Mutation induction throughMutation induction through tissue culture: Stemtissue culture: Stem 

propagated crops (e.g. p p g p ( gCassava, Manihot esculentus)

Plant transfer from field to laboratory

In vivo stem cutting

Stems transplantation

Apical stem Collection

Transfer of the plant  from field to laboratory has advantage to reduce the charge  p y g gof contaminants (fungi, bacteria, Virus,…). Stems can be pre‐treated with Clorox before transplanting .

Samples collection from the field or laboratorylaboratory

Apical stem

collection

Healthy meristem Unhealthy meristem

Appical 

merite

m 

ollection

Good enter A m co

Healthy appical meristem nodes sizeis collected with 4‐8 

auxillairy meristems  with good internodes si e for Bad enter 

node size

good internodes size for tissue culture.

Isolation of the meristem

Si d tiSize reduction2‐3 nodes

ater 

g

The stem is reduced  to the size 2 to 3 nodes (ideal size for  250ml flask). Tape water rinsing is to remove any soil or solid contaminants Spilt to 10 to 15

Tape

 wa

washing

any soil or solid contaminants. Spilt to 10 to 15 cutting per flask for better in vitro sterilization

Id l t

Spilting

Ideal amount

Too many cutting

In vitro sterilisation: Cleaning with ethanol

70% EthanolIncubation10‐20s10 20s

Rinsingwith sterile

1X

water

70% ethanol is for surface sterilisation, therefore ,treatment should be as short as possible. if not, it will cause tissue burn.

In vitro sterilisation: Clorox cleaningIn vitro sterilisation: Clorox cleaning

Incubation20 min

20% Clorox 1-2 trops

tween 20 mintween

Rinsingwith 3Xsterilewater

Treatment is done with solution Cloroxcontaining 20% tween (1‐2 drops). The incubation is performed by hand shaking every 5 min.

Buds cleaning and transfer to culture media

Rinsing with sterile water

Forceps cleaningg

3  es 

ing

2 to 

node

cutti

Growth chamber incubation

Transfer to media

After Clorox treatment  and  rinsing with sterile water, the necrotic end of each sample are removed and cut to 2‐3 nodes and then transfer to solid medium which allowsremoved and cut to 2‐3 nodes and then transfer to solid medium which allows contaminants visualisation after 2 to 3 days.  The contaminated samples are discarded  and the clean ones are used for further for multiplication.  

Buds multiplicationp

Transfer to  Media

Forceps cleaning

3-4 weeks

tion 

haker 

gweeks

After 3 to 4 weeks new shoots from

Incuba

ton

 a sh

Stem

cutting Subculture  

4 weeks

After 3 to 4 weeks, new shoots from clean samples are isolated and subculture (2-3 nodes) in liquid

di f f t lti li timedium for fast multiplication

Subculture

Mutation induction: Radiation test

Sample Stemstti preparationcutting

Dose assessmentassessment

A radiation test is performed to determine the optimal dose for mutation induction. F b tt l ti t tti l 2 d ( ith t) tti ill b d It i

0 Gy 5 Gy 10 Gy 15 Gy 20 Gy 30 Gy

For better evaluation stem cuttings only 2 nodes (without) cuttings will be used It is assessed by plant  biomass evaluation (height, weight, number of nodes…) and multiplication ratio.

Mutation induction : Bulk irradiation

Buds  preparation

ion 

al 

e

Irrad

iat

optim dos e

Incubation 4 weeks

The optimal dose is used to irradiate high amount of samples (X 1000) for mutagenic population which will further be screened for trait of interest.

Mutation induction: Chimerism dissolutiondissolution

Transfer toTransfer to  Medium2 -3 nodes

cuttings

cuba

tion 

n shaker 

era  

ution

Subculture1‐2X

Inc on

Chim

edissol Subculture  

4 weeksSamples ready for shipment

Transfer 

Subcultureto rooting medium

Chimerism dissolution is done by isolation of any new buds and cleaning off external leaf layers for at least 3 round and then transfer to solid medium for rooting and shipment .

Acclimatization

Cutting in wet soil with 1(one) node covered by the soilW t il d f t l t ti Cutting in wet soil with 1(one) node covered by the soil for roots development.Wet soil ready for transplantation

with in vitro material in tube. Cutting with 2 to 3 nodes ready to be planted in wet soil.

Pot containing the cutting kept in plastic bag with little water in the bag to maitaim high moisture.

Cassava plant ready to be planted without any micro-climate ( time to remove the plastic bag away).

The plantlet is cut into 2 to 3 nodes (mature stem special the bottom part to allow straight cutting during water lost period when adapting to soil culture)  and transfer to a best soil (well aerated )  and well irrigate to keep in plastic bag with high moisture and seal the bag.

Acclimatization

Gl hGlasshouse maintenance

A least two months plant3-4 months plant

s St

em

cutti

ngs

c

Field screening

Acclimatized plants are transferred in the field  (as cutting that way it p ( g yallows replication and also multiple trials) for screening after at least 2 to 4 month glasshouse maintenance. 

Acknowledgements

This protocol was developed by:

Souleymane Bado, Abdelbagi MA Ghanim,Gilbert Seballos Gunther & BertholdAndreaGilbert Seballos, Gunther & BertholdAndreaDraganitsch.

Edited and compiled by Souleymane Badoand Abdelbagi MA Ghanim, Brian P. Forster,and Abdelbagi MA Ghanim, Brian P. Forster,Plant Breeding and Genetics Laboratory,Joint FAO/IAEA Division, Vienna, Austria, ,