Micropropagation via nodal explants of ancient local olive ecotypes (Olea europaea L.) in

Lebanon

-Ahmad ELBITAR-Lebanese Agricultural Research Institute

24 May 2021abitar@lari.gov.lb

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

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gth

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vit

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(cm

)

Mu

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nra

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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