A rapid plant regeneration system by using stem and leaf explants of Olea europea L. cv. Koroneiki; a multipurpose bioenergy tree

doi:10.21203/rs.3.rs-2211677/v1

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A rapid plant regeneration system by using stem and leaf explants of Olea europea L. cv. Koroneiki; a multipurpose bioenergy tree

https://doi.org/10.21203/rs.3.rs-2211677/v1

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Olea europea L. cv. koroneiki is a multipurpose tree which belongs to family Oleaceae. Koroneiki is famous for its virgin oil production and suitable for high density growing system around the world. Being grafted and recalcitrant in nature, Koroneiki demands an efficient in vitro protocol for its propagation and conservation. The aim of the present study was to set up an efficient protocol for in vitro regeneration of this beneficial olive cultivar. Three different basal media, Murashige and Skoog (MS), Olive Medium (OM) and Woody Plant Medium (WPM) were tested for two types of explants, leaf and nodal segments for callus induction and shoot regeneration. To find out the best explant disinfestation method during this study, explants were soaked in detergent for 15 minutes with continuous agitation. After washing, explants were rinsed with 70% ethanol for 10 seconds and finally treated with 0.1% mercuric chloride for 3 minutes and rinsed 4–5 times with autoclaved distilled water under the laminar air flow cabinet considered as most suitable for cv. Koroneiki. Media were either used alone or supplemented with 6-benzylaminopurine (BAP) (8.8 and 17.7 µM) and zeatin (4.56 and 9.12 µM) to find out suitable PGR for callus induction and shoot proliferation. Maximum callus induction (70%) on nodal explants was observed on OM medium containing 9.12 µM zeatin + 17.7 µM BAP under both dark and light conditions. The WPM containing the combination of BAP and zeatin showed 67% callus induction under light by using nodal explants. However, in case of leaf explants, 50% callus induction response was observed under dark conditions, but no callus was observed under light. The OM supplemented with combination of BAP (17.7 µM) and zeatin (9.12 µM) induced highest shoot length (1.5) and maximum number of leaves (3.5). This study might helpful for commercial propagation of cv. Koroneiki at mass scale under in vitro conditions to fulfill the virgin oil demand for rapidly growing population.

Callus induction

In vitro

Olea europea

Regeneration

Olea europea L. cv. koroneiki is member of the family Oleaceae also known as Queen of olives (Kabbash et al., 2019). It is an ever green, medium sized fruit bearing plant. At maturity, olive tree is almost 15 meter in height and have a lifespan of more than 500 years (Baldoni and Belaj, 2009). It is originated from Greece and now dispersed in different places over the five continents such as Asia, Africa, Europe and Australia but most abundantly grown in Mediterranean basin (Khan et al., 2019). Olea europea L. cv. koroneiki is the most prominent and popular among olives because of virgin oil. Olive oil is also included in World Intangible Cultural Heritage by UNESCO (Litz et al., 2020). It covers almost 60% of area among Greek olive trees due to its beneficial effects on human health (Kandylis et al., 2011). Within last three decades, world olive oil production increased from 1874 to 2881 thousand tons and that of table olives from 960 to 2564 thousand tons (Gökdoğan and Erdoğan, 2018). Approximately 1250 cultivars of olive plants are being cultivated in 54 countries on an area of 10.8 million hectares (Cavaca et al., 2020).

Olea europea L. cv. koroneiki is distinguished by its oblong, coriaceous leaves having tapered ends and upper surface of leaf is dark green whereas lower surface appears silvery or grey (Bartolini and Petruccelli, 2002). The flowers of the olive are of two types; small, white, and creamy in color, sweet scented and appear at axil position of leaf. They are bisexual, blooms in spring and are pollinated by wind. Many cultivars of Olea europaea L. are self-incompatible (Rhizopoulou, 2007). The olive fruit is like a drupe and found in different shapes with a pulp/stone ratio of almost 3.0–6.5 g (Galanakis, 2011). When it is fully ripened in autumn, its color changes from green to black and purple while oil is obtained from fleshy part of the fruit (Rhizopoulou 2007). Total weight of a seed lies between 0.5 to 20 g but commonly falls between 3 to 10 g. Olive oil contains rich amount of oleic acid which increases its shelf life (Galanakis 2011). The olive oil is used as a medicine in treating obesity, cancer, metabolic syndrome, cardiovascular diseases, and type-2 diabetes (Lambardi et al., 2012; Goenka and Simon, 2021).

