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International Journal of Agricultural Science and Research (IJASR) ISSN 2250-0057 Vol.

3 Issue 2, Jun 2013 163-174 TJPRC Pvt. Ltd.

ISSNRESPONSE 22496939

OF SEED PRIMING WITH BORON ON GERMINATION AND SEEDLING SPROUTS OF BROCCOLI

Vol.2, Issue 2 (2012) 1-16 TJPRC Pvt. Ltd.,

NOOR-UN-NISA MEMON1, MOULA BUX GANDAHI1, VAJANTI MALA PAHOJA2 & NASIM SHARIF3
1 2

Department of Horticulture, Sindh Agriculture University, Tandojam, Sindh, Pakistan


3

Department of Crop Physiology, Sindh Agriculture University, Tandojam, Sindh, Pakistan Ayub Agriculture Research Institute, Faisalabad, Punjab, Pakistan

ABSTRACT
Better germination and vigor of the seedlings are main problems for the success of stand establishment of crop plants. The present study was therefore conducted on seed priming with boron to observe the efficacy of priming on germination and growth related attributes of the broccoli seedlings. Broccoli seeds (cultivar Marathon) of SAKATA Seed Company were soaked in boric acid solution at 0.01, 0.05, 0.5 and 1% (w/v) for 18 hours. Seeds were also soaked in distilled water (hydropriming) and unprimed seeds were taken as control. All primed and non-primed seeds were grown in earthen pots contained sandy clay loam soil with EC 0.74 dS/m, pH 7.8, organic matter (0.71%), total nitrogen content (0.06%), available phosphorus 3.6 mg kg-1, exchangeable potassium 168 mg kg-1 and Boron 0.48 ppm. The results revealed that Germination percentage (GP), Mean germination time (MGT), Germination index (GI), Seedling vigor index (SVI), Chlorophyll content, Shoot and root related attributes were significantly influenced by primed seeds as compared to unprimed seeds. The highest germination index (6.289), seedling vigor index (1753.3), chlorophyll content (4.137 mg ml-1) and less mean germination time (3.23 days), maximum length of shoot (5.97 cm), root (11.57 cm), weight of the shoot (15.35 g) and root (2.68 g) were observed from the treatment where seeds were primed with boron solution at the lowest concentration of 0.01%.

KEYWORDS: Seed Priming, Boron, Germination, Seedling Vigor INTRODUCTION


Broccoli (Brassica oleracea) is one of the main cole crops of Brassicaceae or cruciferae family. It is supposed to be the first of the Cole crops evolved from the wild species of cabbage or kale (Rubatzky and Yamaguchi, 1997). Broccoli, a rich source of vitamin C, E, B1, carotenoids, phenolic and possess anticancer properties (Goncalves et al., 2011). Consumption of broccoli is increasing day by day as staple food due to its health promoting properties. Broccoli is green in color due to chlorophyll within the sepals of the floral buds and grown as an annual crop for its immature flower buds (Gray, 1982; Jett et al., 1995). In most of the annual crops yield losses occur due to poor germination rate and stand establishment of the crop (Eskandari and Kazemi, 2012). Uniform and better stand establishment of the crop may allow grower to reduce costs and increases harvest efficiency (Heather and Sieczka, 1991). Better germination and vigor seedlings are the main basic foundations for the success of stand establishment of any crop plant. Various seed technologies including priming, coating and conditioning of the seeds are applied to improve germination and emergence in seeds of many crops (Arif et al., 2008; Taylor et. al., 1998). Seed priming is a technique in which seeds are soaked in solutions of low water potential that initiates pre-germinative metabolic activity but prevents radical protrusion (Bradford, 1986; McDonald, 2000; Ashraf and Foolad, 2005; Farooq et al., 2006; Janmohammadi et al., 2008). However, the beneficial effects of seed priming have been well documented in many research based published data

