Effect of Priming on Brassica rapa subsp. chinensis (Bok Choy) Seeds Germination

Brassica rapa subsp. Chinensis, commonly known as Bok Choy, is a nutrient-rich vegetable with substantial antioxidant content. Therefore, this study aimed to evaluate the effect of hydropriming and bio-nutri-priming using Sandwich compost leachate on seed germination, SPAD reading, and dry matter accumulation in 280 dwarf variants of Bok Choy seeds sourced from Green Eagle. The experimental process involved hydropriming with tap water, bio-nutri-priming using 0.2% Sandwich compost leachate, and a control group cultivated in soil without priming. A complete randomization design (CRD) with three replications assessed seed germination performance, SPAD, root and shoot dry matter, and root-to-shoot ratio. While there was no significant difference in the germination percentage (88.35±1.13%), the entire priming seeds exhibited a 2-day peak germination period, compared to 3 days for the non-priming counterparts. Bio-nutri-priming seeds showed faster median and mean germination times due to enhanced nutrient uptake. They further displayed high SPAD readings, suggesting a lack of toxic compounds. The dry matter production of all treated Bok Choy was similar because administered treatments did not interfere with plant growth and development. Therefore, applying bio-nutri-priming using Sandwich compost leachate positively affected seed germination performance, warranting its recommendation as a seeds priming solution.


INTRODUCTION
Food waste is a persistent challenge worldwide, specifically in Malaysia, necessitating effective management strategies.Addressing food waste disposal requires optimizing its use at the source.Digestion methods, including fermentation, offer a promising approach (Usmani et al., 2021).Sandwich compost, derived from Bokashi, has positively affected plant initial growth.Employing raw and cooked food waste in preparing Sandwich compost presents an alternative for waste management.
The potential of Sandwich compost leachate extends to enhancing seed germination and substantial growth, contributing to food security.As a sustainable resource, Sandwich compost leachate holds promising applications in food production.For example, priming tomato seeds with Sandwich compost leachate led to a 13% increase in transplant stem diameter, facilitating nutrient uptake (Olle, 2020).Similarly, passion fruit treated with 16% Sandwich compost priming solution exhibited superior initial growth (Bócoli et al., 2020).Humic acids extracted from Sandwich compost proved advantageous for the initial growth of maize (Baldotto & Baldotto, 2016).
Seeds priming has been recognized for improving germination rates by facilitating rapid water absorption to activate messenger ribonucleic acid (mRNA) and promote germination (Johnson & Puthur, 2021).For instance, biopriming of corn triggers DNA, RNA, and protein production (Nciizah et al., 2020).Timely germination is a critical concern for farmers, leading to the adoption of various priming techniques such as hydropriming, chemo-priming, osmo-priming, hormopriming, solid matrix priming, and nutri-priming to mitigate abiotic stress (Deshmukh et al., 2020;Lutts et al., 2016).
Brassica sp.possesses high sulfur-and nitrogencontaining secondary metabolites (Miao et al., 2021;Park et al., 2020), which are vital for plant defence against microbial pathogens and herbivorous insects (Chhajed et al., 2020).In Malaysia, Brassica sp.production reaching 0.15 Mt, accounted for 15% of total vegetable production in 2019 (Jabatan Pertanian Malaysia, 2019).Considering the high demand for Bok Choy, the application of priming is essential to help enhance seed germination, uniformity, and final stand (Ruttanaruangboworn et al., 2017).
To address food waste challenges, this study employed Sandwich compost leachate for seed priming, investigating seeds emergence and subsequent growth.Additionally, this study provided insight into agricultural food waste management.The inefficiently used potential of Sandwich compost leachate prompted the examination of its impact on Bok Choy seed germination, SPAD reading, and dry matter accumulation compared to hydropriming and dry seed germination.The major aim was to evaluate the effect of hydropriming and bio-nutri-priming using Sandwich compost leachate on seed germination, SPAD reading, and dry matter accumulation in 280 dwarf Bok Choy seeds.

