Cultivo in-vitro de in vitro

Desarrollo aseptico de protoplastos, clulas , tejidos u rganos vegetales en un medio d t l di de cultivo , tan definido como sea posible y que se mantenga bajo condiciones ambientales controladas .

Explant Inoculum

a piece of plant material isolated from mother plant to be cultured a subculture of plant material which is already in culture

Explanto
Clula, Clula tejido u rgano vegetal usado para iniciar un cultivo in-vitro in vitro La eleccin del p p explanto depende del propsito del cultivo. Por ejemplo las yemas axilares y los il l meristemas son apropiados para micropogacin. i i

Eleccin del explanto

Fcil de esterilizar Juvenil Que responda al cultivo

Explantos

Partes de rganos Hojas Tallos R Races vstagos Yemas axilares Semillas Hypoctilos Ti Tipos celulares especficos l l fi Polen Endosperma

Las partes areas son mas limpias que las subterrneas Las Cuanto ms pequeo sea el explanto ms posibilidades de d evitar problemas fit it bl fitopatolgicos ( i t l i (virus, microplasmas, i l bacterias), pero disminuye su posibilidad de desarrollo Los tejidos internos suelen estar menos contaminados que los ms externos Explantos similares puede reaccionar de forma diferente por influyen: Su localizacin en la planta madre Su condicin de juvenilidad

Juvenilidad
Frecuentemente asociada a buena habilidad para enraizar y/o ausencia de floracin La iniciacin de cultivos a partir de explantos no juveniles es problemtica Dnde se localizan los tejidos juveniles?

Juvenil es diferente de jven

Jven se refiere a la edad, es el opuesto de viejo. Juvenil es el opuesto de adulto, adulto Puede estar referido a: Al perodo de desarrollo del vegetal en cual el rgano se inici (las yemas latentes en la base de un tallo se iniciaron en los seedling); La proximidad al sistema radicular

Juvenility in several hard-woods: Density of crosshatching indicates degree of juvenility. In the juvenile zone, note the single trunk, retention of leaves close to the trunk in winter, and obtuse branch angle. In the mature zone note the forked trunk and acute branch zone, angles.

Juvenility in a regulary shaped conifer: the degree of juvenility of an apical meristem is inversely proportional to the distance (along trunk and branches) between the root-shoot junction (A) and the meristem. meristem The distance AB>AC>AD>AE>AF, and therefore, meristem B is t e most mature, e ste s the ost atu e, and meristem F is the most juvenile. juvenile

Polarity
Cultured explants frequently express polarity in cell proliferation and morphogenesis. morphogenesis This can be related with the position and/or to its orientation which the e plant had on the hich explant mother plant, and also to its or orientation within the culture vessel.

Example of polarity: Shoot initiation happens only at one side of the leaf of Saintpaulia

Asepsis
Tissue culture is an aseptic technique

Terminology avoiding contaminations using sterilization asepsis procedures axenic free from association with other living organisms killing or excluding microorganisms or their spores sterilization with heat filters chemicals or other sterilants heat, filters,

Stage I - Sterilisation
Bacteria and fungi will overgrow the explant on the medium unless they are removed Pre-treatments to clean up the explant Detergents Sterilants and Antibiotics

Stage I - Sterilisation
Pre treatments Pre-treatments Transfer plants to a greenhouse to reduce endemic contaminants Force outgrowth of axillary buds Washing removes endemic surface contaminants

Surface sterilization of plant material

Base protocol
1. Wash 1 W h explants in a mild detergent before treatment with the l t i ild d t tb f t t t ith th disinfecting solution. (Herbaceous material may not require this step). 2. Rinse explants thoroughly under running tap water for 10-30 minutes. 3. Submerge explants into the disinfectant solution. Seal bottle and gently agitate. 4. Under sterile conditions, decant the solution and rinse explants several times with sterile distilled water.

