ABSTRACT
The effect of gamma irradiation and gibberellic acid (GA3) treatment on different responses of fennel was investigated. Combined effect of -irradiation at 2 Krad dose and GA3 treatment at 50 mgL -1 concentration resulted in the maximum germination percentage, seed production, essential oil yield, as well as the best quality of fennel (Foeniculum vulgare) oil because of its high content of the flavour compounds, e.g. trans-anethole and fenchone. GA3 application reduced the harmful effects of higher irradiation doses (5-20 krad).
PDF Abstract XML References Citation
How to cite this article
DOI: 10.3923/pjbs.2001.805.808
URL: https://scialert.net/abstract/?doi=pjbs.2001.805.808
INTRODUCTION
The medicinal and aromatic plants are of special importance all over the world. The genus Foeniculum (family Umbelliferae) includes some varieties among which sweet fennel (Foeniculum vulgare var. Dulce) is a perennial plant growing wild in several localities. The main characteristic of fennel oil is the high content of trans-anethole, limonene, estragele, fenchone, γ-terpinene, and α-pinene (Akgul, 1986). Akgul and Bayrak (1988) reported that Turkish bitter fennel have lesser value than sweet fennel because of their low yield of oil, low percentage of trans-anethole and large amount of hydrocarbons.
Gamma radiations have been used in storage, mutation, induction, stimulation, and other purposes. Sjodin (1962) reported that the material and energy necessary for initial growth are already available in the seed, and so the young embryo has no need to form new substances, but only to activate those already stored in the cotyledons. The role of low doses of γ-radiations may be the increasing enzymatic activation and awakening the young embryo, which result in an increasing rate of cell division, which affects not only germination, but also vegetative growth and flowering. Exposing the dry seeds to low γ-irradiation doses resulted in increasing yield of some plants such as safflower (Abo-Hegazi et al., 1988), and Ammi visnage (El-Shafie et al., 1993). Plant hormones play an important role in cell division, elongation, differentiation, and the various metabolic processes, thus could be expected to influence the biosynthesis of essential oils in aromatic plants. Hegazy et al., (1990) observed that irradiation of air dry cowpea seeds with 10 Krad dose increased the amount of the growth promoter, Indole- 3- acetonitrile (IAN), as well as the amount of the growth inhibitor, Abscisic aicd (ABA). The effect of irradiation in stimulating the formation of the growth inhibitor was greatly lessened when the seeds were soaked in water before irradiation. Soaking the seeds in GA3 solution (40 mg L-1) increased the amount of the growth promoter, but was less effective than distilled water in decreasing the amount of growth inhibitor. Atia and Ebaid (1990) also indicated a significant interaction effect of gamma irradiation and GA3 application to increase the number of open bolls/plant and boll weight of Egyptian cotton.
Therefore, in the present investigation, gamma irradiation and gibberellic acid (GA3) used individually or in combination for improving yield, seed germination percentage, quality and composition of the essential oil were investigated.
MATERIALS AND METHODS
The air dried seeds of fennel (Foeniculum vulgare) were exposed to different doses of gamma rays (0, 2, 5, 10, 20, 30, 40, 50 Krad) in the National Center for Radiation Research and Technology, Nasr City, Cairo, Egypt. A preliminary experiment was carried out using a series of concentrations of gibberellic acid (GA3), cycocel (CCC), and indole-3-acetic acid (IAA) to determine the most favorable plant growth regulating substance. GA3 (50 mg L-1) was the most effective one.
The field experiment was carried out in the garden of Helwan University. The soil was carefully prepared and divided into plots. Each plot included 3 rows. The seeds were sown in hills (4-6 seeds per hill) at 20 cm distances. After 30 days from sowing, the plants were thinned to one plant per hill. Other cultural practices such as weed control and irrigation were performed whenever it was necessary. The first five plots were subjected to γirradiation of 0, 2, 5, 10, and 20 Krad doses, while the second five plots were subjected to the same doses and sprayed with GA3 (50 mg L-1) two times. The first spraying was applied after 45 days from sowing, and the second at the flowering stage (90-d-old) by an atomizer until dripping the treated plants. Tipple was used as a wetting agent in order to minimize the surface tension between leaves and growth regulating substance. The spraying process was always performed early in the morning. After about seven months, the fruits were harvested before full ripening. The plants were cut off at the soil surface and left in a shaded place about two weeks, then hammered for fruit separation.