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include early germination; improves germination rate (Dahal et al., 1990; Jett et al., 1996; Bradford, 1986; Taylor and Harman, 1990; Ghassemi-Golezani et al., 2008); breaks dormancy (Cantliffe et al., 1984; Wurr and Fellows, 1984) improves seedling vigor (Harris, 1996; Saber et al., 2012); strengthens stand establishment (Khan et al., 1992; Jett et al., 1995; Arif et al., 2005; Ali et al., 2007; Diniz et al., 2009) and increase yield (Rengel and Graham, 1995a, 1995b; Yilmaz et al., 1998). Besides all these beneficial effects, seed priming also reduce leakage of metabolites, repairs deteriorated seed parts, improve RNA and protein synthesis (McDonald, 2000). Number of priming treatments are generally in use including hydro, halo, osmo, thermo and matrix priming (Khan, 1992; McDonald, 2000; Basra et al., 2005; Ghiyasi et al., 2008). Every technique responds differently in various crops. These various priming technique have been successfully applied for their beneficial effects in many agronomic crops including wheat, sugar beet, maize, soybean and sunflower, rice (Khajeh-Hosseini et al., 2003; Sadeghian and Yavari, 2004; Iqbal et al., 2012; Rehman et al., 2012; Ahmad et al., 2012). However very few studies have also been conducted in vegetable crops including summer squash (Atta Aly, 1998); common beans (Mohondas, 1985); water melon (Elmstrom, 1985); carrot and onions (Brocklehurst and Dearman, 1983); okra (Mereddy et al., 2000; Shah et al., 2011); tomato (Afzal et al., 2011; Nawaz et al., 2011); hot pepper (Amjad et al., 2007); lentil (Saglam et al., 2010) and Dil (Mirshekari, 2012). Nutripriming is now recently focused by using macro or micronutrient enriched seeds as reported by Rehman et al. (2012) and Mirshekari (2012). Generally nutrients are delivered to plants as soil applications, fertigation or foliar spray (Johnson et al., 2005; Rober, 2008). Applying nutrients as seed treatment, through seed coating and seed priming, is another option which avoids aforementioned risks (Farooq et al., 2012). The use of micronutrient-enriched seeds (seed priming) has been reported to be a better strategy in overcoming micronutrient deficiencies (Musakhandov, 1984; Harris et al. 1999). Priming the seeds with micronutrients makes them able to rapidly imbibe water and revive metabolism and germination. This then results in a higher germination rate (Rowse, 1995), improved stand establishment, increased drought and pest tolerance, and ultimately higher yields (Harris et al., 1999). Boron is one of the essential micronutrient for all vascular plants (Warington, 1923). It is absorbed from the soil by plants as borate. Since boron is non-mobile in plants, and continuous supply from soil or planting media is required in all plant meristems. Deficiency or toxicity of boron causes severe reduction in crop yield, which is due to disturbance in metabolic events involving boron (Brown et al., 2002). Boron has vital role in the formation of proteins, nitrogen metablolism, cell division, cell membrane integrity, cell wall formation, nucleic acids, and antioxidative systems (Parr and Loughman, 1983; Gupta et al., 1985; Cakmak and Romheld, 1997; Goldbach and Wimmer, 2007; Bonilla et al., 2009; Koshiba et al., 2009). It has also vital role in the process of transpiration for the movement of potassium to the stomata of the leaf. Boron also maintains stable balance between sugars and starches, pollination and seed production (Gupta et al., 1985). Seed priming with macro or micro nutrient application is recently done on various agronomic crops including dill (Mirshekari, 2012), rice (Kalita et al., 2002; Johnson et al., 2005), maize (Harris et al., 2007); wheat and lentil (Jhonson et al., 2005, Iqbal et al., 2012). There is no report reported in vegetables except dill (Mirshekari, 2012).

