PLANT TISSUE CULTURE: CONCEPTS, VOCABULARY AND APPLICATIONS

MUDIT MISRA
B.TECH(BIOTECHNOLOGY) V SEMESTER SECTION-A
Email: mudit.misra@gmail.com

PLANT TISSUE CULTURE

VERSUS

BIOTECHNOLOGY ???!!!!!

WHAT IS BIOTECHNOLOGY???
BIO BIOLOGICAL SYSTEM(S) TECHNOLOGY INDUSTRIAL SCALE VENTURE

WHAT FOR?

FOR THE BENEFIT OF MANKIND

Cell/Tissue Culture

Instrumentation

Breeding

Natural Product Chemistry P h y si o l o g y

Chemical Engineering

BIOTECHNOLOGICAL ACTIVITY
Biochemistry Microbiology

G e n e t i c s

Molecular Biology (Proteomics, Genomics, Metabolomics)

Genetic / Recombinant Engineering

Organic Chemistry

Taxonomy

Cultivation know how

BIOTECHNOLOGY PRODUCT
DOWN-STREAM PROCESSING POST HARVEST PROCESSING/STORAGE

REGULATORY FORMALITIES CONCERNING ENVIRONMENT SAFETY PACKAGING AND LABELLING

TOXICITY/ALLERGY TESTING

SAFE TRANSPORT
NATIONAL/INTERNATIONAL POLITICS

CONSUMERS

WORKING DEFINITION OF BIOECHNOLOGY

INDUSTRIAL EXPLOITATION OF BIOLOGICAL SYSTEMS OR PARTS THEREOF FOR THEIR COMMERCIAL UTILIZATION IN THE SERVICE OF SOCIETY

BIOTECHNOLOGY IS AN OLD SCIENCE BEING PRACTICED BY NATURE AND MAN SINCE A LONG TIME TODAY WE ARE BETTER EQUIPPED IN TERMS OF KNOWLEDGE, EXPERIMENTAL TOOLS, INSTRUMENTATION AND CONTROLS TO UNDERSTAND, MANIPULATE AND HASTEN THE BIOTECHNOLOGICAL EVENTS AT CELLULAR, MOLECULAR, GENETICAL AND BIOCHEMICAL LEVELS ON REAL TIME SCALE WITH PRECISION

WHY WE NEED BIOTECHNOLOGICAL INTERVENTIONS IN PLANTS ??

yPopulation rise yEnhanced diseases yLess land for agriculture yMalnutrition yDesire to learn the secretes of nature

Demand

yWe need more plants yWe need quality plants yWe need healthy plants yWe need to conserve plants yWe need more natural products based medicines

yWhat dictates a cell to divide and follow various developmental patterns of growth yHow cellular tasks are performed through the dialects of genetics and metabolite chemistry yHow can the fate of a living cell be modulated experimentally yHow alternate strategies to obtain desired plant products (natural compounds) in large quantity be devised at various level of cellular differentiation

We must
We dream & aspire to know
yDesign novel plants superior in traits and productivity yUpgrade the genetic machinery through novel gene combination in an otherwise acceptable genomic background yAdd value to our existing crop usage

PLANT TISSUE CULTURE CONCEPTS VOCABULARY APPLICATIONS

PLANT TISSUE CULTURE

A general term that encompasses and is concerned with the study of cells, tissues and organs maintained or grown in an artificial environment (in vitro) for more than 24 hours on a synthetic substrate.

Cell culture:Culture of isolated unorganised cells in vitro. Tissue culture: Culture of tissues in vitro in a way that may
allow preservation of original derivatization of new tissues. architecture/ function or

Organ culture: The culture of organ primordial, or the whole or

part of an organ in vitro that may allow preservation or modification of the original architecture/ function.
Plant tissue culture is therefore a set of in vitro manipulation techniques by which single plant cells, unorganised group of cells or organised mass of cells can be stably (preferably indefinitely) maintained , preserved or regenerated to accomplish a defined morphogenic or metabolic task under suitable artificial physical and chemical environments.

PRE-REQUISITES FOR RAISING PLANT TISSUE CULTURES

HEALTHY AND YOUNG COMPETENT CELLS WITH MORPHOGENIC PLASTICITY ASEPTIC ENVIRONMENT

GROWTH SUPPORTING FOOD SUPPLIMENTED WITH APPROPRIATE HORMONE COMBINATION & CONCENTRATION

A cellular characteristic in which the capacity of a higher organism cell to form all the cell types of an adult organism and hence ability to differentiate into an entire individual is retained.
It implies that all differentiated living cells of a plant, except those that have been exclusively programmed into narrow path of specialization, possess an ability to display full genetic programme and embark upon a developmental pathway similar to that of a zygote, leading ultimately to the formation of a new individual.

