Plant Tissue Culture Application I plan to go into more technical detail with tissue culture techniques than I do with some of the other molecular biology techniques. The reason is that there is much more breeder support available for molecular biology than for tissue culture techniques, and if you want to apply some of these techniques, you are much more likely to be on your own than you are if you want to apply some of the molecular biology techniques in your breeding program.

Development of superior cultivars Germplasm storage Somaclonal variation Embryo rescue Ovule and ovary cultures Anther and pollen cultures Callus and protoplast culture Protoplasmic fusion In vitro screening Multiplication

Tissue Culture Applications Micropropagation Germplasm preservation Somaclonal variation Haploid & dihaploid production In vitro hybridization – protoplast fusion

Micropropagation

Features of Micropropagation Clonal reproduction Way of maintaining heterozygozity Multiplication stage can be recycled many times to produce an unlimited number of clones Routinely used commercially for many ornamental species, some vegetatively propagated crops Easy to manipulate production cycles Not limited by field seasons/environmental influences Disease-free plants can be produced Has been used to eliminate viruses from donor plants

Microcutting propagation It involves the production of shoots from pre-existing meristems only. Requires breaking apical dominance This is a specialized form of organogenesis

Steps of Micropropagation Stage 0 – Selection & preparation of the mother plant sterilization of the plant tissue takes place Stage I  - Initiation of culture explant placed into growth media Stage II - Multiplication explant transferred to shoot media; shoots can be constantly divided Stage III - Rooting explant transferred to root media Stage IV - Transfer to soil explant returned to soil; hardened off

COMPARISON OF CONVENTIONAL & MICROPROPAGATION OF VIRUS INDEXED REGISTERED RED RASPBERRIES Duration: 6 years 2 years Labor: Dig & replant every 2 years; Subculture every 4 weeks; unskilled (Inexpensive) skilled (more expensive) Space: More, but less expensive (field) Less, but more expensive (laboratory) Required to prevent viral Screening, fumigation, spraying None infection:

Ways to eliminate viruses Heat treatment. Plants grow faster than viruses at high temperatures. Meristemming. Viruses are transported from cell to cell through plasmodesmata and through the vascular tissue. Apical meristem often free of viruses. Trade off between infection and survival. Not all cells in the plant are infected. Adventitious shoots formed from single cells can give virus-free shoots.

Elimination of viruses Plant from the field Pre-growth in the greenhouse Active growth Heat treatment 35oC / months Adventitious Shoot formation ‘Virus-free’ Plants Virus testing Meristem culture Micropropagation cycle

Indirect Somatic Embryogenesis Explant → Callus Embryogenic → Maturation → Germination Callus induction Embryogenic callus development Maturation Germination

Induction Auxins required for induction Proembryogenic masses form 2,4-D most used NAA, dicamba also used

Development Auxin must be removed for embryo development Continued use of auxin inhibits embryogenesis Stages are similar to those of zygotic embryogenesis Globular Heart Torpedo Cotyledonary Germination (conversion)

Maturation Require complete maturation with apical meristem, radicle, and cotyledons Often obtain repetitive embryony Storage protein production necessary Often require ABA for complete maturation ABA often required for normal embryo morphology Fasciation Precocious germination

Germination May only obtain 3-5% germination Sucrose (10%), mannitol (4%) may be required Drying (desiccation) ABA levels decrease Woody plants Final moisture content 10-40% Chilling Decreases ABA levels

Plant germplasm preservation In situ : Conservation in ‘normal’ habitat rain forests, gardens, farms Ex Situ : Field collection, Botanical gardens Seed collections In vitro collection: Extension of micropropagation techniques Normal growth (short term storage) Slow growth (medium term storage) Cryopreservation (long term storage DNA Banks

In vitro Collection Use : Recalcitrant seeds Vegetatively propagated Large seeds Concern: Security Availability cost

Ways to achieve slow growth Use of immature zygotic embryos (not for vegetatively propagated species) Addition of inhibitors or retardants Manipulating storage temperature and light Mineral oil overlay Reduced oxygen tension Defoliation of shoots

