Summary:
The olive tree is a plant species of great economic importance for the economy of many countries that the majority of which spread around the Mediterranean basin. It is a long-lived, evergreen plant which bears fruit and adapted to adverse conditions, even and poor crop care. The olive tree is cultivated in unsuitable soils for other plants, prevents soil erosion, and contributes to the income of thousands of farmers. At the same time
olive tree varieties can be exploited, not only for their main products (olive oil and olives) but also for their by-products (leaves, wood, etc.) for pharmaceutical and other purposes.
Unlike the majority of fruiting plants with huge economic and nutritional value,
cultivation of olive tree still requires laborious and not always efficient processes. The non-competitiveness of the cultivation of olive tree is mainly due to low productivity,
limited photosynthetic ability of crop varieties, and traditional farming methods.
Therefore alternative regenerative approaches (i.e. in vitro regeneration) are required.
Somatic embryogenesis is a promising in vitro regenerative process of plant tissues in aseptic conditions. The induction of high levels of somatic embryogenesis from
immature and mature tissues of olive tree is crucial for the implementation of fast, easy, and efficient propagation methods of olive tree with reduced production costs.
As research efforts to induce somatic embryos in olive tree explants were limited and the rates of induction were low too, we considered essential to conduct research to identify the factors required for the induction of high levels of somatic embryogenesis,
development, and survival of somatic embryos.
During our initial experimental approach, different explants (radicles and
cotyledonary segments) of mature zygotic embryos of Koroneïki olive tree were tested. Koroneiki olive tree was chosen as the most suitable experimental material, as
preliminary experiments with other Greek varieties (Tzonati, Kalamata, Amfissa and
Angouromana) showed that displays the highest embryogenic potential.
The morphogenic capacity of four different culture media supplemented with 25.0 μΜ ΙΒΑ and 2.5 μΜ 2iP were tested;
• OMc (Cañas et al. 1988).
• ΟΜc without hydrolyzed casein.
• MS (Murashige et al. 1962).
• MS with 1.0 g/L hydrolyzed casein.
The results proved that ΟΜc is the most suitable nutrient medium to induce
somatic embryos in olive tree tissue cultures. Similarly, the presence of hydrolyzed casein is crucial for the induction and expression of embryogenic capacity of the explants.
The dependence of somatic embryogenesis on the age of explant, was studied by inoculating immature olive zygotic embryos, collected from July to August, in OMc
supplemented with 25.0 μΜ ΙΒΑ and 2.5 μΜ 2iP. Calli from radicles exhibited a greater morphogenetic capacity which increased proportionally with age. The embryogenic
capacity of calli from proximal and distal segments of immature zygotic cotyledons was much lower than that of calli from proximal and distal segments of mature zygotic
cotyledons. In any case however, higher rates of somatic embryogenesis were recorded in tissue explants of mature zygotic embryos than those in tissue explants of immature
zygotic embryos.
We investigated the change of the embryogenic capacity of radicles which were cut into two halves;
• The upper half radicle (towards the cap).
• The bottom half radicle (towards the cotyledons).
Explants were inoculated in ΟΜc supplemented with 25.0 μΜ ΙΒΑ and 2.5 μΜ 2iP. The lower half of the radicle gave higher somatic embryogenesis than the intact
radicle, while the upper half radicle inhibited somatic embryogenesis, suggesting that there is “a gradient of embryogenic potential” from the proximal to the distal region of the radicle.
Subcultures of radicles, proximal and distal cotyledonary segments of mature
zygotic embryos in OMc supplemented with 25.0 μΜ ΙΒΑ and 2.5 μΜ 2iP, every 21 days, resulted in a gradual decrease and finally zeroing of the embryogenic capacity of radicles during 4th subculture and of proximal and distal cotyledonary segments during 2nd
subculture.
In another experimental test five concentrations of sucrose were used;
• 5.0 g/L sucrose (0.015 M)
• 20.0 g/L sucrose [0.058 M (control experiment)]
• 40.0 g/L sucrose (0.12 M)
• 80.0 g/L sucrose (0.23 M) and
• 160.0 g/L sucrose (0.46 M).
