Micropropagation via nodal explants of ancient local olive ...
Transcript of Micropropagation via nodal explants of ancient local olive ...
Micropropagation via nodal explants of ancient local olive ecotypes (Olea europaea L.) in
Lebanon
-Ahmad ELBITAR-Lebanese Agricultural Research Institute
24 May [email protected]
Kingdom Green Plants
Subkingdom Tracheobionata - vascular plants
Superdivision Spermatophyta - seed plants
Division Magnoliophyta - flowering plants
Class Magnoliopsida - Dicotyledons
SubClass Asteridae
Order Scrophulariales or Lamiales
Family Oleaceae - ash, privet, lilac and olives
Genus Olea
Species Europa
Subspecies Olea europaea
Taxonomy
Olive cultivation area in Lebanon
➢ Olive holds a major place in LebaneseAgriculture. As to the latest olive mapestablished in 2010, the area under olivecultivation in Lebanon was estimated at 45,000ha (nearly 18% of the total cultivated area)
➢ Olive trees are cultivated in the plain and onhills and mountains, from 45 to 1200 m abovesea level (a.s.l.). The best areas are thenorthen (40%) and southern (38%) regions ofLebanon that benefit from exposure to thehumid air of Mediterranean sea
➢ Family plantations are characterized by beingfragmented to small sized orchards of 0.2 to2.0 ha, while large size orchards are somewhatcommon in the main area of cultivation in theSouth and the North.
In Lebanon, oleoculture is millennia.The age of some olive trees exceeds 1500 years.
The average age of olive trees in the country is around 150 years.
• heterogeneous plantsSeeds
• Labor for grafting, low germination rate and Time consuming
Grafting
• Long time to enter in production, risk of reproducing the rootstocks.
Cutting by nutsedge
Traditional propagation of olive trees
• Material agronomically very heterogeneous or attacked by various diseases.
Woody cuttings
• Difficult for very old tree “millennial tree”Semi-woody
cuttings
Plant tissue cultureMicropropagation for large scale production
Strategy developpement for the micropropagation of the ancient trees for the most common olive variety in
Lebanon called
Baladi
Plant material
Twigs of the year taken during
mid-May to mid-June, on four
trees representing four ecotypes
of the local variety called "Baladi".
Ecotype Origin Description of the tree
Bchaaleh Bchaaleh
(1200 m a.s.l)Millennial ecotype
Tyr Tyr
(11 m a.s.l)Ecotype selected by LARI, for its oil content (23%)
Qana Qana
(680 m a.s.l)Ecotype selected by LARI, for its oil content (23%)
Hidab Hidab
(840 m a.s.l)Ecotype selected by LARI, for the quality of its olives
6- Agitation with HgCl2 (0.01%) for 12 min
7- At least three times rinsing with sterile dH2O
4- Soaking in alcohol (70%) for 1min
3- Agitation with NaOCl (5%) + Tween 20 for 5 min
2- Agitation with Fungicid (10 g/l) for 30 min
1- Washing with soap and tap water for 45 min
5- Three times rinsing with sterile dH2O
Disinfection of plant material
Mineral composition (mg/l)
MS OM P
NH4NO3 1650 412 6650
KNO3 1900 1100 950
MgSO4.7H2O 370 732.6 185
KH2PO4 170 340 85
CaCl2.2H2O 440 332.16 740
Ca (NO3)2 4H2O - 416.92 -
KCl - 500 -
Culture mediaChemical composition of MS (Murashige and Skoog 1962), OM (Rugini
1984) and P (Fiorino and Leva 1986)
✓Vitamins and Iron MS ✓PVP (Polyvinylpyrrolidone)✓ Mannitol 30 g.l-1
✓ pH 6.00✓ Agar 7.5 g.l-1
Initiation and Multiplication
Mineral composition Kinetin Zeatin BAP NAA
M1 MS 0 0 0 0
M2 OM 0 0 0 0
M3 P 0 0 0 0
M4 MS 2 2 0 0
M5 MS 0 2 2 0
M6 MS 0 4 0 0
M7 OM 2 2 0 0
M8 OM 0 2 2 0
M9 OM 0 4 0 0
M10 P 2 2 0 0
M11 P 0 2 2 0
M12 P 0 4 0 0
Different growth regulators types and concentrations (mg.l-1) used for the in vitro culture of olive.
