Artificial seeds and Cryopreservation.pptx
- 1. IIIM-CSIR Artificial Seeds and Cryopreservation Synthetic Seeds :Prospects and Limitations
- 2. IIIM-CSIR Artificial Seeds and Cryopreservation Definition Synthetic seeds are encapsulated somatic embryos, shoot buds, cell aggregates or any other tissue that can be used for sowing as a seed or that possesses the ability to convert into a plant under in vitro or ex vitro conditions and that can retain this potential also after storage.
- 3. IIIM-CSIR Artificial Seeds and Cryopreservation What makes a synthetic seed? Synthetic seeds are produced by encapsulating a plant propagule in a matrix which will allow it to grow into a plant. Plant propagules consist of shoot buds or somatic embryos that have been grown aseptically in tissue culture. In culture, these plant propagules easily grow into individual plants as we have the capacity to control its growth using chemicals provided in the culture media. In the production of Synthetic seeds , an artificial endosperm is created within the encapsulation matrix. The encapsulation matrix is a hydrogel made of natural extracts from seaweed (agar, carageenan or alginate), plants (arabic or tragacanth), seed gums (guar, locust bean gum or tamarind) or microrganisms (dextran, gellan or xanthan gum.). These compounds will gel when mixed with or dropped into an appropriate electrolyte (copper sulphate, calcium chloride or ammonium chloride). Ionic bonds are formed to produce stable complexes. Useful adjuvants such as nutrients, plant growth regulators, pesticide and fungicide can be supplied to the plant propagule within the encapsulation matrix.
- 4. IIIM-CSIR Artificial Seeds and Cryopreservation Need for Synthetic seed production technology Characteristics of Clonal Propagation Systems •Micropropagation Low volume, small scale propagation method Maintains genetic uniformity of plants Acclimatization of plantlets required prior to field planting High cost per plantlet Relatively low multiplication rate •Greenhouse cuttings Low volume, small scale propagation method Maintains genetic uniformity of plants Rooting of plantlets required prior to field planting High cost per plantlet Multiplication rate limited by mother plant size •Artificial seeds High volume, large scale propagation method Maintains genetic uniformity of plants Direct delivery of propagules to the field, thus eliminating transplants Lower cost per plantlet Rapid multiplication of plants.
- 5. IIIM-CSIR Artificial Seeds and Cryopreservation Ease of handling while in storage Easy to transport Has potential for long term storage without losing viability Maintains the clonal nature of the resulting plants Serves as a channel for new plant lines produced through biotechnological advances to be delivered directly to the greenhouse or field Allows economical mass propagation of elite plant varieties Synthetic seeds have the potential for providing an inexpensive plant delivery system. It also provides rapid bulking up for the production of individual genetically engineered plants. Advantages of Artificial or Synthetic Seeds over Somatic Embryos for Propagation
- 6. IIIM-CSIR Artificial Seeds and Cryopreservation Basic Requirements for Production of Synthetic Seeds • Production of high quality, vigorous somatic embryos that can produce plants with frequencies comparable to natural seeds. • Inexpensive production of large number of high quality somatic embryo with synchronous maturation and high conversion frequency.
- 7. IIIM-CSIR Artificial Seeds and Cryopreservation Steps of Synthetic Seeds Production Establishment of somatic embryogenesis Maturation of somatic embryos Synchronization and singulation of somatic embryos Mass production of somatic embryos Standardization of encapsulation Standardization of artificial endosperm Mass production of synthetic seeds Green house and field planting
- 8. IIIM-CSIR Artificial Seeds and Cryopreservation What are somatic embryos? Somatic embryos are bipolar structures with both apical and meristematic regions, which are capable of forming shoot and root respectively. A plant derived from a somatic embryo is sometimes referred to as an ‘embling’.
- 9. IIIM-CSIR Artificial Seeds and Cryopreservation Somatic Embryos vs Zygotic Embryos and Their Advantages Somatic Embryos Zygotic Embryo Develop from somatic cells Can be used to produce duplicates of a single genotype. It does not involve sexual recombination, thus allow specific and directed changes to be introduced into desirable elite individuals by inserting isolated gene sequences into somatic cells. Develop from zygote (i.e. fusion product of male and female gametes) Cannot be used. It involves sexual recombination.
