Delivery of Genetic Materials: Introducing genetic material into plant cells.
Regeneration: Regenerating plants from the transformed cells using tissue culture.
Selection: Differentiating transformed cells from non-transformed cells.
Delivery Methods
Challenges
Plant cells are surrounded by a cell wall, making it difficult to deliver DNA.
Main Methods
Gene Gun (Particle Bombardment): Used with calli.
Protoplasts: Utilizes PEG or other methods for DNA delivery.
Agrobacteria-mediated Transformation: A biological method using Agrobacterium.
Gene Gun Method
Process:
DNA-coated gold particles are loaded onto a plastic disk.
Helium gas is used to propel the disk.
The gold particles are shot through a screen.
The particles bombard the target plant cells.
Caveats of Biolistic Transformation
Genome Damage:
Extreme genome damage can occur, including chromosome truncations, large deletions, partial trisomy, and chromothripsis.
Transgenic events may contain megabase-scale arrays of introduced DNA mixed with genomic fragments.
These fragments are assembled by nonhomologous or microhomology-mediated joining.
Regulatory Challenges: Due to the potential for significant genome damage, gaining regulatory approval for commercial applications can be difficult.
Tissue Culture
Definition
Regenerating plants in vitro using tissues, cells, or single cells.
Process
Tissues/cells are cultured on a nutrient medium in a sterile environment.
Carefully controlled environmental conditions, such as temperature, light, and humidity, are essential.
Applications
Plant Propagation: Widely used for mass clonal propagation.
Conservation: Elite varieties can be clonally propagated, and endangered plants can be conserved.
Disease Elimination: Virus-free plants can be produced by meristem culture.
Germplasm Conservation: Used for germplasm conservation.
Stress Tolerance: Stress-tolerant plants (e.g., drought and salinity) can be produced.
Research: Serves as a powerful tool for studying plant growth, development, and metabolism at the cellular and molecular levels.
Production of Pharmaceuticals: Used in the production of pharmaceuticals, phytochemicals, and secondary metabolites of medicinal and industrial importance.
Transgenic Plant Production: An indispensable tool for transgenic plant production and gene editing.
MS Media
Development: In 1962, Toshio Murashige and Folke Skoog published the composition of MS medium, the most widely used medium for tissue culture.
Background: Murashige was a doctoral student in Skoog’s lab, and they developed MS medium while trying to discover new hormones from tobacco leaf extracts to enable better growth.
Significance: MS medium seems to be ideal for many cultures since it has all the nutrients that plants require for growth and contains them in the proper relative ratios.
Components: The medium has high macronutrients, sufficient micronutrients, and iron in a slowly available chelated form.
Impact: The success of tobacco culture using MS medium laid the foundation for future tissue culture work.
Legacy: The article by Murashige and Skoog (1962) remains one of the most highly cited papers in plant biology.
Liquid and Solid Culture Medium
Tissue cultures can be grown in both liquid and solid culture media.
Filter paper bridges can be used in liquid media.
Plant Regeneration
Brassica juncea plants can be produced from hypocotyl explants.
Shoots are produced when a combination of auxin and cytokinin is used.
Key stages include callus formation, shoot development, elongation, and transfer to pots.
Organogenic Callus-Based System
Stages:
Callus formation
Callus with early shoot development
Callus with more developed shoots.
Anther/Microspore Culture
Definition: The culture of anthers or isolated microspores to produce haploid plants.
Use: Microspore culture has become a powerful tool in plant breeding.
Process:
Embryos can be produced via a callus phase or direct recapitulation of zygotic embryo development.
Late uninucleate to early binucleate microspores are the best explants for embryogenesis.
Somatic embryos develop into haploid plants.
Doubled haploids can be produced by chromosome-doubling techniques.
Advantages: Microspore culture enables the production of homozygous plants in a short period compared to conventional breeding techniques.
Applications: Useful in mutant isolation, gene transfer, studies of storage product biochemistry, and physiological aspects of embryo maturation.
Somatic Embryos
Somatic embryos can be regenerated from an anther in culture.
Meristem Culture
Use: Production of pathogen-free plants using apical meristem tips.
Nomenclature: Referred to as meristem culture, meristem tip culture, or shoot tip culture.
Application: Commonly used in the elimination of viruses in many species (e.g., potato).
Rationale: Apical meristems in plants are suitable explants for the production of virus-free plants since the infected plant’s meristems typically are either nearly or totally virus-free.
Enhancement: Meristem culture in combination with thermotherapy has resulted in successful production of virus-free plants when meristem culture alone is not successful.
Carlos Miller, a postdoc in Skoog’s laboratory, is credited with the discovery of cytokinin.
Miller and his co-workers (1954) isolated and purified the cell division substance in crystallized form from autoclaved herring fish sperm DNA.
