Plant regeneration and somaclonal variations

Plant regeneration and somaclonal variations

1. PLANT REGENERATION

2. PLANT REGENERATION The process of growing an entire plant from a single cell or group of cells. Regeneration is possible because plant cells can be made totipotent using hormones . Differentiated tissue: stems, leaves, roots, etc. Undifferentiated (embryonic) cells are totipotent : can become a whole new plant by differentiating into a whole new plant.

3. THE PLANTS CAN BE REGENERATED BY ORGANOGENESIS It refers to the formation of organs from the cultured explants. The shoot buds or monopolar structures are formed by manipulating the ratio of cytokinins to auxins in the cultures. SOMATIC EMBRYOGENESIS In Somatic embryogenesis, the totipotent cells may undergo embryogenic pathway to form somatic embryos which are grown to regenerate into complete plants .

4. PLANT MATERIAL—THE EXPLANT Any part of a plant taken out and grown in test tube under sterile conditions in special nutrient media is called explant .

5. HORMONES USED IN PLANT TISSUE CULTURE 1. Auxins 2. Cytokinins 3. Gibberellins 4. Abscisic Acid 5. Polyamines

6. ENVIRONMENTAL CONDITIONS: NUTRIENT MEDIUM inorganic salts , a carbon source ,vitamins ,amino acids and growth regulators (ii) ASEPTIC CONDITIONS The microbes compete with growing tissue and finally kill it. It is essential to maintain aseptic conditions of tissue culture. Thus sterilization means complete destruction or killing of.

7. microorganisms so that complete aseptic conditions are created for in vitro culturing. (iii) AERATION OF THE TISSUE Proper aeration of the cultured tissue is also an important aspect of culture technique. It is achieved by occasionally stirring the medium by sterring or by automatic shaker.

8. REGENERATION OF PLANTLETS 1. Preparation of Suitable Nutrient Medium: Suitable nutrient medium as per objec­tive of culture is prepared and transferred into suitable containers. 2. Selection of Explants: Selection of explants such as shoot tip should be done. 3. Sterilization of Explants: Surface steriization of the explants by disinfectants and then washing the explants with sterile distilled water is essential. 4. Inoculation: Inoculation (transfer) of the explants into the suitable nutrient medium (which is sterilized by filter-sterilized to avoid microbial contamination) in culture vessels under sterile conditions is done .

9. 5. Incubation: Growing the culture in the growth chamber or plant tissue culture room, having the appropriate physical condition (i.e., artificial light; 16 hours of photoperiod), temperature (-26°C) and relative humidity (50-60%) is required. 6. Regeneration: Regeneration of plants from cultured plant tissues is carried out. 7. Hardening: Hardening is gradual exposure of plantlets to an environmental condi­tions. 8. Plantlet Transfer: After hardening plantlets transferred to the green house or field conditions following acclimatization (hardening) of regenerated plants

10. PLANT PROPAGATION

11. INTRO: Propagation of plant is defined as production of new individuals from a selected plant having all the characters of the original one.   New  plants  are created from a variety of sources: seeds,  cuttings , bulbs, tissue culture etc. Plant propagation in done sexually and asexually as well as on artificial platform using TISSUE CULTURE TECHNEQUIES.

12. PRINCIPLE: The basis of plant propagation is  totipotency ,  the capability of cells to regenerate missing parts and, subsequently, an entire organism.

13. TYPES : Sexual propagation  is with the use of seed or spore that is separated from the parent plant. This method is so termed "sexual" because there is the involvement of the sexes, referring to the contribution of both the male and female gametes in the production of new plants. Pollen is transferred from anther to stigma. Fertilization occurs and seeds are produce.

14. Asexual propagation , also called  vegetative propagation , is with the use of planting materials which are vegetative parts of any plant rather than seeds or spores which are reproductive parts. E.g. cutting, layering, grafting etc.