Study Site and Experimental Design
The seeds germination tray study was conducted in Field 10 net house at the University of Putra Malaysia (UPM), located at the coordinates with latitude 2°59'34.0"Nand longitude 101°42'52.3"E.The experimental process employed a completely randomized design (CRD) with three replications, each consisting of 104 seeds.The Sandwich compost preparation method uses accumulated food waste (Phooi et al., 2022).A Sandwich taster was created by inoculating effective microbe (EM)-1 (commercially available) into rice bran and fermenting it anaerobically for two weeks.EM-1 comprised various microbes in a liquid culture with a pH below 3.5 (Higa, 2001;Higa & Parr, 1994).Cooked and raw food waste was alternately layered with the bran to create the compost.
On the 34 th day of fermentation, leachate was collected to serve as a bio-nutri-priming solution.Commercial 280 dwarf Bok Choy seeds were purchased from Green Eagle (Green Eagle Seeds, 2022).The seeds were bio-nutri-priming with 0.2% Sandwich compost leachate following a previously described method (Phooi et al., 2022), where 1 g seeds were soaked in 250 mL solution for 3 hours.A separate treatment involved hydropriming the seeds with tap water for 3 hours (Alias et al., 2018).
The control group was left unprimed, while the seeds were sown in 104-hole germination trays on moist peat moss.The diameter of the holes was 3.7 cm, and the peat moss was procured from Kekkila Professional.Top irrigation was carried out twice daily using tap water delivered through a hose to prevent soil water from drying out.The seeds were subjected to a 16:8 hours light/dark photoperiod at 32 °C (day) and 28 °C (night) and cultivated until day 30.Additionally, the samples were collected in two holes away from the tray margin.
The physicochemical properties of the priming solutions (tap water and Sandwich compost leachate) were determined, as presented in Table 1.Kjeldahl methods determined total nitrogen (Campbell & Hanna, 1937).A total of 1 g of 2 mm-sized soil and 5 mL of concentrated sulphuric acid with 5% salicylic acid were put in a digestion tube overnight.Subsequently, 0.3 g Na 2 S 2 O 3 and a single Kjeldahl tablet were added and digested until the mixture turned greyish-white.The resulting digest was diluted with distilled water up to 100 mL.A mixture of 10 mL of 30% NaOH and 10 mL extractant was subjected to distillation with 10 mL of 2% boric acid containing an indicator.The initial purple colour of the boric acid-indicator mixture changed to green.The green solution was collected after distilling it to a volume of 50 mL for titration with 0.01 N HCl.
Available phosphorous (P) was evaluated with the colourimetric method described by Murphy and Riley (Murphy & Riley, 1962).The extracting solution for available P was prepared by combining 60 mL of 1 N H 4 F and 200 mL of 1 N HCl in a 2000 mL volumetric flask.Reagent A was created by stirring with 12 g of ammonium molybdate and 148 mL H 2 SO 4 , following cooling, 0.2908 g antimony K tartaric was added.Reagent B was prepared by dissolving 1.32 g ascorbic acid and combining it with reagent A up to a volume of 250 mL.In this process, 2 g air-dried soil featuring a particle size <2 mm was mixed with 14 mL extracting solution in a 50 mL falcon tube, which was then shaken for 45 seconds at a constant speed.The resulting filtrate (2 to 5 mL) was added to 8 mL reagent B and made to reach 50 mL using distilled water in a volumetric flask.The solution was allowed to stand for 15 minutes and read spectrophotometrically at 885 nm.The nutrient contents, including (Ca), sodium (Na), manganese (Mn), iron (Fe), and zinc (Zn), were subsequently extracted (Mehlich, 1984;Minca et al., 2013) and analyzed with Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) (Optima 8300 ICP-OES, Perkin Elmer, Waltman, MA, USA).