Rooting
Mineral composition
Kinetin Zeatin BAP NAA
R1 MS 0 0 0 0
R2 MS 0 0 0 1
R3 MS 0 0 0 2
Different growth regulators types and concentrations (mg.l-1) used for the in vitro culture of olive.
For each ecotype, a set of 480 sections of 1 cm, eachcarrying a pair of axillaries buds were cultivatedhorizontally on 12 media with 4 replicates per medium and4 explants per replicate.
Cultures were incubated at 23±2°C under 16 h
photoperiod of 3000 lux.
1- Effectiveness of disinfection treatment
2- Average lenghts and number of regenerated shootlets
Multiplication
One month after cultures initiation, regenerated shootletswere transferred onto the 12 multiplication media formultiplication .
Cultures were incubated at 23±2°C under 16 h photoperiod of 3000 lux.
1- Multiplication rate was calculated after 30 days
(Number of total explants / Number of initial explants)
Rooting
Vitroplantlets of the third subcultures were transferredonto the rooting media R1, R2 and R3.
1- Percentage of rooting
Cultures were incubated at 23±2°C under 16 h photoperiod of 3000 lux.
2- Average length and number of roots
Results
Ecotype Number of explants cultured
Number (%) of explants lost by Number of surviving
explants (%)Bacteria Fungi Oxydation
Bchaaleh 480 20(4.16)
0(0)
288(60)
172(35.84)
Tyr 480 0(0)
164(34.16)
96(20)
220(45.84)
Qana 480 32(6.67)
144(30)
96(20)
208(43.33)
Hidab 480 48(10)
32(6.6)
160(33.4)
240(50)
Survival explants following the disinfection treatment, after 40 days of cultures initiation .
Initiation phase
Ecotype Medium Average number of regenerated vitroplantlets
Bchaaleh M9 (OM + 4 Zeatin) 1.3 ± 0.4
Tyr M6 (MS + 4 Zeatin) 1.6 ± 0.5
Qana M12 (P+ 4 Zeatin) 1.7 ± 0.4
Hidab M6 (MS + 4 Zeatin) 1.4 ± 0.4
Qana / M12 Tyr/ M6 Hidab/ M6
Effect of ecotype and culture medium on the average number of vitroplantlets regenerated after 40 days of culture.
Initiation phase
Ecotype Medium Average length (cm)
Bchaaleh M12 (P+ 4 Zeatin) 1.0 ± 0.1
Tyr M6 (MS + 4 Zeatin) 1.2 ± 0.1
Qana M6 (MS + 4 Zeatin) 2.1 ± 0.4
Hidab M6 (MS + 4 Zeatin)M9 (OM + 4 Zeatin)
3.0 ± 0.2
Bchaaleh/ M12 Hidab/ M6
Effect of ecotype and culture medium on the average length of the regenerated vitroplantlets
Initiation phase
0
1
2
3
4
5
6
7
Bchaaleh Tyr Qana Hidab
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Bchaalet Tyr Qana Hidab
Ecotype effect on the average length of vitrplantlets ( medium M6:MS + 4 mg.l-1 Zeatin)
Ecotype effect on the multiplication rate of vitrplantlets ( medium M6:MS + 4 mg.l-1 Zeatin)
Av
era
ge l
en
gth
of
vit
rpla
ntl
ets
(cm
)
Mu
ltip
lica
tio
nra
te
Multiplication phase
Vitroplantlets of Hidab ecotype having between 2 and 3 pairs of leaves were tested.
Rooting phase
R1 (0 mg NAA)
R2(1 mg NAA)
R3 (2 mg NAA)
Number of vitroplantles 25 25 25
Number of rooted vitroplantlets
2(8%)
9(36%)
19(76%)
Average number of roots per vitroplantlet
1.6 5.9 5.3
Average length of roots (cm) 0.9 0.5 0.4
✓Macro and microelements of MS✓Mannitol 20 g.l-1
✓ pH 6.00✓ Agar 7.5 g.l-1