Olive trees are propagated by various methods including cutting, grafting and sprouted roots etc. It is reported that cuttings are useful only when taken from healthy mother plant and on onset of spring (Fabbri et al., 2009). Rooting of olive plant highly depends upon the cultivar and nutritional material available to them. To resolve the rooting issue, grafting is an applicable technique, but it is more difficult than the other propagation methods and demands skilled persons (Mangal et al., 2014). In addition to this, olive trees have a long juvenile period due to which they show recalcitrant nature for vegetative propagation methods (Micheli et al., 2018; Rkhis et al., 2011). Therefore, there is a need of such propagation method which can resolve all issues related to traditional propagation methods of olive.

Micropropagation technique is more advantageous as compared to conventional methods because of rapid propagation rate of true-to-type plants which is helpful in conserving the genetic resources (Mazri et al., 2011). Micropropagation of olives by means of axillary buds has been profitably used to propagate many olive cultivars commercially used in Italy and Spain (Jain and Gupta, 2018). Micropropagation is playing significant role in the production of many fruit species like apple, plum and peach but olive industry is still far behind (Balla and Mansvelt, 2012) because of recalcitrant nature of olive plants, high contamination rate (Yancheva and Kondakova, 2018) and long period of shoot elongation which needs a constant supply of medium. These problems made the olive micropropagation very challenging (Golmohamadi, 2019).

Olive micropropagation success rate depends upon the cultivar specific measures because each cultivar demands specific supply of nutrients. Hence, it is perhaps necessary to have a more or less cultivar-specific protocols (Bayraktar et al., 2020). Now, due to an increased demand of olive oil and fruits, a kind of competition has begun for which there is a need to solve all associated problems related to its propagation on priority basis to overcome the demand of olive oil and table olives. Considering the current challenges, the need was felt to initiate the development of an efficient in vitro technique for a rapid proliferation of this important Olea species through which this highly economical tree can be conserved. Thus, the aim of the present study was to optimize explant types and disinfection protocol, culture conditions as well as development of an efficient protocol for callus induction and shoot proliferation of Olea europaea L. cv. koroneiki. This might be helpful in future for plant multiplication and preservation of precious germplasm of this plant species.

2.1. Procurement of plant material, disinfection, and its establishment under in vitro conditions

For this research work, healthy, disease-free, and certified Olea europea L. cv. koroneiki plants were procured from Barani Agricultural Research Institute (BARI), Chakwal, Pakistan (Latitude: 32° 55' 29.39" N; Longitude: 72° 51' 11.99" E) and was raised in open field at Institute of Botany, University of the Punjab, Lahore. For callus induction and micropropagation, leaves and nodal portion were used as the explants. The explants were collected in early December 2020 from 5-year-old tree.

The first step of the present experiment was to optimize explant disinfection method, for which healthy shoots and leaves were collected and nodal portions were separated from the shoot. To optimize disinfection method two different approaches were used. In the one, explants were washed thoroughly with running tap water for 15 minutes. Then they were washed in water containing 15% (w/v) detergent (Surf excel; Unilever Pakistan) for 15 minutes with continuous agitation. After this, the explants were again washed with running tap water to remove all the traces of detergent. Remaining steps were carried out under the laminar air flow cabinet. The explants were rinsed with a 70% ethanol for 10 seconds afterwards and washed with autoclaved distilled water. Finally, the explants were treated with 0.1% (w/v) mercuric chloride (Sigma Aldrich) solution for 3 minutes and rinsed 4–5 times with autoclaved distilled water. Subsequently, the explants were ready for inoculation purpose. In the second method of disinfection, explants were washed thoroughly with autoclave distilled water for 30 minutes and rinsed with 70% ethanol for 10 seconds in laminar air flow cabinet. They were disinfected with a 0.75% (v/v) solution of NaClO (Sigma Aldrich) for 10 minutes with continuous agitation and finally rinsed 4–5 times with autoclaved distilled water.