Seeds Germination Test
Seed germination was identified when a visible protrusion with at least 0.2 cm length of the seedling radicle emerged.Germinated seeds were counted daily for seven days.The data obtained were analyzed using the "germinationmetrics" package in R statistical software by comparing germination traits (Aravind et al., 2019).Parameters assessed included germination percentage (GP), peak germination time, median germination time (t 50 ) (Coolbear), mean germination time ( Fe (ppm) 160±42.5 0.306±0.015* mean ± standard error.
The physicochemical properties of the priming solutions (tap water and Sandwich compost leachate) were determined, as presented in Table 1.Kjeldahl methods determined total nitrogen (Campbell & Hanna, 1937).A total of 1 g of 2 mm-sized soil and 5 mL of concentrated sulphuric acid with 5% salicylic acid were put in a digestion tube overnight.Subsequently, 0.3 g Na2S2O3 and a single Kjeldahl tablet were added and digested until the mixture turned greyish-white.The resulting digest was diluted with distilled water up to 100 mL.A mixture of 10 mL of 30% NaOH and 10 mL extractant was subjected to distillation with 10 mL of 2% boric acid containing an indicator.The initial purple colour of the boric acid-indicator mixture changed to green.The green solution was collected after distilling it to a volume of 50 mL for titration with 0.01 N HCl.
Available phosphorous (P) was evaluated with the colourimetric method described by Murphy and Riley (Murphy & Riley, 1962).The extracting solution for available P was prepared by combining 60 mL of 1 N H4F and 200 mL of 1 N HCl in a 2000 mL volumetric flask.Reagent A was created by stirring with 12 g of ammonium molybdate and 148 mL H2SO4, following cooling, 0.2908 g antimony K tartaric was added.Reagent B was prepared by dissolving 1.32 g ascorbic acid and combining it with reagent A up to a volume of 250 mL.In this process, 2 g air-dried soil featuring a particle size <2 mm was mixed with 14 mL extracting solution in a 50 mL falcon tube, which was then shaken for 45 seconds at a constant speed.The resulting filtrate (2 to 5 mL) was added to 8 mL reagent B and made to reach 50 mL using distilled water in a volumetric flask.The solution was allowed to stand for 15 minutes and read spectrophotometrically at 885 nm.The nutrient contents, including (Ca), sodium (Na), manganese (Mn), iron (Fe), and zinc (Zn), were subsequently extracted (Mehlich, 1984;Minca et al., 2013) and analyzed with Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) (Optima 8300 ICP-OES, Perkin Elmer, Waltman, MA, USA).

Seeds Germination Test
Seed germination was identified when a visible protrusion with at least 0.2 cm length of the seedling radicle emerged.Germinated seeds were counted daily for seven days.The data obtained were analyzed using the "germinationmetrics" package in R statistical software by comparing germination traits (Aravind et al., 2019).Parameters assessed included germination percentage (GP), peak germination time, median germination time (t50) (Coolbear), mean germination time ( ̅ ), variance germination time (S 2 T), standard error of germination time (s T ̅), coefficient of variation of the germination time (CVT), speed of accumulated germination (Saccumulated), mean germination percentage (%), Geoge's index (GR), and germination index (GI) (Melville).
The germination percentage was employed to measure the germination capacity (ISTA, 2015), as shown in Equation 1.
), variance germination time (S The physicochemical properties of the priming solutions (tap water and Sandwich compost leachate) were determined, as presented in Table 1.Kjeldahl methods determined total nitrogen (Campbell & Hanna, 1937).A total of 1 g of 2 mm-sized soil and 5 mL of concentrated sulphuric acid with 5% salicylic acid were put in a digestion tube overnight.Subsequently, 0.3 g Na2S2O3 and a single Kjeldahl tablet were added and digested until the mixture turned greyish-white.The resulting digest was diluted with distilled water up to 100 mL.A mixture of 10 mL of 30% NaOH and 10 mL extractant was subjected to distillation with 10 mL of 2% boric acid containing an indicator.The initial purple colour of the boric acid-indicator mixture changed to green.The green solution was collected after distilling it to a volume of 50 mL for titration with 0.01 N HCl.
Available phosphorous (P) was evaluated with the colourimetric method described by Murphy and Riley (Murphy & Riley, 1962).The extracting solution for available P was prepared by combining 60 mL of 1 N H4F and 200 mL of 1 N HCl in a 2000 mL volumetric flask.Reagent A was created by stirring with 12 g of ammonium molybdate and 148 mL H2SO4, following cooling, 0.2908 g antimony K tartaric was added.Reagent B was prepared by dissolving 1.32 g ascorbic acid and combining it with reagent A up to a volume of 250 mL.In this process, 2 g air-dried soil featuring a particle size <2 mm was mixed with 14 mL extracting solution in a 50 mL falcon tube, which was then shaken for 45 seconds at a constant speed.The resulting filtrate (2 to 5 mL) was added to 8 mL reagent B and made to reach 50 mL using distilled water in a volumetric flask.The solution was allowed to stand for 15 minutes and read spectrophotometrically at 885 nm.The nutrient contents, including (Ca), sodium (Na), manganese (Mn), iron (Fe), and zinc (Zn), were subsequently extracted (Mehlich, 1984;Minca et al., 2013) and analyzed with Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) (Optima 8300 ICP-OES, Perkin Elmer, Waltman, MA, USA).

