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International Journal of ChemTech Research CODEN (USA): IJCRGG ISSN: 0974-4290 Vol.8, No.12 pp 471-476, 2015
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In vitro germination and development of Arbequina and Coratina olive cultivars
A.M. Abd allatif1, S.A.M.Hassan2 and T.F.El-Sharony3
1Pomology Department, Faculty of Agriculture, Cairo University, Giza, Egypt
2Biotechnology Lab., Pomology Dept., National Research Centre, Dokki, Giza, Egypt
3Hort. Crops Technology Dept., National Research Centre, Dokki, Giza, Egypt
Abstract: In vitro culture method was carried out during 2014-2015 seasons at the laboratory of Pomology Department, Faculty of Agriculture Cairo University and Biotechnology Lab., Pomology Dept., National Research Centre, to evaluate the germination percentage and development of embryos isolated from seeds of Arbequina and Coratina olive cultivars. Embryo germination and development success were determined by in vitro culture using 1/3 strength MS medium. The embryo cultured within the cut seed portion of the seed containing the embryo freeing from remaining seed tissue was used for culture the basal media (Agar +Sucrose +MS+GA3 solely or in combinations each other). The most effective during development of embryo culture stage as it recorded the high germination percentage (72.66% in Coratina with Agar). The mean germination started in short period of time (6 days) with agar media for Coratina cv. The two tested cultivars showed high growth performance after germination, while Arbequina Superiority with Agar +Sucrose +MS in leaf number (5.66). Also, Agar+ Sucrose +MS media gave the highest plant height (3.50 cm) with Coratina cv.
Keywords: Olive, In vitro, Embryo culture, Germination percentage.
Introduction
Olive (Olea europaea L.)one of the most ancient cultured trees. It was originated in the eastern side of the Mediterranean basin, and spread to other countries. Until recently the genetic improvement of olive tree basedon theselectionofindividuals adapted to local conditions and constrains 1. To date the used method to improve olive cultivars is by classical crosses in breeding programs between existing cultivars. A few cultivars from these breeding programs have been reported recently2, 3, 4.
Barranco et al5 stated that the main cause of the few programs to improve cultivars conducted to date is the prolonged duration of juvenile phase in olive6, and the slow, and low germination in seeds of olive Lagarda et al 7.
Due to the heterozygosis of the generated individuals and the added possibility of genetic recombination after controlled cross pollination, seed is a great important for breeding in olive Troncoso et al 8.
However, seed germination in olive is very slow, for example seeds of Manzanillo cv. take 3 years for germination of only 17% Acebedo et al 9.This could be attributed to the nature of olive seeds which are covered by thick stony endocarp. Also, embryo dormancy has been reported by many authors Lagarda et al 10and Crisosto and Sutter11.
Numerous treatments have been applied to improve seed germination, including mechanical and chemical scarification 11 and Bandino et al 12and soaking in solutions of phyto-hormones Lagarda and Martin 13.However, low success had been obtained 12.
In vitro embryo culture has been used successfully in many fruit species to overcome such germination problems 14. Germination of isolated embryo of olive cultivars had higher germination percentage in comparison with stonless seeds Acebedo et al 9; Garcia et al. 15 and Maalej et al 16. Also, Acebedo et al 9 reported that the isolated olive embryos germinated uniformly within 10-14 days.
Therefore this study aim to evaluate the germination percentage and development in vitro of embryos isolated from seeds of Arbequina and Coratina olive cultivars.
Materials and Methods
The present study was carried out during 2014-2015 seasons at the laboratory of Pomology Department, Faculty of Agriculture, Cairo University and Biotechnology Lab., Pomology Dept., National Research Centre.
Fruits of olive cultivars (Arbequina and Coratina) were harvested when the color begin to change from yellow green to violet. The fruit pulp was removed and the sclarified endocarps were broken according to Sotomayor-Leon and Caballero 17.
After elimination of the endocarp, the stoneless seeds were surface sterilized under sterile conditions with 20% commercial sodium hypochlorite solution for 15 min. then the seeds were transferred to 0.1% HgCl2 for 10 mint Maalej et al 18 and Kiani et al 19.Finally, the seeds were washed three times with sterile water.
The sterilized seeds were soaked for 48h in sterilized water to promote swelling and faciliting the embryo isolation. Embryo was isolated by cutting off two lateral sections, and freeing the embryo from remaining seed tissue 9.
