tema 4 iniciacion de cultivos in vitro

8/6/2019 TEMA 4 Iniciacion de Cultivos in Vitro

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-

Desarrollo aseptico deprotoplastos, clulas , tejidos u

rganos vegeta es en un m e o e

cultivo , tan definido como sea

condiciones ambientales

controladas .


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Explant

a piece of plant material

isolated from mother plant tobe cultured

Inoculum a subculture of plant material


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Eleccin del explanto

Fcil de esterilizar

Que responda al cultivo


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Partes de rganos

Tallos

a ces

vstagos

Explantos Yemas axilares

Hypoctilos

pos ce u ares espec cos Polen

Endosperma


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Cuanto ms pequeo sea el explanto ms posibilidadese ev ar pro emas opa o g cos v rus, m crop asmas,

bacterias), pero disminuye su posibilidad de desarrollo

Los tejidos internos suelen estar menos contaminados

que los ms externos

Explantos similares puede reaccionar de forma diferentepor influyen:

Su localizacin en la planta madreSu condicin de juvenilidad


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Juvenilidad

Frecuentemente asociada a buena habilidad para

La iniciacin de cultivos a partir de explantos nojuveniles es problemtica


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Juvenil es diferente de jven

Jven se refiere a la edad, es el opuesto de viejo.

uven es e opues o e a u o,

Puede estar referido a:

Al perodo de desarrollo del vegetal en cual el rgano se

en los seedling);

La proximidad al sistema radicular


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Juvenility in several hard-woods:

Density of crosshatching indicates degree of juvenility.

close to the trunk in winter, and obtuse branch angle.

,angles.


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Juvenility in a regulary shaped conifer:

the degree of juvenility of an apical

meristem is inversel ro ortional to

the distance (along trunk and branches)between the root-shoot junction (A)

.

The distance AB>AC>AD>AE>AF,

and therefore,

meristem B is the most mature

and meristem F is the

.


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Polarity

Cultured explants frequently express polarity.

This can be related with the position and/or tos or en a on w c e exp an a on e

mother plant,

and also to its or orientationwithin the culture vessel.

Shoot initiation happens only at one

side of the leaf of Saintpaulia


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AsepsisTissue culture is an aseptic technique

Terminology

asepsisavoiding contaminations using sterilization

proceduresaxenic free from association with other living organisms

sterilizationkilling or excluding microorganisms or their spores

, ,


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Stage I - Sterilisation

the medium unless they are removed

Pre-treatments to clean up the explant


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Stage I - Sterilisation-

reduce endemic contaminants

Force outgrowth of axillary buds

Washing removes endemic surface


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Surface sterilization of plant material

Base protocol

. as exp an s n a m e ergen e ore rea men w e

disinfecting solution. (Herbaceous material may not requirethis step).

2. Rinse explants thoroughly under running tap water for 10-30

minutes.

3. Submerge explants into the disinfectant solution. Seal bottleand gently agitate.

4. Under sterile conditions, decant the solution and rinseexplants several times with sterile distilled water.


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FOR PLANT TISSUE CULTURE

Disinfectant Concentration (%) Exposure (min)

a c um ypoc or te - -

Sodium hypochlorite* 0.5-5 5-30

- -

Ethyl alcohol 70-95 0.1-5.0

Silver nitrate 1 5-30

Mercuric chloride 0.1-1.0 2-10

Benzalkonium chloride 0.01-0.1 5-20

*Commercial bleach contains about 5% sodium hypochlorite, and thus may beused at a concentration of 10-20% which is e uivalent to 0.5-1.0% sodiumhypochlorite.


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An elaborated example of surface sterilization

Cut the plant material to an appropriate size to fit the containerwhich will be used during the sterilsation procedure

nse p ant mater a un er runn ng tap water

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


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Operations in the Sterile Cabinet:

1. Tie back your hair, roll your sleeves up and remove your watch and.

solution suitable for skin application. If allergic to any disinfectantwash your hands with water and wear a pair of surgical gloves.

2. Sterilise the inside of the cabinet by spraying with 70% alcohol andwiping dry with sterile tissue.

.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 steriliseyour instruments by dipping them in alcohol between eachmanipulation and flaming them.


