Cas9 is the most widely used Cas protein variant in Class 2. It was initially discovered in Streptococcus pyogenes and has become synonymous with CRISPR/Cas technology. Cas9 is an RNA‑guided endonuclease that generates DSBs at target DNA sites. The two main domains, known as the RuvC and HNH nuclease domains, are responsible for cleaving the DNA strands.17 These domains are guided to the target DNA sequence by a single guide RNA (sgRNA), which is a fusion of a CRISPR RNA (crRNA, carrying a sequence complementary to the target DNA) and a trans‑activating crRNA (tracrRNA, helping in the processing and stability of the crRNA) and provides the necessary speci ficity by base‑pairing with the complementary DNA sequence (namely protospacer). In addition, a short DNA sequence located immediately adjacent to the protospacer, i.e., PAM, is also essential for Cas9 binding and cleavage. The specific PAM sequence recognized by Cas9 varies depending on the Cas9 ortholog and species.18–20 For the commonly used S. pyogenes Cas9 (SpCas9), the PAM sequence is NGG (where N can be any nucleotide). Over the years, researchers have identified, char acterized, and even modified numerous Cas protein variants of Class 214,16 (Table 1.1). These Cas variants exhibit distinct features and functionalities, which greatly serve versatile applications and the ever‑increasing need for editing specificity, efficiency, targeting range, etc.
1. nCas9 (nickase Cas9): nCas9 is a modified version of the Cas9 nuclease protein that retains its ability to bind to target DNA but has lost one of its two nuclease domain activi ties. Unlike the wild‑type Cas9, which creates a DSB in the target DNA, nCas9 induces a single‑strand break (SSB) or nick in one of the DNA strands within the protospacer region. The nicking activity of nCas9 is achieved through specific point mutations in the nuclease domains of the Cas9 protein. The most commonly used mutation is the D10A mutation, which disrupts the endonuclease activity of RuvC domain of Cas9. By using a pair of gRNAs, one targeting each DNA strand, nCas9 can be employed to create a pair of nicks on opposite DNA strands.21 This generates a DSB at the desired genomic locus. The paired nicking strategy reduces off‑target effects, as the nicks can be repaired by error‑free mechanisms such as homologous recombination rather than relying on error‑prone NHEJ repair pathways. The use of nCas9 offers several advantages over wild‑type Cas9, includ ing improved specificity and reduced potential for off‑target effects. It allows for precise genome editing with minimal disruption to the DNA sequence, making it particularly useful in applications that require high accuracy and minimal impact on the target locus.21
2. dCas9 (deactivated Cas9 or dead Cas9): dCas9 is a modified version of the Cas9 protein that lacks both of its nuclease activities. Through point mutations, such as D10A and H840A,
Popular Cas Variants in Class 2
Cas Type Cas Variant
IICas9 (i.e., SpCas9 from Streptococcus pyogenes)
Cas9‑VQR NGA
Cas9‑EQR NGAG
DNA NGG The activity of both RuvC and HNH nuclease domains create a DSB at the targeted location on dsDNA
Cas9‑VRER NGCG
Cas9‑NG NGN
Cas9‑xCas9 NG/GAA/GAT
Cas9‑SpG NGN
Cas9‑SpRY PAM‑less
Numerous applications involving dsDNA/genome editing such as gene knockout, replacement, etc.
St1Cas9 (from S. thermophilus) NNAGAA 18
NmCas9 (from Neisseria meningitidis) NNNNGATT
TdCas9 (from Treponema denticola) NAAAAN
FnCas9 (from Francisella novicida) NGG
FnCas9‑RHA YG
nCas9 NGG Lack one nuclease activity (usually RuvC) through mutation, creating a nick (single‑strand break) at a specific location on dsDNA Link with various effectors for precise editing (base editing, prime editing, etc.) or gene regulations
dCas9 NGG Lack both RuvC and HNH nuclease activities through mutations, no cleavage but can still bind to a specific location on dsDNA. Link with various effectors for precise editing (prime editing, etc) or gene regulations
VCas12a
TTN, or TTTN Staggered cutting Auto‑processing pre‑crRNA activity for multiplex gene regulation 14–16,24,37,38
VICas13 RNA A, U, C (no G in PFS) Non‑specifically cleaves non‑target
which alter critical residues necessary for endonuclease activity, dCas9 is rendered inactive. It can still bind to target DNA sequences, guided by the associated sgRNA, but does not induce DNA cleavage. This binding alone is often sufficient to attenuate or block the transcription of the targeted gene, especially when the sgRNA positions dCas9 in a manner that obstructs the access of transcription factors and RNA polymerase to the DNA. Instead of introducing permanent genetic changes, dCas9 can be utilized for gene regulation purposes.22 The modi fiable regions of dCas9, typically located at the N‑ and C‑terminus of the protein, can be uti lized to attach transcriptional activators, repressors, or other effector domains. When dCas9 is fused with transcriptional activators, it is referred to as CRISPR activation (CRISPRa), enabling targeted upregulation of gene expression. Conversely, when dCas9 is fused with transcriptional repressors, it is known as CRISPR interference (CRISPRi), allowing targeted downregulation of gene expression. This approach offers a powerful tool for studying gene function, gene networks, and regulatory elements in plants.22,23
3. Cas12a (previously known as Cpf1): Cas12a is an alternative RNA‑guided endonucle ase that was first identified in the bacterium Francisella novicida. Cas12a exhibits a key distinction from Cas9 in its ability to create staggered breaks (i.e., sticky ends).24 When Cas12a cleaves DNA, it produces overhangs of five base pairs, which proves advanta geous for specific applications that necessitate the generation of single‑stranded DNA, like HDR experiments. Moreover, Cas12a is well‑suited for targeting genomic regions with a high adenine‑thymine (A‑T) content since its PAM does not rely on the presence of guanine‑cytosine (G‑C) pairs.24
4. Cas13 (previously known as C2c2): Cas13 is an RNA‑guided nuclease that distinguishes itself from Cas9 by its ability to target RNA instead of DNA, and it has been found in different bacterial species. This unique characteristic makes Cas13 particularly valu able for generating transient modifications in signaling molecules, specifically RNA, as opposed to permanent alterations in the genome. Similar to Cas14, Cas13 exhibits non‑ specific cleavage of single‑stranded RNA when bound to its RNA target sequence.25 This RNA‑targeting capability renders Cas13 highly effective in targeting mRNA and serves as a useful mechanism for temporarily suppressing gene expression. As a result, this system can be employed as an alternative to small interfering RNA (siRNA) or short hairpin RNA (shRNA) for efficient and multiplexable knockdown of RNA.
