Slide 1: MICROPROPAGATION & STAGES By- JWALANT LOHIYA MICROPROPAGATION : MICROPROPAGATION MICROPROPAGATION:- It is the in vitro regeneration of plants from organs ,tissues cells or protoplasts & true to type propagation of selected genotype using in vitro culture technique for micro propagation . It is important to use healthy ,vigorously growing plants as source material of selected genotype using in vitro culture technique. Factor affecting source tissue :- : Factor affecting source tissue :- a) Healthiness b) Bud dormancy :- * All temperate woody perennials show a seasonal cycle of bud activity. * Selection should be done of buds or tissue from nondormant plants. * Use buds that are all at the same stage of flushing. * It is known that period when the bud is emerging from the temporary period of dormancy is likely to be the period of maximum response. Slide 4: C) Effect of juvenility and maturity :- Stage of maturity of woody plants also affects the response of the tissue or a tissue culture medium. * As the age of tree increase its juvenility goes down and maturity increases so tendency or potential to regenerate form callus is decreases. Slide 5: Reducing effect of maturity:- 1. Pruning 2. Repeated Grafting of buds from mature scion wood onto seedling rootstocks. 3. Alternative approach is to establish molecular markers ,which make use of band patterns from restriction fragment length polymorphism(RFLP), RAPDs & micro satellite analysis that correlates with particular character. Slide 6: METHODS OF MICROPROPAGATION Methods used in micro propagation are based on the production of sterile tissue ,stimulation of regeneration ,rapid growth of the young plantlets , rooting of plantlets and their weaning onto normal soil conditions. Shoot tip and axillary bud culture. Meristem culture. Adventitious bud production. (A) Axillary budding:- Development of shoots from preexisting meristems on nodal region ensures genetic stability of the regenerants is termed as ax illary budding.Most reliable method of in vitro propagation producing about 90% of the current production of the micro propagated plants. Slide 7: (B) Meristem culture:- Cultivation of axillary or apical shoot meristems is known as meristem culture. Shoot apical merited is the portion lying distal to the youngest leaf primordium. (C) Adventitious bud production:- Occurrs where non meristematic cells become meristematic in response to growth regulators in the external medium and nutrient gradients with in the tissue ,meristemoids is formed. These meristemoids then develop as stem or roots based on auxin & cytokinin ratio. Slide 8: Stages of Micro propagation At present ,micro propagation is a five stage process:- Selection and preparation of mother plants (stage0) Culture initiation(stage1) Multiplication(stage 2) Rooting of shoots(stage3) transfer of plants to soil Slide 9: Selection & preparation of mother plants(stage 0):- Stage was introduced by Edberg and Maine in 1981. Consist of identification of mother plants and their preparation for establishment of contamination free cultures. In case of woody plants ,bulbs tubers ,corms etc ., suitable temperature & photo period use to overcome bud dormancy and provide more responsive explants. While taking explants from the field , age of mother plant , yield potential & healthiness should be considered. Slide 10: Some of the explants commonly used for micro propagation Mode of micro propagation Type of explant Remarks Enhanced axillary proliferation Explant having preexisting meristems a)Shoot tips This may be the only explant in rosette plants. b)Nodal segments c) Pieces of inflorescence In case of cauliflower ,pieces of curd are used Adventitious shoot bud formation Explants devoid of meristems Explant drived from the root , stem leaf are nucellus. Somatic embryogenesis a)Immature zygotic embryos cereals ,woody spp. b)Nucellus In case of citrus and mango. Virus elimination Apical meristem A minimum of surrounding tissue included. Slide 11: Multiple shoot formation in shoot tip culture Slide 12: Initiation of aseptic culture(Stage 1):- * Consist of surface sterilization of explants & establishing them in vitro. * When the objective of in vitro culture is virus elimination ,it is desirable to culture the shoot tip meristem. * Sub terminal & older segments withstand the toxic effects of sterilizing agents much better than the terminal cuttings. * Sterilization technique depends on the concentration of sterilizing agents & plant material used for micro propagation Slide 13: Multiplication(stage 2):- * The stage consists of production of few to several shoots/somatic embryos from each culture. * Multiplication can be achieved by the following three approaches a) Enhanced proliferation of axillary shoot bud . b) Induction of adventitious buds ,bulbs. c) Somatic embryogenesis. This stage aims st obtaining a rapid increase on no. of shoots or asexually embryos which can ullimate be used to provide the large no.of plants. Slide 14: Three main routes for micro propagation Mother plant Explants having pre- Explants devoid of Explants having embryogenic existing meristems pre-existing meristems potential 1.Shoot tips 2.Nodal segments Adventitious bud induced by an anxin 3.Young inflorescence pieces regeneration unually ,2 ,4-d Enhanced axillary branching by Multiple shoots Somatic embryos(SEs) cytokinin Rooting Multiple shoots SEs Artificial seeds Plantlets germinated Rooting a. In vitro b. ex vitro