15. Tissue culture , also called  in vitro  culture and micropropagation , is the technique of propagating plants indoor under aseptic or sterile conditions in artificial growth media. The growth medium, also called  nutrient solution , is a mixture of  essential elements . The initial propagule in tissue culture is called  explant

16. MICROPROPAGATION Micro-propagation is the propagation of plants through tissue culture It is a proven means of producing millions of identical plants under a controlled and aseptic condition independent of seasonal constraints Small pieces of plant tissues ( ex plants )use for regeneration ( shoot system rooting and growing into full size plant)

17. STARTING MATERIAL Tip bud Leaf Axillary bud Internode Root Seed

18. STEPS OF MICROPROPAGATION Selection and preparation of mother plant Initiation of culture Multiplication Rooting Transfer to soil

19. 1.ESTABLISHMENT: The objective of this stage is to get clean cultures that can begin the process of shoot multiplication The explant is usually sterilized with a combination of detergent and bleach. In difficult situations alcohol or a fungicide may be used. 2. INITIATION: Explant placed into growth media.

20. 3. MULTIPLICATION: Explant transferred to shoot media where shoots can be constantly divided. The objective of the shoot multiplication stage is to increase the number of shoots produced by the original explant .

21. The culture or a portion of the culture is removed from the jar and placed on a sterile paper towel. A scalpel and forceps are used to cut and separate the larger culture into smaller pieces for transfer to a new jar to complete the subculturing procedure. A high cytokinin to auxin ratio is used during the multiplication stage to induce axillary or adventitious shoot formation

22. This ratio is decided upon by preliminaryresearch . Too high a concentration of cytokinin will result in a high number of adventitious shoots that do not elongate. Common cytokinins used in culture are benzyladenine and kinetin.

23. 4. ROOTING: Shoots multiplied in culture must be rooted in order to create a new plantlet. In the rooting stage, microcuttings are induced to form roots - usually by application of auxin . Micrcuttings can either be rooted in vitro or ex vitro . In general, microcuttings rooted ex vitro have a more normal root system and acclimatize to a normal growing environment better than cuttings rooted in vitro . However, the propagator has more control over the rooting environment in vitro and this method may fit their production scheme better.

24. Microcuttings are inserted directly into the rooting substrate often using forceps to handle the small cuttings. 5. TRANSFER TO SOIL: Finally, after roots have become well established on the microcutting , plantlets must be acclimatized to a normal growing environment This involves gradually moving to open-air conditions where the humidity is reduced and the light levels increased

25. APPLICATIONS: Rapid multiplication Seasonal independent production Use to conserve rare or endangered species Greater output

26. SOMACLONAL VARIATION

27. SOMACLONAL VARIATIONS: The term somaclonal variation was first used by Larkin and Scowcroft in 1981. Somaclonal variation is the variation seen in plants that have been produced by plant tissue culture. The term somaclonal variation is used for genetic variability present among all kinds of cells or obtained from cells cultured in vitro. Plants derived from such cells or progeny of such plant is called somaclones . These variations can be detected as genetic or phenotypic traits.

28. BASIC FEATURES: Variations in number and structure of chromosomes are commonly observed. Regenerated plants with altered chromosomal changes often show changes in leaf shape and colour , growth rate and habit, and sexual fertility. It is generally heritable mutations and persist in plant population even after plantation into the field.

29. CAUSES OF S.C VARIATIONS: The causes may be; Physiological Genetic Biochemical

30. 1. GENETIC Pre-existing variations in the somatic cells of explant that are Caused by mutations and other DNA changes. Typical genetic alterations are; changes in chromosome numbers ( polyploidy  and  aneuploidy ), Change in chromosome structure (translocations, deletions,  insertions  and duplications). DNA sequence (base mutations). Occur at high frequency

31. 2. Physiological Exposure of culture to plant growth regulators. Culture conditions 3. BIOCHEMICAL Lack of photosynthetic ability due to alteration in carbon metabolism Antibiotic resistance

32. ADVANTAGES: • Rather stable and high frequencies.  New alleles or even novel mutations may be isolated which were not available in the germplasm . Can be performed in all types of plants.  Reduce the time required for the release of new variety by two years as compared to mutation breeding.  A very effective selection method. 

33. DISADVANTAGES: Applicable only to those species cell cultures which regenerate into complete plants.  Selected cell lines often show reduced or no regeneration potential.  Show undesirable features such as reduced fertility, growth rate and even overall performance of plant.  Selected clones are unstable not always stable and heritable.