Seeds Germination Test
Seed germination was identified when a visible protrusion with at least 0.2 cm length of the seedling radicle emerged.Germinated seeds were counted daily for seven days.The data obtained were analyzed using the "germinationmetrics" package in R statistical software by comparing germination traits (Aravind et al., 2019).Parameters assessed included germination percentage (GP), peak germination time, median germination time (t50) (Coolbear), mean germination time ( ̅ ), variance germination time (S 2 T), standard error of germination time (s T ̅), coefficient of variation of the germination time (CVT), speed of accumulated germination (Saccumulated), mean germination percentage (%), Geoge's index (GR), and germination index (GI) (Melville).
The germination percentage was employed to measure the germination capacity (ISTA, 2015), as shown in Equation 1.
The germination percentage was employed to measure the germination capacity (ISTA, 2015), as shown in Equation 1.The physicochemical properties of the priming solutions (tap water and S leachate) were determined, as presented in Table 1.Kjeldahl methods determin (Campbell & Hanna, 1937).A total of 1 g of 2 mm-sized soil and 5 mL of concentra with 5% salicylic acid were put in a digestion tube overnight.Subsequently, 0.3 g Na2 Kjeldahl tablet were added and digested until the mixture turned greyish-white.Th was diluted with distilled water up to 100 mL.A mixture of 10 mL of 30% NaOH and was subjected to distillation with 10 mL of 2% boric acid containing an indicator.The in of the boric acid-indicator mixture changed to green.The green solution was collecte to a volume of 50 mL for titration with 0.01 N HCl.
Available phosphorous (P) was evaluated with the colourimetric method des and Riley (Murphy & Riley, 1962).The extracting solution for available P was prepared mL of 1 N H4F and 200 mL of 1 N HCl in a 2000 mL volumetric flask.Reagent A was c with 12 g of ammonium molybdate and 148 mL H2SO4, following cooling, 0.2908 g an was added.Reagent B was prepared by dissolving 1.32 g ascorbic acid and combinin A up to a volume of 250 mL.In this process, 2 g air-dried soil featuring a particle size < with 14 mL extracting solution in a 50 mL falcon tube, which was then shaken for constant speed.The resulting filtrate (2 to 5 mL) was added to 8 mL reagent B and m mL using distilled water in a volumetric flask.The solution was allowed to stand fo read spectrophotometrically at 885 nm.The nutrient contents, including (Ca), sodium (Mn), iron (Fe), and zinc (Zn), were subsequently extracted (Mehlich, 1984; Minca analyzed with Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) ( OES, Perkin Elmer, Waltman, MA, USA).

