In Vitro Evaluation Of Seed Vigour Enhancement Techniques Biology Essay


Impact of pre-sowing seed treatments priming on the germination and seedling vigour of mungbean were tested under laboratory conditions. The seeds were invigorated by traditional soaking, osmoconditioning using, KH2PO4, Manitol, Polyethylene glycol (PEG), Na2MO4.2H2O and hormonal priming (salisylic acid) for 4 h. All the invigoration treatments significantly affected the germination percentage, time required for fifty percent seeds to germinate (T50%), mean germination time (MGT), root and shoot length. Osmopriming using P @ 0.60% applied in the form of KH2PO4 significantly improve seed vigour and final germination percentage. Overall all the seed priming techniques significantly improved the vigour of mungbean seedling.

In Pakistan, mungbean was grown on an area of 245,000 hectares in 2007-08 with production of 177,000 tonns under rainfed and irrigated conditions (Anonymous, 2009) while 1199 tonns of yield recorded in Potohar. Yields for the rainfed area are generally low and variable due to sparse, erratic rainfall and marginal soils. In Punjab province, mungbean production is dependent mainly on surface irrigation but it is also grown under rainfed conditions. In the Southern region of Pakistan rainfall is scanty and mungbean is grown with surface irrigation only. Poor crop establishment is a major restraint for mungbean production (Naseem et. al., 1997; Rahmianna et al., 2000) and high yields can be associated with early vigor (Kumar et al., 1989).

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Seed priming is a technique by which seeds are partially hydrated to a point where germination processes begin but radical emergence does not occur. Seed priming can be found effective for legumes i.e., yields of Mungbean and Chickpea were increased substantially by priming seeds for 8 h before sowing (Harris et al., 1999; Musa et al., 2001; Rashid et al., 2004).

Improved seed invigoration techniques are being used to reduce the germination time, to get synchronized germination, improve germination rate, and better seedling stand in many horticultural (Bradford et al. 1990; Rudrapal & Nakamura 1998) and field crops like wheat, maize (Dell Aquilla & Tritto 1991; Basra et al. 2002) and more recently rice (Farooq et al. 2004, 2004a, 2005, 2006, 2006a). These invigoration techniques include hydropriming, osmoconditioning (Basra et al. 2005), osmohardening (Farooq et al. 2006a) and hardening (Farooq et al. 2004a). These treatments can also be employed for earlier and better nursery stand establishment (Lee et al. 1998), which will result in the improved performance of traditional transplanting rice production system.

Seed priming sometimes reduces the base water potential towards more negative values, increasing the ability of the seed, under low water accessibility, to germinate (Bradford, 1990). However, there is substantiation in tomato seeds that without lowering the base water potential priming shortens the time to germination (Dahal and Bradford, 1990).

This study was initiated to explore the effects of aerated hydration, hormonal priming and osmoconditioning on mungbean (vigna radiata) seed germinability and seedling vigour under laboratory conditions.

Material and method

Seed materials

Seeds of mungbean cultivar Chackwal Mung-97 (CH-MUNG 97) were obtained from Barani Agricultural Research Institute (BARI), Chackwal. The seeds were sterilized by using 30% hypochlorite for five minutes and then washed three time with distilled water and allowed to dry under forced air.

Selection of proper priming compounds

Seeds were treated with different priming compounds viz. Salisylic acid (SA), KH2PO4, Manitol, Polyethylene glycol (PEG) and sodium molybdate (Na2MO4.2H2O). There has been two levels of each priming agents used. Four hour priming duration was determined as suitable time for mungbean seeds for all the priming materials. The primed seeds were set to germinate in an incubator at 27 oC. The selection was made on the basis of germination percentage and seedling vigor etc.

