Introduction to Plant Biotechnology

Course Content Overview

• This is an introduction to plant biotechnology
• Topics covered include plant genome organization and expression, plant tissue cultures, and molecular biology techniques
• Molecular mapping, markers, plant breeding, and production/analysis of plant metabolites will be examined
• Covers gene/protein expression regulation, plant development/transport, abiotic stress responses, and biotic interactions

Plant Biotechnology Introduction

• Biotechnology uses living organisms' metabolic properties to produce valuable products
• Plants are a key source of primary and secondary metabolites
• These metabolites are used in pharmacy, biotechnology, and food technology
• Plant biotechnology can improve the quality/quantity of agricultural, ornamental, and horticultural plants
• It can also manipulate plants for better agronomic performance

Agricultural Biotechnology Uses and Aims

• Food security
• Stress-resistant crops help ensure supply
• Increases shelf life through genetic modification
• Land conservation
• Biofuels are a renewable energy source
• Carbon sequestration
• Genetically modified trees can absorb more CO2
• Reduced waste
• Fermentation produces useful byproducts
• Water preservation
• Agriculture on acid soils promotes conservation
• Reduced emissions
• Enzymes can break down pollutants
• Development of disease-free plants and medicines
• Use of animal feed, enzymes, and soil microbes
• Improved aquaculture and enhanced taste of foods

Primary vs. Secondary Metabolites

• Primary metabolites are involved in plant development
• Examples include fatty acids, lipids, sugars, amino acids, and nucleotides
• Secondary metabolites are produced in response to environmental interactions
• Examples include phenolic compounds, terpenes, carotenoids, and flavonoids

Challenges and Considerations of Plant Genomics

• Realizing the potential of lignocellulosic biomass requires the development of high-yielding crops/trees
• These should efficiently use resources and minimize disruption to food and fiber markets
• Bioenergy feedstock production must withstand changing climatic conditions and environmental impacts.
• This includes drought, extreme temperatures, pathogen pressure, which may threaten crop productivity
• Plant research faces challenges:
• Genetic diversity
• Frequent segmental and whole genome duplications
• Large gene families
• Sub-functionalization of member genes
• Plant genome features add complexity to eukaryotic gene expression and regulatory networks
• Complexity increases plants' adaptability but poses challenges
• Challenges exist in identifying and characterizing functional roles of genes responsible for plant phenotypes
• Approximately 40% of all genes in any sequenced plant genome have unknown functions

Plant Genomes

• Plant genomes are described by genome size, gene content, repetitive sequences, and polyploidy/duplication events
• Plants have mitochondrial and chloroplast genomes, but their nuclear genome is largest/most complex
• Nuclear genome size varies significantly without obvious functional significance
• The nucleus contains genetic material that governs cell heredity
• Mitochondria and chloroplasts have their own genomes
• These genomes govern their heredity, and they have their own ribosomes "mitochondrial and chloroplast ribosomes"
• 'Ribosomes' in eukaryotes refers refers to cytosolic ribosomes

Chloroplast Genome

• All plastids have a "chloroplast genome”, a circular DNA molecule
• Ranges in size from 120 to 217 kb
• Plant cells usually have many copies of chloroplast DNA, depending on cell type/differentiation
• Mesophyll cells in growing leaves have 20-40 copies of the chloroplast genome
• Amyloplasts in potato tubers / chromoplasts in tomato fruits have fewer copies
• Amyloplasts, chromoplasts, and chloroplasts of the same plant have identical genetic information

Mitochondrial Genome

• Mitochondrial DNA is a circular chromosome in the cellular organelles called mitochondria
• Mitochondria are located in the cytoplasm
• Sites of the cell's energy production/metabolic functions
• Offspring inherit mitochondria (and mtDNA) from their mother

Polyploidy

• Polyploidy is the heritable condition of possessing more than two complete sets of chromosomes
• Polyploids are common among plants, as well as among certain groups of fish and amphibians
• The polyploid offspring of two diploid progenitors is more vigorous and healthier than either parent
• Gene gives rise to the existence of multiple genes that perform the same function
• Polyploidy allows gene function diversification over time

Nuclear Genome Size Examples

• Wheat nuclear genome size = 15,966 Mb
• Onion nuclear genome size = 15,290 Mb
• Garden pea nuclear genome size = 3,947 Mb
• Corn nuclear genome size = 2,292 Mb
• Asparagus nuclear genome size = 1,308 Mb
• Tomato nuclear genome size = 907 Mb
• Sugar beet nuclear genome size = 758 Mb
• Apple nuclear genome size = 743 Mb
• Common bean nuclear genome size = 637 Mb
• Cantaloupe nuclear genome size = 454 Mb
• Grape nuclear genome size = 483 Mb
• Human nuclear genome size = 2,910 Mb
• 70-80% of flowering plants result from at least one polyploidization event
• Economically important plant species like corn, wheat, potato, and oat are either ancient/recent polyploids
• Some, like wheat, contain three different homologous genomes