The isolated embryos were cultured on one of the following media:
2- Agar+Sucrose.
3- Agar+Sucrose+MS.
4- Agar+Sucrose+MS+GA3
MS medium Murashige and Skoog 20 was used at 1/3 strength and the sucrose was added at 30 g per liter. The pH was adjusted at 5.8. The media was solidified by adding 6% agar and autoclaved.
The isolated embryos were placed individually in sterile jars each containing 30 ml of culture media. The jars were placed in growth chamber at 25°C with 16 h photoperiod. 100 jars were used for each cultivar.
The following parameters were recorded.
Including seed weight (g), stoneless weight (g), and the percentage of empty seeds.
The emergence of roots and opening of cotyledons was the sign of germination, the emerged platelets were counted and germination percentage was calculated.
Growth measurements were performed for each plant; stem and root length (cm) and plant fresh weight (g) were measured.
Experimental design and data analysis
This study followed the randomized complete design with 5 replicates, 20 jars for each one and data were subjected to analysis of variance (ANOVA) according to Snedecor and Cochran 21 using MSTAT C statistical package Freed et al 22 software, and means of the treatments were compared by Least Significant Difference (L.S.D.) according to Duncan 23 at significance level of 0.05.
Results and Discussions
Data presented in Table (1) showed that Coratina cv. had higher seed weight (0.46 g) compared with Arbequina (0.26 g). Also Coratina had higher stonless seed weight (0.050 g) compared with Arbequina (0.041 g).
The number of the empty seeds also varied among the studied cultivars, Arbequina cv. had low percentage of empty seeds (10.66%).
Table 1. Seed characteristics of Arbequina and Coratina olive cultivars.
Cultivar |
Seed weight |
Stoneless weight |
Empty seed % |
Arbequina |
0.26 |
0.041 |
10.66 |
Coratina |
0.46* |
0.050* |
16.33* |
Means within each column are significant (*) or not significantly (NS) at P < 0.05
Acebedo et al 9 reported that there is a high variation in seed characteristics i.e. seed weight, stoneless seed weight and empty seed percentage. These characteristics explain partially the difference in seed germination capacity between olive cultivars.
As shown in Table (2) Coratina olive cv. recorded high germination percentage (52.58%) compared with Arbequina that showed the lowest germination percentage (49.08%). while the highest germination percentage obtained from Agar + Sucrose + MS media (62.83% ) flow it Agar+Sucrose + MS +GA3 media (59.5%) while the lowest germination percentage obtained from Agar +Sucrose media (21.83%) .
Table 2. Effect of different media on Germination %, of Arbequina and Coratina olive cultivars.
Cultivars |
|
|
Media |
|
|
Agar |
Agar+ Sucrose |
Agar+ Sucrose+ MS |
Agar+Sucrose+MS+GA3 |
Mean |
|
Arbequina |
45.66 |
15.66 |
71.00 |
64.00 |
49.08 B |
Coratina |
72.66 |
28.00 |
54.66 |
55.00 |
52.58 A |
Mean |
59.16 B |
21.83 C |
62.83 A |
59.50 AB |
|
Means within each column with the same letter are not significantly different at P < 0.05
Concerning the time required for germination process, data in Table (3) showed that, embryo germination started in short period of time (5days for Coratina with Agar +Sucrose media and 6 days for
Arbequina with Agar media without any addition). However, the highest long period were recorded with Agar +Sucrose +MS+GA3 media 9 days with both Arbequina and Coratina cultivars. Generally Coratina took low period to emergence 7.08 days.
Table 3. Effect of different media on first emergence of germination of Arbequina and Coratina olive cultivars .
Cultivars |
|
|
Media |
|
|
Agar |
Agar+Sucrose |
Agar+Sucrose+MS |
Agar+Sucrose+MS+GA3 |
Mean |
|
Arbequina |
6.00 |
9.00 |
8.00 |
9.00 |
8.00 A |
Coratina |
7.00 |
5.00 |
7.00 |
9.00 |
7.00 A |
Mean |
6.50 B |
7.00 AB |
7.50 AB |
9.00 A |
|
Means within each column with the same letter are not significantly different at P < 0.05
Table (4) showed that, the mean germination time (time for complete germination) were shorter in Coratina cv. (8 day) compared with Arbequina cv. (14.5 day). Whereas, long time in both cultivars Arbequina and Coratina cvs. 16 days with Agar + Sucrose+MS media, while a short time with Agar without any addition7.5 days.