TOTIPOTENCY

Totipotency is therefore:
Genetically (endogenously) controlled Developmentally dependent Strongly regulated by external factors (light, Temperature, wounding etc.)

Totipotency can be:

Morphogenic, Physiological, Biochemical,Metabolic and Regenerative

MORPHOGENESIS Morpho = shapes; genesis = beginning

The process of growth and development of differentiated structures from an undifferentiated state of organisation Inception of growth, division, anatomical specialization and multiplication through diverse developmental patterns leading to formation of specialized forms (organs) from earliest to final status. Morphogenesis involves first the fating of cells to undergo morphological change and then carrying out that fating through differentiation and growth.

DIFFERENTIATION
Cyto-specialization of cultured cells/ tissues leading to development of permanent tissues / organs or structures showing diverse growth, morphology and function. The formation and development of definite structures/ organs from their primitive conditions (meristemoids/ primordia)

ORGANOGENESIS
ORGANOID An organ of apparently unknown function ORGANOPHYSIOLOGY The requisite modification in a structure to enable a species to settle in a given situation

REGENERATION
Vegetative growth after a wound, amputation ordrying of the surface

PLASTICITY
The condition of a cell characterized by degree of ease or ready response to a given stimulus for a desired out-put.

INDUCTION
In its wider developmental sense, is a process seen by its end results. It is the earliest change in the fate or destiny of a cell or a group of cells. It is this change in fate that is what is known as cellular determination.

COMMITMENT/ DETERMINATION
Event which initiate a specific developmental path by singling out from among the various possibilities for which a cellular system might be competent.

Totipotency (Plasticity andCompetence)

cytodifferentiation cellular specialization evolutionary hierarchy

POLARITY
The condition of having distinct cellular poles in terms of an established gradient of growth substances. It is essential for differentiation and is achieved by orientation. Polarity level Differentiation
1

Growth rate
It is loss of this sense of polarity and orientation that help cells in suspensions to multiply at a much faster rate than organised structure on static medium.

SOME MORE TERMS IN THE LANGUAGE OF PTC

EXPLANT
A plant part or piece of tissue(s) excised from a parent plant to initiate a culture

SHOOT TIP CULTURE

Culture of a shiny, dome-like structure consisting of a shoot apical meristem plus one to few primordial leaves (0.1-1.0mm)

MERISTEMOID
A localized group of meristematic cells that arise in a cultured tissue (usually callus) and give rise to an organised structure (shoot and/or roots)

CELL GENERATION TIME

The interval between consecutive division of a cell.

CELL DOUBLING TIME

Time required for a cell population to double either in number or cell mass if all the cells in the cultured population are capable of deviding and dont undergo lysis

CLONE
A group of genetically identical cells or organisms produced asexually from a common ancestor; they may or may not be homogenous

CLONING
Asexual multiplication from a single cell/organism. In molecular biology refers to the replication of a small DNA molecule or a gene in a vector

CELL (SUSPENSION) CULTURE

Culture of single cells or small aggregates of similar cells in liquid medium

AXENIC
A culture without any foreign or undesired life forms. It may include the purposeful co-cultivation of different types of cells, tissues, or organisms

ASEPSIS
Without infection or contaminating micro-organisms

CELL LINE
Cells derived from common ancestral cell that acquire the ability to multiply indefinitely in culture

AUTOTROPHIC CULTURES
A culture capable of self nourishment

ADVENTITIOUS
Development from unusual points of origin

PROTOPLASTS Single cells with their walls stripped off; may or may not contain active nucleus CYBRIDS A viable cell resulting from fusion of a cytoplasm with a cell; nucleus of one and cytoplasm of both the fusing partners CYTOPLAST Intact cytoplasm remaining following enucleation of a cell SOMACLONAL VARIATION Expression (spontaneous as well as induced) of a phenotypic variation, either genetic or epigenetic in origin displayed by cultured somatic cells in vitro

CHEMOSTAT An apparatus for maintaining cells (mostly of microbial systems) in the exponential phase of growth over prolonged periods BIOREACTOR A vessel in which a bioprocess takes place BIOCONVERSION A chemical conversion using a biocatalyst ( organism, cell or part there from IMMOBILIZATION Holding of plant cells so that their metabolic and growth machinary is immobilized by entrappment in polymeric matrices and using such cells for continuous conversion of substrate into products

SPHEROPLAST A cell from which most of the cell wall has been removed VARIANT A cell line or plant exhibiting a stable phenotypic change that may be genetic (mutant) or epigenetic in nature ACCLIMATIZATION Biological process whereby an organism adapts to a new environment