All explants were inoculated in ΟMc supplemented with 25.0 μΜ ΙΒΑ and 2.5 μΜ 2iP. Morphogenetic primacy (55.0% induction of somatic embryos) of the medium
containing 160.0 g/L sucrose was recorded only 14 days after inoculation although the explants were still in the “callogenesis” medium. The replacement of sucrose by three concentrations of mannitol [10.0 g/L (0.05 M), 42.0 g/L (0.23 M) and 85.0 g/L (0.46 M)] indicated no correlation of high percentage of somatic embryogenesis (in the medium supplemented with a high concentration of sucrose) with an increased osmotic pressure of the medium.
For the investigation of the dependence of somatic embryogenesis on auxins, OMc supplemented with 2.5 μΜ 2iP and one of the following auxins was tested:
i) ΙΒΑ: 0.39 / 1.56 / 6.25 / 12.5 / 25.0 / 50.0 / 100.0 and 200.0 μΜ.
ii) ΝΑΑ: 0.1 / 0.39 / 1.56 / 6.25 / 12.5 / 25.0 and 50.0 μΜ.
iii) 2,4-D: 0.01 / 0.05 / 0.1 / 0.39 and 1.56 μΜ.
The results indicated that both NAA and 2,4-D, in all concentrations and the types of explant, induce lower somatic embryogenesis than those of the control experiment (4.0-15.0%). IBA induced much higher somatic embryogenesis than all other experiments in radicles (23.0-31.0%) and in proximal and distal cotyledonary segments especially at the highest concentrations (17.0%).
In parallel we studied the effect of four combinations of auxins on somatic
embryogenesis supplemented with 2.5 μΜ 2iP in OMc medium:
0.195 μΜ ΝΑΑ / 0.78 μΜ ΙΒΑ
0.39 μΜ ΝΑΑ / 1.56 μΜ ΙΒΑ
6.25 μΜ ΝΑΑ / 12.5 μΜ ΙΒΑ
12.5 μΜ ΝΑΑ / 25.0 μΜ ΙΒΑ
The combined supply of auxins reduced both direct and indirect somatic
embryogenesis (15.0%), probably due to the inhibitory effect of NAA, compared to IBA (23,0% direct somatic embryogenesis and 31.0% indirect somatic embryogenesis). These data suggest neither “an additive” effect of the two auxins nor the existence of different target sites of auxin in the cells of olive tree.
The morphogenetic effect of three different synthetic cytokinins (TDZ, 2iP and BA) at various concentrations (0.625 / 2.5 / 10.0 or 40.0 μΜ), when were supplied to OMc medium containing 25.0 μΜ IBA, was tested. In radicle calli 0.625 μΜ 2iP induced about 9.0% direct somatic embryogenesis, while the other concentrations induced higher rates of indirect somatic embryogenesis, and 2.5 μΜ 2iP the highest (15.0% somatic
embryogenesis). In proximal and distal cotyledonary segments the induction of somatic embryos ranged from 0.0-10.0% in medium supplemented with 20.0 μM 2iP. Supply of BA in radicles partially inhibited the somatic embryogenesis (2.0-5.0%) and TDZ fully.
The use of four combinations of cytokinins in medium OMc containing 25.0 μM IBA did not advance rates of indirect somatic embryogenesis. The combined supply of 2iP and BA (2.5 μM / 10,0 μM and 1.25 μM / 5.0 μM) inhibited the embryogenic induction against individual supply of 2iP and ranged in corresponding levels as individual supply of ΒΑ. The combined supply of 2iP and TDZ (2.5 μM / 10,0 μM and 1.25 μM / 5.0 μM) reduced the proliferative effects of individual supply of 2iP.