Surface sterilization of plant material

COMMONLY USED DISINFECTANTS FOR PLANT TISSUE CULTURE

Disinfectant Calcium hypochlorite C l i h hl i Sodium hypochlorite* Hydrogen peroxide Ethyl alcohol Silver nitrate Mercuric chloride Benzalkonium chloride

Concentration (%) 9-10 9 10 0.5-5 3-12 3 12 70-95 1 0.1-1.0 0.01-0.1

Exposure (min) 5-30 5 30 5-30 5-15 5 15 0.1-5.0 5 30 5-30 2-10 5-20

*Commercial bleach contains about 5% sodium hypochlorite, and thus may be used at a concentration of 10 20%, which is equivalent to 0.5-1.0% sodium 10-20%, 0.5 1.0% hypochlorite.

Sterilization procedures may be enhanced by:

1. Placing the material in a 70% ethyl alcohol solution prior to treatment with another disinfectant solution. The use of a two-step (twosource) sterilization procedure has proven beneficial with certain species. i 2. Using a wetting agent, such as Tween 20 or 80 can be added to the d s ecta ts disinfectants to reduce surface te s o a d a o bette surface educe su ace tension and allow better su ace contact. 3. Conducting the sterilization process under vacuum. This results in the removal of air bubbles and provides a more efficient sterilization process.

Surface sterilization of plant material

An elaborated example of surface sterilization Cut the plant material to an appropriate size to fit the container which will be used during the sterilsation procedure Rinse plant material under running tap water Ri l t t i l d i t t Shake for a few seconds in alcohol HgCl2 (0.1 - 1%) + Teepol 2 drops/100ml: 3-5 min Rinse in autoclaved distilled water Commercial bleach 7-15% + Teepol 2 drops/100ml: 10-30 min Rinse several times in autoclaved distilled water

Operations in the Sterile Cabinet:

1. Tie back your hair, roll your sleeves up and remove your watch and other jewellery Wash your hands thoroughly with the disinfectant jewellery. solution suitable for skin application. If allergic to any disinfectant wash your hands with water and wear a pair of surgical gloves. 2. Sterilise the inside of the cabinet by spraying with 70% alcohol and wiping dry with sterile tissue. 3. 3 Collect and organise all the items you will need close to or inside the cabinet. 4. Working in the cabinet, take a sterilised piece of biomass with a pair of forceps (do not touch the plant material with your hands). Also sterilise your instruments by dipping them in alcohol between each manipulation and flaming them.

El tamao ptimo de un explanto es del orden de 0,5 a 1cm2.

El medio de cultivo en relacin a las caractersticas del explanto p

When you make an explant like lik an axillary b d you ill bud, remove it from the sources of many chemicals and have to re supply re-supply these to the explants to allow them to grow.

Shoot tip - Auxins p and Gibberellins

Leaves sugars, GAs

Roots - water, vitamins mineral salts and cytokinins

Azcar: A
La concentracin de sacarosa depende fundamentalmente de la edad del material vegetal a cultivar cultivar. Los tejidos jvenes requieren concentraciones de azcar. menores

En trminos generales el crecimiento y desarrollo de un cultivo se incrementa con el aumento de azcar. azcar

Agua: g
En algunas especies un exceso de agua en los medios de cultivo (cultivos lquidos o slidos con bajas concentraciones de agar) provoca la vitrificacin del cultivo.

p pH is normally adjusted between 5.5 and 6.0 before

autoclaving
Para la mayora de la especies el pH ptimo de un cultivo est comprendido entre 5.0 y 6.5.

it governs the solubility of the salts influences the uptake of medium ingredients affects the gelling efficiency of agar
Los valores pH bajos traen aparejados la baja estabilidad de algunos reguladores de crecimiento (auxinas y giberelinas) y vitaminas la vitaminas, precipitacin de sales del medio y la disminucin de la absorcin del amonio.

pH is altered by autoclaving and during the culture period

El acondicionamiento del mismo se realiza antes de la esterilizacin del medio, y el mismo sufrir una disminucin del orden de 0,3-0,5 unidades.

