2DefinitionsPlant cell and tissue culture: cultural techniques for regeneration of functional plants from embryonic tissues, tissue fragments, calli, isolated cells, or protoplastsTotipotency: the ability of undifferentiated plant tissues to differentiate into functional plants when cultured in vitroCompetency: the endogenous potential of a given cell or tissue to develop in a particular way
3DefinitionsOrganogenesis: The process of initiation and development of a structure that shows natural organ form and/or function.Embryogenesis: The process of initiation and development of embryos or embryo-like structures from somatic cells (Somatic embryogenesis).
4Basis for Plant Tissue Culture Two Hormones Affect Plant Differentiation:Auxin: Stimulates Root DevelopmentCytokinin: Stimulates Shoot DevelopmentGenerally, the ratio of these two hormones can determine plant development: Auxin ↓Cytokinin = Root Development Cytokinin ↓Auxin = Shoot DevelopmentAuxin = Cytokinin = Callus Development
6Factors Affecting Plant Tissue Culture Growth MediaMinerals, Growth factors, Carbon source, HormonesEnvironmental FactorsLight, Temperature, Photoperiod, Sterility, MediaExplant SourceUsually, the younger, less differentiated the explant, the better for tissue cultureGeneticsDifferent species show differences in amenability to tissue cultureIn many cases, different genotypes within a species will have variable responses to tissue culture; response to somatic embryogenesis has been transferred between melon cultivars through sexual hybridizationEmphasize the implications for genetic involvement: Could there be undesirable genes linked to genes influencing tissue culture response?
7Micropropagation The art and science of plant multiplication in vitro Usually derived from meristems (or vegetative buds) without a callus stageTends to reduce or eliminate somaclonal variation, resulting in true clonesCan be derived from other explant or callus (but these are often problematic)Basic definition of micropropagation: The art & science of plant multiplication in vitro; an important aspect is that usually meristematic tissue is used as the explant, and a callus stage is avoided. If you avoid the callus stage, you can avoid the consequences often associated with callus culture: somaclonal variation, and true clones are a result; micropropagation can be done with other explants and even callus tissue, but other explants are usually more difficult to increase and as mentioned, a callus stage often has problems.
8Steps of Micropropagation Stage 0 – Selection & preparation of the mother plantsterilization of the plant tissue takes placeStage I - Initiation of cultureexplant placed into growth mediaStage II - Multiplicationexplant transferred to shoot media; shoots can be constantly dividedStage III - Rootingexplant transferred to root mediaStage IV - Transfer to soilexplant returned to soil; hardened off
10Features of Micropropagation Clonal reproductionWay of maintaining heterozygozityMultiplication Stage can be recycled many times to produce an unlimited number of clonesRoutinely used commercially for many ornamental species, some vegetatively propagated cropsEasy to manipulate production cyclesNot limited by field seasons/environmental influences
11Potential Uses for Micropropagation in Plant Breeding Eliminate virus from infected plant selectionEither via meristem culture or sometimes via heat treatment of cultured tissue (or combination)Maintain a heterozygous plant population for marker developmentBy having multiple clones, each genotype of an F2 can be submitted for multiple evaluationsProduce inbred plants for hybrid seed production where seed production of the inbred is limitedMaintenance or production of male sterile linesPoor seed yielding inbred linesPotential for seedless watermelon productionElimination of viruses is a standard application of micropropagation. Micropropagation has also been used to maintain a population of heterozygous individuals for marker development, especially when you’re trying to create a map (we did this at Seminis with both an F2 population and also with an F1 of an interspecific cross in tomato). A more novel use has to do with seed production. At Seminis, we set up a lab in Baja Mexico for multiplying plants using cauliflower curds where we could get several 100 plants from each curd, but typical seed production of these inbreds was much more limited. A potential use that I intend to investigate has to do with seed production of seedless watermelon. We currently have a very narrow genetic base of tetraploid breeding material because of generally low seed production. All current tetraploid inbreds have been selected for good seed production, which has limited the genetic pool.