Seed germination percentage was expressed as:
Yield production was assessed as number of umbels/ plant, fresh and dry mass of umbels/ plant, weight of 100 seeds, percentage of oil content, and oil yield/ 100 plants.
Statistical analysis was carried out according to Snedecor and Cochran (1980) using analysis of variance (Completely Randomized three-factor Design) and the significance was determined using L.S.D. values at p= 0.05 and 0.01.
Electrophoretic protein banding was examined in pre-sowing gamma-irradiated and unirradiated seeds, as well as in the seeds of plants developed from 20 Krad γ-irradiated seeds in comparison with control un-irradiated seeds.
Fig. 1: | Protein profile of A: the pre-sowing Υ- irradiated seeds, and B: the produced seeds by fennel plants. |
Separation of proteins was performed using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS- PAGE), in the central laboratory of Faculty of Agriculture, Ain Shams University, Cairo, Egypt.
The seed content of essential oil was determined by steam distillation according to the method of Guenther (1961). The amount of oil obtained from plant material was calculated according to the following equation: Oil % = Observed volume of oil in graduated tube (ml)/ weight of sample (g.)x 100. The yield of oil produced/plant was calculated by multiplying the average of fresh weight of seeds/plant by the average of oil percentage/plant for different treatments. GLC Model HP- 5890 Gas- Liquid Chromatograph equipped with flame ionization detector was used for the separation of essential oil components, in the National Center of Research, Cairo, Egypt.
RESULTS AND DISCUSSION
Germination percentage of fennel seeds under irradiation conditions was expressed as Germination Irradiation Index (GII) (Table 1). The data reveal a stimulatory effect of gamma radiations at 2 Krad dose on germination of fennel seeds by 2% as compared with un-irradiated seeds. Higher doses of gamma rays exhibited a gradual retarding effect on seed germination to become 0.7% of the control at 50 Krad treatment. GA3 application led to a marked rise in germination percentage of un-irradiated and γ-rradiated seeds up to 20 Krad treatment, above which there is no valuable effect of GA3 on germination. The highest germination percentage of fennel seeds was recorded for 2 Krad γ-irradiated seeds, treated with 50 mg L-1 of GA3; followed by GA3- treated un-irradiated seeds, and 2 Krad γirradiated seeds. The positive effect of GA3 application on seed germination may be due to its effect on enzymatic activity and synthesis of RNA and protein (Jacobson et al., 1979), as well as on the endogenous phytohormones (Hegazy et al., 1990).
Table1 shows that gamma irradiation doses of 5, 10 and 20 Krad were accompanied by a progressive decrease in number of umbles per plant, as well as the fresh and dry mass of umbles. Nevertheless, the pre-sowing treatment with 2 Krad dose of gamma rays resulted in increasing the number of umbels produced per plant, fresh and dry mass of umbels and seed mass. Application of GA3 resulted in a considerable improvement in quality of the produced seeds, as well as the number, and mass of the fresh and dry umbels per plant, particularly at 2 Krad γirradiation treatment. Georgieva (1987) attributed the inhibitory effect of high doses of γirradiation on yield to the enhanced effect on peroxidase and polyphenoloxidase activity, and the increased content of quinones which may affect cytochrome oxidase activity, pollen germination, and pollen tube growth.
The gene changes during irradiation might be reflected in the storage protein of the plants, as the seeds are enriched with such altered proteins. The present investigation is concerned with the qualitative and electrophoretic studies of protein profiles in control and pre-sowing 20 Krad γirradiated fennel seeds as well as in the produced seeds (Table 2 and Fig. 1). The protein bands showed a wide variation in 20 Krad treated seeds as compared to control. The control seeds showed 23 bands with mobility from 0.011 to 0.974. The molecular masses ranged approximately from 269.113 to 11.761 KDa. In the pre-sowing γ-irradiated seeds, some bands were missing, like those with molecular masses of 269.113, 109.896, 99.317, 43.254, and 15.797; meanwhile some new bands appeared with molecular masses of 220.085, 117.721, 94.787, 40.577, and 13.433. The notable changes in the protein profile of 20 Krad γ-irradiated seeds were the subtraction of protein having high molecular weight and addition of new low molecular weight protein. This may be due to the partial degradation of protein by the high γirradiation dose. The produced seeds by control plants showed 24 bands, and the mobility ranged between 0.075 and 0.978 (approx. mol. mass 140.528 to 11.65 KDa). The seeds produced by plants which developed from pre-sowing 20 Krad γ-irradiated seeds, showed 25 bands. Some of bands disappeared as compared to control, such as those with molecular masses of 103.296, 75.678, and 39.794, while some new bands appeared with molecular masses of 230.008, 101.221, 17.532, and 13.241. Except one new protein with high molecular weight (230.01 KDa), the appearance of proteins having relatively lower molecular weight was the marked feature in the produced seeds after the pre-sowing treatment with 20 Krad dose of gamma rays.