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


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El medio de cultivo en relacin a las

caractersticas del ex lanto

When you make an explant Shoot ti - Auxinsi e an axi ary u , you

remove it from the sources ofmany chemicals and have to

-

and Gibberellins

explants to allow them togrow.

Leaves -

sugars, GAs

-

mineral salts and cytokinins


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z car:

La concentracin de sacarosa dependefundamentalmente de la edad del material.

Los tejidos jvenes requieren menores

concentraciones de azcar.

En trminos generales el crecimiento y desarrollode un cultivo se incrementa con el aumento de

.


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A ua:En algunas especies un exceso de agua en los

con bajas concentraciones de agar) provoca lavitrificacin del cultivo.


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His normally adjusted between 5.5 and 6.0 before

autoclavingPara la mayora de la especies el pH ptimo de un cultivo est comprendido entre

5.0 6.5.

it governs the solubility of the salts

affects the gelling efficiency of agar

Los valores pH bajos traen aparejados la baja estabilidad de algunos,

precipitacin de sales del medio y la disminucin de la absorcin del

amonio.

pH is altered by autoclaving and during the culture

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.


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FITORREGULADOR PROPOSITO RANGO

AUXINAS

IAA Induccin de callos 10-30 MEstimulacin de organognesis 1-10 M

IBA Induccin de callos 10-30 MRegeneracin e races 1-10 M

2,4-D Induccin y mantenimiento de callos y

cultivos sumergidos en estado indiferenciado

1-50 M

pCPA

cido

p-clorofenoxiactico

Induccin y mantenimiento de callos y

cultivos sumergidos en estado indiferenciado

1-50 M

NAA Induccin de callos 2-20 M

Induccin de races 0,2-2 M


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CITOQUININAS

K

6-furfurilaminopurina

Induccin y crecimiento de callos y suspensiones 120 MInduccin de la multi licacin de vsta os, 550 M

yemas axilares y meristemas

BAP Induccin y crecimiento de callos y suspensiones 0,55,0 Mn ucc n e mor og nes s v s agos

Multiplicacin de yemas y meristemas 550 M

2iP Induccin de callos 210 M( - sopenten am nopur na Induccin de morfognesis 1025 M

Multiplicacin de yemas, vstagos y meristemas 3050 M

Zea Induccin de morfo nesis 0,0510 M


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Hormone Balance

Auxin C tokinin

High Low

Root formation on explants

EmbryogenesisAdventitious root formation in callus

Callus initiation

Adventitious shoot formationAxillary shoot growth

Low High


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GIBERELINAS

Gib A3 Promocin de crecimiento de vsta os 0,314 MIncremento del desarrollo de embriones y

cultivo de vulos

0,3-48 M

ABA Prevencin de germinacin precoz y

promocin del desarrollo de embriones

0,0410 M


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Explanto

CitoquininasTallos adventicios

Races

Callos

Auxinas


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Temperature

Practically it is impossible to optimize temperature for eachspecific plant and each specific stage.

or mos p an s e se po n or room empera ure ur ng e

day is 22 2C.

el desarrollo de un cultivo es de 3 a 4C ms elevada que lamedia 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 inthe 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.


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Photoperiod Usual daylength is 16h.

approx. 30 mol m-2 s-1(PAR).

Under this condition the cultures aremixotrophic.

g t ntens ty For autotrophic tissue cultureintensities up to 250 mol m-2 s-1 are

.

Depending on the type of closure light

n ens y n e con a ner s re uce o a

lower or higher extend.


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Container types and closure devices


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Callus Unorganized, growing mass of cells

Dedifferentiation of explant Loosely arranged thinned walled, outgrowths from

No predictable site of organization or differentiation

Often can be maintained indefinitely bysubculture, but may lose ability to redifferentiate

Compact vs friable

Habituation


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Maintenance of Callus

General - Callus induction and maintenance media

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

.

This interval is referred to as a subculture orassa e.


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Callus mor holo -icells may be tightly joined and the tissue mass is

one solid piece (compact)

or ce s are oose y o ne an n v ua ce s rea y

separable (friable), which is affected by the genotype

.