Storage of living tissues at ultra-low temperatures (-196°C) Cryopreservation Storage of living tissues at ultra-low temperatures (-196°C) Conservation of plant germplasm Vegetatively propagated species (root and tubers, ornamental, fruit trees) Recalcitrant seed species (Howea, coconut, coffee) Conservation of tissue with specific characteristics Medicinal and alcohol producing cell lines Genetically transformed tissues Transformation/Mutagenesis competent tissues (ECSs) Eradication of viruses (Banana, Plum) Conservation of plant pathogens (fungi, nematodes)

Cryopreservation Steps Selection Excision of plant tissues or organs Culture of source material Select healthy cultures Apply cryo-protectants Pre-growth treatments Cooling/freezing Storage Warming & thawing Recovery growth Viability testing Post-thawing

Cryopreservation Requirements Preculturing Usually a rapid growth rate to create cells with small vacuoles and low water content Cryoprotection Cryoprotectant (Glycerol, DMSO/dimetil sulfoksida, PEG) to protect against ice damage and alter the form of ice crystals Freezing The most critical phase; one of two methods: Slow freezing allows for cytoplasmic dehydration Quick freezing results in fast intercellular freezing with little dehydration

Cryopreservation Requirements Storage Usually in liquid nitrogen (-196oC) to avoid changes in ice crystals that occur above -100oC Thawing Usually rapid thawing to avoid damage from ice crystal growth Recovery Thawed cells must be washed of cryo-protectants and nursed back to normal growth Avoid callus production to maintain genetic stability

Somaclonal Variation Variation found in somatic cells dividing mitotically in culture A general phenomenon of all plant regeneration systems that involve a callus phase Some mechanisms: Karyotipic alteration Sequence variation Variation in DNA Methylation Two general types of Somaclonal Variation: Heritable, genetic changes (alter the DNA) Stable, but non-heritable changes (alter gene expression, epigenetic) The only other source for genetic variation that I am aware of is polyploidy. As a breeder, you have to be aware of epigenetic variation because it can look like a good source to use for breeding, but since it’s not heritable, it can only be useful for vegetatively propagated crops. Sources for epigenetic variation include gene aplification, DNA methylation, and increased transposon activity. Since using somaclonal variation is the same as mutation breeding, I would like to take a look at mutation breeding in more detail.

Epigenetic The three main mechanisms for regulation are: the study of gene regulation that does not involve making changes to the SEQUENCE of the DNA, but rather to the actual BASES within the nucleotides and to the HISTONES The three main mechanisms for regulation are: CpG island methylation (…meCGmeCGmeCGmeCGmeCGmeCGmeCGmeCG…) acetylation and methylation of histone H3 the production of antisense RNA CpG island methylation: CpG islands are located in the promoter sequences of about 40% of all genes. They are strings of ~500 bp of CG repeat. This is very specific methylation of these strings. CpG islands are recognized by specific enzymes called methylases - they add the methyl group to the 5 position of the cytidines (all of them in the island) This methylated DNA is recognized by methyl binding proteins, which BLOCK binding of transcription factors to the site (i.e.- it will shut down transcription of that gene!) Modification of histones: Previously, we talked about acetylating histones to “open up” the DNA so that transcription can occur. In epigenetic regulation, we are actively REMOVING histones by the process of HISTONE DEACETYLASES (HDACs) Methylation of histones can also occur - this PRECLUDES the histones from be acetylated! Production of antisense RNA: Antisense RNA can be transcribed and can then BIND to mRNAs and keep them from being translated (think about how the microRNAs worked!) Production of antisense RNA can also provide steric hindrance for the production of SENSE mRNA (promoter is occluded)

Somaclonal Breeding Procedures Use plant cultures as starting material Idea is to target single cells in multi-cellular culture Usually suspension culture, but callus culture can work (want as much contact with selective agent as possible) Optional: apply physical or chemical mutagen Apply selection pressure to culture Target: very high kill rate, you want very few cells to survive, so long as selection is effective Regenerate whole plants from surviving cells

