40.0 Introduction

1. Plant Genome Organization Can Be Very Complex
1. Complexity Observed at DNA and Chromosomal Levels
1. Plants can have duplicate sets of entire chromosomes
2. Variable ploidy exists even within a given genus

2. Plant Cells Display Totipotency
1. Under appropriate conditions differentiated cells can produce whole plants
2. Unique plant characteristics helped develop plant genetic engineering fig 40.1

1. Traditional Ways to Study Plant Genomes
1. Plant Genomes Are More Complex than Other Organisms
1. Analysis reveals evolutionary changes in DNA over time
2. Plants show varied chromosome numbers and ploidy levels
1. Confer selective advantages under different environmental conditions fig 40.2

3. Great amount of genetic variation contributes to survival and proliferation

2. Size of Plant Genomes Shows Tremendous Variation
1. In both number of chromosomes and total number of nucleotide base pairs
2. Example: Tulips have 170 times as much DNA as Arabidopsis tbl 40.1
3. Plant DNA contains special regions
1. Sequence repeats, sequence inversions, transposable element insertions
2. Further modify genetic content

4. Study of evolutionary history of plant species
1. Traditionally examine variation in chromosome inversions and ploidy
2. Currently study organization of DNA sequences

2. Organization of Plant Genomes
1. Determination of Plant Relatedness
1. Examine conserved gene arrangements in related species: Synteny
2. Determine close relations and divergent ones
3. Plan genomes vary in several ways
1. Number of repeated sequences and sequence inversions
2. Effects of transposable elements

4. Large amount of DNA can mask effects when they occur in noncoding regions

2. Low-, Medium-, and High-Copy-Number DNA
1. Most higher plants contain more DNA than needed for coding and regulatory functions
1. Very small part of genome actually encodes genes for protein production
2. Portion of genome called low-copy-number DNA
3. DNA sequences are present in single or small numbers of copies

2. Medium-copy-number DNA
1. Includes DNA sequences encoding ribosomal RNA (rRNA)
2. rRNA involved in translation of messenger RNA into protein
3. Plant rRNA genes repeated several hundred to several thousand times
4. Number of genes and mutations most useful in studying evolutionary patterns

3. High-copy-number DNA
1. Highly repetitive sequences
2. Function yet unknown

3. Sequence Replication and Inversion
1. Nucleotide numbers in high-copy-number DNA is variable
1. May be short as in GAA sequence
2. May be several hundred nucleotides long

2. Number of copies varies from 10,000 to 100,000
3. Organization of high-copy repetitive sequences fig 40.4a
1. Simple tandem array: Several copies present together in same orientation
2. Repetitive sequences dispersed in single-copy DNA
1. Repeat/single copy interspersion: Sequences have same orientation
2. Inverted repeats: Sequences have opposite orientation

3. Groups of repetitive sequences can occur together
1. Compound tandem array
2. Repeat/repeat interspersion

4. Can be difficult to find and characterize single-copy genes
5. Variety of mechanisms account for repetitive sequences
1. Generated by DNA sequence amplification, multiple rounds of DNA replication
2. Generated by unequal crossing-over during mitosis or meiosis (translocation)
3. Unequal crossing-over due to action of transposable elements

6. Sequence inversions result from chromosome breakage, reinsertion in reverse direction

4. Transposable Elements
1. Special sequences that can move from one location to another
1. Can excise and reinsert at unpredictable times
2. Commonly called jumping genes

2. Can insert into coding or regulatory regions of a gene fig 40.4b
1. Affect gene expression
2. Result in mutation that is detectable or not
3. Detectable mutation may revert if transposable element dissociates from gene
4. Recognition by McClintock resulted in Nobel Prize
1. Importance of discovery in corn not realized for many years
2. Occurred after discovery and analysis of transposons in bacteria

3. Can replicate independently and move through genome
4. Can be involved in generating repetitive DNA sequences
1. Mutation in transposable element to prevent transposition
2. Result in retention of repetitive sequence

5. Chloroplast Genome and Its Evolution
1. Chloroplast functions in photosynthesis, replicates independently
2. Possess own DNA separate from that in cell nucleus
1. DNA is maternally inherited
2. Encodes for unique chloroplast proteins, many involved in photosynthesis