A friable callus is often used to initiate a li uid cell

suspension culture


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Genotypic Effects on Callus Morphology

Arabidopsis Tobacco3.0 mg/L 2,4-D

Compact Callus Friable Callus


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Medium Effects on Tobacco Callus Morphology

0.1 mg/L kinetin 2.0 mg/L IAA

. mg , - . -


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Cells of callus are genetically very heterogeneous and the

e erogene y ncreases ur ng cu ure

Regenerated plants will reflect this genetic variation

(somaclonal variation).

somatic (non-germ line) cells

However, morphogenetic competence is more associated

with genetically stable (e.g. meristematic) cells


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The cytogenetic changes that occur are polyploidy/aneuploidy,rans oca on, amp ca on, me y a on, ep gene cs e c,

although the exact genetic basis for most somaclonal variation isunknown

Cytogenetic variation can be minimized by choosing explants

division


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

II. Growth patterns leading to continued proliferation of

unorganized callus maintenance


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Callus Growth Is Predominantly at the Periphery of

the Tissue


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E. Sutton, UC Davis


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Senescencia

Clulasmeristemticas

Divisin

aumento deroto lasma

Diferenciacin Clula madura

Muerte celula

Expansin

I Growth Patterns Leading to Continued Proliferation of


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I. Growth Patterns Leading to Continued Proliferation ofUnorganized Callus

A. Induction of growth (manifested as a lag) - Fresh medium induces, .

Cells in G1 phase proceed through S (DNA/RNA synthesis) phase and

B. Division phase - rapid increase in cell number through periclinal (parallel

to nearest surface divisions sub acent to the eri her of the callus andfollowed by anticlinal (perpendicular to surface) divisions.

Division >> fresh weight gain resulting in substantial reduction in cell

volume (regressive growth), cells dedifferentiate (become meristematic-like),

C. Cell expansion - no differentiation

II Growth patterns leading to organized development


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II. Growth patterns leading to organized development

. n uc on o grow ag

B. Division phase

C. Differentiation - cell division slows, during this perioddifferentiation occurs which is then followed by cell expansion

resu ng n e eve opmen o an organ ze s ruc ure.

morphogenesis (adventitious organogenesis or somatic

embryogenesis)

J l A ti h k T b C ll


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Jerusalem Artichoke Tuber Callus

Cell number increases 10-fold in the first 7 days

and cells dedifferentiate into meristematic cells


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Differentiation

Or ano enesis

Somatic embryogenesis


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S ti E b i f C t


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Somatic Embryogenesis of Carrot

2,4-D (mg/L)

O i


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Organogenesis

The formation of organs (such as

leaves, shoots, roots) on a plant


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The rocess of initiation and develo ment of astructure that shows natural organ form and/orfunction.

-form various organs de novo.

These organs may arise out of pre-existing

meristems or out of differentiated cells.

This, like embryogenesis, may involve a callusintermediate but often occurs without callus.

Plant Organogenesis


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Plant Organogenesis

Indirect:

This pathway includes a callus

stage. Callus: Undifferentiated tissue

a eve ops on or aroun an

injured or cut plant surface or

in tissue culture.

It bypasses a callus stage. The

cells in the explant act as

rec precursors o a new

primordium

An organ or a part in its most

development


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-

development of shoots from cell clusters in theabsence 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

pro i eration o ce s ca us prior toadventitious shoot formation.


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Organogenesis indirecta

Ex lanto Callos desarrollo meristemoide Primordio


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-

roots.

Auxin/cytokinin 1:10-1:100 induces

Intermediate ratios around 1:1 favor

callus growth.


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non-germ cells.

Usually involves a callus intermediate stage


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El desarrollo de los


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El desarrollo de los

embriones somticospasa por los mismos

de desarrollo que unembrin ci tico:

proembrin globular,

trapezoidal,

embrin cordiformey torpedo.

Cleavage Polyembryony- conifers


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g y y y

Cleava e len thwa s

Embryo

Suspensor

Embyro

Secondary embryo formation


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y y

- Most dicots

Abundant

Secondary

Embryos

Early embryo

tema 4 iniciacion de cultivos in vitro

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