Seeds Germination Test
Seed germination was identified when a visible protrusion with at least 0.2 seedling radicle emerged.Germinated seeds were counted daily for seven days.The d analyzed using the "germinationmetrics" package in R statistical software by compa traits (Aravind et al., 2019).Parameters assessed included germination percen germination time, median germination time (t50) (Coolbear), mean germination ti germination time (S 2 T), standard error of germination time (s T ̅), coefficient of germination time (CVT), speed of accumulated germination (Saccumulated), mean germin (%), Geoge's index (GR), and germination index (GI) (Melville).
The germination percentage was employed to measure the germination capa as shown in Equation 1.
The physicochemical properties of the priming solutions leachate) were determined, as presented in Table 1.Kjeldahl (Campbell & Hanna, 1937).A total of 1 g of 2 mm-sized soil and with 5% salicylic acid were put in a digestion tube overnight.Sub Kjeldahl tablet were added and digested until the mixture turne was diluted with distilled water up to 100 mL.A mixture of 10 mL was subjected to distillation with 10 mL of 2% boric acid containing of the boric acid-indicator mixture changed to green.The green s to a volume of 50 mL for titration with 0.01 N HCl.
Available phosphorous (P) was evaluated with the colour and Riley (Murphy & Riley, 1962).The extracting solution for avail mL of 1 N H4F and 200 mL of 1 N HCl in a 2000 mL volumetric fla with 12 g of ammonium molybdate and 148 mL H2SO4, following was added.Reagent B was prepared by dissolving 1.32 g ascorb A up to a volume of 250 mL.In this process, 2 g air-dried soil featu with 14 mL extracting solution in a 50 mL falcon tube, which w constant speed.The resulting filtrate (2 to 5 mL) was added to 8 mL using distilled water in a volumetric flask.The solution was read spectrophotometrically at 885 nm.The nutrient contents, inc (Mn), iron (Fe), and zinc (Zn), were subsequently extracted (Me analyzed with Inductively Coupled Plasma Optical Emission Spectr OES, Perkin Elmer, Waltman, MA, USA).
The germination percentage was employed to measure th as shown in Equation 1.
Peak germination represents when the highest frequen (Ranal & Santana, 2006).Median germination time (t50) (Coolbe reach 50% of the maximum germination rate (Coolbear, Francis Equation 2. Where t 50 denotes the median germination time, N is the final number of germinated seeds, and N i and N j stand for the total numbers of germinated seeds identified during adjacent counts at times T i and T j , respectively when N i < Mean germination time ( ̅ ) is an estimation of the average time need germination of a batch of seeds (Czabator, 1962;Ranal & Santana, 2006), as indi Where Ti represents the time from the beginning of the experiment to the <N j .The physicochemical properties of the priming solutions (tap water and Sandwich compost leachate) were determined, as presented in Table 1.Kjeldahl methods determined total nitrogen (Campbell & Hanna, 1937).A total of 1 g of 2 mm-sized soil and 5 mL of concentrated sulphuric acid with 5% salicylic acid were put in a digestion tube overnight.Subsequently, 0.3 g Na2S2O3 and a single Kjeldahl tablet were added and digested until the mixture turned greyish-white.The resulting digest was diluted with distilled water up to 100 mL.A mixture of 10 mL of 30% NaOH and 10 mL extractant was subjected to distillation with 10 mL of 2% boric acid containing an indicator.The initial purple colour of the boric acid-indicator mixture changed to green.The green solution was collected after distilling it to a volume of 50 mL for titration with 0.01 N HCl.
Available phosphorous (P) was evaluated with the colourimetric method described by Murphy and Riley (Murphy & Riley, 1962).The extracting solution for available P was prepared by combining 60 mL of 1 N H4F and 200 mL of 1 N HCl in a 2000 mL volumetric flask.Reagent A was created by stirring with 12 g of ammonium molybdate and 148 mL H2SO4, following cooling, 0.2908 g antimony K tartaric was added.Reagent B was prepared by dissolving 1.32 g ascorbic acid and combining it with reagent A up to a volume of 250 mL.In this process, 2 g air-dried soil featuring a particle size <2 mm was mixed with 14 mL extracting solution in a 50 mL falcon tube, which was then shaken for 45 seconds at a constant speed.The resulting filtrate (2 to 5 mL) was added to 8 mL reagent B and made to reach 50 mL using distilled water in a volumetric flask.The solution was allowed to stand for 15 minutes and read spectrophotometrically at 885 nm.The nutrient contents, including (Ca), sodium (Na), manganese (Mn), iron (Fe), and zinc (Zn), were subsequently extracted (Mehlich, 1984;Minca et al., 2013) and analyzed with Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) (Optima 8300 ICP-OES, Perkin Elmer, Waltman, MA, USA).

Seeds Germination Test
Seed germination was identified when a visible protrusion with at least 0.2 cm length of the seedling radicle emerged.Germinated seeds were counted daily for seven days.The data obtained were analyzed using the "germinationmetrics" package in R statistical software by comparing germination traits (Aravind et al., 2019).Parameters assessed included germination percentage (GP), peak germination time, median germination time (t50) (Coolbear), mean germination time ( ̅ ), variance germination time (S 2 T), standard error of germination time (s T ̅), coefficient of variation of the germination time (CVT), speed of accumulated germination (Saccumulated), mean germination percentage (%), Geoge's index (GR), and germination index (GI) (Melville).
The germination percentage was employed to measure the germination capacity (ISTA, 2015), as shown in Equation 1.
Where t50 denotes the median germination time, N is the final number of germinated seeds, and Ni and Nj stand for the total numbers of germinated seeds identified during adjacent counts at times Ti and Tj, respectively when Ni< +1 2 <Nj.
Mean germination time ( ̅ ) is an estimation of the average time needed for the complete germination of a batch of seeds (Czabator, 1962;Ranal & Santana, 2006), as indicated in Equation 3.
Where Ti represents the time from the beginning of the experiment to the i th interval, Ni is the number of seeds germinated within the i th time interval (not the count, but the count corresponding to the i th interval), and k signifies the total number of time intervals.It is the reciprocal of the mean germination rate ( ̅ ) indicated in Equation 4.
Variance germination time (S 2 T), standard error of germination time (s T ̅), and the coefficient of variation of the germination time (CVT) indicates the germination rate.Speed of accumulated germination (Saccumulated) denotes the rate of cumulative total germinated seeds during the time interval (Santana & Ranal, 2004).Mean germination percentage (G ̅ ) represents the final percentage of germinated seeds per the total time intervals required for final germination time (Czabator, 1962).Geoge's index (GR) quantifies the number of germinated seeds within a specific time interval (George, 1961).The germination index (GI) measures the timing of germinated seeds numbers from the starting day to the interval per the total number of tested seeds (Melville et al., 1980).