2.2. Callus induction from leaf and nodal explants

The second step of the present experiment was the optimization of the medium for callus induction and its proliferation, for which three media were used: Murashige and Skoog medium (MS; Murashige and Skoog, 1962), Olive Medium (OM; Rugini, 1984) and Woody Plant Medium (WPM; McCown and Lloyd, 1981). To determine the suitable PGR for callus induction and multiplication, 6-benzylaminopurine (BAP; Sigma Aldrich) and zeatin were used based on our preliminary experiments. There were six treatments of each basal medium used. One treatment was hormone-free termed as control while remaining five treatments were supplemented with BAP (8.8 µM and 17.7 µM), zeatin (4.56 µM and 9.12 µM) and a combination of BAP (17.7 µM) and zeatin (9.12 µM). There were three replicates for each treatment. Optimum pH of the media (5.7–5.8) was adjusted with 0.1 N HCl or 0.1 N NaOH. The media were sterilized by autoclaving at 15 psi and 121°C for 15 minutes. Leaf (ca. 1 cm) and nodal segments (one explant per culture tube) were inoculated in glass culture tubes (25 × 150 mm; Pyrex) containing 10 mL medium solidified by 8.0 g/L agar. Half of the cultures from each treatment were kept in the dark while the remaining ones were kept under cool white fluorescent 40 µmolm^− 2s^− 1 light with a 16 h photoperiod. The culture room temperature was kept at 25 ± 2°C. For the maintenance, calluses were sub-cultured in the same medium after 30 days. Time of initiation of callus was recorded after inoculation on daily basis and various parameters of callus morphology i.e., percentage callus induction, color and texture were recorded by visual observation after 30 days of inoculation.

2.3 Shoot regeneration from nodal explants

The next step of the experiment was multiple shoot formation from the nodal explants. Basal media mentioned above (MS, OM, WPM) were supplemented with various growth regulators, i.e., BAP (8.8 µM and 17.7 µM), zeatin (4.56 µM and 9.12 µM) and Naphthaleneacetic acid (NAA; 0.123 µM) either alone or in combination. Shoot induction data were collected by observing the days to sprouting, shoot and leaf number per explant (by counting the number of shoots and leaves exhibited by nodal explant) and shoot length which was measured (cm) with the help of scale after 30 days.

2.4. Statistical analysis

The significant difference of mean values was determined by one-way ANOVA by using statistical software SPSS version 20.0.0. Duncan’s multiple range test was also applied to compare the mean values.

3.1. Disinfection and establishment of sterile cultures

To determine the effective disinfection method, two different approaches were followed. The first disinfection method comprised of 0.1 g (w/v) HgCl₂ for 3 minutes showed better contamination control as compared to the second method. In the first method, the contamination rate was 50% while in second method 85% contamination rate was recorded. Therefore, the first method was adopted in the present study to disinfect explants for callus induction and plant regeneration.

3.2. Callus induction on MS and OM media using nodal and leaf explants under dark/light condition

MS medium resulted in 0% callus initiation response on nodal explant. Explant necrosis after 25–30 days and change in color from green to brown was observed in all its tested treatments (data not shown). Thus, MS medium was not used further for callus induction experimentation. Olive medium (OM) supplemented with BAP (8.8 and 17.7 µM) and zeatin (4.56 and 9.12 µM) alone also resulted in no callus. Although the response of nodal explants of cv. Koroneiki on OM for callus initiation was almost similar under dark and light conditions, the medium C6 containing 17.7 BAP + 9.12 zeatin favored 70% callus induction (Fig. 1). When the effect of dark and light conditions was compared for callus induction, it was observed that callus induction was earlier (44 days) under the light conditions compared with 53 days under the dark. The calli induce under dark were creamy and compact while light induced callus cultures were dark green in color (Table 1).

MS medium did not support callus induction from the leaf explants but callus induction was observed using three of OM treatments (C3, C5 and C6). OM containing 9.12 µM zeatin did support 66.7% response while callus induction was observed after 74 to 79 days under dark and light conditions, respectively. Medium supplemented with 17.7 µM BAP did support 33.4% and 30% callus induction after 65 and 68 days under dark or light conditions respectively. OM supplemented with 17.7 µM BAP and 9.12 µM zeatin favored 33% and 30% callus induction after 60 and 61 days of explant inoculation under dark or light, respectively (Fig. 2, 3). Calluses formed on these media were creamy, having compact and granular in texture. The light induced callus cultures were green in appearance, having compact and granular texture (Table 2).