12Germplasm Preservation Extension of micropropagation techniquesTwo methods:Slow growth techniquese.g.: ↓ Temp., ↓ Light, media supplements (osmotic inhibitors, growth retardants), tissue dehydration, etc…Medium-term storage (1 to 4 years)CryopreservationUltra low temperaturesStops cell division & metabolic processesVery long-term (indefinite?)Details to follow on next two slides →There are two basic types of germplasm preservation using micropropagation techniques
13Cryopreservation Requirements PreculturingUsually a rapid growth rate to create cells with small vacuoles and low water contentCryoprotectionGlycerol, DMSO, PEG, etc…, to protect against ice damage and alter the form of ice crystalsFreezingThe most critical phase; one of two methods:Slow freezing allows for cytoplasmic dehydrationQuick freezing results in fast intercellular freezing with little dehydration
14Cryopreservation Requirements StorageUsually in liquid nitrogen (-196oC) to avoid changes in ice crystals that occur above -100oCThawingUsually rapid thawing to avoid damage from ice crystal growthRecovery (don’t forget you have to get a plant)Thawed cells must be washed of cryoprotectants and nursed back to normal growthAvoid callus production to maintain genetic stability
15Somaclonal VariationThe source for most breeding material begins with mutations, whether the mutation occurs in a modern cultivar, a landrace, a plant accession, a wild related species, or in an unrelated organismTotal sources of variation:Mutation, Hybridization, PolyploidyThe 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.
16Somaclonal Variation & Mutation Breeding Somaclonal variation is a general phenomenon of all plant regeneration systems that involve a callus phaseThere are two general types of Somaclonal Variation:Heritable, genetic changes (alter the DNA)Stable, but non-heritable changes (alter gene expression, AKA epigenetic)Since utilizing somaclonal variation is a form of mutation breeding, we need to consider mutation breeding in more detail →There are six basic types of vegetable production which I’ve listed here. Market gardening is generally small scale, usually a few acres but sometimes up to 100 acres. The produce is usually marketed directly to the customer, either through roadside stands, farmers markets or pick your own operations. Because of this, market gardens tend to be local, and a wide variety of produce is usually grown.Truck farms, on the other hand, tend to be much larger.
17Mutation Breeding1927: Muller produced mutations in fruit flies using x-rays1928: Stadler produced mutations in barleyMutation breeding became a bandwagon for about 10 years (first claim to “replace breeders”)Today there are three groups of breeders:Mutation breeding is useless, we can accomplish the same thing with conventional methodsMutation breeding will produce a breakthrough given enough effortMutation breeding is a tool, useful to meet specific objectivesMutation breeding
18Inducing Mutations Physical Mutagens (irradiation) Neutrons, Alpha raysDensely ionizing (“Cannon balls”), mostly chromosome aberrationsGamma, Beta, X-raysSparsely ionizing (“Bullets”), chromosome aberrations & point mutationsUV radiationNon-ionizing, cause point mutations (if any), low penetratingChemical Mutagens (carcinogens)Many different chemicalsMost are highly toxic, usually result in point mutationsCallus Growth in Tissue CultureSomaclonal variation (can be combined with other agents)Can screen large number of individual cellsChromosomal aberrations, point mutationsAlso: Uncover genetic variation in source plant
19Traditional Mutation Breeding Procedures Treat seed with mutagen (irradiation or chemical)Target: 50% killGrow-out M1 plants (some call this M0)Evaluation for dominant mutations possible, but most are recessive, so →Grow-out M2 plantsEvaluate for recessive mutationsExpect segregationProgeny test selected, putative mutantsProve mutation is stable, heritable
20Somaclonal Breeding Procedures Use plant cultures as starting materialIdea is to target single cells in multi-cellular cultureUsually suspension culture, but callus culture can work (want as much contact with selective agent as possible)Optional: apply physical or chemical mutagenApply selection pressure to cultureTarget: very high kill rate, you want very few cells to survive, so long as selection is effectiveRegenerate whole plants from surviving cells
21Somaclonal/Mutation Breeding AdvantagesScreen very high populations (cell based)Can apply selection to single cellsDisadvantagesMany mutations are non-heritableRequires dominant mutation (or double recessive mutation); most mutations are recessiveCan avoid this constraint by not applying selection pressure in culture, but you loose the advantage of high through-put screening – have to grow out all regenerated plants, produce seed, and evaluate the M2How can you avoid this problem?