Requirements for Somaclonal Breeding Effective screening procedure Most mutations are deleterious With fruit fly, the ratio is ~800:1 deleterious to beneficial Most mutations are recessive Must screen M2 or later generations Consider using heterozygous plants? But some say you should use homozygous plants to be sure effect is mutation and not natural variation Haploid plants seem a reasonable alternative if possible Very large populations are required to identify desired mutation: Can you afford to identify marginal traits with replicates & statistics? Estimate: ~10,000 plants for single gene mutant Clear Objective Can’t expect to just plant things out and see what happens; relates to having an effective screen This may be why so many early experiments failed

Embryo Culture Uses Rescuing interspecific and intergeneric hybrids wide hybrids often suffer from early spontaneous abortion cause is embryo-endosperm failure Gossypium, Brassica, Linum, Lilium Production of monoploids useful for obtaining "haploids" of barley, wheat, other cereals the barley system uses Hordeum bulbosum as a pollen parent

H. Bulbosum chromosomes eliminated Bulbosum Method Hordeum bulbosum Wild relative 2n = 2X = 14 Hordeum vulgare Barley 2n = 2X = 14 X ↓ Embryo Rescue Haploid Barley 2n = X = 7 H. Bulbosum chromosomes eliminated This was once more efficient than microspore culture in creating haploid barley Now, with an improved culture media (sucrose replaced by maltose), microspore culture is much more efficient (~2000 plants per 100 anthers)

Excision of the immature embryo: Bulbosum technique H. vulgare is the seed parent zygote develops into an embryo with elimination of HB chromosomes eventually, only HV chromosomes are left embryo is "rescued“ to avoid abortion Excision of the immature embryo: Hand pollination of freshly opened flowers Surface sterilization – EtOH on enclosing structures Dissection – dissecting under microscope necessary Plating on solid medium – slanted media are often used to avoid condensation

Culture Medium Mineral salts – K, Ca, N most important Carbohydrate and osmotic pressure Amino acids Plant growth regulators

Culture Medium Carbohydrate and osmotic pressure amino acids 2% sucrose works well for mature embryos 8-12% for immature embryos transfer to progressively lower levels as embryo grows alternative to high sucrose – auxin & cyt PGRs amino acids reduced N is often helpful up to 10 amino acids can be added to replace N salts, incl. glutamine, alanine, arginine, aspartic acid, etc. requires filter-sterilizing a portion of the medium

Culture Medium natural plant extracts PGRs coconut milk (liquid endosperm of coconut) enhanced growth attributed to undefined hormonal factors and/or organic compounds others – extracts of dates, bananas, milk, tomato juice PGRs globular embryos – require low conc. of auxin and cytokinin heart-stage and later – usually none required GA and ABA regulate "precocious germination“ GA promotes, ABA suppresses

“Wide” crossing of wheat and rye requires embryo rescue and chemical treatment to double the number of chromosomes. Triticale

Haploid Plant Production Embryo rescue of interspecific crosses Creation of alloploids Anther culture/Microspore culture Culturing of Anthers or Pollen grains (microspores) Derive a mature plant from a single microspore Ovule culture Culturing of unfertilized ovules (macrospores)

Specific Examples of DH uses Evaluate fixed progeny from an F1 Can evaluate for recessive & quantitative traits Requires very large dihaploid population, since no prior selection May be effective if you can screen some qualitative traits early For creating permanent F2 family for molecular marker development For fixing inbred lines (novel use?) Create a few dihaploid plants from a new inbred prior to going to Foundation Seed (allows you to uncover unseen off-types) For eliminating inbreeding depression (theoretical) If you can select against deleterious genes in culture, and screen very large populations, you may be able to eliminate or reduce inbreeding depression e.g.: inbreeding depression has been reduced to manageable level in maize through about 50+ years of breeding; this may reduce that time to a few years for a crop like onion or alfalfa

Somatic Hybridization Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties Plants bearing genes of two different species or two different genera have been produced by plant breeders since the 1930s. Scientist in the 1930s and still plant breeders today use traditional plant breeding methods to generate these transgenic plants (e.g., protoplast fusion, embryo rescue, mutagenesis, etc). These practices have not found any resistance from the public and have never been controversial outside certain scientific circles.