3. Origin of chloroplasts in plants
1. Originated from photosynthetic prokaryote via endosymbiosis
2. Chloroplast DNA has many prokaryotic features
1. Chloroplast DNA is a circular loop of double-stranded DNA
2. Contains genes for ribosomes similar to prokaryotic ribosomes

4. All land plant chloroplasts have DNA with similar number of genes
1. Approximately 100 genes
2. Present in same order fig 40.5
3. Chloroplast DNA evolves at much more conservative pace
4. Evolutionary patterns more readily interpreted
5. Not subject to modification by transposable elements or recombination mutations

5. Chloroplast genome has two identical inverted repeats
1. Other sequence inversions or deletions are rare, use to analyze relationships
2. Example: Sunflower family has large inversion not found in any other plant family

6. Compare DNA analysis with traditional examination of anatomy and morphology

3. Comparative Genome mapping and Model Systems
1. New Techniques Provide Greater Knowledge of Plant Genomes
1. Lead to better manipulation of genetic traits
1. Examine genomes of related species to find similarities
2. Finding quantitative trait loci (QTLs) in one species could help find them in others

2. Importance of genomic mapping in model plants

2. RFLP and AFLP as Tools to Map Genomes and Detect Polymorphisms
1. RFLP (restriction fragment length polymorphism) used to identify DNA markers
1. Involves analysis of RFLP map
2. Pattern of DNA fragments made when DNA cut with restriction endonucleases
3. Can identify important regions of DNA at a glance
4. Sequence data requires use of computer-based search and matching systems

2. Compare parent and progeny RFLP maps
1. Indicate heritability of genetic traits
2. Indicate heritable loci characteristic of traits
3. Genetic identification of RFLP markers facilitated with sequencing entire genome

3. AFLP (amplified fragment length polymorphism)
1. Generate maps by hybridizing DNA primers with genomic DNA fragments
2. Fragments cut with specific restriction endonucleases EcoR1 and Mse1
3. Further amplified by polymerase chain reaction (PCR)
4. Products separated by size via gel electrophoresis

4. Band sizes on AFLP gel show more polymorphisms than RFLP maps fig 40.6
5. RFLP and AFLP provide markers for traits from heritable parents to progeny

3. Arabidopsis thaliana as a Model System for Plant Genome Analysis
1. Small weed plant related to mustards has become model system for plants
1. Rapid life cycle, seeds germinate, flower, produce more seed in five weeks
2. Useful to study crosses and production of mutants
3. Plant is small, readily housed in small containers for lab experiments
4. Can be grown via tissue culture
5. Has small genome, repetitive sequences account for 20% of DNA
6. Easier to find single-copy genes

2. Attempting to sequence entire genome to find location and function of every gene
1. Results expected in 1998
2. Will have far-reaching uses in agriculture breeding and evolutionary analysis
3. Finding genes in one plant species will help find them in others
4. Will facilitate gene cloning in many plant species

3. Very useful in production and selection of plants with variety of mutant genes
1. Produced by transposable elements, radiation, UV light, chemical mutagens
2. Mutants produced for developmental and metabolic pathways
3. Has provides means to pinpoint genes responsible for certain phenotypes fig 40.7

4. Genome Sequencing of Rice and Other Grains
1. Similar effort to sequence entire rice genome as monocot model
1. Monocots are major human food source
2. Sequencing rice genome would help understand larger genomes of other grains

2. Rice, corn, barley, wheat diverged more than 50 million years ago
1. Chromosomes show conserved arrangements of segments fig 40.8

3. Analysis important to identify genes associated with disease resistance, nutritional quality, growth capacity
1. Can construct gene map of presumed ancestral cereal genome

1. Overview of Plant Tissue Culture
1. Differentiated Plant Cells Can Dedifferentiate and Regenerate into Whole Plants
1. Most animal cells incapable of such growth and development
1. Animal cells cannot alter developmental pattern once they have matured
2. Cells are locked into particular developmental program

2. Plant cells can express previously unexpressed genes under proper conditions
1. Leads to development of plant from single cell
2. Called totipotency
3. Forms basis of plant tissue culture in artificial media like bacteria and fungi