Table 1: | Effect of γ-irradiation and GA3 treatment on germination, essential oil percentage (A: in the pre sowing γ-irradiated seeds, B: in the produced seeds) and essential oil yield (ml/100 plants) of fennel |
Table 2: | Protein profile of A: the pre-sowing γ-irradiated seeds, and B: the produced seeds by fennel plants in response to 20 Krad γ-irradiation treatment |
Table 3: | GLC analysis of essential oil components of A: the pre-sowing γ-irradiated seeds, and B: the produced seeds by fennel plants in response to γ-irradiation and GA3 treatment |
This may reflect genetic changes during irradiation with high doses. Afify and Shousha (1988) attributed the changes in protein patterns after γirradiation to partial protein degradation, scission of peptide and disulfide bonds and addition to aromatic and heterocyclic amino acid residues. Similar assumption has been suggested by Booth (1970) and Desrosier (1970).
The essential oil content of fennel dry seeds was not affected by the pre-sowing exposure to gamma rays, recording the same value (0.65%), at different doses used (Table 1). Exposing the fennel seeds to pre-sowing gamma irradiation was beneficial at 2 Krad dose, which markedly increased the essential oil percentage and enhanced yield of essential oil/ 100 plants, may be due to its stimulative effect on fresh mass of umbels and seeds, as well as the activation of enzymes involved in the metabolism of essential oil formation.
The essential oil collected from pre-sowing 20 Krad γirradiated seeds, as well as from the seeds by plants developed from seeds subjected to the same treatment was analyzed using Gas Liquid Chromatography (GLC), in comparison with that of the controls (Table 3). The identified flavour components of fennel oil could be generally classified into two categories, namely hydrocarbons (α-pinene, α-phellandrene, and limonene) and oxygenated compounds (fenchone, methylchavicol trans-anethole, and anisaldhyde). Trans-anethole represent the major component, followed by fenchone and methylchavicol. The highest level of total oxygenated compounds (96.56%) and lowest content of hydrocarbons (1.67%) were detected by the combined effect of GA3 (50 mg L-1) and gamma irradiation (2 Krad). Meanwhile, the highest level of total hydrocarbons (5.98%) and lowest level of oxygenated compounds (83.27%) were recorded in plants treated with 20 Krad γ-irradiation dose. The obtained results agree with that of Guenther (1961), who reported that the main components in fennel seed oil are anethole and fenchone, which define the oil quality. Accordingly, the best quality of the essential oil of fennel seeds was obtained by 2 Krad γirradiation treatment combined with GA3 application because of their high content of trans-anethole and fenchone, and low content of αpinene, α-phellandrene and limonine, as a result of these treatments. Similar results have been obtained by El-Geddawy and Rashwan (1993), and Farag et al., 1996) who reported that the fruit oil with a high percentage of oxygenated compounds and low amount of hydrocarbons seems to be a valuable flavoring agent for various food products.
REFERENCES
- Afify, A.M.R. and M.A. Shousha, 1988. Effect of Low-Dose Irradiation on Soybean Protein Solubility, Trypsin Inhibitor Activity, and Protein Patterns Separated by Polyacrylamide Gel Electrophoresis. J. Agric. Food Chem., 63: 810-813.
CrossRefDirect Link - Akgul, A. and A. Bayrak, 1988. Comparative volatile oil composition of various parts from Turkish bitter fennel (Foeniculum vulgar var. vulgare). Food Chem., 30: 319-323.
CrossRef - Farag, Z.S., N.H. Aziz and A. Ali, 1996. Comparing effects of washing, thermal treatments and gamma irradiation on quality of spices. Nahung, 40: 32-36.
Direct Link