FITORREGULADOR AUXINAS IAA

PROPOSITO

RANGO

Induccin de callos Estimulacin de organognesis

10-30 M 1-10 M 10-30 M 1-10 M 1 10 M

IBA

Induccin de callos Regeneracin d races R i de

2,4-D pCPA cido p-clorofenoxiactico NAA

Induccin y mantenimiento de callos y 1-50 M cultivos sumergidos en estado indiferenciado Induccin y mantenimiento de callos y 1-50 M cultivos sumergidos en estado indiferenciado Induccin de callos Induccin de races 2-20 M 0,2-2 M

CITOQUININAS K 6-furfurilaminopurina BAP Induccin y crecimiento de callos y suspensiones 120 M Induccin de la multiplicacin de vstagos, 550 M p g , yemas axilares y meristemas Induccin y crecimiento de callos y suspensiones 0,55,0 M Induccin de I d i d morfognesis ( t f i (vstagos) ) Multiplicacin de yemas y meristemas 2iP Induccin de callos (N-isopentenilaminopurina) Induccin de morfognesis (N i t il i i ) Multiplicacin de yemas, vstagos y meristemas Zea g Induccin de morfognesis 110 M 1 10 M 550 M 210 M 1025 M 3050 M 0,0510 M ,

Hormone Balance
Auxin High Cytokinin y Low

Root formation on explants Embryogenesis Adventitious root formation in callus Callus initiation Adventitious shoot formation Axillary shoot growth Low High

GIBERELINAS Gib A3 Promocin de crecimiento de vstagos g 0,314 M , Incremento del desarrollo de embriones y 0,3-48 M cultivo de vulos ACIDO ABSISICO ABA Prevencin de germinacin precoz y 0,0410 M promocin del desarrollo de embriones somticos

Explanto Tallos adventicios Races Callos Citoquininas

Auxinas

Temperature
Practically it is impossible to optimize temperature for each specific plant and each specific stage. For F most plants the set point for room temperature during the t l t th t i tf t t d i th day is 22 2C. En general es aplicable que la temperatura ms adecuada para el desarrollo de un cultivo es de 3 a 4C ms elevada que la media que soporta la planta in vivo. Temperature in some culture container is higher (up to 5C) than room temperature due to the greenhouse effect. One of the major problems is to have a uniform temperature in the culture room. There are gradients. Safety device: a maximum thermostat needs to be installed, which switches off the lights in case of too high temperature.

Photoperiod

Usual daylength is 16h.

An appropriate light intensity is approx. 30 mol m-2 s-1(PAR). Under this condition the cultures are mixotrophic.

Light i t Li ht intensity it

For autotrophic tissue culture intensities up to 250 mol m-2 s-1 are required. required Depending on the type of closure light intensity i th container i reduced t a i t it in the t i is d d to lower or higher extend.

Container types and closure devices

Callus

Callus
Unorganized, growing mass of cells Dedifferentiation of explant
Loosely arranged thinned walled, outgrowths from explant No predictable site of organization or differentiation

Often can be maintained indefinitely by subculture, but may lose ability to redifferentiate Compact vs friable Habituation

Maintenance of Callus
General - Callus induction and maintenance media contain the same basal constituents. Most callus requires auxin and cytokinin in the maintenance medium, particularly after prolonged culture (except habituated cells). Callus is re-cultured after 4 to 6 cell doublings, when growth becomes nutrient limited in a batch culture culture. This interval is referred to as a subculture or p passage. g

Ca us o p o ogy Callus morphology icells may be tightly joined and the tissue mass is one solid piece (compact) or cells are l ll loosely j i d and i di id l cells readily l joined d individual ll dil separable (friable), which is affected by the genotype or the medium composition composition. q A friable callus is often used to initiate a liquid cell suspension culture

Genotypic Effects on Callus Morphology

Arabidopsis
3.0 mg/L 2,4-D

Tobacco

Compact Callus

Friable Callus

Medium Effects on Tobacco Callus Morphology

2.0 mg/L IAA 3.0 3 0 mg/L 2 iP 2-iP

0.1 mg/L kinetin 3.0 3 0 mg/L 2,4-D /L 2 4 D

friable callus

compact callus

Cytogenetic/genetic variation Cells of callus are genetically very heterogeneous and the heterogeneity increases d i culture h t it i during lt Regenerated p g plants will reflect this g genetic variation (somaclonal variation). Somaclonal variation genetic variation that arises in somatic (non-germ line) cells However, morphogenetic competence is more associated with genetically stable (e.g. meristematic) cells

The cytogenetic changes that occur are polyploidy/aneuploidy, translocation, amplification, methylation, epigenetics etc, t l ti lifi ti th l ti i ti t although the exact genetic basis for most somaclonal variation is unknown Cytogenetic variation can be minimized by choosing explants that are meristematic and maintain callus in media that favor cell division

