The Study of Increasing Roots Shoots and Hairy Roots Production by Different Extracts of Saffron (Crocus sativus L.) in Mungbean (Vigna radiata) Seedlings
A.O. Ataei, B.D. Hashemloian
Deportment of Biology
Islamic Azad University of Saveh branch
Saveh
Iran
Keywords: allelopathy, germination, growth stimulator
Abstract
Mungbean (Vigna radiata) is a dicotyledonous plant from leguminosea family. Recent pharmacological studies have demonstrated that saffron (crocus sativus L.) extract has antitumor and hypolipidemic effects but in this investigation, we studied effects of extracts of saffron on growth of mungbean seedlings. In the maceration method, stigma was macerated in warm distilled water for three days. The extract was subsequently filtered and remains of stigmas were washed for 10 times. The remains of stigmas was macerated in 100 ml acetone for one day and it was filtered and concentrated under reduced pressure at 35 ˚C. The acetonic extract was solved in 100 ml distilled water. The seeds of mungbean (100 seeds) were cultured in 15-20 ml of these two extracts with different concentrations. After two week, results of these experiments indicated that: as treatment results increasing the rate of shoots, roots growth and hairy roots production but diameter of shoots and roots were reduced. The extracts of saffron are growth stimulator in mungbean seedlings.
Introduction
Saffron (Crocus sativus L.) is a perennial plant with succulent corm, belonging to Iridaceae family. Saffron is considered to be the most expensive spice in the world. The flower of saffron is a light purple, with thread-like reddish coloured stigma that is valued both as a spice and as a natural dye. Saffron is the dried stigma of a crocus which originated in Greece and Asia Minor but which is now widely cultivated in a band from Western Europe through temperate and sub-tropical Asia to China. The domesticated saffron crocus (C. sativus) is a fall-flowering perennial plant unknown in the wild, and is a sterile triploid mutant of the eastern Mediterranean fall-flowering Crocus cartwrightianus (Deo, 2003). Saffron-based pigments have been found in 50,000 year-old depictions of prehistoric beasts in what is today Iraq. Later, the Sumerians used wild-growing saffron in their remedies and magical potions (Willard, 2001; Humphries, 1998). Saffron was thus an article of long-distance trade before the Minoan palace culture's 2nd millennium BC peak. Saffron was also honored in the Herbrew Song of Solomon (Humphries, 1998). Ancient Persians cultivated Persian saffron (Crocus sativus 'Hausknechtii') in Derbena, Isfahan, and Khorasan by the 10th century BC. Saffron contains more than 150 volatile and aroma-yielding compounds. It also has many nonvolatile active components, many of which are carotenoids, including zeaxanthin, lycopene, and various α- and β-carotenes. Crocus sativus linneaus contains crocin, the source of its strong coloring property, bitter-crocin, which offers the distinctive aroma and taste and essential oils which are responsible for its therapeutic properties (2005 Greekproducts.com). This crocin is trans- crocetin di-(β-D-gentiobiosyl) ester. This means that the crocin underlying saffron's aroma is a digentiobiose ester of the carotenoid crocetin. Crocins themselves are a series of hydrophilic carotenoids that are either monoglycosyl or diglycosyl pollyene esters of crocetin (Abdullaev, 2002). Mungbean (Vigna radiata) is a dicotyledonous plant from leguminosea family. The germination of seeds in nature conditions is often influenced by the presence of other plants (Rice, 1984). Allelopathy is an important mechanism of plant mediated by the release of plant produced phytotoxins to the plant environment (Singh et al., 2002). The toxicity of allelochemicals released in the environment is the function of their concentration as well as age and metabolic stage of the donor-plant (Rice, 1984; Wardle et al., 1993). There were some allelopathic effects of saffron corms on germination of all tested species except wheat, but this effect was more pronounced for cotton and soybean. Although, there is strong evidence for allelopathic effects of some plants on others (Rice, 1984; Putnam, 1994; Moyer and Huang, 1997). A glycoconjugate has been characterized from saffron corms (Crocus sativus L.) that inhibits the growth of roots of Nicotiana tabacum and Arabidopsis thaliana (Fernándezet al., 2000). The effects of Corm water extract of saffron on seed germination and seedling of important crop species such barley (Hordeum vulgaris), wheat (Triticum sativus), corn (Zea mays), canola (Brassica napus), cotton (Gossypium hirsutum), and soybean (Glycine max) is studied. Results indicated allelopathic effects but the magnitude for each species and the various germination indices were different. Radicle length was more affected than other parameters (Abbassi, 2005). Mungbean (Vigna radiata) is a dicotyledonous plant from leguminosea family. Many studies have been conducted on the nutritional quality of mungbean protein. Recent pharmacological studies have demonstrated that saffron (crocus sativus L.) extract has antitumor and hypolipidemic effects but in this investigation, we studied effects of saffron extracts on seed germination and seedlings growth of mungbean, as a plant regulator.