The effect of the source of inorganic nitrogen in the induction of somatic embryos was another parameter studied. Radicles of mature zygotic embryos were inoculated into OMc containing 25.0 μM IBA, 2.5 μM 2iP and NH4NO3 or KNO3, or NH4Cl at the following concentrations; 5.0 / 20.0 / 80.0 or 160.0 mM. The addition of NH4NO3 as the only source of inorganic nitrogen in any concentration, did not affect the rate of differentiation of somatic embryos (32.0-36.0%) compared with the control experiment, except 160.0 mM that significantly inhibited both somatic embryogenesis and callogenesis (19.0%). The supply of low concentrations of KNO3 reduced drastically the production of somatic embryos (18.0%) and yet more strongly the higher concentrations (4.0%), while no
reduction of callogenesis was observed. The supply of NH4Cl (5.0 / 20.0 or 80.0mM)
increased somatic embryogenesis (52.0-58.0%) and much higher (62.0%) the
concentration of 160.0 mM. Callogenesis was decreased by the concentration of 80.0 mM (60.0%) and the concentration of 160.0 mM (40.0%).
The effects of two combinations of inorganic nitrogen sources KNO3:NH4NO3 and KNO3:NH4Cl in different ratios (1:9, 3:7, 5:5, 7:3 and 9:1) on somatic embryogenesis were tested too. Radicles of mature zygotic embryos were inoculated in OMc supplemented with 25.0 μΜ ΙΒΑ and 2.5 μΜ 2iP and combinations of inorganic nitrogen sources. The results of all the combinations demonstrated the inhibitory effect in the induction of
somatic embryos of KNO3 and the stimulating effect of NH4NO3 and NH4Cl. Furthermore, they verified the inhibitory effect on callus induction of NH4Cl at high concentrations. For combinations of KNO3 and NH4NO3 induction of somatic embryogenesis was recorded ranging from 16.0-3.0%. Similar results have been observed in ratios KNO3:NH4Cl. In 1:9 ratios a high percentage of somatic embryogenesis (38.0%) was recorded while the rest of the ratios reduced it drastically (27.0-1.0%).
As a conclusion of all experimental tests we prepared a medium that combined all those compounds and growth regulators which were proved to be more appropriate for inducing somatic embryos from olive explants in vitro. Radicles of mature zygotic embryos were inoculated in OMc supplemented with 25.0 μΜ ΙΒΑ, 2.5 μΜ 2iP, 20.0 mM NH4Cl and 160.0 g/L sucrose. The induction of somatic embryogenesis in this "ideal" medium was impressive (62.0%) compared to OMc (24.0%) which was proposed by Cañas et al. (1988) as the most appropriate medium for tissue cultures in olive tree. Noteworthy is the appearance of the first somatic embryos in callogenesis medium even before calli were transferred to differentiation medium.
During the molecular approach of somatic embryogenesis in olive tissue cultures, calli from radicles of zygotic embryos of Koroneiki olive tree were used in an attempt to identify in the genome of olive tree the genes Dc8 and Dc59. The study of
autoradiographs made evident the existence of both genes. Specifically, for Dc8 no
polymorphism in the genome of olive tree was observed, as has been found in carrot, which is indicated by the appearance of a single band (approximately 3 Kb). Regarding the gene Dc59, were identified numerous hybridized zones (4.4-6.6 Kb), suggesting the
existence of polymorphism in this gene in the olive tree genome like in carrot and many other plant species.
We studied the expression of Dc8 in different tissues too. The results showed
expression of this gene in intact zygotic embryo and endosperm of olives, in full zygotic embryos of carrot, but not in the endosperm, which is inconsistent with the existing
literature data. Probably this is due to lower expression in the endosperm (about 2 times lower than in zygotic embryos), in combination with the low concentration of total RNA (8.0-10.0 mg/l) that was isolated and used to detect the transcript of Dc8.
Finally, we investigated the expression of Dc8 in eight different types and ages of calli of mature zygotic embryos of olive tree, which were in different morphogenetic - embryogenic stages. The Dc8 expression was significantly during the early days of
inoculation, throughout the culture of radicles in callogenesis medium (21-day-old calli) and when were transferred to the differentiation medium in which were inoculated for at least 60 days. The amount of transcripts was relatively stable in 7- day-old calli and 31-day-old calli and increased in the last two RNA samples from 36 day-old calli and 42 day-old calli. Increased accumulation of Dc8’s transcripts coincides with the period during which the rate of induction of somatic embryos in tissue culture is stabilized at the
maximum level and the majority of the somatic embryos are either in the heart-shaped and the torpedo stage either start to mature and evolve further into plantlets.