The first naturally occurring cytokinin was isolated and crystallized simultaneously by Miller and D.S. Lethum (1963–65) from the milky endosperm of corn (Zea mays) and named zeatin.
Lethem (1963) proposed the term cytokinins for such substances.
Zeatin Identification
David Stuart Letham and co-workers (Letham et al. 1964) identified zeatin (Z), the first cytokinin identified in extracts from plants.
Auxin Overproduction Mutant yuc1-D
Mutant yuc1-D exhibits auxin overproduction.
Role of Flavin Monooxygenase-Like Enzymes
YUCCA encodes a flavin monooxygenase-like enzyme that catalyzes hydroxylation of the amino group of tryptamine, a rate-limiting step in tryptophan-dependent auxin biosynthesis.
Description of Discovery
Zhao et al. (2001) characterized a dominant Arabidopsis mutant, yucca, which contains elevated levels of free auxin.
yucca mutants possessed long hypocotyls and displayed other phenotypes typical of elevated auxin levels.
yucca plants contained at least 50% more free IAA than wild-type plants.
Floral Dipping
Floral dipping has revolutionized Arabidopsis transformation.
Antibiotics such as Kanamycin or herbicides (Basta) are used to kill non-transformed plants.
Controversial Reports of Plant Transformation
DNA Uptake in Pollen (Hess 1980)
Pollen from a white-flowering petunia was soaked in DNA extracted from a red-flowering petunia.
Some resulting seeds produced plants with partially or fully red flowers.
The author concluded that the DNA must have been taken up by pollen and passed onto the seedling from the fertilization process.
No one has been able to repeat this work, and the most plausible explanation is pollen contamination.
Pollen Tube Pathway (Luo and Wu 1988)
DNA is inoculated into the hollow pollen tube, where it finds its way to the freshly fertilized egg for incorporation into the DNA of the young zygote.
Transformation was confirmed using reliable molecular techniques, but the patterns of DNA hybridization were unusual.
Shou et al. (2002) performed an extensive study and concluded that it was not reproducible.
Rye Floral Tiller Injection (de la Pe˜na et al. 1987)
Young floral tillers of rye were injected with DNA carrying a kanamycin resistance gene.
Molecular analysis seemed to show the presence of an intact transgene, but the most important results were only briefly described as “data not shown.”
This work has never been repeated.
Protoplasts
Protoplasts are cells which have had their cell wall removed, usually by digestion with enzymes.
Tape Arabidopsis Sandwich protoplast isolation
Time tape for the upper epidermis and the lower epidermis was stuck to clear 3 M tape.
Quick protoplast isolation
Only need 25 mg leaves for one transfection.
Use leaves within 30 min.
Both young and older leaves are fine.
Arabidopsis protoplasts transfected with an mRFP gene
High transfection efficiency >70%.
History of Protoplast regeneration and transformation
1971, from protoplasts to whole plants
Advantages:
Very easy to deliver DNA or protein into protoplasts
Methods
Nanoparticles
Cell-penetrating peptides
Particle bombardment
RNP
PEG
PEG-mediated transfection
Lipofection
Disadvantages
Very difficult to regenerate plants from protoplasts
Plant transformation
Delivery of genetic materials into plant cells
Regenerate plants from the transformed cells
Differentiate transformed from non- transformed (selection).
Improve transformation efficiency
Optimize the existing platforms
Using morphogenic factors
Effective selection markers
QuickCorn Method
An Improved Agrobacterium-Mediated Transformation
Original vs the improved methods: 165 days vs 51 days
Morphogenic Regulators
Baby boom and Wuschel Improve Monocot Transformation
GRF-GIF chimeric protein
A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants.
Across 15 experiments, the average regeneration efficiency of the GRF4-GIF1 chimera (65.1 \pm 5.0 %) was 7.8-fold higher than the empty vector control (8.3 \pm 1.9, P < 0.001)
A faster wheat transformation protocol: 56 days vs 91 days
Agrobacterium expressing a type III secretion system
Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation.
Agrobacterium-mediated delivery of AvrPto increases transient and stable transformation efficiency in multiple tissues of Arabidopsis and Nicotiana benthamiana. Similarly, enhanced AMT was observed when two other T3Es, AvrPtoB and HopAO1, were delivered via a T3SS. Moreover, this strategy works well in crop species (namely wheat, alfalfa and switchgrass), increasing AMT efficiency by 260%–400%.
Cut–dip–budding delivery system
Cut–dip–budding delivery system enables genetic modifications in plants without tissue culture
The cut-dip-budding (CDB) delivery method is tissue-culture free and does not need sterile condition. The CDB method is extremely simple and potentially applicable to many plants.