MATERIAL AND METHODS


The experiment was carried out at horticulture garden, Department of Horticulture Sindh Agriculture University, Tandojam, Pakistan located at 25o 2560N 68o31 60E, altitude 19.5 m asl. Broccoli seeds CV. Marathon of SAKATA seed company were used in the present study to observe the efficacy of seed priming with boron on germination and growth attributes of seedlings. Twenty broccoli seeds were soaked in solution of 0.01, 0.05, 0.5 and 1% boric acid solution

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(w/v) for 18 hours. Seeds were also soaked in distilled water (hydropriming) and unprimed seeds were taken as control. The seeds were surface washed thrice with distilled water and dried at room temperature. All primed and unprimed seeds were planted in earthen pots containing soil. The soil was analyzed for soil texture by Bouyoucos Hydrometer method, pH of 1:5 soil-water extract by pH meter, EC of 1:5 soil-water extract by EC meter, organic matter by Walkley-Black, total nitrogen by Kjeldahl's method, available phosphorus by Olsen, exchangeable potassium by extraction with ammonium acetate and boron by hot water method. The soil was sandy clay loam, non-saline in nature with EC 0.74 dS/m and slightly alkaline in reaction with pH 7.8. The soil was also low in organic matter (0.71%), total nitrogen content (0.06%), available phosphorus 3.6 mg kg-1, exchangeable potassium 168 mg kg-1 and Boron 0.48 ppm. Monthly mean minimum and maximum temperatures and other related data of weather was also recorded and presented in Table 1. The data was recorded for Germination Percentage (GP), Mean Germination Time (MGT), Germination Index (GI), Seedling Vigor Index (SVI), length and fresh weight of shoot and root, electrolyte leakage of leaf and chlorophyll content of broccoli sprouts. Germination percentage -Germination was checked on every alternative day for six days and the germination percentage was calculated by using following equation as described by Larsen and Andreasen (2004). GP = n/ N 100 where n is number of germinated seeds at each counting and N is total seeds in each treatment. Mean Germination Time (MGT) was calculated by using following equation of Ellis and Roberts (1981) MGT = Dn n where n is the number of seeds germinated on day D and D is the number of days as counted from the beginning of germination. Germination index (GI) was calculated by the formula given by the Association of Offi cial Seed Analysts (1983) GI = Number of germinated seeds + ---------- + ---------- + Number of germinated seeds Days of first count Days of last count

Seedling Vigor Index (SVI) was calculated by using following formula, Abdul-Baki and Anderson (1970) Vigor index (VI) = [seedling length (cm) germination percentage] One and half months old seedlings were kept under observation for length and weight of shoot and root, electrolyte leakage of leaf and chlorophyll content of leaf. Electrolyte leakage of leaf was measured by taking leaf discs of size 1 cm2 and weight 0.5 g from random samples of leaf. The leaf discs were rinsed well in deionized water prior to incubation in 25 ml of deionized water for 3 h at room temperature. After incubation, the conductivity (value A) of the bathing solution was measured with the conductivity meter. The petal discs were boiled with bathing solution for 15 min to lyse all cells. After cooling to room temperature, the conductivity (value B) of the bathing solution was again measured. The electrolyte leakage was expressed as percent value according to the formula given below. Electrolyte leakage of leaf % = (Value A/Value B) 100 Chlorophyll content leaf sample of 0.5 g was grinded in 10 mL of 80% acetone and filtered with Whatman paper 1

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by adding more 10 ml of acetone. Samples were poured in cuvettes at 663 and 645 nm using UV-Vis spectrophotometer (Bruinsma, 1963) The experiment was carried out in Completely Randomized Design with three replications. The obtained data was statistically analyzed by using software Statistixs 8.1 and interpreted on the basis of Duncans Multiple Range Test (DMRT). Table 1: Monthly Mean Temperatures and Other Related Weather Data Recorded During the Study Temperatures (00C) Minimum Maximum December, 2012 0 10.6 25.7 January, 2013 0.03 10.5 25 Source: Regional Agro Met Centre Tandojam, Sindh, Pakistan Months/Year Rainfall (mm) Relative Humidity (%) 55 54 Cloudness (Octas) 0.3 0.7 Sunshine (Hours) 7.9 8.1

