Ctifl

CONTEXT

In vitro walnut micropropagation Development of lateral-bearing varieties (breeders)

"Juglans regia L. application" Selection of vigourous rootstock tolerant to Cherry

Leaf Roll Virus (CLRV)
Selection of Juglans regia L. seedlings (Ctifl)
Laurence Bourrain
Difficults traditional propagation techniques
Ctifl in vitro laboratory

Ctifl has developed an in vitro walnut

micropropagation technique
COST 873 – Murcia, March 2009
COST 873 – Murcia, March 2009

Ctifl in vitro protocole In vitro establishment:

for Juglans regia L. "Mother plant"

• 1-2 year old

seedlings
• or 1 year old
grafted plants
• development in
greenhouse
• phytosanitary
treatment if necessary

COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

In vitro establishment: In vitro establishment:

"Mother plant" "Disinfection protocole"
young herbaceaous shoot, semi lignified or not • shoots are washed with
Mercryl (combination of
chlorhexidine gluconate and
benzalkonium chloride) and
with
70° alcohol for few minutes
• sodium hypoclorite
solution:
differente balance possibilities
– concentration / time
For example : 1-2 %, 20 minutes and 10-12 % 2 minutes or 10-20%, 10-20 minutes
+ 3 rinses with sterile distilled water

COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

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 In vitro establishment:
In vitro establishment "5 weeks after disinfection"
disinfection"
Culture conditions
• establishment better • 25-28 °C
between March and May,
or all round a year if good • 16 h photoperiod
quality material
One shoot and Medium
directly start One
• single bud microcutting of an axillary apex • DKW medium:
bud 0.5-1 mg/l BA ;
0.01-0.05 mg/l IBA ;
One single bud 30 g/l glucose ;
per tube 9-10 g/l agar ;
pH = 6
COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

Multiplication
In vitro phase In vitro shoot proliferation
establishment:
"Disinfection control" Shoots at the beginning of
subculture
Basal part of
the explant Culture cycle: 5 weeks

• After visual control each

microcutting is tested one or
twice for bacterial and/or fungal
contamination on a microbiological medium
Shoots at the end of
the contaminated explants are eliminated subculture
basal explant apex
COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

In vitro shoot proliferation In vitro shoot proliferation

One single shoot Shoots coming from apex Shoots coming from basal explant
for rooting Too
small
shoot for
rooting

1 to 4 shoots good for rooting per tuft

Axillary buds development for next cycle But less material for next cycle, and lower quality
COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

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 In vitro shoot proliferation In vitro rooting:
rooting:
5 good shoots for rooting but "Phase 1 = Induction "
few material for next cycle Culture conditions
MS medium:
DKW medium supplemented with:
1 to 3 mg/l IBA
0.7-1 mg/l BA ;
0.01 mg/IBA ; 30 g/l sucrose
30 g/l glucose ; 9-10 g/l agar
9-10 g/l agar ; pH = 6
pH = 6 darkness = 7 days
temperature : 23-
Temperature: 26-28 °C 24°C
Photoperiod: 16 h 2 to 5 cm long shoots
culture cycle: 5 weeks
COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

In vitro rooting:
rooting:
"Phase 2 = Rooting expression"
In vitro rooting:
rooting: "Root
"Root quality"
quality"

MS medium : No secondary roots

Expression Induction macronutrients diluted 1/4
Callus
no growth regulator
30 g/l glucose Spongeous roots
9-10 g/l agar
pH = 6
Agar rooting
vermiculite Vermex M
(rate = 150 ml medium/190 Secondary
ml vermiculite)
photoperiod = 16 h
roots
Modified Jay-Allemand technique temperature = 23-24°C Vermiculite/agar
rooting

COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

In vitro rooting Other important in vitro aspects

Rooted JAR
plants Le parfait / Meli
(apex in Multiplication/elongation Le
active Aeration system, gas exchange
Meli Parfait
growth) Hyperhydricity
ready for GELLING AGENT
weaning
CARBON SOURCE Agar brand/Gelrite/Phytagel
Glucose for proliferation Multiplication/elongation
Sucrose for induction rooting Hyperhydricity
COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

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 Weaning Weaning
7*7*8 Substrat = Bud
Steckmedium growth
Klasman
"peat perlite +
fertilisation"

Emerged
roots

9*9*9.5
3-4 weeks 2 months
Classical conditions of weaning in greenhouse Peat or plastic pots
COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

Weaning:
Weaning:
"Mychorrize effect"
effect"
Weaning:
Weaning: "Key factors"
factors"
Endomychorrize: Glomus sp • Quality of plants to wean: well
Inoculation with rooted, apex in active growth
With Without arbuscular mycorrhizal • Quality of substrate: well
fungi (AMF) aerated
"Endorize-iv"
from Biorize company
(Agrauxine group)
• Climatic conditions of
Superior survival greenhouse: 22-24 °C, light,
hygrometry controled
Weaning of a difficult-to-wean clone Faster regrowth
• Mycorrhize: faster regrowth,
one month after acclimatization superior survival
COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

Weaning:
Weaning: Problems Nursery
After treatment with growth Behaviour of 2 different in vitro clones
regulator: Promalin
(19 g/l BA + 19 g/l gibberellin A4-A7)

Growth stop of
the terminal bud

COST 873 – Murcia, March 2009 COST 873 – Murcia, March 2009

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 for

COST 873 – Murcia, March 2009