5. Cas14 (or Cas12f): In contrast to other Cas enzymes, Cas14 exhibits a preference for single‑stranded DNA (ssDNA) rather than double‑stranded DNA (dsDNA). It displays a remarkable ability to cleave ssDNA with exceptional accuracy, as even a single mismatch in the target sequence can be detected. Similar to Cas12, Cas14 is considerably smaller in size compared to the conventional Cas9 protein. Its small size offers advantages for packaging into viral vectors, potentially facilitating its delivery into cells for genome edit ing applications. In addition, akin to Cas12a, Cas14 demonstrates non‑specific cleavage of non‑complementary ssDNA upon engagement with its target sequence.26
1.2.2 PreCise editing teChnologies
The efficacy of gene targeting technology largely depends on HDR for introducing desired sequence modifications, but the limited efficiency of HDR has hindered its application.40,41 To address this constraint, alternative genome editing technologies have emerged, including base editing and prime editing. These CRISPR/Cas‑based technologies offer precise sequence editing without requir ing DSBs or donor DNA, making them more efficient than HDR in plants. Initially developed for human cells, cytosine base editors (CBEs) and adenine base editors (ABEs) laid the foundation for the advancement of dual base editors and precise DNA deletions specifically tailored for plants. In this section, we provide a brief overview of the recently developed CRISPR/Cas‑based technologies used to achieve precise editing of plant genomes (Table 1.2).
Summary of CRISPR/Cas‑based Precise Editing Technologies in Plants
Editing Technology
APOBEC1‑CBE2/ CBE3/CBE4 Rat APOBEC1 nCas9 (D10A), dCas9 (D10A and H840A), or nCas9‑NG
hAID‑CBE3 Human AID nCas9, nSpCas9‑NG, nScCas9, or nCas9‑SpRY
ABE7.10 TadA‑TadA7.10 nCas9, nSpCas9‑NG, nSaCas9, or nScCas9
ABE‑P1S
ABE8e TadA8e nCas9, nCas9‑NG, nCas9‑SpG, or nCas9‑SpRY
ABE9 TadA9 nCas9, nSpCas9‑NG, nCas9‑SpRY, or nScCas9
or nCas9‑NG
PE2 M‑MLV‑RT (H9Y/D200N/ T306K/W313F/T330P/L603W) nCas9 (H840A) all kinds of base substitutions, precise insertions, deletions, and combinations of these sequence modifications
PEmax nCas9 (R221K/N394K/ H840A)
or C1‑C1786.10
1. CBE: T he initial version of CBE, known as CBE1, was created by linking a rat cyti dine deaminase, rAPOBEC1, to a dCas9.42 CBE1 induces the substitution of cytosine (C) to thymine (T) by deaminating C to uracil (U) on the non‑target DNA strand, which is then converted to T during DNA repair and replication. However, the presence of ura cil N‑glycosylase (UNG) in the cellular base excision repair (BER) pathway leads to low editing efficiency of CBE1 due to the elimination of uracil.42 To enhance efficiency, the second‑generation CBE, CBE2, was developed by adding a uracil DNA glycosylase inhibi tor (UGI) to the C‑terminal of CBE1, preventing UNG activity.42 CBE2 improves edit ing efficiency threefold and reduces unexpected indels.43 To further enhance efficiency, the third‑generation CBE, CBE3, was created by adding rAPOBEC1 and UGI to nCas9 (D10A).42,44,45 Although CBE3 does not cleave dsDNA, it creates an incision in the target strand to initiate the repair process. The fourth‑generation CBE, CBE4, improves deami nation activity by linking two UGIs to nCas9, resulting in increased base editing efficiency and reduced incidents of undesired C to A or G transversions compared to CBE3.46–48 In addition, the Mu Gam protein from bacteriophage was linked to CBE4, resulting in CBE4‑Gam, which further enhances product purity and reduces indel occurrences.48,49
2. ABE: A BE7.10 was the first ABE created by linking nCas9 (D10A) to a dimer compris ing a wild‑type adenine deaminase called TadA and an evolved adenine deaminase called TadA7.10.50 ABE7.10 allows the precise conversion of adenine (A) to inosine (I), which is then recognized as guanine (G) during DNA repair and replication processes.51 The editing window of ABE7.10 is located at positions 4–8 nt in the protospacer region. To enhance editing efficiency, ABE7.10 underwent codon optimization to improve its performance. In addition, a nuclear localization sequence (NLS) was added to facilitate the transport of ABE7.10 into the cell nucleus.52 ABEmax was developed by adding NLS sequences at both ends of ABE7.10. This modification further increased the editing efficiencies to less than 50% at most targets.50,53,54 ABEmax demonstrated successful A‑to‑G conversions in various target genes in rice plants. A simplified version called ABE‑P1S was devel oped (i.e., TadA7.10‑nCas9 vs. commonly used TadA‑TadA7.10‑nCas9), and it showed significantly higher editing efficiency in rice, indicating its potential for precise editing in plants.55 Later, by using another adenine deaminase variant TadA8e (V106W), the new ABE8e exhibited significantly improved A‑to‑G conversion and reduced off‑target effects.56,57 Further, TadA9, an improved adenosine deaminase by introducing V82S and Q154R mutations into TadA8e, was found to be compatible with various Cas variants. Thus, ABE9 with TadA9 should have quite relaxed PAM restrictions and demonstrate stronger editing capabilities with an expanded editing window.58
3. CGBE (C‑to‑G base editor): CBEs and ABEs are limited to inducing base transitions rather than base transversions. To overcome this limitation, a novel base editor known as CGBE has been developed. CGBE consists of a variant of the rAPOBEC1 cytidine deami nase (R33A), a nCas9 (D10A), and a UNG. Recent studies have demonstrated the success ful use of CGBE in achieving efficient C‑to‑G transversion in bacteria and mammalian cells.59,60 Further, a rice‑specific base editor called OsCGBE03 was created through codon optimization on UNG, which showed efficient C‑to‑G editing in five rice genes.61 The development of CGBE expands the repertoire of base editing tools, offering a powerful approach for generating diverse base substitution types in precise crop breeding and the creation of novel germplasm resources.