Materials and Methods
Study of the effects of water extracts from saffron stigma on seed germination and seedlings growth of Mungbean (Vigna radiata) was conducted under lab conditions in a completely randomized design with factorial arrangement and in four replications. The stigmas were grounded (5 g/l). In the maceration method, stigma was macerated in warm distilled water for three days. The extract was subsequently filtered and remains of stigmas were washed for 10 times. In another experiment, saffron powder (0.5 g) was extracted by 100ml absolute acetone and chloroform. Chloroform extract was isolated with water. Water remain was named chloroform residue. The acetonic remains of stigmas were macerated in 100 milliliter distilled water for one day and it was filtered. The acetonic and chloroformic extracts were concentrated under reduced pressure at 35 ˚C and solved in 100 ml distilled water. The seeds of mungbean (10-15 seeds) were cultured in 15-20 ml of water extracts with different concentrations (5-10 replications), but acetonic and chloroformic and their residue with 1 concentration (5 g/l). Treatments containing stigmas extracts in different concentrations: 0 (distilled water - control), 0.5, 1, 2.5 and 5 g/l in temperature; 25 ºC for 6 days condition and 0, 2.5 and 5 g/l in 35 ºC for 2 days. The extracts were deposited on Petri dishes with 12 seeds of mungbean. After 3-6 days the percentage of seed germination, and; roots and shoot systems length and number of lateral roots of seedlings were measured. Germination percentage is an estimate of the viability of a population of seeds. The equation to calculate germination percentage is: GP = seeds germinated/total seeds x 100.
The end, segments of hypocotyls and epicotyls of mungbean were cultured in agar medium accompanied 0.5 g/l saffron (12 % agar and 1.5 % sugars). Segments of hypocotyles and epycotylesis obtained from mungbean seedlings in control treatment (Table 3). Statistical analysis of the results was carried out by SPSS software. Duncan Multiple Range Test (DMRT) for comparison of means was used.
Results and discussion
The effects of different concentration of saffron water extracts on seed germination and seedling growth of mungbeen in 25 ºC, after 6 days, showed that water extract from 2.5 g/l of saffron have maximal activity, and it increased mungbean seed germination, length of main root and shoot systems, and the number of lateral roots (Table 1). The most concentrations of the water extracts of saffron increased mungbaen seed germination and seedling growth, but decreased length of both roots and shoots systems in 5 g/l of saffron. The zone of lateral roots formation changed from root (in 0-0.5 g/l) to hypocotile with increasing concentration of saffron (in1-5 g/l). The high number of lateral roots was showed in 0 and 5 g/l saffron (Table 1. and Fig. 1).
Table 2 shows that water extract of saffron no affect on percentage of germination of mungbean, but significantly increasing shoot and root systems length, at 5 g/l concentration in 35 ºC after 2 days. The results also showed that with increasing extract concentration, lateral roots number are increased. Short and thick roots in control treatment changed to long and thin roots in 5 g/l saffron (Fig. 2).
Percentage of seedgermination mungbean was not suppressed by any of the extracts, but decreased by chloformic extract (Table 3). Table 3 indicates that there was significant effect of water and chloformic-residue of extracts of saffron on hypocotyls length, whereas acetonic-residue on radicles length was stimulated. Acetonic and chloroformic extracts decreased the radicles growth. In acetonic extract treatment, seedlings of mungbean produced the most lateral roots on hypocotyls (Table 3, Fig. 3). The hypocotyls with bud and hypocotyls segments of mungbean produced lateral roots in agar medium contain 0.5 g/l saffron, whereas wasn’t produced any roots in control agar medium (Fig. 4).