RESULTS
Germination percentage was significantly influenced by seed priming with boron in relation to number of days Figure 1. Unprimed seeds took more time for germination as compared to hydro and boron primed seeds producing 75% germination on 6th day of plantation with no germination on 2 nd day of plantation. However, no germination was also observed from primed seeds on 2nd day of plantation but most part of the germination was covered on 4th day of plantation producing the highest germination (98.33%) from seeds primed with boron solution at concentration of 0.01% as compared to unprimed seeds (13.33%). On the same 4th day of plantation, germination was observed decreased with increasing concentrations of boron i.e. from 0.05 to 1.0%. However 100% germination observed form the seeds primed with boron at concentration of 0.01 and 0.05% each on 6 th day of plantation. These results were at par with the results obtained from hydroprimed seeds. On the basis of mean germination as presented in Figure 2, the highest mean germination (66.11%) was observed from the seeds primed with boron at concentration of 0.01 Figure 2. These results were at par with the results obtained from hydroprimed seeds (61.11%) and primed seeds with boron at concentration of 0.05% (61.67%). The lowest mean germination (25%) was recorded from unprimed seeds. Germination index (GI), mean germination time (MGT), seedling vigor index (SVI) and chlorophyll content was highly influenced by boron seed priming Table 2. The highest germination index (6.289), seedling vigor index (1753.3), chlorophyll content (4.137 mg ml-1) and less mean germination time (3.23 days) were observed from the treatment where seeds were primed with boron solution with the lowest concentration of boron i.e. 0.01%. The results of all these traits are statistically at par with the results obtained from the treatment where seeds were primed with boron solution at concentration of 0.05%. Moreover, results were observed decreased with increasing concentration of boron solution i.e. 0.5 to 1.0%. Better results were also observed from hydroprimed seeds as compared to unprimed seeds. No effect of priming was observed on electrolyte leakage of leaf. Shoot and root related parameters were significantly influenced by seed priming with boron Table 3 . The maximum length of shoot (5.97 cm), root (11.57 cm), weight of the shoot (15.35 g) and root (2.68 g) was observed from the treatment where seeds were primed with the lowest concentration solution of boron (0.01%). However only weight of the shoot and root had non-significant results with the results produced from the seeds primed with boron solution at 0.05% Table 3.

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HP= Hydroprimed Seeds; BP= Boron Primed Seeds Figure 1: Effect of Priming on Germination Percentage of Broccoli Seeds in Relation to Number of Days

HP= Hydroprimed Seeds; BP= Boron Primed Seeds Figure 2: Effect of Priming on Mean Germination of Broccoli Seeds Table 2: Influence of Seed Priming with Boron on Germination Related Parameters, Chlorophyll and Electrolyte Leakage of Leaf Content Electrolyte Leakage of Leaf (%) 11.83 10.82 14.51 11.28 13.1 12.57

Priming Treatments (Hydro and Boron Primed)

GI

MGT (Days)

SVI

Chlorophyll Content (mg ml-1) 2.999 bc 3.605 ab 4.137 a 3.867 a 2.885 c 3.018 bc

Unprimed seeds (control) 2.989 e 5.097 a 753.67 e Seeds primed with Distilled water 5.761 bc 3.65 bc 1260.0 bc Seeds primed with B @ 0.01% 6.289 a 3.23 c 1753.3 a Seeds primed with B @ 0.05% 6.017 ab 3.45 c 1413.3 b Seeds primed with B @ 0.5% 5.450 c 3.62 bc 1124.0 c Seeds primed with B @ 1.0% 4.555 d 4.03 b 946.50 d MGT= Mean Germination Time, GI = Germination Index, SVI = Seedling Vigor Index

Table 3: Response of Seed Priming with Boron on Length and Weight of the Shoot and Root of the Seedlings Treatments Unprimed seeds (control) Seeds primed with Distilled water Seeds primed with B @ 0.01% Seeds primed with B @ 0.05% Seeds primed with B @ 0.5% Seeds primed with B @1.0% Shoot Length (cm) 3.67 b 3.97 b 5.97 a 4.37 b 3.83 b 3.90 b Root Length (cm) 6.17 e 8.63 c 11.57 a 9.77 b 7.77 cd 7.50 d Shoot Fresh Weight (g) 7.92 bc 10.46 b 14.11 a 15.35 a 7.30 bc 6.52 c Root Fresh Weight (g) 1.31 b 1.50 b 2.68 a 2.33 a 1.65 b 1.42 b