Callus Growth Patterns

I. Growth patterns leading to organized development morphogenesis (adventitious organogenesis or somatic embryogenesis)

II. Growth patterns leading to continued p p g proliferation of unorganized callus maintenance

Callus Growth Is Predominantly at the Periphery of the Tissue

Division

E. Sutton, UC Davis

Senescencia
Clulas meristemticas aumento de p o op as a protoplasma Divisin

Diferenciacin

Clula madura Muerte celular

Expansin

I. Growth Patterns Leading to Continued Proliferation of

Unorganized Callus g
A. Induction of growth (manifested as a lag) - Fresh medium induces quiescent cells (stationary phase) to enter the cell cycle G1SG2M cycle, G1SG2M. Cells in G1 phase proceed through S (DNA/RNA synthesis) phase and then through a short G2 phase prior to mitosis B. Division phase - rapid increase in cell number through periclinal (parallel to nearest surface) divisions subjacent to the p p y of the callus, and ) j periphery , followed by anticlinal (perpendicular to surface) divisions. Division >> fresh weight gain resulting in substantial reduction in cell volume (regressive growth), cells dedifferentiate (become meristematiclike), C. Cell expansion - no differentiation

II. Growth patterns leading to organized development

A. Induction f A I d ti of growth (lag) th (l ) B. Division phase C. Differentiation - cell division slows, during this period differentiation occurs which is then followed by cell expansion resulting i th d lti in the development of an organized structure. l t f i d t t morphogenesis (adventitious organogenesis or somatic embryogenesis)

Jerusalem Artichoke Tuber Callus

Cell number increases 10-fold in the first 7 days and cells dedifferentiate into meristematic cells

Induction

Cell division

Differentiation
Organogenesis g g Somatic embryogenesis

Shoot Organogenesis of Tobacco

High cytokinin

Low cytokinin

Somatic Embryogenesis of Carrot

2,4-D (mg/L)

Organogenesis g g
The formation of organs (such as leaves, shoots, roots) on a plant organ, usually of a different kind.

Organogenesis
The process of initiation and development of a p p structure that shows natural organ form and/or function. the ability of non meristematic plant tissues to non-meristematic form various organs de novo. the production of roots, shoots or leaves. These organs may arise out of pre-existing meristems or out of differentiated cells. This, like embryogenesis, may involve a callus intermediate but often occurs without callus.

Plant Organogenesis
Indirect:
This pathway includes a callus stage.
Callus: Undifferentiated tissue that develops on or around an th t d l d injured or cut plant surface or in tissue culture.

Direct:
It bypasses a callus stage. The cells in the explant act as direct di t precursors of a new f primordium
An organ or a part in its most rudimentary form or stage of development

Organogenesis
Adventitious shoot formation is the de-novo de novo development of shoots from cell clusters in the absence of pre-existing meristems. In some species (e.g. Saintpaulia), many shoots can be induced (3000 from one leaf). In other species (e.g. coffee), it may be necessary to induce an unorganised mass proliferation of cells (callus) prior to lif i f ll ( ll ) i adventitious shoot formation.

Organogenesis indirecta g g

Explanto Callos desarrollo meristemoide p Primordio

 Auxin/cytokinin 10:1-100:1 induces roots. Auxin/cytokinin 1:10-1:100 induces shoots Intermediate ratios around 1:1 favor callus growth.

Embriognesis somtica

Somatic Embryogenesis
The production of embryos from somatic or non-germ cells. Usually involves a callus intermediate stage

Somatic Embryos
Tissue culture conditions can be modified to cause to somatic cells to reprogram into a bipolar structure These bipolar structures b h Th bi l t t behave lik a like true embryo - called somatic embryos

El desarrollo de los embriones somticos pasa por los mismos estados morfolgicos de desarrollo que un embrin cigtico: g

proembrin globular, trapezoidal, embrin cordiforme y torpedo.

Cleavage Polyembryony- conifers

Cleavage lengthways le gt ways Embryo

Suspensor

Normal Embyro

Lateral division

New embryos

Secondary embryo formation - Most dicots

Abundant Secondary Embryos

Early embryo