Table 1

**Effect of OM supplemented with growth regulators on callus induction and morphology using nodal explants of** Olea europea **L. cv. koroneiki under dark and light conditions.**
Media	OM + PGRs (µM)	Callus induction		Days to initiation		Callus morphology
		Dark	Light	Dark	Light	Dark	Light
C1	Without PGR	0%	0%	0	0	-
C2	8.8 BAP	0%	0%	0	0	-
C3	17.7 BAP	0%	0%	0	0	-
C4	4.56 zeatin	0%	0%	0	0	-
C5	9.12 zeatin	0%	0%	0	0	-
C6	17.7 BAP + 9.12 zeatin	70%	70%	53 ± 1.1^a	44 ± 1.2^a	Creamy, compact, granular, non-embryogenic	Dark green, compact, granular, non-embryogenic
Data represented here are the means (± S.D) of 3 replicate cultures per treatment.

Different alphabetic letters in each column show significant difference at p ≤ 0.05 according to Duncan’s multiple range test. Here “C1-C6” represents callus induction medium (OM + PGR).

Table 2

**Effect of OM supplemented with growth regulators on callus induction and morphology using leaf explants of** Olea europea **L. cv. koroneiki under dark and light conditions.**
Media	PGRs (µM)	Callus induction		Days to initiation		Callus morphology
		Dark	Light	Dark	Light	Dark	Light
C1	0	0%	0%	0	0	-	-
C2	8.8 BAP	0%	0%	0	0	-	-
C3	17.7 BAP	33.4%	30.0%	65 ± 1.3^ab	68 ± 2.3^ab	Creamy, compact, granular, non-embryogenic	Creamy, compact, green, non-embryogenic
C4	4.56 zeatin	0%	0%	0	0	-	-
C5	9.12 zeatin	66.7%	66.7%	74 ± 2.2^a	79 ± 1.2^a	Creamy, compact, granular, non-embryogenic	Creamy, compact, green, non-embryogenic
C6	17.7 BAP + 9.12 zeatin	33%	30%	60 ± 1.4^b	61 ± 1.3^b	Creamy, compact, granular, non-embryogenic	Creamy, compact, green, non-embryogenic
Data represented here are the means (± S.D) of 3 replicate cultures per treatment.

Different alphabetic letters in each column show significant difference at p ≤ 0.05 according to Duncan’s multiple range test. Here “C1-C6” represents callus induction medium (OM + PGR).

3.3. Callus induction response on WPM media using nodal/leaf explants under dark/light conditions

The response of nodal explants in terms of callus induction on WPM under light or dark was quite interesting (Table 3). Some 13.4% whitish, granular and friable callus was induced on PGR-free WPM in dark but none under light conditions. The medium supplemented with 9.12 µM zeatin induced 20% creamy white and compact callus under dark but none was observed in light. WPM with 17.7 µM BAP and 9.12 µM zeatin, on the other hand produced 67% lush green, compact callus under light but no callus induction was recorded under dark. The medium supplemented with 8.8 µM BAP, 17.7 µM BAP and 4.56 µM zeatin separately did not support callus induction in either dark or light. Using leaf explants, only WPM containing 9.12 µM zeatin alone or in combination with 17.7 µM BAP supported 30% and 50% callus induction, respectively under dark (Table 4; Fig. 4, 5). Overall, it was observed that the effect of zeatin alone (9.12 µM) on callus induction response was better compared with BAP both under dark and light conditions. However, WPM medium supplemented with 17.7 µM BAP and 9.12 µM zeatin supported best callus induction response by using leaf explants under light conditions in Olea europea L. cv. koroneiki.

Table 3

Effect of WPM supplemented with growth regulators on callus induction and morphology using nodal explants of *Olea europea* L. cv. koroneiki under dark and light conditions.
Media	PGRs (µM)	Callus induction %		Days to initiation		Callus morphology
		Dark	Light	Dark	Light	Dark	Light
M1	0	13.4%	0%	70 ± 1.1^a	0	Whitish, friable, granular, non-embryogenic
M2	8.8 BAP	0%	0%	0	0
M3	17.7 BAP	0%	0%	0	0	-
M4	4.56 zeatin	0%	0%	0	0
M5	9.12 zeatin	20%	0%	69 ± 1.3^a	0	Creamy white, compact, granular, non-embryogenic
M6	17.7 BAP + 9.12 zeatin	0%	67%	0	60 ± 0.19^a	-	Lush green, compact, granular, non-embryogenic

Data represented here are the means (± S.D) from 3 replicate cultures per treatment.