Seed treatment

Selection of suitable priming compounds was made on the basis of findings of different research workers (Basra et al., 2002; Kaur et al., 2006; Marcelo Grandi et al., 1999; farooq et al., 2006). The seeds were set to germinate in an incubator at 27oC. The range of osmotic potential (ψs) of all the solution were between -0.5 to -1.5 M Pa. A non-soaked, non-dried treatment was included as a control. The number of seeds germinated was recorded after every twelve hours and the final germination percentage and time for 50 % of seeds to germinate (t50) was calculated. The shoot and root length of the seedlings was also calculated (farooq et al., 2006).

Post priming operations.

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After soaking, seeds were given three surface washings with distilled water (Khan et al., 1992) and re-dried under shade, near to original weight with forced air. The seeds were then sealed in polythene bags and stored in refrigerator till further use (Basra et al., 2002).

Germination test

Germination potential of mungbean seeds was estimated in accordance with the AOSA method (AOSA, 1990). In an incubator three replicates of 25 seeds, each were sown in 12 cm diameter petri dishes, between the layers of moist Whatman-45 filter papers at 27°C. The petri dishes were arranged in a CRD with three replicates. Starting on the first day of imbibitions, counts of germinating seeds were made at 12 hour intervals as far maximum germination was attained.

The time to reach 50% germination (T50) of final germination was calculated according to the following formula (Coolbear et al., 1984) and modified by Farooq et al. (2005):

T50 = ti + (N/2 - ni) (tj - ti) / nj - ni

Where N is the final number of germination and ni, nj cumulative number of seeds germinated by adjacent counts at times ti and tj when ni < N/2 < nj.

Mean emergence time (MGT) was calculated according to the equation of Ellis and Roberts (1981) as under:

MGT = ∑ Dn/∑n

Where n is the number of seeds, which were germinated on day D and D is the number of days counted from the beginning of germination.

Result and Discussion

Time for 50 percent germination (T50 %)

Different seed priming techniques had significant (p<0.05) impact on time required for fifty percent germination (t50 %) of treated seed lot. Time required for 50 percent seed to germinate showed that T1 took maximum time (1.39 days) to germinate followed by T10 (1.30 days) figure-1. The data also depicts that T5 showed minimum T50 value (1.08days) as compare to all other treatments as well as control.

Fig 1. Impact of different seed priming techniques on T50 (%) value of mungbean.

T1 = Control, T2 = Hydropriming, T3 = Molybdenum @ 0.02%, T4 = Molybdenum @ 0.04%, T5 = Phosphorous @ 0.06%, T6 = Phosphorous @ 1.20%, T7 = Salicylic acid @ 10 ppm, T8 = Salicylic acid @ 20 ppm, T9 = Manitol @ 2 %, T10 = Manitol @ 4 %, T11 = Polyethylene glycol @ 5%, T12 = Polyethylene glycol @ 10%

Widely reported, seed priming has been contribute to plant growth and development (Harris et al., 2005). Ajouri et al. (2004) reported a stimulation of P and Zn uptake, as well as an enhanced germination and seedling growth in barley after soaking seeds in water and in solutions containing 5-500 mM P.

These results are in line with the findings of Ozbingol et al. (1999) who reported priming tomato seeds with PEG 8000 (-1.0 M Pa) significantly reduced T50. Significant reduction in T50 may be attributed to early reserve breakdown and early reserve mobilization. It might also be due to possible early activation or de novo synthesis of cell wall degrading enzymes (Hisashi and Francisco, 2005). However Nascimento and West (1999) reported early germination of primed seeds but not recorded any improvement in the growth of seedlings in muskmelon seeds under laboratory conditions. Contradictory results, where priming did not show any beneficial results also reported, by different scientists (Mwale et al., 2003; Giri and Schillinger, 2003).

The enhancement of the mungbean seed vigor may be because following priming , seeds have completed phases 1 (hydration) and 2 (lag phase) of germination and only require a favorable water potential gradient for water uptake in order to begin radicle growth (Pill, 1995).

Treated seeds had high germination percentages and quicker germination time. One hypothesis is that benefits of priming can be due to metabolic repair of damage during treatment and that change in germination events i.e., changes in enzyme concentration and formation and reduces lag time between imbibition and radicle emergence (Bradford et al., 1990). Along with metabolic advancement, Nerson et al. (1985) found that KNO3 priming also increased embryo length in tetraploid watermelon seeds. Treated seeds had stronger embryos that were able to more easily emerge from seeds.