- 10. IIIM-CSIR Artificial Seeds and Cryopreservation Somatic Embryogenesis A Young Plant Isolated single cells from leaves Doublet formation after first mitosis in culture Cell colony following many mitosis Globular embryo Heart shape embryo Torpedo stage embryo
- 11. IIIM-CSIR Artificial Seeds and Cryopreservation Encapsulation Encapsulation is necessary to produce and to protect synthetic seeds. The encapsulation is done by various types of hydrogels which are water soluble. The gel has a complexing agent which is used in varied concentrations. Gelling agent (% w/v) Complexing agent (μM) Sodium alginate (0.5 – 5.0)* Sodium alginate (2.0) with Gelatin (5.0)* Carragenan (0.2 – 0.8) Locust beam gum (0.4-1.0) Gelrite (0.25) Calcium salts (30 –100) Calcium chloride (30 –100) Potassium chloride Ammonium chloride (500) temperature lowered *Alginate hydrogel is frequently selected as a matrix for synthetic seed because of its moderate viscosity and low toxicity for somatic embryos and quick gellation, low cost and biocompatibility characteristics.
- 12. IIIM-CSIR Artificial Seeds and Cryopreservation Principle and Conditions for Encapsulation with Alginate Matrix • The major principle involved in the alginate encapsulation process is that the sodium alginate droplets containing the somatic embryos when dropped into the CaCl2.2H2O solution form round and firm beads due to ion exchange between the Na+ in sodium alginate with Ca2+ in the CaCl2 . 2H2O solution. • The hardness or rigidity of the capsule mainly depends upon the number of sodium ions exchanged with calcium ions. Hence the concentration of the two gelling agents i.e., sodium alginate and calcium CaCl2.2H3O and the complexing time should be optimized for the formation of the capsule with optimum bead hardness and rigidity. • Sodium alginate (3%) upon complexation with CaCl2. 2H2O (1%) for half an hour gives optimum bead hardness and rigidity for the production of viable synthetic seeds.
- 13. IIIM-CSIR Artificial Seeds and Cryopreservation Utilization of Synthetic Seeds • Multiplication of non-seed producing plants, hybrids or propagation of polyploid plants with elite traits. • Propagation of male or female sterile plants for hybrid seed production. • Germplasm conservation of recalcitrant species. • Conservation of transgenic plants. • Production of disease free plants Crops: Synthetic seed production and plant conversion Reported In vitro propagules Crops Somatic embryos Alfaalfa, celery, brinjal, carrot, brassica, lettuce, potato, sweet potato, cucumber, asparagus, rice, horse raddish, wheat, triticale, maize, sorghum, soyabean, sugarcane, coffee, tobacco and cotton Axillary or adventitious buds mulburry, eucalyptus and grape Shoot tips Banana, cardamom and Carum carvi
- 14. IIIM-CSIR Artificial Seeds and Cryopreservation Preparation of Artificial Seeds With Hairy Root Cultures
- 15. IIIM-CSIR Artificial Seeds and Cryopreservation Potential Uses of Artificial Seeds Delivery Systems: • Reduced cost of transplants • Direct greenhouse and field delivery of: • Elite, select genotypes • hand-pollinated hybrids • Genetically engineered plants • Sterile and unstable genotypes • Large-scale mono cultures • Mixed-genotypes plantations • Carrier for adjuvants such as microorganisms, plant growth regulators, pesticides, fungicides, nutrients and antibiotics • Protection of meiotically unstable, elite genotypes. • Can be conceivably handled as seed using conventional planting equipment. • Germplasm conservation and cryopreservation • Propagation with a low cost, high volume capabilities of seed propagation. • Can be produced with in short time ( one month) at anytime and in any season of a year • Dormancy period can be reduced to a great extent thereby shortening the life cycle of plant. • Production and transport of pathogen free propagules across the international borders avoiding bulk transportation of plants, quarantine and spread of diseases. • Storage of genetically heterozygous plants or plants with unique gene combination. Analytical Tools: • Comparative aid for zygotic embryogeny. • Production of large numbers of identical embryos. • Determination of role of endosperm in embryo development and germination • Study of seed coat formation. • Study of somaclonal variation.