Arabidopsis for Genetic Studies

• Model organisms like Drosophila, C. elegans, and S. cerevisiae provide insights into the biology of complex species
• The plant scientific community adopts model organisms
• Most plant species' genomes are too large/complex to be analyzed
• Model organisms share features like being diploid and appropriate for genetic analysis, being amenable to genetic transformation, having a small genome/short growth cycle, available tools/resources, and being the focus of a large scientific community. Tobacco and petunia formerly filled this role with the advent of tissue culture
• Rice and Arabidopsis currently models currently used for mono-/dicotyledonous plants

Arabidopsis Characteristics

• Arabidopsis is a small Cruciferae plant without agricultural use, seeds in 6 weeks
• Has a small genome of 120 Mb and five chromosomes
• Extensive tools exist for its genomic analysis, including the whole genome sequence and EST collections
• Characterized mutants and mutagenized populations (with transposons/T-DNA of Agrobacterium) are available
• Arabidopsis can be transformed, and genetic/physical maps for this plant are saturated

Arabidopsis vs Rice

• Rice is a major cereal crop (over 500 million tons produced annually with a huge population relying on it)
• Two main rice subspecies: Japonica (Japan) and indica (China-Asia Pacific)
• Rice has:
• Saturated genetic maps
• Physical maps
• Whole genome sequences
• EST collections from different tissues/developmental stages
• Twelve chromosomes/genome size of 420 Mb
• Like Arabidopsis it can be transformed through biolistics and A. tumefaciens.
• Efficient transposon-tagging systems for gene knockouts lack
• Gene detection is not yet available for saturation mutagenesis, although some recent successes have been reported

Drawbacks to earlier genetics models

• Mendel's work on Pisum sativum and Zea mays brought these crops into use for crop genetics studies
• These often fail to develop into genetics models
• Long generation time
• Complex genomes

Arabidopsis (cont.)

• Arabidopsis thaliana, a dicot flowering weed (Brassicaceae family) gained recognition in the 20th century
• Initially ignored until Friedrich Laibach sought a plant with suitable chromosomes for cytogenetic analysis
• It became a genetic model
• Short generation time
• Small size
• Large progeny
• Self-pollinating lifestyle with possibility of outcrossing

Arabidopsis Genome Info

• Its relatively smaller genome size (approx. 125 Mb) led to it being chosen for first plant genome sequencing project
• Brassica napus (rapeseed mustard) and Brassica juncea (Indian mustard), are about ten times that of Arabidopsis.
• Rice, maize, and wheat genomes are much more complex
• About 3x, 45x, 100x, respectively, compared to Arabidopsis
• First sequenced plant genome (year 2000)
• Each chromosome is represented as a colored bar
• Sequenced portions are red, telomeric and centromeric regions are light blue
• Heterochromatic knobs are black and the rDNA repeat regions are magenta
• Pseudo-colour assignments, from red (high density) to deep blue (low density) present
• Gene, EST and transposable element frequency ranges vary
• Mitochondrial/chloroplast (MT/CP) insertions are black/green tick marks
• Transfer RNAs/small nucleolar DNAs were assigned black & red ticks

Plant Tissue Culture Overview

• Plant tissue culture is a popular technique used in biotechnology with diverse applications across various fields

Tissue Culture Applications

• Conservation
• In situ and Ex situ
• Micropropagation
• Cryopreservation
• Synthetic seeds
• Metabolite engineering (Agrobacterium-mediated gene transformation, etc.)
• Enhanced production of active components
• PGRs
• Bioreactors
• Additives
• Elicitors
• Genome editing / Synthetic biology (CRISPR/Cas9, etc.)
• Antimicrobial/antioxidant activity assessment
• Haploidy and artificial polyploidy induction
• Hairy root induction
• Change the chemical profile

The role of plants

• Addressing challenges in food, energy, and environment
• Research in plant biotechnology provides solutions
• Modern plant biotechnology has made achievements (agriculture, pharmaceuticals, germplasm conservation, food security/plant breeding)
• Genetic engineering, molecular biology, and nanotechnology methods support this
• Plant cell culture is an important tool for producing economically important plants