2. Basics of Plant Cell Culture
1. Requires several conditions fig 40.9
1. Use proper plant starting material
2. Possess appropriate nutrient medium
3. Time hormonal treatments to maximize growth potential, drive differentiation

2. Plant cell cultures usually start with explants, section of tissue from intact plant
1. Removed under sterile conditions
2. Placed in growth medium with nutrients, vitamins, growth regulator combinations
3. Cells begin to divide and proliferate
4. Form organs, roots, shoots, embryos, leaf primordia
5. Cam regenerate a whole plant

3. Growth of whole plant needed for success in production of genetically engineered plants
1. Genetic manipulation can begin at level of single cells
2. Whole plants produced bearing introduced genetic trait

4. Also used to mass produce genetically identical plants (clones)
1. Clonal propagation used in commercial production of ornamental plants
2. Examples: Chrysanthemums and ferns

5. Various cultures made by using different tissues for explants and different media

2. Types of Plant Tissue Cultures
1. Callus Culture
1. Grow unorganized masses of plant cells in culture
2. Explant contains region of meristematic cells
3. Culture process fig 40.10
1. Explant incubated on growth medium
2. Medium contains growth regulators, auxin and cytokinin
3. Cells grow and divide forming undifferentiated mass called a callus
1. Mass of cells analogous to plant tumor
2. Proliferate indefinitely if periodically transferred to fresh medium

4. Transferred to medium with different growth regulators to initiate differentiation
1. Cells differentiate into roots and/or shoots
2. Process called organogenesis

4. Plantlet transferred to soil when sufficiently large

2. Cell Suspension Culture
1. Growth of single or small group of cells in liquid culture
2. Culture process fig 40.11
1. Transfer cells from callus into liquid medium
2. Medium contains growth regulators, chemicals to promote cell disaggregation
3. Cultures shaken to promote aeration and chemical exchange with medium

3. Used when important to access single cells
1. Select out cells with certain desirable traits
2. All cells respond to chemical conditions due to uniform exposure

4. Used to produce cell chemical secretions
5. Produce whole plants via somatic cell embryogenesis fig 40.12
1. Regenerate whole plants after single cell genetic engineering
2. Special medium drives differentiation of cells
3. Cells organize to form embryos
4. Embryos transferred to new growth medium, become whole plants

3. Protoplast Isolation and Culture
1. Protoplast: Plant cell lacking cell wall
1. Cell wall removed by enzymatic process
2. Leaves plant cell enclosed only be plant cell membrane

2. Useful in research on plant cell membrane, synthesis of cell wall
3. Aids in transforming cells with foreign DNA by electroporation
4. Protoplasts can be caused to fuse, producing unique genetic combinations
1. Provide additional means to accomplish genetic engineering
2. Allow incorporation of genetic material across species

5. Culture process fig 40.13
1. Protoplasts transferred to growth medium
2. Cell wall regenerates, cell divides
3. Cells form callus
4. New plants formed by organogenesis or somatic cell embryogenesis

4. Anther/Pollen Culture
1. Anthers contain pollen, shed to disperse to other flowers
2. Culture process fig 40.14
1. Anthers excised, transferred to appropriate growth medium
2. Pollen cells manipulated to become tiny plantlets
3. Plantlets grown in culture to form whole plants through formation of embryos

3. Resulting plants may be haploid, sterile, not useful for breeding or genetic engineering
1. Treat with colchicine to promote chromosome duplication
2. Can become fertile diploid organism, homozygous for all traits

4. Useful for breeders to introduce normally recessive trait

5. Plant Organ Culture
1. Plant organs grown under culture conditions
2. Useful to study plant organ development
3. Example: Tomato flowers
1. Pollinated flowers excised, transferred to growth medium
2. Ovular portion develops into tomato fruit that fully ripens

4. Example: Root portions fig 40.15
1. Roots excised, transferred to liquid growth medium
2. Roots proliferate, form primary and secondary root branches

3. Applications of Plant Tissue Cultures
1. Suspension Cultures as Biological Factories
1. Large-scale suspension cultures used to produce various chemical compounds
1. Include antimicrobials, antitumor alkaloids
2. Also produce vitamins, insecticides, food flavors