- 16. IIIM-CSIR Artificial Seeds and Cryopreservation Limitations Although results of intensive researches in the field of synthetic seed technology seen promising for propagating a number of plant species. Practical implementation of the technology is constrained due to the following reasons: • Limited production of viable micropropagules useful in synthetic seed production. • Anomalous and asynchronous development of somatic embryos. • Improper maturation of the somatic embryos that makes them inefficient for germination and conservation into normal plants. • Lack of dormancy and stress tolerance in somatic embryos that limit the storage of synthetic seeds. • Poor conservation of even apparently normally matured somatic embryos and other micropropagules into plantlets that limit the value of the synthetic seeds and ultimately the technology it self . • Somaclonal variation is the genetic variation arising through tissue and cell culture. If it happens, the basis of uniformity of synthetic seed will be defeated.
- 17. IIIM-CSIR Artificial Seeds and Cryopreservation Applicability and Feasibility of Artificial Seed Production Technology • In order to be useful, synthetic seed must either reduce production costs or increase crop value. • The relative benefits gained, when weighed against development costs, will determine whether its use is justified for a given crop species. • Considering a combination of factors, including improvement of the existing embryogenic systems, relative cost of seed as well as specific application for synthetic seed of seedless water melon would actually cost less than conventional seed, providing a benefit at the outset of crop production. • Although embryogenic systems for this crop do not exist, the benefit that could be conferred by use of synthetic seed would be very great. • Value added aspects that would increase crop worth are numerous and include cloning of elite genotypes, such as genetically engineered varieties, that cannot produce true seed.
- 18. IIIM-CSIR Artificial Seeds and Cryopreservation Cryopreservation
- 19. IIIM-CSIR Artificial Seeds and Cryopreservation • Cryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as 77 K or −196°C. • Any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. • First successful cryopreservation of fish sperm was reported in 1950s. • In fish farming and seed production sector, storage of milt can facilitate, 1)selective breeding 2)hybridisation 3)commercial seed production. Cryopreservation
- 20. IIIM-CSIR Artificial Seeds and Cryopreservation Risks associated with cryopreservation Damage to cells during cryopreservation occur during the freezing stage, and include, Solution effects • As ice crystals grow in freezing, water solutes are excluded, causing them to become concentrated in the remaining liquid water. • High concentrations of some solutes can be very damaging Extracellular ice formation • When tissues are cooled slowly, water migrates out of cells and ice forms in the extracellular space. • Too much extracellular ice can cause mechanical damage to the cell membrane due to crushing. Dehydration • The migration of water causing extracellular ice formation can also cause cellular dehydration. • The associated stresses on the cell can cause damage directly. Intracellular ice formation • Some organisms and tissues can tolerate some extracellular ice, but any appreciable intracellular ice is almost always fatal to cells.
- 21. IIIM-CSIR Artificial Seeds and Cryopreservation Prevention of Risks • Lethal intracellular freezing can be avoided if cooling is slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. • The rate differs between cells of differing size and water permeability. • A typical cooling rate around 1°C/min is appropriate for many fish cells after treatment with glycerol or dimethyl sulphoxide (DMSO)
- 22. IIIM-CSIR Artificial Seeds and Cryopreservation Cryopreservation Methods
- 23. IIIM-CSIR Artificial Seeds and Cryopreservation Steps Involved in Cryopreservation
- 24. IIIM-CSIR Artificial Seeds and Cryopreservation Steps Involved in Cryopreservation
- 25. IIIM-CSIR Artificial Seeds and Cryopreservation Physical Events Occurred During Freezing and Thawing
- 26. IIIM-CSIR Artificial Seeds and Cryopreservation Addition of Cryoprotectant • Cryoprotectants are chemicals that allow cells to survive freezing protocols. • Cryo-injury is the degeneration of the sperm membrane during cooling and thawing. • It is regarded as the principal cause of reduced post-thaw viability. Cryoprotectants are of two categories: 1) Permeating cryoprotectants – DMSO, Methanol, Glycerol. 2) Non-permeating cryoprotectants -Glucose or sucrose, dextran, milk and egg proteins, antifreeze proteins such as those found in polar fishes. • Cryoprotectants are added to extenders to minimize the stress on cells during cooling and freezing. • Methanol is highly toxic for carps but used in tilapia. For carps, DMSO is considered to be the best.