Different alphabetic letters in each column show significant difference at p ≤ 0.05 according to Duncan’s multiple range test. Here “M1-M6” represents Callus induction medium (WPM + PGR).

Table 4

Effect of WPM supplemented with growth regulators on callus induction and morphology using leaf explants of *Olea europea* L. cv. koroneiki under darks and light conditions.
Media	PGRs (µM)	Callus induction %		Days to initiation		Callus morphology
		Dark	Light	Dark	Light	Dark	Light
M1	0	0%	0	0	0	-	No response
M2	8.8 BAP	0%	0	0	0	-	0
M3	17.7 BAP	0%	0	0	0	-	0
M4	4.56 zeatin	0%	0	0	0	-	0
M5	9.12 zeatin	30%	0	60 ± .88^b	0	Light brown, compact, granular, non-embryogenic	0
M6	17.7 BAP + 9.12 zeatin	50%	0	71 ± .78^a	0	Light brown, compact, granular, non-embryogenic	0
Data represented here are the means (± S.D) of 3 replicate cultures per treatment.

Different alphabetic letters in each column show significant difference at p ≤ 0.05 according to Duncan’s multiple range test. Here “M1-M6” represents callus induction medium (WPM + PGR).

3.4. Shoot proliferation of Olea europea L. cv. koroneiki on MS, OM and WPM media

Pre-sterilized nodal segments were selected and dissected into small segments (1 cm each) for shoot formation. Six treatments were designed by using MS medium i.e., Control (PGR’s free), 8.8 µM BAP, 17.7 µM BAP, 4.56 µM zeatin, 9.12 µM zeatin, 17.7 µM BAP + 9.12 µM zeatin for shoot regeneration and multiplication. However, no regeneration response was observed on any treatment with MS medium either alone or in combination with growth regulators. After 20 days, the explants turned brown and became necrotic and thus not used for further experimentation with respect to shoot formation.

OM supported shoot regeneration on all its tested treatments (Table 5). OM supported shoot formation on hormone-free medium with 1.3 shoots after 84 days of explants transferred. A gradual increase on hormone-free medium was observed for all the tested parameters, however, it took more time for sprouting and bud break as compared to PGRs containing media. OM when supplemented with zeatin (9.12 µM) was found more effective as compared to OM with BAP alone as it showed higher shoot length (1.3 cm) and number of leaves (3.3). OM supplemented with BAP (17.7 µM) and zeatin (9.12 µM) induced highest shoot length (1.5) and maximum number of leaves (3.5). The medium supplemented with 9.12 µM zeatin exhibited highest shoot number (2) and highest leaves number ((3.3) (Table 5; Fig. 6)).

Shoots were induced using nodal explants when transferred to WPM containing BAP (17.7 µM), zeatin (9.12 µM) or in combination with BAP (17.7 µM) and zeatin (9.12 µM). The highest number of leave (3.6) and shoots per explant (1.3) were observed in medium having zeatin (9.12 µM). The maximum shoot length (1.2 cm), number of shoots (1.5), and leaves (3.3) were observed on medium supplemented with zeatin (9.12 µM) + BAP (17.7 µM) after 33 days of explant inoculation (Fig. 7). Minimum number of leaves (1.6) and shoots per explant (1) was recorded on medium containing zeatin (9.12 µM) as compared to medium having BAP only. The response of bud break and days needed for leaves to emerge were minimum in case of WPM supplemented with combination of BAP and zeatin (Fig. 8).