2. Roots grown in culture, produce useful plant compounds

2. Horticulture Uses
1. Mass propagation of plants with valuable traits
2. Produce genetically identical plants by vegetative asexual propagation
3. Useful in flower industry
4. Can also produce disease-free plants
1. Propagate tissue in sterile environment
2. Produce cultures from meristematic tissue untouched by viruses or disease
3. Important in producing disease-free orchids

3. Somaclonal Variation
1. Problematic side effect used to an advantage
2. Occurs during extended periods of growth in callus or suspension cultures
1. Various parts of genome become active, released from control of gene expression
2. Transposable elements may become active
3. Chromosome rearrangements may occur

3. Provides new source of genetic diversity, results in expression of novel traits fig 40.16
1. Traits identified in tissue culture stage, like disease resistance or heat tolerance
2. Traits identified in whole plants

4. Problematic if the expectation was to produce identical plant clones

1. World Population in Relation to Advances Made in Crop Production
1. Recent Increases in World Food Production
1. Food production doubled since 1960, due to crop breeding, farming techniques
1. Productivity from land and water usage has tripled
2. Genetic improvements via crop breeding is a slow process

2. Shortfall in genetic potential of plants
3. Plants unable to tolerate or adapt to environmental stresses fig 40.17

2. Greater Food Production Required with Expected Population Increases
1. Population expected to double by 2030
2. Current pace of food production may not keep up with population
3. Agriculture must become more productive and less taxing to environment

2. Plant Biotechnology for Agricultural Improvement
1. Resolve Problem of Feeding the World via Genetic Engineering
1. Biotechnology improving quality of seed grains
1. Increase protein levels in crops
2. Improve resistance to disease, insects, herbicides, viruses

2. Engineer plants with higher tolerance to environmental stresses, heat or salt
3. Improve nutritional quality, protect people from health problems

2. Advantages of Genetic Engineering Compared to Plant Breeding
1. Compresses time frame
2. Overcomes normal genetic barriers
1. Resolves problems with pollen incompatibility to pistil
2. Easier to transfer single gene rather than multiple gene traits like nitrogen fixation

3. Plant transformation
1. Incorporation of foreign DNA into existing plant genome
2. Approaches include use of Agrobacterium if plant is a dicot
3. Important food crops include cereal grains, monocots
4. New approaches developed for monocots and other dicots

3. Plant Transformation Using the Particle Gun
1. Literally blast foreign DNA into plant genome through cell wall
2. Utilizes microscopic gold particles coated with foreign DNA
1. Acceleration to high velocity via high pressure helium gas fig 40.18a
2. Can also use electrical discharge

3. Only a few cells receive DNA and survive treatment, identify with selectable marker
1. Marker present on foreign DNA
2. Ensures that only cells with DNA will survive on growth medium fig 40.19
3. Markers include genes for herbicide or antibiotic resistance

4. Growing cells further tested for presence of desired foreign genes

4. Plant Transformation Using Electroporation
1. Foreign DNA shocked into cells lacking cell walls
2. Uses pulse of high-voltage electricity in a solution of protoplasts and DNA
1. Opens small pores in protoplast plasma membrane fig 40.18b
2. Foreign DNA enters pores

3. Protoplasts transferred to growth medium, reform cell walls, grow into whole plants
4. Also uses selectable marker system
5. Regenerated whole plants tested for presence of desired trait

3. Useful Traits that Can Be Introduced into Plants
1. Improved Nutritional Quality of Food Crops
1. Trend toward utilizing plant oils instead of animal fats
2. Genetically engineer seeds to produce edible and non-edible "designer oils" fig 40.20
1. Modify canola oil to replace cocoa butter as source of saturated fatty acids
2. Modify enzyme ACP desaturase to create mono-unsaturated fatty acids in plants

3. Modify amino acid content of various plants
1. Present more complete nutritional diet to consumer
2. Attempts to develop high-lysine corn seed for livestock, reduce lysine supplements

4. Engineer fruits and vegetables with more vitamins A, C and beta-carotene
5. Develop biodegradable plastics more cheaply in plants than transgenetic bacteria

2. Plants Bearing Vaccines for Human Diseases
1. Introduce "vaccine genes" into edible plants
1. Genes coding for antigen introduced into edible plant via transformation
2. Antigen then present in plant cells
3. Human eating cells would develop antibodies to the antigen