- 27. IIIM-CSIR Artificial Seeds and Cryopreservation Cryopreservation Non cryogenic preservation (short-term) • It is a short duration preservation for 3-4 days. • The milt sample is diluted with extender and stored in a thermocol chamber with ice or in refrigerator at 4ºC. • This is helpful for designing different sib breeding out of different male and female traits. • Milt samples are brought to room temperature before inseminating the egg sample for better result. Cryogenic preservation (long term) • Spermatozoa can be stored for years together in liquid nitrogen (-196ºC). • At this temperature all the biochemical activities of a living cell ceases. • The milt is diluted in extender and mixed with cryoprotectant.
- 28. IIIM-CSIR Artificial Seeds and Cryopreservation Cryopreservation Equilibration • It is a time interval between mixing the milt in the diluent and putting the mixture into liquid nitrogen. • For effective protection during cooling sufficient time must be allowed to facilitate the penetration of cryoprotectants into cells. • The longer the equilibration time the lower the fertility, because the cryoprotectant may become toxic to cells. Ampouling • The diluted samples within the equilibration period is filled in French straws (250ul) and plugged with sealing powder. • The sealed and polypropylene vials frozen straws are stored under liquid nitrogen. Labeling of straws • The straws used should be labeled to indicate fish identification number, cryoprotectant, and cryoprotectant concentration. • A simple method for labeling is to use straw colour for identification. • The basic information that should be recorded is given below. Fish : species, sex, origin, number. Preservation details : Date, pre-frozen quality, diluent, dilution rate(s), cooling/warming
- 29. IIIM-CSIR Artificial Seeds and Cryopreservation Cryopreservation Labeling of goblets Straws can be stored in LN2 in plastic containers called goblets. • Goblets should be labeled to identify species, date, technician, type of study and additional pertinent information. • To avoid problems in removing frozen straws do not pack them too tightly in the goblets. Labeling of cans • Goblets are usually attached to aluminium canes for storage in LN2. • Cans should be labeled on the top for easy identification. • Labeling decreases excessive searching of cryocan contents for necessary straws, thus helps to protect the straws from warming during handling. Freezing • The milt should be frozen immediately after collection. • Freezing should be rapid to minimise thermal shock and not so fast as to allow the formation of cellular ice crystals. • The sealed visitubes require to be cooled at 15ºC/minute by programmable cooling chamber. • Straw freezing is possible by manual vapourisation over Li-N2 in thermocol chamber.
- 30. IIIM-CSIR Artificial Seeds and Cryopreservation Cryopreservation Storing • Liquid nitrogen (-196ºC) is used as a cryogen. • The freezed milt straws/visitubes are immediately immersed in liquid nitrogen and kept undisturbed. • The evaporation loss of liquid nitrogen is compensated by regular filling to the container. • To manage cryobanks efficiently, it is essential to keep comprehensive records of all stocks preserved. Thawing • Fast thawing is preferable as slow thawing can recrystalize the small intercellular crystals which may damage the cells. • The cryomilt samples of carps can be thawed in warm water of 38+1ºC for 7-9 seconds.
- 31. IIIM-CSIR Artificial Seeds and Cryopreservation Importance of Cryopreservation • Under asynchronous conditions, gametes can be collected and held under appropriate storage conditions for later use. • Few male broodstock can be maintained and effective population size can be increased. • Cryobanks can play a crucial role in the genetic management and conservation of wild resources and endangered wild or cultured stocks. • Sperm cryobanks can also be used to manage the genetic integrity of farmed stocks. • Through frozen sperm banks, genes from valuable stocks which have desirable characteristics, can be preserved for future use and development. • In selection programmes, milt from original stocks can be cryopreserved and used to make half-sibling comparisons at a later date. • Periodic freezing of milt from disease free males can help to buffer farms against disease outbreaks. • In instances of scarcity of males, e.g., protoandrous hermaphrodites, milt can be stored for later use. • Retrieval of the whole genome of endangered or extinct stock from cryopreserved milt can be done through androgenesis.
- 32. IIIM-CSIR Artificial Seeds and Cryopreservation Importance Steps and Factors Involved in Successful Root Cryopreservation