2. PLANT TISSUE CULTURE.pptx

Btech 110: Basic Biotechnology and Society Plant Tissue Culture  Standard TCL facilities - stated b the Phil. Agricultural Structures (PAES 416:2002)  Agric’l Structures PTCL - specify the minimum requirements for PTCL  Department Circular No. 03, Series of 2020, Revised General G...

Btech 110: Basic Biotechnology and Society Plant Tissue Culture  Standard TCL facilities - stated b the Phil. Agricultural Structures (PAES 416:2002)  Agric’l Structures PTCL - specify the minimum requirements for PTCL  Department Circular No. 03, Series of 2020, Revised General Guidelines for the Accreditation of Plant Tissue Culture Facility (PCTF) for the Production of Quality Planting Materials.  What is Plant tissue culture? - broadly refers to the cultivation in vitro of all plant parts (single cells, tissues and organs i.e. leaves, roots, shoot tips) in artificial medium under aseptic condition - also referred to as in vitro (meaning literally in a ‘glass’) culture, micropropagation or clonal propagation http://cpbhisar. org/ 3 Fundamental Abilities of Plants 1. Totipotency -potential or inherent capacity of a plant cell to develop into an entire plant if suitably stimulated - implies that all the information necessary for growth and reproduction of organism is contained in the cell 2. De-differentiation capacity of mature cells to return to meristematic condition and development of a new growing point Differentiation  process by which meristematic cells (e.q SAM & RAM) undergo changes in structure, shape to perform the specific function ; cannot divide De-differentiation - act leading to maturation;  process by which the already differentiated or mature cells became meristematic again ; can divide and produce new cells Redifferentiation –  ability of cells/ tissues to reorganize to new organ; or cells/tissues which lose the ability to divide and perform specific function again; lose power to divide 2. De-differentiation capacity of mature cells to return to meristematic condition and development of a new growing point 3. Competency and Determination Competency - endogenous potential of a given cells or tissue to develop in a particular pathway Determination, in which cells become irreversibly committed to a particular pathway Types of in vitro culture 1. Culture of intact plants e.g. seed culture in orchids 2. Organ culture e.g. meristem culture, shoot tip culture root culture, anther culture 3. Embryo culture e.g. immature embryo culture 4.Callus culture 5.Cell culture 6.Protoplast culture  Advantages a. Rapid method of mass producing planting materials b. Not seasonal hence ensuring year-round availability of planting materials c. Planting materials produced could be disease-free d. Saves economic yield (e.g. potato tubers) which is otherwise used as planting materials for next cropping e. Crops have higher growth and yield potential than those propagated by seeds or  Limitations 1. Requires equipment/facility for aseptic condition. 2. Need skilled micropropagator. 3. Specific protocols for each plant species. 4. Requires high initial capital. 5. Propagules may be expensive.  Application of tissue culture 1. Elimination of diseases and production of clean planting materials (e.q. from virus-infected stock). 2. Production of pathogen free plants. 3. Micropropagation 4. Crop improvement, plant breeding 5. Production of biochemicals, secondary products expression in vitro. 6. Embryo rescue. 7. Germplasm preservation. 8. Plant genetic engineering  Important historical development in the plant tissue culture  1878 Hermann Vochting - discovered that cells along the stem length are capable of forming roots as well as shoots, depending upon their relative portion in the cutting (e.q. young secondary phloem capable of reverting into meristematic tissues as root initials)  1902 GOTTLIEB HABERLANDT - Father of Plant tissue culture - conducted tissue culture experiments which gave insight to the properties and potential which a cell possess - attempt to culture palisade tissues and epidermis of E. crasspes and grew these on Knop’s solution with sucrose, however none of cells divided nor differentiated - failure due to lack of plant growth regulators required for cell division and differentiation - expresses the concept of TOTIPOTENCY 1962 MURASHIGE & SKOOG : developed the famous media on tobacco tissue culture: - with mineral salts, vit B, sugar, auxin and cytokinin - concentration of some salts were 25 times that of Knop’s solution particularly, the level of NO3− and NH4+ Philippine Tissue Culture DR. EMERITA DE GUZMAN -noted for her research on the propagation of pure makapuno trees DR. RAMON BARBA DR. EMERITA DE - pioneered the induction of mango flowering G - development of micropropagation techniques for UZ M banana and abaca A N DR. HELEN VALMAYOR -Micropropagation of orchids DR. RAMON DR. HELEN B VALMAYOR A Plant development In whole seed plants  denotes the gradual and progressive changes in size, structure and function which collectively comprise the transformation of a zygote into a mature reproductive plant In tissue culture  gradual process involving appearance of organs from initials or primordia or of whole plants from single cells or tissues Development encompasses interrelated process: Growth - irreversible increase in size - generally, includes cell division and cell enlargement Cellular differentiation - refers to the transformation of cells into diversified cells with various biochemical, physiological and structural specialization 2 kinds of plant growth are possible in vitro: Organized Growth - occurs either when plant organs such as growing point : SAM&RAM, leaf initials etc are transferred to culture (where they continue to grow with structures preserved), - The process of de novo organ formation is called organogenesis or morphogenesis. Unorganized Growth - seldom found in nature, and occurs frequently