Kader and Jutzi (2002) suggested that under favorable thermal conditions, the suctions forces although they are not the same between unprimed and primed seeds, are sufficiently high to allow the uptake of water from medium at a similar rate. Conversely, seed priming has been demonstrated to partially overcome the negative effect of low temperature on seed metabolism by increasing the imbibition speed (Patane et al., 2006). However, a faster water imbibition at low temperature influences the beginning of seed respiration but not the subsequent metabolic activity preceding germination, due to biochemical reactions controlled by temperature (Patane et al., 2006).

1.2 Mean germination time (MGT)

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Different seed priming techniques had significant (p<0.05) effect on mean germination time (MGT) in vitro studies showed. The mean germination time decreased with the application of different seed priming techniques. Maximum mean germination time (1.90 days) observed in T1 (control) where dry untreated seeds were sown.

Minimum (1.39 days) MGT was observed in T5 (P @ 0.06 % applied in the form of KH2PO4). All the treatments were significantly lower from control (1.90 days).

During pre-sowing seed treatments the dormancy of the seed is broken and the seed bio-chemical processes begins, which lead to faster germination as well as emergence (Farooq et al., 2006). Seed priming ensured the proper hydration, which resulted in enhanced activity of a-amylase that hydrolyzed the macro starch molecules in to smaller and simple sugars. The availability of instant food to the germinating seed gave a vigorous start as indicated by lower E50 and MET in treated seeds (Farooq et al.,2006) during priming de novo synthesis of a-amylase is also documented (Lee and Kim, 2000). More the a-amylase activity the higher will be the metabolic activity in seeds, which indicates the higher vigor of the seeds (Farooq et al., 2006). This was plausibly due to dormancy breakdown in fresh seed (Basra et al., 2005). These results also with line with the previous studies that pansy seed primed with CaCl2 (-1.0 MPa) for three days 23 oC had significantly higher germination than non-primed seeds (Yoon et al., 1997).

Early emergence as indicated by lower T50 and MET in treated seeds may be due to the faster production of germination metabolites (Saha et al. 1990; Lee & Kim 2000; Basra et al. 2005) and better genetic repair, i.e. earlier and faster synthesis of DNA, RNA and proteins (Bray et al. 1989). Gray and Steckel (1983) also concluded that priming increased embryo length, which resulted in early initiation of germination in carrot seeds.

Fig 2. Impact of different seed priming techniques on mungbean mean germination time (days) under controlled conditions.

T1 = Control, T2 = Hydropriming, T3 = Molybdenum @ 0.02%, T4 = Molybdenum @ 0.04%, T5 = Phosphorous @ 0.06%, T6 = Phosphorous @ 1.20%, T7 = Salicylic acid @ 10 ppm, T8 = Salicylic acid @ 20 ppm, T9 = Manitol @ 2 %, T10 = Manitol @ 4 %, T11 = Polyethylene glycol @ 5%, T12 = Polyethylene glycol @ 10%

Germination percentage

There was significant (p<0.05) effect of seed priming techniques on germination percentage (fig-3). Maximum (95.33%) germination was observed in T5 (P @ 0.06%). There was an increase of 19 percent was observed in T5. The germination percentage decreased in T4, T7, T9 upto T12 (fig).

Seed treatments other than hydropriming and some osmopriming treatments resulted in lowering the germination vigor and seedling growth. Reduction in germination and seedling vigor in osmopriming treatments might be the result of toxicity of the solutes use, as earlier found in KNO3 osmopriming in rice (Basra et al., 2003; 2005). In contradiction to present findings, Al-Mudaris and Jutzi (1999) reported germination enhancement from seeds primed with nitrogen, phosphorus and potash fertilizer treatments in Sorghum bicolor and Pennisetum laucum.

