Plant Tissue Culture

DefinitionRefers to a process in which we culture a part of a grown plant. That part could be a cell, protoplast, tissue or an organ which are called the (explants). This process is meant to be done in a microbe-free plant material, supplied by all the nutrients needed for survival of the living tissue, in an aseptic environment.DefinitionWhy?What is Needed?A Little From The PastHow it is all be done?ApplicationsPlant Tissue CultureDefinitionWhy?What is Needed?A Little From The PastHow it is all be done?ApplicationsDetermination of phenolic content and antioxidant activity of extracts obtained from Rosmarinus officinalis. calliOzlem Yesil-Celiktas, Department of Bioengineering, Faculty of Engineering, EGE University, Bornova - Izmir, Turkey

Why?Plant Tissue CultureDefinitionWhy?Rare Plants ExtinctionTitan Arum(Amorphophallus titanum.)What is Needed?A Little From The PastHow it is all be done?Applications

Plant Tissue CultureDefinitionWhy?Why?Climate StressCold, Drought & SalinityWhat is Needed?A Little From The PastHow it is all be done?Applications

Plant Tissue CultureDefinitionWhy?Why?Artificial Medicine Side-effectsThe Way To Herbal RemediesWhat is Needed?A Little From The PastHow it is all be done?Applications

A Little From The PastPlant Tissue CultureDefinitionWhy?A Little From The PastThis technology relies on two main concepts:DifferentiationTotipotencyIt implies that every cell of the plant contains all the information necessary for growth and it is capable of developing into an entire plant if suitably stimulated. (the meristemic cells are best able to express this ability)The ability of mature cells to return to the meristemic condition and reorganize into new organs.What is Needed?How it is all be done?Applications

A Little From The PastPlant Tissue CultureDefinitionWhy?A Little From The PastHaberlandt .. early 1900sproposed concept of Totipotency(Cells cultured under right conditions)Gautheret, Nobecourt, Whire in the 1930s.(Cells kept alive but did not develop)The first commercial use of plant clonal propagation on artificial media was in the germination and growth of orchid plants, in the 1920sIn the 1950s and 60s there was a great deal of research, but it was only after the development of a reliable artificial medium (Murashige & Skoog, 1962) that plant tissue culture really took off commerciallyWhat is Needed?How it is all be done?Applications

(a)-Apical meristem(b)-Leaf Primordium(c)-Axillary/Lateral meristem(d)-Grown Leaf(e)-PithWhat is Needed?Plant Tissue CultureDefinitionWhy?A Little From The PastWhat is Needed?1) Appropriate TissueHow it is all be done?Applications(Some tissues culture better than others)

What is Needed?Plant Tissue CultureDefinitionWhy?A Little From The PastWhat is Needed?How it is all be done?Applications2) A Suitable Growth MediumCulture Medium ConstituentsInorganic salt formulationOrganic supplementSource of carbohydratesWaterPlant HormonesSolidifying/Gelling Agent(This can be either liquid or solid forms, depending on the type of culture being grown)

What is Needed?Plant Tissue CultureDefinitionWhy?A Little From The PastWhat is Needed?How it is all be done?Applications2) A Suitable Growth MediumCulture Medium ConstituentsInorganic salt formulationSalt formulations are some of the elements important for plant nutrition and their physiological function. These elements have to be supplied by the culture medium in order to support the growth of healthy cultures in vitro.(This can be either liquid or solid forms, depending on the type of culture being grown)

ElementFunctionNitrogenproteins, nucleic acidsCalciumcell wall synthesis, membrane functionMagnesiumcomponent of chlorophyllPhosphorusnucleic acids, energy transferChlorineRequired for photosynthesisIronElectron transfer as a component of cytochromes

What is Needed?Plant Tissue CultureDefinitionWhy?A Little From The PastWhat is Needed?How it is all be done?Applications2) A Suitable Growth Medium(This can be either liquid or solid forms, depending on the type of culture being grown)Culture Medium ConstituentsOrganic supplementsupplying vitamins and/or amino acidsOnly two vitamins, thiamine (vitamin B1) and myoinositol are considered essential for the culture of plant cells in vitro.Amino acids provide a source of reduced nitrogen and uptake causes acidification of the medium. Casein hydrolysate can be used as a relatively cheap source of a mix of amino acids.