Table 5

Effect of OM supplemented with growth regulators on shoot regeneration by using nodal explant of *Olea europea* L. cv. koroneiki
Media	PGRs (µM)	Shoot length (cm)		Shoot number		Number of leaves		Days of sprouting
		OM	WPM	OM	WPM	OM	WPM	OM	WPM
S1	0	1.06 ± .06^a	0	1.3 ± .33^b	0	2.3 ± .33^b	0	84 ± .88^a	0
S2	8.8 BAP	1.1 ± .07 ^a	0	1.0 ± .31^c	0	2.1 ± .32^b	0	65 ± 2.3^b	0
S3	17.7 BAP	1.2 ± .14^a	0.8 ± .08^b	1.3 ± .03^b	1.3 ± .33^a	2.0 ± .57^b	2.6 ± .33^b	43 ± 1.23^d	55 ± .30^a
S4	4.56 zeatin	1.1 ± .10 ^a	0	1.0 ± .31^c	0	2.1 ± .32^a	0	55 ± 1.3^c	0
S5	9.12 zeatin	1.3 ± .16^a	1.1 ± .14^a	1.4 ± .10^b	1 ± .10^ab	3.3 ± .88^a	1.6 ± .31^a	42 ± 1.4^d	45 ± 1.5^b
S6	17.7 BAP + 9.12 zeatin	1.5 ± .29^a	1.2 ± .24^a	2 ± .13^a	1.5 ± .40^a	3.5 ± .57^a	3.3 ± .30^b	33 ± .30^e	38 ± 1.0^c

Data represented here are the means (± S.D) of 3 replicate cultures per treatment.

Different alphabetic letters in each column show significant difference at p ≤ 0.05 according to Duncan’s multiple range test. Here “S1-S6” represents shoot induction medium (OM/WPM + PGR).

In the present study, healthy, young, and soft nodal segments and leaf portions were used as a source of explants. Olive in vitro propagation is very challenging due to its recalcitrant nature and contamination. It was reported that olive plants either in vitro cultures or field grown are extremely contaminated. To achieve disinfected tissue and their establishment is a very crucial as reported by several researchers (Roussos and Pontikis, 2002; Debnath, 2018). During this investigation, for both types of explants, surface sterilization with 70% ethanol for 15 sec. and then with 0.1% (w/v) HgCl₂ solution for 3 minutes was proved best as compared to all other tested methods. Therefore, HgCl₂ is usually used to disinfect the explant in several previous studies (Zacchini and De Agazio, 2004). Although different types of explants were used since 90s but only a few olive cultivars were successfully micropropagated (Fabbri et al., 2009). Therefore, it is mandatory to select such an explant having maximum potential of callus induction and plant regeneration. In few studies, explants obtained from seedlings or embryos were preferred but nodal segments were recommended as the most suitable ones for olive micropropagation (Roussos and Pontikis, 2002; Yancheva and Kondakova, 2018). The leaves and petiole explants were preferred for callus induction in previous studies (Golmohamadi et al., 2019). Similarly, shoot tips and axillary buds were also chosen as an explant for in vitro propagation of different Olea europea cultivars (Zacchini and De Agazio, 2004).

Several authors have reported that every species has different nutrient demands because it depends upon the species as well as on the genotypes so it is necessary to determine the nutrient medium for each genotype (Peixe et al., 2007; Zuccherelli and Zuccherelli, 2000). In the present study, three types of basal media were used namely MS, OM and WPM to find the appropriate medium for callus induction and in vitro plant regeneration of Olea europea cv. koroneiki. It was observed in the present study that on MS medium, both types of explants showed no response at all and turned brown after 10 to 15 days of inoculation. The reason behind the explant browning may be the higher levels of organic or inorganic salts concentration in MS medium (Bayraktar et al., 2020). that might cause toxicity to the explant used. Looking at the negative response, this medium was not used further for callus induction and shoot regeneration. It was studied that the rate of callus induction depends not only on the type of explant but also on the medium and concentration of suitable PGRs (Brito et al., 2009). OM when supplemented with PGR’s in combination (17.7 µM BAP and 9.12 µM zeatin) showed overall best response in callus induction (70%) on nodal explant under both light and dark conditions while remaining all treatments of OM showed no callus initiation. However, when leaf explant was used, OM + 9.12 zeatin showed best results but with slightly low percentage of callus induction (66.7%) under dark and light conditions, respectively and took more time (74 and 79, respectively) to induce callus as compared to nodal explants. As compared to OM, WPM induced 67% callus on nodal explant when supplemented with 17.7 µM BAP and 9.12 µM zeatin under light condition. Similar media combination induced 50% callus on nodal explant under dark condition on leaf explant. Overall, the response of OM was better towards callus induction as compared to WPM. In previous studies, results indicates that OM is more effective as compared other medias (Bayraktar et al., 2020). Nodal segments are recommended as best explant source for callus induction.