when pieces of whole plants are cultured in vitro - tissues formed typically lack recognizable structure  Application of Tissue Culture Techniques to Horticultural Crops and Definition of Terms Tissue culture of ornamentals Anthuriu m Aglaonem a, Orchi Alocasia, ds etc..  a b In vitro culture of Gladiolus (a), Chrysanthemum (b) Different tissue culture-derived orchid varieties sold commercially Tissue culture of fruit crops  Micropropagation techniques may replace vegetative propagation techniques employed  example: majority of all commercially produced strawberries are produced from in- vitro derived plantlets Commercializa tion of date palm; pomengranate , etc. Tissue culture of vegetable crops Source: https ://www.vubhb.cz/en/laboratory-of- Source: tissue-cultures https://potatoes.colostate.edu/p otato- certification-service/  Tissue culture of plantation crops Source: https:// www.pinterest.ph/pin/banana-tissue-culture-- 739997782493896556/ Source: https://manoxblog.com/2020/07/12/cannabis-tissue-culture-technology- company-raises-over- 15-million/ Tissue culture of woody species Types of tissue culture can be grouped also by the structures formed in culture. 1. Plantlet formation - e.q. meristem culture, shoot culture, adventitious shoot formation Source: https:// www.researchgate.net/figure/Different-stages-of- meristem-culture-in-Abelmoschus-esculentus-A- Growth-of- isolated_fig1_209843881 2. Seedling formation - seed culture, embryo culture, embryo rescue, ovule and ovary culture Source: https:// www.researchgate.net/figure/n-vitro-organogenesis-from-culture-of- mature-zygotic-embryos-of-Passiflora-edulis-a_fig1_268743307 3. Callus formation - callus culture, protoplast suspension culture 4. Somatic (vegetative) embryo formation - somatic embryogenesis, synthetic seed 1. Organ cultures - involve culture of isolated plant organs and manipulation of cultural conditions so it remains organized throughout all of the cultural +procedures - e.q. shoot tips, buds, leaves, nodal segments, root tip, stem with one to four nodes 1.1Shoot culture -most common form of micropropagation Direct 1.2. Nodal culture -used with species with strong apical dominance - long shoots are cut into single nodes (with lateral buds) and placed vertically in the medium - the new shoot elongates and the process is repeated 1.3 Meristem culture - cultures initiated with small apical meristem (meristem culture), with 2 or more leaf primordia (shoot tip culture) - usually forvirus eradication Source: ww.ndsu.edu/pubweb/chiwonlee/plsc368/lecture/chpt9/cpt9clones. html 2. Embryo Culture - excision of a fertilized or unfertilized seed embryo and germinating it in aseptic culture - usually for propagation and breeding http://www2.bioversityinternational.org/publications/Web_version/3 3. Seed culture  In nature, orchid seeds rely on symbiotic mycorrhizal fungi associations to germinate.  However, they can germinate and develop into seedlings on the proper tissue culture medium without the 4. Callus culture– begin with pieces of tissues and cells from an organized part of plant (e.q. leaves) but cultural call conditions are manipulated us to create an unorganized unorganized state e.g. a callus, single state or aggregate cells 5. Suspension or Cell Culture - started by placing a piece of friable callus or homogenized tissue in liquid medium so that cells dissociate from each other  6. Anther and Pollen Culture - culture of anther and/or immature pollen grain in an effort to obtain a haploid cell or callus line (usually for breeding) 7. Protoplast culture- protoplasts are isolated single cells the from which wall has been removed by cell -fungal when enzymes cultured, a new wall is synthesized thus restoring original state of the plant cell - however, when protoplast is wall-less, there is apossibility to: 1.add foreign material to protoplast 2. fuse protoplasts of 2 cultivars (somatic hybridization) General procedures in plant tissue culture /micropropagation: 1.Isolation of plant part or explant 2.Placing of excised plant part in appropriate environmental condition - includes inoculation and placing culture vessel in incubation room with right physical conditions 3.Regeneration of Plants - through SE or Organogenesis  PRINCIPLES OF MICROPROPAGA Basic plant regeneration and TION propagation procedures I. Modes of Plant Regeneration 1.Embryo culture - aseptic culture of zygotic embryo in nutrient medium 2.Plant development through organogenesis -formation from outgrowth of shoot from callus or initiation of axillary buds from cultured tips -shoot is unipolar structure and physically connected 3. Somatic or asexual embryogenesis -production of embryo like structures from somatic cells Somatic embryo is an independent bipolar structure and is not physically attached to the tissue of origin Somatic embryos from nucellus tissues - frequently formed on nucellus tissue of cultured ovules  MODE OF PLANTLET REGENERATION Organogenesis Embryogenesis Changes leading to Production of production of bipolar structure unipolar structure (Root+shoot) (root or shoot) with attached to the parent independent tissue vascular system Origin-group of cells Origin- single cell Direct regeneration Direct Indirect regeneration regeneration Indirect regeneration Somatic embryogenesis - process of a single cell or a group of cells initiating the developmental pathway that leads to reproducible regeneration of non-zygotic embryos capable germinating 2 Ways of Somatic Embryogenesis: Direct embryogenesis  Indirect embryogenesis Embryoge nesis - the process of initiation and development of an embryo from a zygote (zygotic embryogenesis) or a somatic cell (somatic embryogenesis) Both types of embryos develop as passing through typical developmental stages: globular, scutellar and coleoptilar stages for monocots, or globular, heart, torpedo and cotyledonary stages for Thank you for Listening!