Somatic hybridization technique 1. isolation of protoplast 2. Fusion of the protoplasts of desired species/varieties 3. Identification and Selection of somatic hybrid cells 4. Culture of the hybrid cells 5. Regeneration of hybrid plants

Isolation of Protoplast (Separartion of protoplasts from plant tissue) 2. Enzymatic Method 1. Mechanical Method

Mechanical Method Plant Tissue Cells Plasmolysis Microscope Observation of cells Release of protoplasm Cutting cell wall with knife Collection of protoplasm

Mechanical Method Used for vacuolated cells like onion bulb scale, radish and beet root tissues Low yield of protoplast Laborious and tedious process Low protoplast viability

Enzymatic Method Leaf sterlization, removal of epidermis Plasmolysed cells Plasmolysed cells Pectinase +cellulase Pectinase Protoplasm released Release of isolated cells Protoplasm released cellulase Isolated Protoplasm

Enzymatic Method Used for variety of tissues and organs including leaves, petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores Mesophyll tissue - most suitable source High yield of protoplast Easy to perform More protoplast viability

(Fusion of protoplasts of two different genomes) Protoplast Fusion (Fusion of protoplasts of two different genomes) 1. Spontaneous Fusion 2. Induced Fusion Intraspecific Intergeneric Chemofusion Mechanical Fusion Electrofusion

Uses for Protoplast Fusion Combine two complete genomes Another way to create allopolyploids In vitro fertilization Partial genome transfer Exchange single or few traits between species May or may not require ionizing radiation Genetic engineering Micro-injection, electroporation, Agrobacterium Transfer of organelles Unique to protoplast fusion The transfer of mitochondria and/or chloroplasts between species

Spontaneous Fusion Protoplast fuse spontaneously during isolation process mainly due to physical contact Intraspecific produce homokaryones Intergeneric have no importance

Chemofusion- fusion induced by chemicals Induced Fusion Chemofusion- fusion induced by chemicals Types of fusogens PEG NaNo3 Ca 2+ ions Polyvinyl alcohol

Induced Fusion Mechanical Fusion- Physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette Electrofusion- Fusion induced by electrical stimulation Fusion of protoplasts is induced by the application of high strength electric field (100kv m-1) for few microsecond

Possible Result of Fusion of Two Genetically Different Protoplasts = chloroplast = mitochondria Fusion = nucleus heterokaryon cybrid hybrid cybrid hybrid

Identifying Desired Fusions Complementation selection Can be done if each parent has a different selectable marker (e.g. antibiotic or herbicide resistance), then the fusion product should have both markers Fluorescence-activated cell sorters First label cells with different fluorescent markers; fusion product should have both markers Mechanical isolation Tedious, but often works when you start with different cell types Mass culture Basically, no selection; just regenerate everything and then screen for desired traits

Advantages of somatic hybridization Production of novel interspecific and intergenic hybrid Pomato (Hybrid of potato and tomato) Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters Production of heterozygous lines in the single species which cannot be propagated by vegetative means Studies on the fate of plasma genes Production of unique hybrids of nucleus and cytoplasm

Problem and Limitation of Somatic Hybridization Application of protoplast technology requires efficient plant regeneration system. The lack of an efficient selection method for fused product is sometimes a major problem. The end-product after somatic hybridization is often unbalanced. Development of chimaeric calluses in place of hybrids. Somatic hybridization of two diploids leads to the formation of an amphiploids which is generally unfavorable. Regeneration products after somatic hybridization are often variable. It is never certain that a particular characteristic will be expressed. Genetic stability. Sexual reproduction of somatic hybrids. Inter generic recombination.

TYPICAL SUSPENSION PROTOPLAST + LEAF PROTOPLAST PEG-INDUCED FUSION

NEW SOMATIC HYBRID PLANT

True in vitro fertilization A procedure that involves retrieval of eggs and sperm from the male and female and placing them together in a laboratory dish to facilitate fertilization Using single egg and sperm cells and fusing them electrically Fusion products were cultured individually in 'Millicell' inserts in a layer of feeder cells The resulting embryo was cultured to produce a fertile plant

In Vitro Fertilization

Requirements for plant genetic transformation Trait that is encoded by a single gene A means of driving expression of the gene in plant cells (Promoters and terminators) Means of putting the gene into a cell (Vector) A means of selecting for transformants Means of getting a whole plant back from the single transformed cell (Regeneration)