Hardening Planted in soil SEs are germinated Plant lets transfer to soil & give rise to plants hardening Slide 15: Somatic Embryogenesis:- Somatic embryos have been used for micro propagation of several plant species and in some species they are the only route available for this purpose. E.g. oil palm ,date palm etc. Advantages of somatic embryogenesis:- Amenable to scaling up. Fastest multiplication rate. Maintained for long period of time. SEs are used to produce artificial seeds. There is no need for a rooting stage Limitations of SE route:- . SE quality is often poor & artificial seeds are low(15-25%). . Synchronisation of SE is inadequate. . Risk of somaclonal variations. . Field conversion frequencies of SEs is also poor. Slide 16: Developmental Pattern of SEs Slide 17: Rooting of shoots(Stage 3):- * Stage involves preparation of shoots for rooting. The proliferated shoots are transferred to rooting medium. * A rooting medium which is different from the shoot multiplication medium. Particularly in hormonal & salt composition. Rooting stage contain high auxin level. Kinds of Rooting:- * In vitro rooting :- Shoots rooted in an agar medium. Ex vitro/in vivo rooting:- Shoots are rooted directly in vermiculite mixture. Rooting medium has low concentration of salts & contain NAA or IBA as growth regulator. Slide 18: In vitro rooting Ex vitro/In vivo rooting Labor saving & expensive. . Require more labor & not much expensive. Accounting for 35-75% of the total . In vivo rooting combine the rooting & cost of micro propagation. Acclimatization stages & reduces the aseptic handling. . In vitro roots are often thick ,lack root . In vivo formed roots are structurally & fun- hairs. tonally of better quality . Poorly developed vasculature. . They Often die during transfer to soil. . No risk of root damage during transfer to . Often callus may form at the cut ends soil. of the shoots in vitro leading to poor connection b/w the shoots & roots. Slide 19: Transfer of plantlets to soil (stage 4):- Describe as hardening ,in this process ,the plantlets under aseptic condition of lab have to be shifted to glasshouses. Hardening typically involves slowly weaning the plantlets from a high humidity ,low light warn environment to what would be considered to normal growth environment. Plantlets are removed from the plant media & transferred to soil(potting compost) for continued growth by conventional method. Hardening procedures are of the following three types:- In vitro hardening. Ex vitro hardening. Formation of tubers. Tissue Culture Stages: Tissue Culture Stages Slide 22: Problems arise in vitro during micro propagation Due to its commercial enterprise ,hence aim is to reduces the problem that arises in tissue culture practice. Removal of phenolic compounds :- Phenolic compounds are produced by the plant in response to stress ,many of these compounds are phototoxic & will lead to death of plant tissue if released on to cells. Oxidation of polyphones by polyphone oxides when the tissue is excised caused increase level of browning in the shoot tips. Methods to reduces the effect of phenolics:- Source plant must be healthy & disease & pest free. Phenolics are either absorbed or leached out of the explants. Absorbs of phenolic compound- charcoal ,or polyuinylyrrolidme added to reduces tissue black eniring. Disadvantage:- Charcoal also absorbs growth eegulators. Leaching of phenolic :- Leach into the medium by washing excised tissue in running water for 2-3 hr. The activity of polyphone oxidize is inhibited by inclusion of a chelating agent such as ethylene demine tetra acetic acid. Slide 23: Avoiding of endogenous contamination The evidence that endogenous contaminates are present is that the infection only becomes apparent at a late stage in the initiation of culture or after a series of subculture ,or the tissue continues to loaf brown despite changes to media composition method to reduce over come. Shoot tips or explants are isolated. Bacteria are removed from the culture by the use of antibiotics added to the medium. Viruses may be vested by the in clusion of antiviral agents such as virazol. Vitrification of plantlets in vitro:- Misshapen & thickened leaves & skins have been referred to collectively as vitrify action. Slide 24: Advantage of micropropagation:- √ Rapid multiplication rate of easity generated spp. ,the cycle of axillary bad rapid multiplication of rare genohybe & shoot multiplication soon provides very large no. of plants or mass clonal propagation of desired. √ Lack of seasonal restriction on plant production since the propagation stage is laboratory based. √ Maintenance of self incompatible inbred lines used in hybrid seed production. √ The production of virus free stocks such as stawberries & potato by meristem culture. √ In case of dioecious spp.plants of one sex may be more desirable than the other. Slide 25: Disadvantages of micro propagation * Micro propagation methods through use of tissue culture involves capital intensive expensive materials like auto ,laminar air flow bench ,controlled culture rooms. This is a technically skilled work. Contamination is a serious threat & cause severe damage to material. No uniform standard method ,that is diff. spp. Give diff. response to the growth hormones applied. Small delicate plantlets are produced ,which take longer initial time to grow. Genetic stability is doubtful in certain methods. Browning of media. Hyperhydration. Slide 26: T hank you.