Discussion
In this experiment, the water extracts of saffron were induced different effects on germination and seedling growth (Table 1). The seed germination and lateral root formation on hypocotyls increased with increasing of water extract concentration (0-5 g/l). The concentration of 2.5 g/l of saffron is shooting and root growth stimulation. The concentration of 5 g/l of saffron is seedlings growth inhibitor. Results of table 2 indicated concentration of 2.5 g/l is seedling growth stimulation. Previous reports indicate the ability of some plants to affect the germination and growth of other plants (Putnam 1994). In agricultural crop production the main concern has usually been the effect of toxins from one crop on the yield of next crop (Francis, 1986). But in the literature there are detailed reports on allelopathic effects of saffron corm on germination or growth of next crops (Farhoodi et al., 2003). There were not report about the effect of stigma of saffron on seed germination and seedling growth. In this study, significant effect of different extracts of saffron on percentage of germination and seedlings growth of mungbean was observed. Chloroformic and acetonic extracts of saffron reduced mungbean seed germination. The shoot systems growth, in control medium was similar to acetonic and chloformic extracts. In acetonic-residue and chloformic-residue increased roots length but both decreased shoot growth. Results indicate that water extract of saffron, induced root formation on seedlings segments. However, Hosseini and Rizvi (2003) reported that at higher concentration of water extract from saffron corms (20 %) the percentage and rate of germination of wheat as well as its dry weight of radicle and plumule were strongly reduced but this investigation was determined that seed germination and seedling growth, in depended to kind of extract and concentration. Results of this investigation showed stigma of saffron extract has stimulatory effects on seed germination and seedling growth of mungbean. There were some effects of saffron corms on germination of all tested species except wheat, but this effect was more pronounced for cotton and soybean. Although there is strong evidence for effects of some plants on others (Rice, 1984; Putnam, 1994; Moyer and Huang, 1997), these effects have not, until now, been investigated for bulb plants such as saffron or other similar crops.
Literature Cited
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Table 1. The effects of different concentration of saffron water extracts on seed germination and seedling growth of mungbeen (25 ºC, after 6days).
(g/l) / %G / HypL(cm) / EpyL(cm) / RL (cm) / N. LaR
/ Another
1 / 0 / 75a / 11.9433c / 2.9533b / 3.8867b / 5-10 / Root
2 / 0.5 / 80a / 9.1600b / 1.1633a / 1.9133a / 3-4 / On root
3 / 1 / 95b / 11.2167c / 4.6067c / 4.4967b / 3-4 / On hypocotyle
4 / 2.5 / 98.33b / 12.3300c / 7.2200d / 6.1633c / 5-10 / On hypocotyle
5 / 5 / 98.33b / 6.0033a / 2.3300ab / 2.4967a / 10-15 / On hypocotyle
Note the following pertains to the above table, %G: % Germination; HypL: Hypocotyle Length; EpyL: Epycotyle Length, RL: Radicle Length, and N. LaR: Number of Lateral Root.
*Values with same letters in every column there are not significantly different.
Table 2. The effects of different concentration of saffron water extracts on seed germination and seedling growth of mungbeen (35 ºC, after 24 hours).
N / Concentration(g/l) / %G / HypL (cm) / EpyL (cm) / RL
(cm) / N. LaR
/ Another
1 / 0 / 96.67 / 1.1900a / 2.9533b / 1.3800a / 2 / Root thick and short
2 / 2.5 / 96.67 / 1.9433b / 1.1633a / 3.4433c / 4 / Root normal
3 / 5 / 100.00 / 1.0267a / 4.6067c / 2.1033b / 7 / Root long and thin with hairy roots
Note the following pertains to the above table, %G: % Germination; HypL: Hypocotyle Length; EpyL: Epycotyle Length, RL: Radicle Length, and N. LaR: Number of Lateral Root. *Values with same letters in every column there are not significantly different.