Fig 3. Impact of different seed priming techniques on germination percent under controlled conditions.

T1 = Control, T2 = Hydropriming, T3 = Molybdenum @ 0.02%, T4 = Molybdenum @ 0.04%, T5 = Phosphorous @ 0.06%, T6 = Phosphorous @ 1.20%, T7 = Salicylic acid @ 10 ppm, T8 = Salicylic acid @ 20 ppm, T9 = Manitol @ 2 %, T10 = Manitol @ 4 %, T11 = Polyethylene glycol @ 5%, T12 = Polyethylene glycol @ 10%

Shoot and root length

The data regarding length of plumule and radical has been presented in the table-1. Maximum shoot length was achieved in T5 (P @ 0.60 %) as compare to other priming agents. The data also depicts that the lower level of P concentration is more beneficial than higher (P @ 1.2 %) level in T6 as shown in figure. The minimum root and shoot length was in control (7.44 cm).

There was similar trend observed for root length of mungbean as affected by different seed priming techniques. Maximum (10.5 cm) shoot length was observed in T5 (P @ 0.06%) followed by T9 (manitol @ 2%). Maximum root length was observed in T8. All the priming techniques improved shoot as well as root length of mungbean plants. Overall an increase of 24 percent observed in soot length due to seed priming techniques.

Unlike other vigour enhancement treatments where hydration is stopped before germination is completed, pre-germination (which is very much similar to traditional soaking practice in Pakistan) is characterized by seed hydration to the point of radical protrusion. The result is more uniform, faster germination and almost 100% seedling establishment (McDonald, 2000).

These findings support the earlier work on canola [Brassica compestris] (Zheng et al., 1994), wheat [Triticum aestivum] (Nayyar et al., 1995) and rice [Oryza sativa] (Lee and Kim 2000; Basra et al., 2003) who reported improved germination rate and percentage in seeds subjected to hydropriming and seed hardening for 24 h. The earlier and better-synchronized germination is associated with increased metabolic activities in the hardened seeds (Lee and Kim, 2000). Faster emergence rate after hardening and traditional soaking may be explained by an increased rate of cell division in the root tips as previously found for wheat (Triticum aestivum) (Bose and Mishra, 1992; Basra et al., 2002) and fine rice [Oryza sativa] (Basra et al., 2003). The enhanced activity of α-amylase during the pre-sowing treatments may be attributed to hydration during treatment, resulting in increased starch hydrolysis, increased contents of total and reducing sugars and lower contents of non-reducing sugars. The benefit of increased starch hydrolysis following hydration treatments was not lost during the redrying process, as shown by the better rate and spread of germination. These agree with the findings of Lee and Kim (1999) and Lee and Kim (2000) who reported that increased α-mylase activity was associated with higher germination in coarse rice [Oryza sativa].

In conclusion the results of the present study suggest that seed priming is very effective tool for seed invigoration in mungbean. There is a direct relationship between the germination and seedling vigour. The plausible basis of seed invigoration by these seed treatments are increased membrane repair and enzymatic activities (Farooq et al., 2006)

Table 1. Impact of different seed priming techniques on shoot and root length of five days old seedlings





7.1 g

6.2 g


8.6 cd

7.3 f


8.1e f

7.5 ef


8.4d e

8.0d ef


10.5 a

8.8 abc


9.5 b

7.3 f


8.5 cd

9.2 ab


8.7 c

9.5 a


9.7 b

8.9 abc


8.6 c

8.4 cd


8.0 f

8.6 bcd


7.9 f

8.2 cde




T1 = Control, T2 = Hydropriming, T3 = Molybdenum @ 0.02%, T4 = Molybdenum @ 0.04%, T5 = Phosphorous @ 0.06%, T6 = Phosphorous @ 1.20%, T7 = Salicylic acid @ 10 ppm, T8 = Salicylic acid @ 20 ppm, T9 = Manitol @ 2 %, T10 = Manitol @ 4 %, T11 = Polyethylene glycol @ 5%, T12 = Polyethylene glycol @ 10%