4. Dual base editor: A novel gene editing tool called the “saturated targeted endogenous mutagenesis editor” (STEME) has been developed for the simultaneous editing of cytosine and adenine bases in plants using a sgRNA.62 This system consists of a cytidine deami nase (APOBEC3A), an adenosine deaminase (ecTadA–ecTadA7.10), a nCas9 (D10A), and a UGI. STEME enables the conversion of cytidines to uridines and adenosines to inosines within the editing window, which are then replicated and repaired by the plant’s DNA
repair mechanisms, resulting in dual substitutions of C:G>T:A and A:T > G:C. To expand the editing capacity and target a wider range of sequences, an SpCas9–NG PAM variant is used in the STEME system, which recognizes NG PAM sequences.28 This allows for the editing of a larger number of target sites. The ability to perform dual base editing with STEME opens up possibilities for the directed evolution of endogenous plant genes in their native context. In addition, STEME can be utilized for modifying cis‑regulatory elements in gene regulatory regions and for high‑throughput genome‑wide screening in plants.
5. Mu ltinucleotide deletion: APOBEC–Cas9 fusion‑induced deletion systems (AFIDs) are a type of genome editing tool that combines the activity of APOBEC and Cas9 proteins to induce targeted deletions in DNA.63 APOBEC proteins are naturally occurring enzymes that can induce DNA mutations, particularly cytosine‑to‑uracil changes. AFIDs utilize two cytidine deaminases, namely hAPOBEC3A and the C‑terminal catalytic domain of hAPOBEC3B (hAPOBEC3Bctd), which could generate DNA deletions spanning from the targeted cytidine or a preferred TC motif to the DSB induced by Cas9, respectively.63 These predictable deletions ensure more consistent editing outcomes. AFIDs could be applied to create predictable multinucleotide deletions and study DNA regulatory regions and protein domains.
6. PE (prime editor): PEs are a class of CRISPR/Cas‑based genome editing tools that offer precise and versatile editing capabilities for targeted genetic modifications. They are designed to generate all 12 types of base substitutions, precise insertions, deletions, and combinations of these sequence modifications without the requirement for DSBs or the reliance on donor DNA templates. The prime editing system consists of a fusion pro tein composed of a nCas9 (H840A) (or dCas9), a Moloney murine leukemia virus reverse transcriptase (M‑MLV‑RT), and an engineered prime editing guide RNA (pegRNA). The pegRNA consists of a reverse transcriptase template (RTT, harboring designed edits) and a primer‑binding site (PBS, initiating reverse transcription) at the 3ʹ end of a sgRNA (target ing the specific location). PBS pairs with the single‑strand DNA nicked by nCas9, which allows reverse transcription and incorporation of the designed edits from the RTT into the genome. Subsequently, a series of processes, including equilibration, ligation, and repair, lead to the desired edit. Since its first introduction in 2019,64 several generations of PEs (PE1, PE2, PE3, PE3b, and subsequent iterations) have been developed to expand their functionality and improve editing efficiency, and the engineering of core editor protein and the redesigning of pegRNA have played a crucial role in the gradual improvement. Initially, PE1 was created by adding a wild‑type virus reverse transcriptase to nCas9 (H840A) to enable prime editing. To increase the editing efficiency, PE2 was developed by changing the wild‑type reverse transcriptase in PE1 with a modified version containing six specific mutations. Subsequently, by incorporating an additional nicking sgRNA to induce another cut, PE3 was introduced to further increase editing efficiency. However, the PE3 system, inducing two SSBs on complementary DNA strands, could lead to higher indel frequencies through the NHEJ repair pathway. To mitigate undesired indels, PE3b employs a specific sgRNA to induce the second nick after the incorporation of the designed edit into the targeted genome locus is done. In addition, inhibiting critical components of the DNA mismatch repair (MMR) pathway, like MLH1, has been shown to effectively enhance prime editing efficiency.65 PE4 and PE5 were generated by adding a dominant negative MMR effector (i.e., MLH1dn) to PE2 or PE3, respectively, which can help avoid MMR and thus enhance prime editing capacity and significantly increase editing efficiency by several folds. Furthermore, the ability to create nicks in the non‑target strand is essential for efficient prime editing. By switching the nCas9 (H840A) of PE2 with the SpCas9max variant containing two additional mutations (i.e., R221K and N394K), PEmax has achieved significantly improved prime editing efficiency.65–67 Prime editing has several advantages over conventional HDR strategies, including improved precise genome editing efficiency
and the ability to generate combinations of various sequence modifications at a relatively wide range of positions, which reduces constraints imposed by its PAM.64,68–72 However, the editing efficiency of PEs in plants is still unsatisfying, even with the utilization of various strategies like alternative reverse transcriptase orthologs, ribozymes for precise pegRNA production, temperature optimization, enhanced sgRNA scaffold modifications, and selective markers for cell enrichment.65,73–79 In addition, the capacity of PEs to induce larger genetic edits spanning hundreds of nucleotides and their level of specificity have yet to be verified in plants.68,72,80,81
1.2.3 deliVery MeChanisMs
To effectively utilize CRISPR/Cas9 technology in plants, it is crucial to have a reliable and universally applicable method for delivering the necessary CRISPR/Cas components into plant cells. However, the two commonly used delivery strategies, i.e., Agrobacterium ‑mediated trans formation and biolistics, are both inadequate to meet our needs. First of all, both strategies require tissue culture, which is time‑consuming and laborious. The recipient plant species avail able for Agrobacterium infection is increasing, but the editing efficiency still varies significantly among different genotypes, especially in monocots.101 Further, the intrinsic feature of random insertion of foreign DNA into the host genome stirs concerns over safety risks. Regarding biolis tics, the mechanical force used for delivery may cause damage to the host genome, and the deliv ery efficiency is still unsatisfying. As a result, there is an urgent need for innovative delivery strategies to overcome these challenges and enhance the application of CRISPR/Cas9 in plant research.