Acebedo et al 9 studied the germination of isolated embryo of 10 olive cultivars; isolated embryos of all cultivars had higher germination percentage. Similar results were obtained by Maalej et al 16 and Kiani et al 19. However, some studies reported that Olive seed germination may continue erratically for 3 years Sotomayor-Leon and Caballero17 and Acebedo et al 9.Even the increase of germination rate and uniformity were obtained from stonelss seeds after removing the hard endocarp 11. However germination rate of stonless seeds of many olive cultivars still slow reported 24.Acebedo et al 9 reported that the mean germination time was longer in the stoneless seeds (55 to 95 days) while the isolated embryos germinated uniformly within 10-14 days. Zienkiewicz et al 25 studied in the complete course of seed germination, they reported that olive cotyledons started to greenish around the 4th day, 26 days later the seedlings were ready for growing in pots.
Table 4. Effect of different media on mean germination time of Arbequina and Coratina olive cultivars.
Cultivar |
|
|
Media |
|
|
Agar |
Agar+ Sucrose |
Agar+ Sucrose+ MS |
Agar+Sucrose+MS+GA3 |
Mean |
|
Arbequina |
9.00 |
15.00 |
21.00 |
13.00 |
14.50 A |
Coratina |
6.00 |
7.00 |
11.00 |
8.00 |
8.00 B |
Mean |
7.50 C |
11 B |
16.00 A |
10.50 B |
|
Means within each column with the same letter are not significantly different at P < 0.05
1- Leaves number
Data in Table (5) showed that, the two tested of olive cultivars showed good growth over the period of the experiment. Arbequina cv. had high number of leaves (3.72) compared with Coratina (3.21). The highest leaf number showed with Agar+Sucrose+ MS media followed by Agar+Sucrose+MS+GA3, while Agar media recorded the lowest value in leaves number.
Table 5. Effect of different media on leaf number of Arbequina and Coratina olive cultivars
Cultivars |
|
|
Media
|
|
|
Agar |
Agar+ Sucrose |
Agar+ Sucrose+ MS |
Agar+Sucrose+MS+GA3 |
Mean |
|
Arbequina |
2.00 |
3.00 |
5.66 |
4.23 |
3.72 A |
Coratina |
2.00 |
2.00 |
4.66 |
4.18 |
3.21 A |
Mean |
2.00 C |
2.5 C |
5.16 A |
4.21 B |
|
Means within each column with the same letter are not significantly different at P < 0.05
2- Plant height
As shown in Table (6) Arbequina olive cv. recorded higher plant height (2.30 cm) compared with Coratina (1.83 cm). While the higher plant height was recorded with Agar+Sucrose+MS media and the lowest plant height was recorded with Agar media. Acebedo et al 9 reported that difference in shoot growth was recorded among the tested cultivars. In vitro cultures help the breeders to overcome the problems associated with seed germination and increase the size of progenies for evaluation state. These results indicted the possibility of obtaining seedling with high survival percentage; Garcia et al15 reported that the survival of olive seedlings after in vitro culture reached up to 85%. Kiani et al19 reported that, percentage of seedlings formation of olive cultivars from in vitro embryo culture ranged from 70.1% in Zard cv. to 79% in Dezful cv. Similar results were reported by Sahijram et al 26 in mango and Jaskani et al 27 in citrus.
Table 6. Effect of different media on plant height of Arbequina and Coratina olive cultivars
Cultivars |
|
|
Media |
|
|
Agar |
Agar+Sucrose |
Agar+Sucrose+MS |
Agar+Sucrose+MS+GA3 |
Mean |
|
Arbequina |
1.00 |
2.16 |
3.03 |
3.00 |
2.30 A |
Coratina |
1.00 |
1.00 |
3.50 |
1.84 |
1.83 A |
Mean |
1.00 C |
1.58 BC |
3.27 A |
2.42 AB |
|
Means within each column with the same letter are not significantly different at P < 0.05
Fig. 1: Effect of different media on development of Arbequina and Coratina olive cultivars . A: Agar,
B: Agar+ Sucrose, C: Agar+ Sucrose+ MS, D: Agar+Sucrose+MS+GA3
References
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