Plant Tissue Culture Research

• Plant tissue culture (PTC) research began with cell theory
• Schleiden and Schwann proposed that cells are the basic structural unit of all living organisms and can regenerate into complete organisms
• Applications can be divided into :
• Cell behavior
• Plant modification and improvement
• Pathogen-free plants & germplasm storage
• Clonal propagation
• Product formation

Tissue Culture Timeline

• 1904: Cultured embryos from several cruciferous species
• 1922: Cultured root and stem tips
• 1926: Discovered Indole acetic acid – first plant growth hormone
• 1934: Continuous growing cultures of tomato root tips
• 1939: Interminable proliferation of callus cultures
• 1941: Coconut water for plant cell division
• 1946: Raised plant by shoot tip culture
• 1952: Developed virus-free from shoot meristem culture
• 1954: Callus tissues broken into single cells
• 1957: Auxin: cytokinin ratio of plant organ formation
• 1959: First evidence of somatic embryogenesis with carrot cell
• 1960:
• Cell suspension filtering / single cell plating
• Test tube fertilization technique development
• 1962: Murashige and Skoog medium developed
• 1964: First haploid plants from pollen grains produced
• 1966: Carrot plants regenerated from single cells of tomato
• 1971: Regeneration of first plants from isolated mesophyll protoplasts
• 1972: First protoplast fusion interspecific hybrid produced protoplast fusion
• 1974: Biotransformation introduced in plant tissue culture
• 1977: Ti plasmid DNA from Agrobacterium tumefaciens integrated into plants
• 1978: Intergenic hybrid between potato and tomato developed
• 1983: Cytoplasmic hybridization between generations in Radish and Grapes
• 1984: Agrobacterium-mediated gene transfer creates transgenic plants
• 1987: Plant biolistic gene transfer method
• 1994: Transgenic crops commercialized
• 2000: First plant genome (A. thaliana) decoded
• 2002: Omics technologies adopted
• 2005: Rice genome sequenced
• 2012: Integrated breeding platform
• 2013: CRISPR applied to plants

Totipotency

• Totipotency has two interpretations
• Capable of developing into a complete organism
• Capable of differentiating into cell types
• Zygotes or one-celled embryos are totipotent in the stricter sense
• Cells which can develop to all the various cell types under different conditions each, are also totipotent
• Embryonic animal stem cells that can produce a wide range (but not all!) cell totipotent

Plant Cells and Totipotency

• Not all plant cells are totipotent
• Certain cells may become totipotent under appropriate conditions is able to develop into a whole plant
• Somatic embryogenesis isn't strictly reliant on cellular totipotency
• Embryos from somatic cells can initiate in 3 ways
• Direct embryogenesis (Single cells through a totipotent (zygote-like?) stage)
• Direct/indirect embryogenesis (Embryo-identity transcription factors (LEC1, LEC2, FUS3)
• Organization of embryos (Groups of cells dependent on auxin and cytokinin gradients linked to meristem organizing Centers

Dedifferentiation and Callus Formation

• Dedifferentiation definitions:
• Mature or specialized cells lose differentiated character
• Contrary to differentiation, dedifferentiation the developmental potency of cells Differentiation, dedifferentiation, increases, transdifferentiation, callus formation are all related
• Dedifferentiation is generally associated with decreased, dedifferentiation with increased developmental potency
• In a strict sense, dedifferentiation can take place only within the same developmental lineage
• Is considered as the reversion of differentiation
• Transdifferentiation describes cell fate changes independent of developmental potency
• In plant biology, transdifferentiation leading to increased developmental potency is often referred to as dedifferentiation, especially during callus formation increases, transdifferentiation. Callus formation is not a step back in the developmental lineage, the result of overproliferation/transdifferentiation
• Transdifferentiation is similarly governs cells from one differentiated state to another
• Plants, “dedifferentiation" is strongly associated with callus formation
• Auxin-induced callus formation on in vitro-cultured Arabidopsis explants follows the lateral root development pathway
• Showed to express root meristem (pluripotency) markers in temporal/spatial organization
• Like primordia (LRPs), auxin-induced callus forms in pericycle cell-like stem cells/ isn't dependent on dedifferentiation differentiated cells
• Plant cell dedifferentiation also believed to form callus in protoplast isolation
• The cells are/separated from each other and exposed to digesting enzymes and released into an artifical meduim
• Stressed cells lose developmental and hormonal constrains
• Events/gene expression in callus are similar to characterizing cellular senescence
• Senescing leaf cells dedifferentiate similarly to the formation of protoplasts
• Senescence is loss of function represents a special and not dedifferentiating

Introduction to plant biotechnology and its applications. Discussion of plant genomes, tissue cultures, and molecular biology techniques. Also covers plant breeding, metabolite production, and responses to stress and biotic factors.