De novo induction of meristem is a promising approach that utilizes morphogenetic regulators to facilitate CRISPR/Cas‑mediated gene editing in plants. These regulators not only assist in trans forming recalcitrant cultivars but can also trigger the formation of new meristems in plants, elimi nating the need for tissue culture.102 A recent study demonstrated the effectiveness of this approach by injecting morphogenetic regulators and sgRNA expression cassettes‑loaded Agrobacterium into pruned sites of Cas9‑overexpressing tobacco plants (Nicotiana benthamiana).103 The resulting shoots directly gave rise to gene‑edited plants with inheritable mutations. This methodology holds poten tial for various plant species and can greatly expedite plant research. Virus‑assisted gene editing is another promising strategy that leverages the capabilities of plant viruses to generate gene‑edited plants without relying on tissue culture. Certain plant viruses can efficiently deliver sgRNA for genome modifications. Several single‑stranded RNA viruses, such as tobacco rattle virus and the single‑stranded DNA cabbage leaf curl virus, have been utilized for this purpose, and the editing efficiencies could reach 80%.104–109 Still, these viruses cannot deliver Cas9 and sgRNA expression cassettes together due to cargo limitations. To overcome this, researchers inserted Cas9 and sgRNA cassettes into the genomes of another two plant viruses with better capacity, which enabled systemic gene editing in tobacco plants.110,111 To address the limitation of heritability, sgRNAs were fused with RNA mobile elements and incorporated into tobacco rattle virus RNA2. These mobile ele ments guided the sgRNAs into shoot apical meristem cells, resulting in high efficiencies of heritable mutations in the offsprings.112
1.3 PART II. APPLICATIONS OF CRISPR/CAS IN PLANT FUNCTIONAL GENOMICS
CRISPR/Cas technology has provided plant scientists with a precise, efficient, and versatile tool for studying gene function, unraveling genetic networks, and accelerating crop improvement efforts. It has significantly advanced our understanding of plant functional genomics and has the potential to revolutionize plant breeding and agriculture. Here are some examples of its vast applications in plant functional studies:
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particularly injurious to the Wickson plums. The flat-headed apple tree borer[182] (Chrysobothris femorata Fabricius) is frequently found in the wood of wild plums and is sometimes a pest of the several cultivated species. It is treated as is the peach borer The shot-hole borer[183] (Eccoptogaster rugulosus Ratzeburg) a diminutive insect which deposits its eggs in the trunks or large branches of various members of the genus Prunus, may be regarded as an effect rather than a cause of disease, for it seldom injures perfectly healthy plum trees. The peach bark-beetle[184] (Phlœotribus liminaris Harris) is somewhat similar in its work to the shot-hole borer and like it is found for most part only in diseased and decrepittrees.
The plum aphis[185] (Aphis prunifolii Fitch) is sometimes very destructive to varieties of the native plums, especially the Americanas, and occasionally injures or even kills the young trees of the Domestica sorts. It is not a formidable foe in New York, and it is the exception when trees must be treated for it, the treatment being any of the contact solutions used against sucking insects. The cherry aphis[186] (Myzus cerasi Linnaeus) and the green peach aphis[187] (Myzus persicæ Sulzer) are much less common than the plum aphis on plum trees, but are sometimes abundant on foliage of this fruit and are combated in the same way as the more common aphis. Gillette enumerates two other aphids as attacking the plum in Colorado—the rusty brown plum louse[188] (Aphissetariæ Thomas) and the mealy plum louse[189] (Hyalopterus arundinis Fabricius).
Several scale insects infest the plum. Chief of these is the dreaded San José scale[190] (Aspidiotus perniciosus Comstock) known and feared by all fruit-growers in the United States. The lime and sulphur solution is now the most common and probably the most effective spray for this insect. The European fruit lecanium[191] (Lecanium corni Bouche) occasionally does a great deal of damage in New York and now and then destroys the whole crop in an orchard. The winter treatment for San José scale is used to control this pest, but usually such treatment is supplemented by a summer spray about July first with such contact sprays as whale oil soap and kerosene emulsion. Of the other scales[192] which feed upon plums and now and then become pestiferous the following may be named: The fruit pulvinaria (Pulvinaria amygdali Cockerell), the mealy bug (Pseudococcus longispinus Targioni), the scurfy scale (Chionaspis furfura Fitch.), the West Indian peach scale (Aulacaspis pentagona Targioni), the Putnam scale (Aspidiotus ancylus Putnam), the cherry scale (Aspidiotus forbesi Johnson), the walnut scale (Aspidiotus juglans-regiæ Comstock), Howard’s scale (Aspidiotus howardii Cockerell), the European fruit scale (Aspidiotus ostreæformis Curtis), the red scale ofCalifornia (Chrysomphalus aurantii Maskell), theoyster-shell scale(Lepidosaphesulmi Linnaeus),andthesoftscale(Coccushesperidum Linnaeus).
Wild plums of several species seem to be favorite feeding grounds for the tent caterpillar[193] (Malacosoma americana Fabricius), but cultivated plums are not so often attacked; the spraying with arsenites usually given for the curculio is fatal to this pest as well. The spring canker-worm[194] (Paleacrita vernata Peck) and the fall canker-worm[195] (Alsophila pometaria Harris) are other caterpillars which often do much damage unless checked by destructive measures, of which the best are the arsenical sprays. The larvæ of a considerable number of other moths and butterflies are often found on plumfoliage butseldom in suchnumbers asto requiresystematic destruction.
The eye-spotted bud-moth[196] (Tmetocera ocellana Schiffermuller) is a rather serious pest in plum nurseries where the larvæ eat into the young buds at the time they are opening, often destroying the shoots. Sometimes the larvæ are found destroying buds on old trees. The arsenical sprays are used to destroy this pest. The peach twig-moth (Anarsia lineatella Zeller) is now and then found in plum twigs butisseldomclassed as a plumpest.
The clover mite[197] (Bryobia pratensis Garman) formerly considered a serious pest of the plum only in the far West, is now abundant in plum orchards in New York. It can be controlled by applications of lime and sulphur The bumble-flower beetle[198] (Euphoria inda Linnaeus) is reported by Goff to be occasionally very injurious to plums in Wisconsin. Remedies seem not to have been tried. The larvæ of the cherry slug[199] (Selandria cerasi Peck) is not infrequently found doing damage to plum foliage and occasionally the larvæ of one or two other saw-flies feed on the plum. These are all overcome by the use of arsenical sprays. At least one of the curious insects know as “case-bearers” attacks the plums. The troublesome one is the pistol case-bearer[200] (Coleophora malivorella Riley), which is not often a
pest but has sometimes done considerable damage. Attempts to check this insect with the arsenical sprays have usually proved successful. The plum tree is a host-plant of the hop aphis[201] (Phorodon humuli Schrank). So much does this insect feed on the plum that the destruction of plum trees in the vicinityof hopfields is recommended to hop-growersby Riley
CHAPTER III
LEADING VARIETIES OF PLUMS.