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DISCUSSIONS
Seed priming with boron (nutripriming) exhibited beneficial effects for seed germination, mean germination time, germination index, seedling vigor index chlorophyll content of leaf, and shoot root related parameters. Priming enhanced seed performances are related to the repair and the buildup of nucleic acid, enhanced synthesis of protein, repair of membranes and improves antioxidant system (McDonald, 1999; Hsu et al., 2003). The beneficial effects of priming with nutrients have been successfully reported by various scientists in various crops (Peeran and Natanasabapathy, 1980, Sherrell, 1984, Wilhelm et al., 1980; Shah et al., 2011; Aboutalebian et al., 2012; Mirshekari et al., 2012; Rehman et al., 2012). However, deficiency or toxicity of these nutrients may damage seed or restrict germination as reported by Roberts (1948). It may also cause abnormal seedlings as reported by Louzada and Vieira (2005) in bean seeds. They observed mortality of the seedlings in bean seeds due to high applications of micronutrients. Diniz et al. (2009) observed reduced percentage of germination when higher doses of micronutreients were used for priming in sweet pepper. The low concentration of boron has been reported to activate key enzymes including phosphorylase, a-amylase etc involved in starch metabolism. High concentration of boron may be toxic as reported by Bonilla et al. (2004). In present study, germination was observed decreased with increased doses of boron i.e from 0.5 to 1%. Rehman et al. (2012) reported no germination from rice seeds primed with boron at 0.5%. While they observed better results for stand establishment of the crop from the seeds primed with boron at 0.001 and 0.1% concentration. Ajouri et al. (2004) observed significantly reduced germination in barley when they applied boric acid exceeding 0.04 M. However in papaya, substantial improvement in germination and early seedling growth was observed when seeds were primed in 2 mg L-1 B solution for 6 h (Deb et al., 2010). The higher doses of micronutrients in soil solution may also slow the establishment of the seedlings as reported by Mershikari (2012). Seedling vigor index is important for rapid stand establishment and early growth of the plants as reported by Tabrizian and Osareh (2007). Mirshekari (2012) observed restricted seedling vigor index of dill when he used boron and iron concentrations beyond 1% and 1.5% respectively. He observed higher SVI at 1% boron solution. However, further SVI increased when boron was used in combination with iron. In present study, SVI was observed higher at the lowest concentration of boron.The significant increase in shoot and root lengths and fresh weight from seeds primed at the lowest concentration of boron may be due to its involvement in cell elongation or cell division and meristematic growth (Bohnsack and Albert 1977; Shelp, 1993; Mouhtaridou et al. 2004; Khan et al. 2006). Boron at lowest concentration of 0.001% has been found successful for improved lengths and fresh weights of shoot and root in rice cultivar as reported by Rehman et al. (2012). Increasing levels of boron may decrease the fresh weight of the shoot as reported by Ayvaz et al. (2012). In contrast, Kumar et al. (2008) reported increased plant height of pea when seeds were primed with boron at 0.5%. This variation in results may be of different genotypes, boron application method, and other soil and environment related conditions. The results of the present study regarding chlorophyll content is the confirmation that boron is necessary for increased chlorophyll content. These results are in agreement with the results of Rehman et al. (2012) and Mouhtaridour et al. (2004). Rehman et al. (2012) reported increased chlorophyll content of leaf from priming solution had very low levels of boron (0.001%). However, deficiency of boron caused reduction in chlorophyll content as reported by Kastori et al. (1995). Deficiency or toxicity of boron levels are very narrow in plants (elik et al.,1998) and may vary from crop to crop.

CONCLUSIONS
It is concluded from the present study that seed priming with boron is beneficial to improve germination and other growth related attributes of the seedlings. However the increased levels of boron caused reduction in most of the evaluated

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parameters of broccoli seedlings. Further experimentation is required to optimize boron concentrations for each specific cop.

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