BASIS FOR PLANT TISSUE CULTURE

Two Hormones Affect Plant Differentiation: Auxin: Stimulates Root Development Cytokinin: Stimulates Shoot Development Generally, the ratio of these two hormones can determine plant development:
Auxin Cytokinin = Root Development Cytokinin Auxin = Shoot Development Auxin = Cytokinin = Callus Development

FACTORS AFFECTING PLANT TISSUE CULTURE Growth Media

Minerals, Growth factors, Carbon source, Hormones

Environmental Factors
Light, Temperature, Photoperiod, Sterility, Media

Explant Source
Usually, the younger, less differentiated the explant, the better for tissue culture

Genetics
Different species show differences in amenability to tissue culture. In many cases, different genotypes within a species will have variable responses to tissue culture.

APPLICATIONS

APPLICATIONS OF PLANT TISSUE CULTURE

Rapid propagation of true-to-type plants in limited space and shorter time (MICROPROPAGATION) Production of disease free or disease tolerant plants (MERISTEM CULTURE) As sources of variation (mutants) with better agronomic/productivity traits (SOMACLONAL VARIATION) Production of haploid plants and pure line via anther, pollen and ovule culture (ANDROGENESIS/GYNOGENESIS) Protoplast culture and fusion to create novel plant types with unique cytoplasmic and nuclear combination across evolutionary limits (SOMATIC HYBRIDIZATION) Industrial production of natural products (METABOLIC ENGINEERING) Ex-situ conservation of biodiversity (TISSUE BANKING) Creation of genetically modified plants using genomic approaches (GENETIC ENGINEERING)

MICROPROPAGATION
IT IS AN IN VITRO CYCLE AIMED TO EXPLOIT THE MORPHOGENETIC COMPETENCE OF CULTURED CELLS/TISSUES TO GENERATE A LARGE NUMBER OF GENETICALLY IDENTICAL, PHYSIOLOGICALLY UNIFORM AND DEVELOPMENTALLY NORMAL POPULATION OF AN ELITE PARENT SELECTION. THE PROGENY MUST POSSESS HIGH PHOTOAUTOTROPHIC CAPABILITY TO SURVIVE IN VIVO The word micro refers to the tiny size of the starting material and lesser space to carry out the operation

A MICROPROPAGATION CYCLE CAN OPERATE THROUGH:

I. MERISTEM CULTURE: Wherein a growing apical or a quiescent axillary meristem (bud) is elicited to elongate and produce many can be individually Wherein de novo differentiation of unipolar shoots and/ or roots is induced in a meristematic tissue, usually a callus (indirect organogenesis) thin layered epidermal or sub-epidermal cortical/ mesophyll tissues (direct organogenesi Wherein growth programming of a dividing haploid or diploid cell is modified in such a manner that it starts behaving like a bipolar zygotic embryo. The subsequent differentiation pattern mimic the steps of a normal embryogenesis.

shoots rooted. II.

that

ADVENTIVE ORGANOGENESIS:

III. SOMATIC EMBRYOGENY:

Micropropagation is relevant where:

Starting material is limited Clonal progeny is required Quality propagules are required in bulk Where speciality plants (sterile hybrids/transgenics) are needed for breeding and R&D work

Efficacy and success of a micropropagation cycle depends on how precisely we answer the following questions
Which is the most competent tissue? What constitutes this competence at cellular level ? What are the optimal stimuli? What is required for best realization of outcome of this programme in terms of final through-put ?

A MICROPROPAGATION PROTOCOL IN ITS STRICT SENSE MUST PROVIDE FOLLOWING DETAILS

y y y y y y y y y y y y y y y y y y y y y

Genotype characteristics Age and agronomic background of the parent plant Optimal season for explant procurement Physiological age of the explant Pre-treatment, if any Explant preparation, sterilization Optimum culture medium and adjuvants Ideal conc. and combination (or sequential) dose of PGR Incubation condition/type of culture vessels Method for overcoming the problems of phenolic leaching and endogenous infection Duration of culture cycle Medium to inoculum ratio Time frame of various morphogenic steps Hardening schedule Stage of transplantation Acclimatization condition Genetic fidelity testing procedures Field performance in terms of desired traits Cost-economics of entire operation and cost of one propagule Possible steps for automation to attain maximum through-put Scientific details of underlying developmental pathway.

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 Multiplication; explant transferred to shoot media; shoots can be constantly divided Rooting in regenerated micro-shoots

Stage II -

Stage III -

Stage IV -

Transfer to soil; plant returned to soil; hardened off