ABUNDANCE
ABUNDANCE
Prunus triflora
1. Am Pom Soc Rpt 96 1887 2. Am Gard 9:360 1888 3. Ga Hort Soc Rpt 51, 52, 53, 99 1889 4. Bailey Ann Hort 103 1889 5. Am Pom Soc Rpt 106, 125 1891 6. Am Gard 13:700 1892 7. Rural N Y
52:666. 1893. 8. Cornell Sta. Bul. 62:19, 27, 32. 1894. 9. Tex. Sta. Bul. 32:488. 1894. 10. Rev. Hort. 160. 1895. 11. Mich. Sta. Bul. 118:52. 1895. 12. Cornell Sta Bul 106:41, 43, 44, 47, 48, 49 1896 13. Va Sta Bul 67:96 1896 14. Cornell Sta Bul 131:195 1897 15. Ibid 139:37, 38, 39, 40 1897 16. Am Pom Soc Cat 26 1897 17. Cornell Sta Bul 175:141, 142, 143 1899 18. Waugh Plum Cult 132, 135 1901 19. Mich Sta Bul 169:242, 248 1899 20. Ont Fruit Exp Sta Rpt 15 1902 21. Ohio Sta Bul 162:254, 255 1905 22. Texas Nur Co Cat 9 1907 23. Ga Sta Bul 68:7, 28 1905
Yellow Fleshed Botan 5 Yellow Japan 12 Yellow Japan 8, 14
Though Abundance has been in America only a quarter of a century, it is now about as well known as any other plum, being probably the best known of the Triflora plums. The two chief assets which have given the variety its great popularity so quickly are adaptability to a wide diversity of soils and climates and, as its name implies, abundance of fruit, for it bears not only heavily but yearly. As a market plum Abundance has been overplanted since it ships and keeps poorly, is much subject to brown-rot, matures unevenly and drops rather too readily as it ripens. Whether for market or home use, the fruit of this variety should be picked before it is quite ripe as it develops in flavor best when so picked and the dropping and rot are thus avoided to some extent. It is an exceedingly variable plum and undoubtedly several well marked strains could be selected, some of which are not as hardy or otherwise as valuable as others. While Abundance has passed the heyday of its popularity it is still one of the most desirable of the Triflora plums.
This variety was imported from Japan by Luther Burbank in 1884, and was introduced by John T. Lovett, Little Silver, New Jersey, under the name Abundance, in 1888. A large number of Japanese plums that have since been introduced have proved to be either identical or so nearly like the Abundance that much confusion has arisen. Abundance was first known as Botan, but that name was
dropped as it refers to a group of plums in Japan rather than to a variety. Babcock, which is said to have been imported by Burbank in 1885 and named for Colonel E. F. Babcock, a nurseryman of Little Rock, Arkansas, has been described by Bailey as indistinguishable from Abundance. Botankio, described in the Georgia Horticultural Society Report for 1889, proved to be the Abundance as tested at the Cornell Experiment Station. The Chase plum, also disseminated in New York under the name Yellow Japan, was bought by the R. G. Chase Company, Geneva, New York, for the Abundance, but as it was thought to blossom and fruit later than that variety, it was distributed as a new plum; in 1897 Bailey considered it the same as Chabot, but in 1899 he stated that it and Abundance were identical. The Douglas plum is also identical. Dr. J. T. Whitaker of Tyler, Texas, imported this variety and introduced it in 1886 under the name of Hytankayo. Bailey, who tested Whitaker’s variety from trees obtained from T. V. Munson, Denison, Texas, found a yellow-fruited strain and to distinguish the purple form named the latter Munson.[202] As this name had been applied to a native plum, R. H. Price, of the Texas Experiment Station, in 1894 renamed the variety calling it Douglas. [203] There have been two types of this Douglas plum disseminated; Bailey, in 1899, found no difference between it and Abundance except that the Douglas seemed to have a little drier flesh; others testing Douglas found it to be identical with the Chabot. Burbank No. 2, imported by Luther Burbank in 1885 and introduced by him in 1889, is very similar if not identical with the Abundance. Oriole, recently introduced by the Texas Nursery Company, Sherman, Texas, is so nearly like Abundance as to be unworthy of a separate name. The American Pomological Society added Abundance to its fruit catalog list in 1897.