What is Needed?Plant Tissue CultureDefinitionWhy?A Little From The PastWhat is Needed?How it is all be done?Applications3) Aseptic (Sterile) Conditions(As microorganisms grow much more quickly than plant and animal tissue and can over run the culture(Surface contaminantseliminated by surface disinfectants (e.g., Na hypochlorite, Ca hypochlorite, ethanol).Internal contaminants(Pathogens) eliminated by thermotherapy, culture of explants free of organisms or by antibiotics.Maintenance of asepsis(During excision and culture) procedures are carried out in sterile laminar air flow

In Plant Tissue Culture:Auxins: Stimulate Root DevelopmentCytokinins: Stimulate Shoot DevelopmentThe General Role Of: Auxins ------------- Cell Enlargement Cytokinins ------------- Cell Division Gibberellins ------------- Cell Elongation Abscisic acid ------------- Inhibits Cell DivisionWhat is Needed?Plant Tissue CultureDefinitionWhy?A Little From The PastWhat is Needed?How it is all be done?Applications4) Growth RegulatorsNaturally occurring or synthetic compounds.Compounds that have main roles in the organizing and controlling all the physiological and metabolic processes in the plant tissues (Also known as the Plant Hormones)Four Main Classes:Auxins, Cytokinins, Gibberellins, Abscisic acidThe ratio of Auxins to Cytokinins, also the interaction between both with each other and between them and other hormones and/or other media components, determine the plant development and the final result or product acquired in the culture process.

What is Needed?Plant Tissue CultureDefinitionWhy?A Little From The PastWhat is Needed?How it is all be done?Applications5) Frequent Subculturing(To ensure adequate nutrition and to avoid the build up of waste metabolites)

What is Needed?Plant Tissue CultureDefinitionWhy?A Little From The PastWhat is Needed?How it is all be done?Applications5) Frequent Subculturing4) Growth Regulators3) Aseptic (Sterile) Conditions2) A Suitable Growth Medium1) Appropriate Tissue

How it is all be done?How it is all be done?Plant Tissue CultureDefinitionWhy?A Little From The PastWhat is Needed?ApplicationsThe Desired PathwayCallus InductionOrganogenesis (Morphogenesis)Embryogenesis

Plant Tissue CultureDefinitionWhy?Callus InductionHow it is all be done?A Little From The PastWhat is Needed?ApplicationsHow it is all be done?The Desired PathwayCallus: Refers to the undifferentiated, unorganized mass of cells produced from the explants cultured due to rapid proliferation of the explants cells.

Plant Tissue CultureDefinitionWhy?Organogenesis (Morphogenesis)How it is all be done?A Little From The PastWhat is Needed?ApplicationsHow it is all be done?The Desired PathwayOrgan - genesisMorph - genesis

Plant Tissue CultureDefinitionWhy?EmbryogenesisHow it is all be done?A Little From The PastWhat is Needed?ApplicationsHow it is all be done?The Desired Pathway

Plant Tissue CultureDefinitionWhy?How it is all be done?How it is all be done?A Little From The PastWhat is Needed?ApplicationsSelection of The Plant TissueSterilizingSelecting & Preparing The Proper MediumEstablishment

Plant Tissue CultureDefinitionWhy?Important Factors Involved During Processing1. Explant SourceUsually, the younger, less differentiated the explant, the better for tissue culture3. Environmental FactorsLight, Temperature, Photoperiod, Sterility, Media2. Growth MediaMinerals, Growth factors, Carbon source, Hormones4. GeneticsDifferent species show differences in amenability to tissue cultureIn many cases, different genotypes within a species will have variable responses to tissue cultureHow it is all be done?A Little From The PastWhat is Needed?ApplicationsHow it is all be done?

ApplicationsApplicationsPlant Tissue CultureDefinitionWhy?MicropropagationGermplasm preservationSomaclonal variation & mutation selectionEmbryo CultureHaploid & Dihaploid ProductionIn vitro hybridization Protoplast FusionIndustrial Products from Cell CulturesWhat is Needed?A Little From The PastHow it is all be done?

