ENHANCEMENT OF SWEET GOURD SEEDLING QUALITIES PRIMING WITH H 2 O 2

A study with three independent experiments for the seeds of three vegetable species was set at the roof top of Sunway Dormitory near the Bus Terminal, Dinajpur, Bangladesh, during March to May, 2019. The aims were to evaluate the physiological fluctuations in the seeds of sweet gourd cv. Monitaraprimed with the aqueous solutions of H 2 O 2 and to find the suitable concentration. Each experiment had eight treatments: T 1 (control/no priming), T 2 (hydro-priming in plain H 2 O) and other six aquatic solutions of H 2 O 2 : T 3 (0.5), T 4 (1.0), T 5 (1.5), T 6 (2.0), T 7 (2.5) and T 8 (3.0%). The seeds were soaked for six hours in those

Seed germination is one of the vital stages in the life cycle of seeded plants. Germination is a very complex process starting with the imbibition of H 2 O and involves events related with the transition of a dry quiescent and/or dormant seed to the metabolically active state (Kranneret al., 2011, andSchopferet al., 2001). The emergence of the embryonic axis through structures surrounding the embryo is the final stage of germination (Weitbrechtet al., 2011). In this link, seed priming is used as a means to enhance seed performance, notably in terms to the rate and the uniformity of germination (Taylor and Harman, 1998). Seed priming is known as the pre-sowing approach to govern seed germination and seedling development by modulating pre-germination metabolic activities prior to emergence of the radicle and usually enhances germination rate and plant growth (Bradford, 1986, andTaylor andHarman, 1998).Various physiological and bio-chemical changes happen in seeds during priming as a result of osmotic conditioning. A wide range of pre-sowing hydration techniques is used to enhance seed germination responses. These include equilibrium under conditions of high humidity (Finnertyet al., 1992), soaking in plain H 2 O (Coolbear and McGill, 1990) or osmotic solutions (Knypl and Khan, 1981) and having equilibrium with a matric potential controlling surface (Hardegree and Emmerich, 1992). Hydro-priming, osmo-priming (with mannitol or PEG 6000) and halo-priming (with KCl, KNO 3 or calcium salts) are effective for seedling establishment under harsh conditions (Toselli and Casenav, 2003). The priming enhances rapid and uniform emergence, high vigor and better yield, which has practical utilities, preferably under water stress conditions (Black et al., 2006). H 2 O 2 is a reactive molecule playing crucial roles in plants, especially under unfavorable germination conditions, developmental processes and in resisting stresses in reactive oxygen species/ROS (El-Maarouf-Bouteau and Bailly, 2008). H 2 O 2 acts as a signaling molecule in the beginning of seed germination involving specific changes at the proteomic, transcriptomic and hormonal levels (Afghani andTaheri, 2012, andDemiret al., 2012). Priming of seeds with H 2 O 2 leads to break primary dormancy (Jann and Amen, 1997); secondary dormancy provoked by salinity (Jiaet al., 2002) and germination inhibitors (Ogawa and Masaki, 2001). It acts as a stress signal in plants and hence exogenous uses of H 2 O 2 in the right dose ameliorates seed germination, reduces time to germinate and seedling growth in many crops (Patadeet al., 2012). So, it has special roles, especially in invigorating seeds with low vigor including long-term stored seeds in gene banks.So, instead of H 2 O, seed priming with aqueous solution of H 2 O 2 with the appropriate dose could be an applicable technology (Copeland and Mcdonald, 1985). The objective of the study was to evaluate the physiological changes of the seedlings of sweet gourd primed with of H 2 O 2 and find the best concentration.

Materials And Methods:-
The present research work was conducted at the roof top of Sunway Dormitory near the Bus Terminal, Dinajpur, Bangladesh.The seeds of sweet gourdcv. Monitara used as the testing seed materials and collected from the Popular Seed Limited, Bangladesh.

Seed flats and their arrangements
The seed were debbled in wooden seed flats (50 cm ×50 cm ×15cm). Firstly; the flats were set on the roof top. Then blue polyethylene sheet was spread at the bottom of the flats to protect washing away of sand from the seed flats. Then flats were filled-in with coarse sand.

Preparation of the required H 2 O 2 solutions
The required solutions were prepared by diluting the required amounts of the H 2 O 2 (30% strength, Plate 1) with H 2 O to get these six concentrations: 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%.