23Successes of Somaclonal/Mutation Breeding Herbicide Resistance and ToleranceResistance: able to break-down or metabolize the herbicide – introduce a new enzyme to metabolize the herbicideTolerance: able to grow in the presence of the herbicide – either ↑ the target enzyme or altered form of enzymeMost successful application of somaclonal breeding have been herbicide toleranceGlyphosate resistant tomato, tobacco, soybean (GOX enzyme)Glyphosate tolerant petunia, carrot, tobacco and tomato (elevated EPSP (enolpyruvyl shikimate phosphate synthase))But not as effective as altered EPSP enzyme (bacterial sources)Imazaquin (Sceptor) tolerant maizeTheoretically possible for any enzyme-targeted herbicide – it’s relatively easy to change a single enzyme by changing a single gene
24Other Targets for Somaclonal Variation Specific amino acid accumulatorsScreen for specific amino acid productione.g. Lysine in cerealsAbiotic stress toleranceAdd or subject cultures to selection agente.g.: salt tolerance, temperature stresses, etc…Disease resistanceAdd toxin or culture filtrate to growth mediaExamples shown on next slide →
25Disease Resistant Success using Somaclonal Variation CropPathogenToxinAlfalfaColletotrichum sp.Culture filtrateBananaFusarium sp.Fusaric acidCoffeePartially purified culture filtrateMaizeHelminthosporium maydisT-toxinOat*Helminthosporium victoriaeVictorinOilseed Rape*Phoma lingamPeachXanthomonas sp.Potato**Phytophthora infestansRice*Xanthomonas oryzaeSugarcane**Helminthosporium sp.Helminthosporium sachariPartially purified HS toxinTobacco*Psedomonas tabaciMethionine-sulfoximineAlternaria alternataPartially purified toxin*Shown to be heritable through sexual propagation**Shown to be stable through vegetative propagation
26Requirements for Somaclonal/Mutation Breeding Effective screening procedureMost mutations are deleteriousWith fruit fly, the ratio is ~800:1 deleterious to beneficialMost mutations are recessiveMust screen M2 or later generationsConsider using heterozygous plants?Haploid plants seem a reasonable alternative if possibleVery 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 mutantClear ObjectiveCan’t expect to just plant things out and see what happens; relates to having an effective screenThis may be why so many early experiments failed
27Questions with Mutation Breeding Do artificial mutations differ from natural ones?Most people agree that they are, since any induced mutation can be found in nature, if you look long enough & hard enoughIf this is true, then any mutation found in nature can be induced by mutation breedingIs it worthwhile, given the time & expense?Still require conventional breeding to incorporate new variability into crop plants (will not replace plant breeders)Not subject to regulatory requirements (or consumer attitudes) of genetically engineered plants
28Reading AssignmentD.R. Miller, R.M. Waskom, M.A. Brick & P.L. Chapman Transferring in vitro technology to the field. Bio/Technology. 9:
30Embryo Culture Uses Rescue F1 hybrid from a wide cross Overcome seed dormancy, usually with addition of hormone to media (GA)To overcome immaturity in seedTo speed generations in a breeding programTo rescue a cross or self (valuable genotype) from dead or dying plant
31Embryo Culture as a Source of Genetic Variation HybridizationCan transfer mutant alleles between speciesCan introduce new genetic combinations through interspecific crossesPolyploidyCan combine embryo culture with chromosome doubling to create new polyploid species (allopolyploidy)
33Embryo Rescue Process Make cross between two species Dissect embryo (usually immature)The younger the embryo, the more difficult to cultureGrow on culture medium using basic tissue culture techniques, use for breeding if fertileMany times, resulting plants will be haploid because of lack of pairing between the chromosomes of the different speciesThis can be overcome by doubling the chromosomes, creating allotetraploidsPolyploids are another source of genetic variation →
