Plant Tissue Culture Application

1. Plant Tissue Culture Application

2. 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

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

4. Micropropagation

5. 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

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

7. 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

8. COMPARISON OF CONVENTIONAL & MICROPROPAGATION OF VIRUS INDEXED REGISTERED RED RASPBERRIES • Conventional Micropropagation • 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:

9. 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.

10. 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

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

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

13. 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)

14. 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

15. 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 • Woody plants

16. 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

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

18. 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

19. 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)

21. 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

22. 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

23. 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

25. 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)

26. Epigenetic 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

27. 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

28. 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

30. 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

31. Bulbosum Method Hordeum bulbosum Wild relative 2n = 2X = 14 Hordeum vulgare Barley 2n = 2X = 14 • 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) X ↓ Embryo Rescue Haploid Barley 2n = X = 7 H. Bulbosum chromosomes eliminated

32. 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

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

34. Culture Medium • Carbohydrate and osmotic pressure • 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

35. Culture Medium • natural plant extracts • 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

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

37. 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)

38. 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

39. Somatic Hybridization Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties

40. 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

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

42. Mechanical Method Plant Tissue CellsPlasmolysis MicroscopeObservation of cells Release of protoplasm Cutting cell wall with knife Collection of protoplasm

43. 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

44. 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

45. Enzymatic Method • Used forvariety 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

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

47. 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

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

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

50. 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