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Plate 1:-The container of the H 2 O 2 (30%)used in the study.

Priming process
At first, only H 2 O and those six solutions were taken in plastic glasses separately. The glasses were marked about the treatments and replications with a permanent glass marker. Then the 200 seeds for each replication were taken in plastic glasses to soak in the desired solutions for six hours in the Laboratory of Horticulture (Plate 2). Dibbling the seeds and caring the seedlings The unprimed and the primed seeds were then dibbling immediately in the seed flats (Plate 3) at the depth of 2cm in lines on the 10 th March, 2019 at the distance of 5cm between rows and seeds too. After sowing, the seeds were covered with hyaline polyethylene sheet and concrete poles (at one foot high) to protect the seeds and seedlings from heavy rainfall. Light watering with a watering cane was done as needed. Hand weeding was also done as per need.

Data collection
The data were collected for % germination and % abnormal seedlings at 10 days of dibbling and shoot length, root length, seedling length, shoot dry matter, root dry matter, seedling dry matter, number of secondary roots (>1cm) per seedling, number of true leaves per seedling, relative growth rate, seedling vigor index, and root shoot ratio (dry weight basis) at 10, 20 and 30 DAD.
Germination (%) and normal seedling (%) were observed and counted as per the ISTA (2010) rules daily up to 10 DAD. For dry matter the normal seedlings were cut and divided into roots and shoots with a razor blade from each treatment and replication wise. Then those were first sundried separately for two days. After that, those were dried at 80°C for 48 hours in an electric oven (Memmrert, ULP 400). Then the dry weights of shoots were recorded up to four decimal places with an electric digital balance (Ohaus, pioneer pro PA214). Finally, the dry weights were expressed in gram per 100-seedling basis. Those processes were repeated with the normal seedlings only obtained from the 10, 20 and 30 DAD. Relative growth rate (RGR)was calculated as per Williams (1946) formula and Seedling vigor index (SVI) was calculated as per Orchard (1977) and Baki and Anderson (1973) viz. SVI = Mean seedling (root + shoot) length (MSL) in cm ×% germination (PG)

Statistical analyses
The analyses of variances (ANOVA) were done and the means were separated using Duncan's Multiple Range Test (DMRT). The MSTAT-C Statistical Package program was used for it.

Results
And Discussion:-Germination % There was significant variation (P≤0.05) in percentage of germination among the treatments (Fig. 1).The maximum germination was recorded in T 4 (90.00) while the minimum was in T 8 (81.33%). Priming of seeds with H 2 O 2 changes the germination mechanism due to improved breaking primary and secondary dormancies and prevents suffocation. In this study, the highest % germination (93.67) was found in seeds primed with 1% H 2 O 2 . Similar results were also obtained by Nandi et al. (2017) in chili seeds found better germination at 2% concentration of H 2 O 2 .That variation could be due to the uses of different seed species. In addition, the higher doses (>1%) of H 2 O 2 probably caused deleterious effects on the germination process and so, it became poor, even than the unprimed and the hydro-primed seeds. The H 2 O 2 levels >2% resulted in severe injury to germinating seeds (

Abnormal seedlings (%)
There were significant differences (P≤0.05) in % abnormal seedlings among treatments (Fig. 2). The highest % abnormal seedlings were documented in T 8 (5.66) while the lowest was noted in T 4 (2.00). The results showed that the incidence of abnormal seedling increased with increasing of H 2 O 2 concentrations which might be due to the harmful effects. Mustafa (2017) andLima (2017) noted the minimum% abnormal seedlings with 1.5% while the maximum at the 3% H 2 O 2 . Kaya et al. (2006) reported that priming reduced the number of abnormal seedlings of sunflower under drought stress.

Shoot, root and seedling length
The results show that the shoot length varied significantly among the treatments at all the three DADs (Table 1). In case of the 10, 20 and 30DAD, the longest shoot was found in T 4 (6.68, 8.94 and 9.73 cm) but the shortest one was observed in T 8 (5.53, 9.