34Polyploids in Plant Breeding Very Brief, General Overview
35DefinitionsEuploidy: An even increase in number of genomes (entire chromosome sets)Aneuploidy: An increase in number of chromosomes within a genomeAutopolyploid: Multiple structurally identical genomes with unrestricted recombinationAllopolyploid: Multiple genomes so differentiated as to restrict pairing and recombination to homologous chromosomes between genomes
37Aneuploid Polyploid Examples AneuploidsSymbolSomatic (2n)Descriptionnullisomic2x-2(AB)(AB)(missing a chromosome set)monosomic2x-1(ABC)(AB)(missing a chromosome)double monosomic2x-1-1(AB)(AC)(missing 2 different chromosomes)trisomic2x+1(ABC)(ABC)(A)(additional chromosome)double trisomic2x+1+1(ABC)(ABC)(A)(B)(2 additional different chromosomes)tetrasomic2x+2(ABC)(ABC)(A)(A)(2 additional chromosomes - same)trisomic-monosomic2x+1-1(ABC)(AB)(A)(missing a chromosome + additional chromosome)
38Polyploids as a Source of Genetic Variation Multiple genomes alter gene frequencies, induce a permanent hybridity, genetic buffering and evolutionary flexibility (esp. Allopolyploids)Autopolyploids typically have larger cell sizes, resulting in larger, lusher plants than the diploid versionChromosome doubling occurs naturally in all plants at low frequency as a result of mitotic failureCan be induced by chemicals (colchicine from Colchicum autumnale) applied to meristematic tissueYoung zygotes respond best; vegetative tissue usually results in mixoploid chimeras
39AutopolyploidsMultiple structurally identical genomes with unrestricted recombinationSource material is highly fertilei.e.: diploidRelatively rare in crop plants:Potato (4x), alfalfa (4x), banana (3x)Typical feature: grown for vegetative productUsually reduced seed fertilityLimited breeding success in seed cropsDespite a lot of effortException: Seedless watermelon →
41AllopolyploidyMultiple genomes so differentiated as to restrict pairing and recombination to homologous chromosomes between genomesFunctionally diploid because of preferential pairing of chromosomesStarting material usually an interspecific hybridF1 usually has a high degree of sterilityFertility of alloploid usually inversely correlated to sterility in source material (F1)
43Uses for Polyploids in Breeding Potential for new crop development (triticale)Move genes between speciesCan get recombination between genomes of alloploids, especially when combined with ionizing radiation (mutation breeding)Can re-create polyploids from diploid ancestors using new genetic variation present in the diploids
44Haploid Plant Production Embryo rescue of interspecific crossesCreation of alloploids (e.g. triticale)Bulbosum methodAnther culture/Microspore cultureCulturing of Anthers or Pollen grains (microspores)Derive a mature plant from a single microsporeOvule cultureCulturing of unfertilized ovules (macrospores)Sometimes “trick” ovule into thinking it has been fertilized
45Bulbosum Method of Haploid Production Hordeum bulbosumWild relative2n = 2X = 14Hordeum vulgareBarley2n = 2X = 14X↓Embryo RescueHaploid Barley2n = X = 7H. Bulbosum chromosomes eliminatedThis was once more efficient than microspore culture in creating haploid barleyNow, with an improved culture media (sucrose replaced by maltose), microspore culture is much more efficient (~2000 plants per 100 anthers)
47Anther/Microspore Culture Factors GenotypeAs with all tissue culture techniquesGrowth of mother plantUsually requires optimum growing conditionsCorrect stage of pollen developmentNeed to be able to switch pollen development from gametogenesis to embryogenesisPretreatment of anthersCold or heat have both been effectiveCulture mediaAdditives, Agar vs. ‘Floating’
48Ovule Culture for Haploid Production Essentially the same as embryo cultureDifference is an unfertilized ovule instead of a fertilized embryoEffective for crops that do not yet have an efficient microspore culture systeme.g.: melon, onionIn the case of melon, you have to “trick” the fruit into developing by using irradiated pollen, then x-ray the immature seed to find developed ovules