Tree large, vigorous, vasiform, open-topped, hardy in New York, very productive, susceptible to attacks of shot-hole fungus; branches rough, dark ash-gray, inclined to split when overloaded, with few, slightly raised lenticels; branchlets slender, short, with short internodes, red early in the season changing to dark brown, glossy, glabrous, with numerous, inconspicuous, small lenticels; leaf-buds small, short, conical, plump, free
Leaves folded upward, narrow-obovate or oblanceolate, peach-like, one and three-eighths inches wide, three and one-quarter inches long, thin; upper surface light green, smooth, glabrous, with grooved midrib; lower surface pale green, pubescent on the midrib and larger veins; apex taperpointed, base cuneate, margin very finely serrate, with small, brownish glands; petiole three-eighths inch long, slightly pubescent along one side, reddish, glandless or with from one to five small, globose, green or reddish glands usually on the stalk
Blooming season early; flowers appearing with the leaves, medium in size; borne in clusters on lateral buds and spurs, in pairs or in threes; pedicels of medium length and thickness, slightly pubescent, greenish; calyx-tube green, obconic, glabrous; calyx-lobes obtuse, with ciliate margins, glabrous, erect; petals broadly oval, entire, abruptly clawed; anthers yellowish; filaments of average length; pistil glabrous, equal to the stamens in length
Fruit early, season short; one and three-eighths inches in diameter, roundish-ovate, halves nearly equal, slightly compressed; cavity medium in depth and width, abrupt, regular; suture shallow, distinct; apex pointed; color pinkish-red changing to darker red, mottled, with thin bloom; dots numerous, of medium size, russet, conspicuous; stem one-half inch long, glabrous, parting easily from the fruit; skin thin, tough, bitterish, separating readily; flesh yellow, very juicy, tender and melting, sweet except next to the pit, pleasantly aromatic; good; stone clinging, three-quarters inch by one-half inch in size, oval, somewhat compressed, pointed, rough, ridged along the ventral suture; dorsal suture grooved
California 16 D’Agen 2, 10, 11, 17, 24 D’Agen 3, 5, 8, 13, 20 D’Ast 13, 17 Date 21 Datte 17 De Brignole 17 D’Ente 13, 17, 24 D’Ente d’Agen 13 Datte Violette 1, 13, 17 Die Blaue Dattelpflaume 17 Die Blaue Dattelpflaume 1 Du Roi 17 French 20 French Prune 15, 18, 23, 25 French Prune (?) 2, 12, 14, 15, 26 Lot d’Ente 18 Petite 20 Petite d’Agen 14, 20, 26 Petite Prune 18 Petite Prune 23 Petite Prune d’Agen 25
Prune d’Agen 5, 6, 7, 8, 15, 17, 19, 25. Prune d’Agen 14, 16, 18, 23. Prune de Brignole (of some) 5, 7, 17. Prune d’Ante 3. Prune d’Ast 5, 7, 10, 11. Prune d’Ente 7, 12, 18. Prunier d’Agen 3. Prunier d’Agen 6. Prune d’Ente 22. Prune du Roi 10. Robe de Sergent 3, 5, 7, 8, 11, 13, 17, 18. Robe de Sargent 10, 12. Roi d’Agen 2. Saint Maurin 2, 4. St. Maurin 5, 7, 10, 17. Saint Mauriniana 4. Violette Dattelzwetsche 17.
Agen is the plum par excellence for prune-making in France and America. Several qualities make it admirably fit for curing into prunes. To begin with, it has a high percentage of sugars and solids so that the plum cures readily into a firm, sweet, long-keeping prune which in cooking needs comparatively little sugar; again, the trees bear regularly, abundantly and the plums are uniform in size,— productiveness, regular bearing and uniformity of size of fruit being necessary attributes of a good prune-making plum; lastly, it hangs well on the tree as it ripens and afterwards so that the curing really begins on the tree. Besides making most excellent prunes, the Agen is a very good dessert plum—one of the best—and ought to be in every home orchard and, where it attains sufficient size, in every commercial plantation. Lack of size is the defect in this variety which has kept it from being more largely grown outside of prune-making
regions. If by pruning, thinning and other cultural treatment the size of the plums could be increased, the Agen should prove a valuable commercial fruit in New York.
The name of this variety is derived from Agen, a region in France where it is extensively grown. Tradition says that on their return from the Crusades, the Benedictine monks brought with them from Turkey or Persia what was then known as the Date plum and planted it in the garden of their abbey on the River Lot, in the vicinity of Bordeaux, France, and that afterwards this became the Agen. Its first recorded importation into the United States was made in 1854 by the United States Patent Office, though it was described by Prince as early as 1832. The most important introduction was made, however, in 1856, when Louis Pellier of San Jose, California, introduced Agen on the Pacific Coast, where it soon became and still is the leading plum, though with curious persistency the fruit-growers there call it the “French Prune” and the “Petite Prune.” In 1862 this variety was added to the fruit catalog list of the American Pomological Society. There are many strains of Agen in America, due to the numerous importations of grafts from various parts of France, where the plum orchards are frequently grown from seedlings or from sprouts; some of these strains are worthy of varietal recognition.
Tree of medium size, upright-spreading, dense-topped, hardy, very productive; branches ash-gray, smooth, with numerous, large, raised lenticels; branchlets slender, short, with short internodes, greenish-red changing to dark brownish-drab, dull, pubescent, with small lenticels; leafbuds of medium size and length, conical, free.
Leaves folded upward, obovate or oval, one and three-quarters inches wide, three and one-quarter inches long, velvety; upper surface with few fine hairs and a narrow, grooved midrib; lower surface pale green, thickly pubescent; apex abruptly pointed, base acute, margin doubly serrate; petiole one inch long, slender, pubescent, tinged red, with two or three small, globose, greenish-brown glands usually on the stalk
Season of bloom intermediate in time and length; flowers appearing after the leaves, one and one-eighth inches across, white; borne on lateral spurs, singly or in pairs; pedicels five-eighths inch long, thick, glabrous except for a few short hairs, greenish; calyx-tube green, campanulate, pubescent; calyx-lobes obtuse, somewhat pubescent within, with
glandular margin, reflexed; petals broadly oval or obovate, entire, tapering to short, broad claws; anthers yellowish; filaments five-sixteenths inch long; pistil glabrous, equal to the stamens in length
Fruit late, season short; one and one-half inches by one and one-eighth inches in size, obovate, the base necked, halves equal; cavity shallow, narrow, flaring; suture very shallow, indistinct; apex roundish or flattened; color reddish or violet-purple, overspread with thin bloom; dots numerous, small, brown, obscure, clustered about the apex and interspersed between russet flecks; stem thick, seven-eighths inch long, glabrous, adhering well to the fruit; skin thin, tough; flesh greenish-yellow, tender, sweet, aromatic; very good to best; stone semi-free or free, seven-eighths inch by one-half inch in size, oval, flattened, with pitted surfaces, rather abrupt at the base and apex; ventral suture somewhat narrow, furrowed, with distinct wing; dorsal suture widely grooved
AITKIN
Prunus nigra
1. Minn Hort Soc Rpt 426 1896 2. Wis Sta Bul 63:24, 27, 28 fig 11, 43 1897 3. Jewell Nur Cat 1899-1906 4. Waugh Plum Cult 169 1901 5. Can Exp Farm Bul 43:29 1903 6. Ia Hort Soc Rpt 227 1904
Aitken 4. Beatty 6 incor. Itasca 1 incor.
Aitkin is very favorably mentioned in the references given above and undoubtedly has value for the Northwest. It was listed in the catalog of the American Pomological Society in 1899. The variety was found growing wild in Aitkin County, Minnesota, by D. C. Hazelton on land adjoining his farm. It seemed to possess merit and was introduced in 1896 by the Jewell Nursery Company of Lake City, Minnesota. Because of having originated near Itasca Lake, it has been confused with the Itasca plum, which preceded it by nearly ten years. The following description is a compilation:
Tree vigorous, productive, ripening its wood very early; fruit earliest in season of its group; large for its class, oval, deep red, with no bloom; skin
thin, not astringent; flesh yellow, juicy, sweet and rich; good; stone large, oval, flattened, clinging.