Determination of phenolic content and antioxidant activity of extracts obtained from Rosmarinus officinalis. calliOzlem Yesil-Celiktas, Department of Bioengineering, Faculty of Engineering, EGE University, Bornova - Izmir, TurkeyJournal of Plant PhysiologyReceived 22 March 2007; Revised 24 April 2007; accepted 14 May 2007

Rosmarinus officinalis.Woody, perennial herb with fragrant evergreen needle-like leaves. It is native to the Mediterranean region. It is a member of the mint family Lamiaceae.

Rosmarinus officinalis.Usage:flavor foods, treat gout, improving memory.The results of a study suggest that carnosic acid, found in rosemary, may shield the brain from free radicals, lowering the risk of strokes and neurodegenerative diseases like Alzheimer.The medical uses of rosemary mainly due to its high content of antioxidants such as carnosic acid and rosmarinic acid. The antioxidant activity of plant extracts is due primarily to phenolic compounds. In rosemary extracts, these are categorized into three groups: phenolic diterpenes, flavonoids, and phenolic acids.

The aim of the studyThe aim of this study was to report the best medium and conditions to obtain high phenolic yield and antioxidant activity of the methanolic extracts of rosemary callus cultures.

Materials & MethodsExplant Source: Three well-grown R. officinalis plants located at Bornova, Izmir, Turkey under Aegean climatic conditions.Explants: sections of young shoots were collected from June 2004 to September 2004.Explants were cultured on the day in which they were collected.Media Used: woody plant medium (WPM) and Murashige and Skoog (MS) media supplemented with 7 g/L agar, 30 g/L sucrose, and 1 and 3 mg/L naphthaleneacetic acid (NAA) for callus initiation. (pH: 5.8)

TreatmentsNamesMSWPM1 mg/L naphthaleneacetic acid (NAA)MS 1WPM 13 mg/L naphthaleneacetic acid (NAA)MS 3WPM 3

Establishment of in vitro callus culturesYoung shootsTap waterHold in distilled water for an hourRemove leavesTake stem sections 5-7 cm lengthStir in 70% ethyl alcohol (5 min)Stir in 0.5% sodium hypochlorite (20 min)Rinse with autoclaved distilled water 3 timesCut stems into 1.0-1.5 cmPut in vessels (150 ml) + 23-25 ml mediumCalli induction was observed within 1015 days on the surface at the cut edges of the sectionsIncubation in the dark

Establishment of in vitro callus culturesAfter calli became 1.01.5 cm in diameter, they were subcultured on the same medium 4 times with intervals of 710 daysThe calli collected from each vessel were gently pressed on filter paper to remove excess water and their fresh weights (FWs) were recordedAfter holding at -18oC for 1 night, they were freeze-dried (lyophilized) and their dry weights (DWs) were recorded

Preparation of methanolic extracts1 g of each lyophilized callus was transferred to a vial and 20.0 ml of methanol was addedAfter that all the samples were sonicated at 35 kHz (Ultrasonic LC 30) for 45 minHalf of the samples sonicated with 50oC and the other half without heat treatmentHPLC analysis for the extracts & Antioxidant assays

Results and discussionCalli culturesOn the basis of these findings, MS medium supplemented with 1 mg/L NAA would prove to be the most effective medium for biomass production among the samples tested.

MediaPercentages of explants producing callus (%)Biomass (fresh weights) (g)MS 165.018.6MS 340.015.4WPM 155.216.7WPM 350.216.0

Results and discussionPhenolic content

Callus extractsContent of rosmarinic acidmg/g dry callusMS 10.5MS 1T2.9MS 31.8MS 3T1.1WPM 13.1WPM 1T5.9WPM 30.2WPM 3T0.8

ConclusionThe extracts from the WPM1 sample treated with temperature provided particularly high amounts of RA, a higher total phenol value, and a higher percentage of radical scavenging activity, although MS1 proved to be the ideal medium for callus inductionTherefore, woody plant medium (WPM) supplemented with 7 g/L agar, 30 g/L sucrose, and 1 mg/L naphthaleneacetic acid (NAA) proved to provide ideal conditions for RA accumulation and higher antioxidant activity.

Great Wishes & Thank You For Your Attention. Thank YouPrepared by: Elmontasser Bellah AhmedUnder Supervision of: Dr. Hoda Elmokadem