In present research work, MS medium showed no response regarding shoot formation as in case of callus induction. However, results indicates that olive media (OM) has played significant role with highest shoot formation, shoot number and shoot length when fortified with PGRs. Similarly, high shoot proliferation response different plant species was observed on OM in previous investigations (Ahmad et al., 2016; Brito et al., 2010). In contrast to this, some studies showed, maximum shoot formation rate and shoot number per explant was on the WPM instead of OM (Bayraktar et al., 2020; Ahmad et al., 2016). This contradiction in results might be due to change of plant species and olive cultivar or environmental conditions. It was observed that hormone free OM was showed lowest shoot regeneration response and WPM showed no response at all. Similarly, it was reported that zeatin induce shoot proliferation significantly (Farahani et al., 2011). It was also reported that each cultivar responds varyingly to plant growth regulators (Grigoriadou et al., 2002). Shoot regeneration via nodal explant in olive has been generally accomplished through zeatin, BAP, TDZ and GA₃ in previous studies (Hamooh and Shah, 2017).

In the present research, nodal explant produced a maximum (2) shoots, when inoculated on OM supplemented with combination of BAP 17.7 µM and zeatin 9.12 µM. In previous studies, highest number of shoots per/plant was reported when using nodal explant. In olive owing to strong apical dominance, formation of less secondary axillary shoot was reported in earlier studies (Santos et al., 2003). In current study, highest shoot length was obtained on OM containing 17.7 µM BAP and 9.12 µM zeatin while highest number of leaves were obtained on WPM containing 9.12 µM zeatin. Likewise, maximum shoot proliferation was found on medium containing zeatin (Rugini et al., 2016). Among different PGRs, combination of BAP and zeatin took less days (33) to initiate bud break and sprouting on OM. However, the response of combination on WPM took more days (38). In both basal media OM and WPM, the treatment containing zeatin resulted next to initiate bud break and sprouting (42 days). However, BAP alone was least effective as compared to other PGRs. It is reported in many studies that zeatin is a fundamental cytokinin to initiate bud breaks in olive cultivars (Rkhis et al., 2011; Zacchini and Agazio 2004; Mendoza-de Gyves et al., 2008; Haddad et al., 2018). A contradictory result was observed in earlier experiments that might be due to change of cultivar as well change of basal medium because WPM was used for shoot proliferation (Rkhis et al., 2003; Micheli et al., 2009).

It is concluded from the present study that among PGRs, zeatin in combination with BAP and among basal media Olive medium is the most effective for callus induction and shoot proliferation. Nodal explant is most suitable for callus induction and shoot proliferation. Dark conditions are most suitable for callus induction while light period is necessary for shoot regeneration in Olea europaea L. cv. Koroneiki in case both media types. Based on these findings, cv. Koroneiki can be regenerated at mass scale under in vitro for commercial propagation and thus to fulfill the virgin oil demand for rapidly growing population.

Acknowledgement: The authors are thankful to the Plant Developmental and Regenerative Biology Research Laboratory, Institute of Botany, University of the Punjab, Lahore for providing an opportunity to conduct this research.

Funding Statement: The authors received no specific funding for this study.

Conflicts of Interest: The authors declare that there are no conflicts of interest to report regarding the present study.

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A rapid plant regeneration system by using stem and leaf explants of Olea europea L. cv. Koroneiki; a multipurpose bioenergy tree

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Procurement of plant material, disinfection, and its establishment under in vitro conditions

2.2. Callus induction from leaf and nodal explants

2.3 Shoot regeneration from nodal explants

2.4. Statistical analysis

3. Results

3.1. Disinfection and establishment of sterile cultures

3.2. Callus induction on MS and OM media using nodal and leaf explants under dark/light condition

3.3. Callus induction response on WPM media using nodal/leaf explants under dark/light conditions

3.4. Shoot proliferation of Olea europea L. cv. koroneiki on MS, OM and WPM media

4. Discussion

5. Conclusion

Declarations

References

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