1. Vt Sta Bul 67:5 1898 2. De Vries Plant Breeding 213 1907
Although it is over a decade since Alhambra was offered to fruitgrowers, it has made little headway in popularity and is chiefly of interest because of its breeding. It is not often that we can trace the pedigree of a plant for more than one or at the most two generations, but in Alhambra we are particularly fortunate. Luther Burbank, the originator, began by crossing Kelsey and Pissardi, and the offspring from this cross was fertilized with Agen pollen. This tri-hybrid was in turn fertilized with pollen from a complex hybrid of a cross of Prunus simonii and Prunus triflora pollinated by a cross of Prunus americana and Prunus nigra. As might be expected, the offspring of this final cross was extremely variable and from it was selected the Alhambra. The variety was named by the originator in 1898.
The fruit as described by Waugh is “egg-shaped, large or very large; cavity medium shallow, abruptly rounded; suture shallow; apex pointed; color dark, dull red; dots many, small, yellowish; bloom thin, purplish; skin firm; flesh yellow inside, reddish outside; stone medium to large, flat, pointed, nearly smooth, clinging; flavor brisk subacid; quality first rate.”
ALTHAM
Prunus domestica
1. Jour. Hort. N. S. 17:228. 1869. 2. Lange Allgem. Garten. 2:419. 1879. 3. Oberdieck Deut. Obst. Sort. 432. 1881. 4. Lauche Deut. Pom. 19, Pl. IV. 1882. 5. Hogg Fruit Man. 692. 1884. 6. Mathieu Nom. Pom. 420. 1889. 7. Gaucher Pom. Prak. Obst. 94, Pl. 1894. 8. Soc. Nat. Hort. France Pom.
Althann’s Reine-Claude 7. Hathen’s Red Gage 1. Reine-Claude Rouge de Hathen 1. Reine-Claude d’Althann 5, 6, 7. Reine-Claude Comte Althan 5, 6. Reine-Claude de Comte Hathem 5, 6. Reine-Claude du Comte Hathem 6, 7. Reine-Claude du Comte d’Althan 8. Reine-Claude Althan’s 5, 6, 7. Reine-Claude Comte d’Althan 6. Reine-Claude d’Althan 8. Reine-Claude d’Althan 6, 7, 10. Reine-Claude Rouge Comte Althan 6, 7. Reine-Claude Rouge du Comte Hethau 6. Reine-Claude rouge du comte Hethan 7. Reinette Claude Comte d’Althan 9 Reinette Claude d’Althan 9
Altham is an excellent plum for dessert or home use. The color is a trifle too dull for market purposes and yet it is better colored than McLaughlin, which sells fairly well. The fruit is the type of the last named plum but is later. In Europe this variety is well known and highly esteemed for its quality, but unfortunately it is almost unknown in America. The variety is well worth trial in this country as a fine plum of the Reine Claude group. Altham is a seedling of Reine Claude, raised by Herr Prochaska, gardener to Count Michael Joseph Althann, of Swoyschitz, in Bohemia. It was noted in the English Journal of Horticulture for 1869 as a new plum sent out by Thomas Rivers.
Tree of medium size, upright-spreading, dense-topped, productive; leafscars prominent; leaves folded upward, obovate, two and one-quarter inches wide, nearly four inches long, very thick, leathery; margin doubly crenate, with few, small, dark glands; petiole thick, with from one to four globose, yellowish-green glands on the stalk; season of bloom intermediate, short; flowers appearing after the leaves, one inch across, yellowish at the apex of the petals; borne on lateral buds and spurs, singly or in twos.
Fruit mid-season; one and one-half inches by one and five-eighths inches in size, oblate, strongly truncate at the base, compressed; color dark purplish-red over a yellow ground, covered with thick bloom; dots russet surrounded with a dark red ring; stem adhering strongly to the pulp; flesh light golden-yellow, firm but tender, sweet, mild, pleasant; very good to best; stone semi-clinging, seven-eighths inch by five-eighths inch in
size, flattened, irregular-oval, with pitted surfaces, contracted at the base into a short oblique neck; ventral suture prominent, heavily furrowed, often with distinct wing; dorsal suture wide, deep
America is illustrated and described in full chiefly because it is the most promising cross between Prunus munsoniana and Prunus triflora. The fruit of the variety is unusually attractive in appearance, golden-yellow with a red cheek and waxy lustre turning currant-red when ripe, ships exceptionally well and is of very good quality for cooking, but is without merit as a dessert plum. The trees are large, very vigorous, as hardy as either of its parents or possibly more so, and enormously productive. The qualities of fruit and tree are such that the variety ought to succeed in commercial plantations where any but the hardiest native plums are cultivated. America is almost phenomenally free from rot, considering its parentage.
This variety is one of Luther Burbank’s productions, grown from a seed of Robinson fertilized by pollen from Abundance. It was introduced by the originator in 1898 and has been since that time well tested at several places in the eastern states and is very generally well spoken of for a plum of its kind for the East.
Tree large, vigorous, spreading, somewhat open-topped, hardy, very productive; branches roughish and with cracked bark, slightly zigzag, dark ash-gray, with numerous, conspicuously raised lenticels; branchlets willowy, long, with short internodes, green with a reddish tinge changing to dark chestnut-red, glossy, glabrous, with numerous, small, raised lenticels; leaf-buds small, short, conical, free.
Leaves folded upward, broadly lanceolate, peach-like, one and one-half inches wide, three and one-fourth inches long, thin; upper surface reddish late in season, smooth and glossy, with deeply grooved midrib; lower surface light green, sparingly pubescent along the midrib and larger veins which are more or less red; apex taper-pointed, base abrupt, margin finely and doubly crenate and with numerous, small, dark glands; petiole one-
half inch long, tinged red, pubescent along one side, glandless or with one or two small globose, reddish glands on the upper part of the stalk.
