Download - 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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Surface sterilization of plant material
FOR PLANT TISSUE CULTURE
Disinfectant Concentration (%) Exposure (min)
a c um ypoc or te - -
Sodium hypochlorite* 0.5-5 5-30
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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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Surface sterilization of plant material
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