Shoot, root and seedling dry matter
There was statistically significant variation (P≤0.05) in shoot, root and seedling dry matter accumulation among the treatments at all the three DADs (Table 2). At the 10, 20 and 30DAD, the maximum dry matter of shoots was observed in T 4 (14.22, 20.97 and 32.07 g) while the minimum was recorded in T 8 (10.79, 17.24 and 25.64g) . The highest dry matter of roots at the 10, 20 and 30 DAD, was found in T 4 (2.60, 7.20 and 9.73 g) but the lowest was recorded T 8 (1.70, 3.99 and 6.85g). At 10, 20 and 30 DAD, the topmost dry matter of seedlings was found in T 4 (16.83, 28.17 and 41.80 g) but the lowest was observed in T 8 (12.70, 21.23 and 32.49g). In addition, the higher doses (>1%) of H 2 O 2 probably caused harmful effects on the shoot and so, it became poor, even than the unprimed and the hydro-primed seeds. However, the applications of H 2 O 2 at doses of 1.8mM each eight days broccoli seedlings, increased the stem length and fresh weight, whereas the dose 1.4m increased the biomass of broccoli seedlings (León-Vargas et al.,2016). In bitter gourd and bottle gourd seeds, Lima (2017) experienced the maximum shoot, root and seedling dry matter primed with the 1.5% H 2 O 2 . In contrast, Mustafa (2017) recorded the maximum shoot, root and seedling dry matter with swampcabbage, radish and Indian spinach seeds at 1% but the minimum shoot, root and seedling dry matter with 3% H 2 O 2. Above 1.0%, others were toxic and hindered most of the parameters (Mustafa, 2017). Kaya et al. (2006) reported that priming increased the soot, root and seedling dry weight of sunflower under drought stress.

Number of secondary roots andtrue leaves per seedling
The number of secondary roots and true leaves was statistically significant variation (P≤0.05) among the treatments at the 10, 20 and the 30DADs ( Table 3). At the 10, 20 and 30DAD, the maximum number of secondary roots and true leaves was counted in T 4 (11.05, 14.35 and 21.53) while the minimum was observed in T 8 (6.00, 9.67 and18.01). At the 10, 20 and 30 DAD, the utmost number of true leaves was experienced in T 4 (0.93, 3.14 and 4.36) while the minimum was recorded in T 8 (0.80, 2.50 and 4.36) . Similar kindsof result were also reported by Lima (2017) in bitter gourd and bottle gourd where the highest number of secondary roots and true leaves was at 1.5% concentration of H 2 O 2. Again, Mustafa (2017) recorded the number of secondary roots and true leaves per seedlingof cucumber, swampcabbage, radish and Indian spinach seeds primed with 1% H 2 O 2. However, when the concentrations of H 2 O 2 came up to 5mM, it played an opposite role to inhibit the growth of adventitious roots and seriously damaged those (Deng et al.,2012).

Seedling vigor index (SVI)
There was significant variation (P≤0.05) in seedling vigor index among the treatments at all the three DADs (Bar diagram.3.11 and Appendix III) judged. At 10, 20 and 30 DAD maximum value was found in T 4 (1083.0, 2016 and 2588) while the minimum value was in T 8 (777.0, 1511 and 1996).Similar types of results were also reported by Nandi et al. (2017) in chili seeds. In addition, the higher doses (>1%) of H 2 O 2 might cause deleterious effects on the seedlings and so, it became less, even than the unprimed and the hydro-primed seeds. While working with yard long bean seeds, Lima (2017) noted seedling vigor index at 1% H 2 O 2.

Relative growth rate (RGR)
There was insignificant difference for the relative growth rate among the treatments compared (Table 4). Mustafa (2017) found highest relative growth rate at 3% (0.22) . Moreover, in bitter gourd seed and bottle gourd seeds, Lima (2017) found relative growth rate in control/no priming (0.11) .

Root: shoot ratio (dry weight basis)
There was insignificant difference for the root: shoot ratio among the treatments at all the three DADs (Table 4). Lima (2017) noted the maximum root: shoot ratio (0.34) in seedlings of yard long been observed from the seeds treated with the 1% H 2 O 2. But the excessive accumulation of H 2 O 2 leads to cellular oxidative damage and even programmed death (Levine et al., 1994 andPrasad et al., 1994) and thus becomes poisonous for seedlings.

Conclusions:-
It canbe concluded thatH 2 O 2 had optimistic effects on the seedling qualities. Again, among the six concentrations of the H 2 O 2 , 1%was the most effective one for sweet gourd and sweet gourd while 1.5% was suitable for radish seeds. Above those concentrations, others were somewhat toxic as those hindered a lot of the parameters evaluated.