Blooming season intermediate and long; flowers appearing after the leaves, one-half inch across, white; borne in clusters on short lateral spurs and buds, in pairs or in threes; pedicels five-sixteenths inch long, slender, pubescent, green; calyx-tube greenish, obconic, glabrous; calyx-lobes obtuse, with a trace of red along the margin, glandular-serrate, glabrous, with marginal hairs, erect; petals small, roundish, entire, tapering abruptly to narrow claws; anthers yellowish; filaments three-sixteenths inch long; pistil glabrous, longer than the stamens.
Fruit early, season of medium length; one and three-eighths inches in diameter, roundish-oval, halves equal; cavity shallow, flaring; suture shallow, a distinct line; apex roundish; color clear, dark, currant-red over golden-yellow, mottled, with thin bloom; dots numerous, small, whitish, inconspicuous; stem slender, one-half inch long, glabrous, adhering to the fruit; skin thin, bitterish, separating readily from the pulp; flesh yellow, juicy, fibrous, somewhat tender, sweet, not high in flavor; fair in quality; stone clinging, seven-eighths inch by one-half inch in size, oval, pointed, with pitted surfaces, broadly ridged along the ventral suture; dorsal suture grooved.
AMERICAN
1. Oregon Sta. Bul. 61:17, 18. 1900.
American Seedling 1.
American originated with a Mr. Peterson of Elkton, Douglas County, Oregon, as a sprout from an old tree. It has never been extensively disseminated, but seems to be a variety of considerable promise. The fruit as grown on the Station grounds resembles Hand rather closely; is large for a plum of its type, is a handsome golden color, is high in quality and will probably keep and ship well. Too little is known of its tree-characters to recommend it unqualifiedly.
Tree above medium in size, vigorous, round-topped, dense, productive; branches numerous; branchlets thick, marked by grayish scarf-skin;
Prunus domestica
leaves flattened, oval or obovate, two and one-quarter inches wide, four and one-half inches long, dark green; margin serrate or crenate; blooming season intermediate, short; flowers appearing after the leaves, one and three-eighths inches across, singly or in twos, fragrant
Fruit mid-season; very large, roundish-oblate, truncate, golden-yellow, indistinctly streaked with green, mottled, covered with thin bloom; flesh light golden-yellow, tender, sweet, pleasant flavor; good to very good; stone clinging, one inch by three-quarters inch in size, broadly oval, flattened, surfaces pitted; dorsal suture wide, deep.
Of the origin of this very good Americana variety little is known except that it probably came from Missouri, as it was introduced, in the fall of 1859, by the Osceola Nursery Company, Osceola, Missouri. Although an old variety it was not listed by the American Pomological Society until 1899. In regions where Americana plums are grown, American Eagle ought to be better known, its chief defect being the dull color of the fruit.
Tree vigorous, spreading; leaves large; petiole glandular Fruit midseason; large, varies from roundish-oval to nearly oblate, dark red, covered with thick bloom; stem short, pubescent; flesh yellow, juicy, fibrous, sweet, aromatic, with characteristic Americana flavor; of good quality; stone clinging, three-eighths inch by one-half inch in size, roundish, turgid, conspicuously winged; surface smooth.
AMES
Prunus americana × Prunus triflora
1. Vt Sta An Rpt 12:220 1899 2. Ia Hort Soc Rpt 112 1899 3. Ia
Am. Hort. Man. 293. 1903. 6. S. Dak. Sta. Bul. 93:9. 1905. 7. Ill. Hort. Soc. Rpt. 422. 1905. 8. Rural N. Y. 65:730. 1906.
De Soto × Oregon No. 3 6. Japan Hybrid No. 3 2.
Though Ames has been known to the public scarcely ten years, its good qualities have given it relatively high rank among Americana plums with which it must be compared. Though supposed to be a cross between Prunus americana and Prunus triflora, the variety shows few, if any, traces of the Triflora parentage, except, possibly in the shape and color of the fruit. The variety is distinguished from other Americana plums by reddish dots on the fruit instead of the yellowish dots commonly found on the plums of this species. The fruit of Ames is very attractive in color, the quality is fair, it keeps and ships well and it is fairly free from rot, characters which make it desirable where the native plums are grown.
This variety was produced by Professor J. L. Budd[204] of Ames, Iowa, by crossing De Soto with pollen of a “large Japanese plum received from Oregon.” For a long while it was known as De Soto × Oregon No. 3 and as Japan Hybrid No. 3, but was named Ames by Professor John Craig, now of Cornell University
Tree of medium size, spreading, dense-topped, hardy, productive; branches roughish, thorny, the trunk shaggy, dark ash-brown, with numerous, large, raised lenticels; branchlets willowy, thick, long, with long internodes, green changing to dark chestnut-red, glossy, glabrous, thickly strewn with conspicuous, large, raised lenticels; leaf-buds small, short, obtuse, plump, appressed.
Leaves falling early, flattened, oval, two inches wide, four inches long; upper surface dark green, glabrous, slightly rugose; lower surface light green, pubescent; apex taper-pointed, base abrupt, margin coarsely serrate, the serrations ending in hair-like tips, eglandular; petiole seveneighths inch long, slender, pubescent, tinged red, glandless or with from one to three globose, greenish-red glands.
Blooming season medium in time and length; flowers appearing after the leaves, nearly one inch across, white; borne in clusters on lateral buds and spurs, in threes or fours; pedicels one-half inch long, slender, glabrous, greenish; calyx-tube green, campanulate, glabrous; calyx-lobes narrow, somewhat acute, reflexed, pubescent on the inner surface, the
margin faintly pubescent and with a trace of red; petals small, oval, somewhat dentate, tapering below to long, narrow, slightly hairy claws; anthers yellowish; filaments five-sixteenths inch long; pistil glabrous, equal to the stamens in length, frequently defective
Fruit mid-season, one and seven-sixteenths inches by one and five sixteenths inches in size, ovate or oval, sides compressed, halves equal; cavity shallow, narrow, flaring; suture a line; apex roundish; color light to dark red over a yellow ground, covered with thin bloom; dots numerous, small, brownish-red; stem slender, glabrous; skin medium in thickness and toughness, adhering; flesh golden-yellow, juicy, coarse, fibrous, tender and melting, semi-sweet; of fair quality; stone nearly free, one inch by fiveeighths inch in size, irregular-oval, flattened and elongated at the base, abruptly pointed at the apex, very smooth; ventral suture winged and furrowed; dorsal suture acute