49What do you do with the haploid? Weak, sterile plantUsually want to double the chromosomes, creating a dihaploid plant with normal growth & fertilityChromosomes can be doubled byColchicine treatmentSpontaneous doublingTends to occur in all haploids at varying levelsMany systems rely on it, using visual observation to detect spontaneous dihaploidsCan be confirmed using flow cytometry
50Uses of Hapliods in Breeding Creation of allopolyploidsas previously describedProduction of homozygous diploids (dihaploids)Detection and selection for (or against) recessive allelesSpecific examples on next slide →
51Specific Examples of DH uses Evaluate fixed progeny from an F1Can evaluate for recessive & quantitative traitsRequires very large dihaploid population, since no prior selectionMay be effective if you can screen some qualitative traits earlyFor creating permanent F2 family for molecular marker developmentFor 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 depressione.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
53Somatic Hybridization using Protoplasts Created by degrading the cell wall using enzymesVery fragile, can’t pipetteProtoplasts can be induced to fuse with one another:Electrofusion: A high frequency AC field is applied between 2 electrodes immersed in the suspension of protoplasts- this induces charges on the protoplasts and causes them to arrange themselves in lines between the electrodes. They are then subject to a high voltage discharge which causes them membranes to fuse where they are in contact.Polyethylene glycol (PEG): causes agglutination of many types of small particles, including protoplasts which fuse when centrifuged in its presenceAddition of calcium ions at high pH values
54Uses for Protoplast Fusion Combine two complete genomesAnother way to create allopolyploidsPartial genome transferExchange single or few traits between speciesMay or may not require ionizing radiationGenetic engineeringMicro-injection, electroporation, AgrobacteriumTransfer of organellesUnique to protoplast fusionThe transfer of mitochondria and/or chloroplasts between species
55Possible Result of Fusion of Two Genetically Different Protoplasts = chloroplast= mitochondriaFusion= nucleusheterokaryoncybridhybridcybridhybrid
56Identifying Desired Fusions Complementation selectionCan be done if each parent has a different selectable marker (e.g. antibiotic or herbicide resistance), then the fusion product should have both markersFluorescence-activated cell sortersFirst label cells with different fluorescent markers; fusion product should have both markersMechanical isolationTedious, but often works when you start with different cell typesMass cultureBasically, no selection; just regenerate everything and then screen for desired traits
57Reading AssignmentEarle, E.D., and M.A. Sigareva Direct transfer of a cold-tolerant ogura male-sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion. Theor Appl Genet. 94:
58Example of Protoplast Fusion Male sterility introduced into cabbage by making a cross with radish (as the female)embryo rescue employed to recover plantsCabbage phenotypes were recovered that contained the radish cytoplasm and were male sterile due to radish genes in the mitochondriaUnfortunately, the chloroplasts did not perform well in cabbage, and seedlings became chlorotic at lower temperatures (where most cabbage is grown)Protoplast fusion between male sterile cabbage and normal cabbage was done, and cybrids were selected that contained the radish mitochondria and the cabbage chloroplastCurrent procedure is to irradiate the cytoplasmic donor to eliminate nuclear DNA – routinely used in the industry to re-create male sterile brassica crops
59One Last Role of Plant Tissue Culture Genetic engineering would not be possible without the development of plant tissueGenetic engineering requires the regeneration of whole plants from single cellsEfficient regeneration systems are required for commercial success of genetically engineered products