SPAD Reading
The chlorophyll concentration was non-destructively measured with a SPAD meter (Konica Minolta SPAD-502 Plus).SPAD reading was used to monitor plant growth and optimize nitrogen fertilization.Furthermore, its mean was immediately calculated before harvesting on day 30.Three mean readings per plant were recorded from the rib of the leaf at the same spot.

Dry Matter and Root-to-Shoot Ratio
Fresh root and shoot samples were washed, air-dried, weighed, and then oven-dried at 45℃ for 60 hours to determine the dry weight.The dry matter was expressed as a percentage using the formula: (fresh weight -dry weight)/dry weight x 100.The root-to-shoot ratio was computed by dividing root dry matter with dry shoot matter.

Statistical Analysis
Recorded data were subjected to the Shapiro-Wilk normality test (p>0.05).Subsequently, a one-way Analysis of Variance (ANOVA) was performed with the "agricolae" package in R statistical software (Mendiburu & Yaseen, 2020).When F values were significant at a p-level of 0.05, treatment means were compared and separated using the Least Significant Difference (LSD) (Mendiburu & Yaseen, 2020).

RESULTS AND DISCUSSION
No significant differences in seeds germination percentage (88.35±1.13%)were observed among the two priming treatments and the control, as presented in Table 2.In this study, seeds treated with bio-nutri-priming and hydropriming exhibited a peak germination time of 2 days, while their counterparts spent 3 days to reach the peak stage.Median germination time was the shortest (1.75 days) for bio-nutri-priming seeds, and the mean time ranged from 2.52 to 2.79 days for priming seeds, (3) Where T i represents the time from the beginning of the experiment to the i th interval, Ni is the number of seeds germinated within the i th time interval (not the count, but the count corresponding to the i th interval), and k signifies the total number of time intervals.It is the reciprocal of the mean germination rate ( in a 2000 mL volumetric flask.Reagent A was created by stirring nd 148 mL H2SO4, following cooling, 0.2908 g antimony K tartaric by dissolving 1.32 g ascorbic acid and combining it with reagent ocess, 2 g air-dried soil featuring a particle size <2 mm was mixed 50 mL falcon tube, which was then shaken for 45 seconds at a (2 to 5 mL) was added to 8 mL reagent B and made to reach 50 tric flask.The solution was allowed to stand for 15 minutes and .The nutrient contents, including (Ca), sodium (Na), manganese subsequently extracted (Mehlich, 1984;Minca et al., 2013) and sma Optical Emission Spectroscopy (ICP-OES) (Optima 8300 ICP-).
ied when a visible protrusion with at least 0.2 cm length of the seeds were counted daily for seven days.The data obtained were ics" package in R statistical software by comparing germination eters assessed included germination percentage (GP), peak on time (t50) (Coolbear), mean germination time ( ̅ ), variance rror of germination time (s T ̅), coefficient of variation of the umulated germination (Saccumulated), mean germination percentage ation index (GI) (Melville).
was employed to measure the germination capacity (ISTA, 2015), when the highest frequency of germinated seeds is observed rmination time (t50) (Coolbear) signifies the duration required to tion rate (Coolbear, Francis, & Grierson, 1984), as expressed in ) indicated in Equation 4. Mean germination time ( ̅ ) is an estimation of the average time needed for the complete germination of a batch of seeds (Czabator, 1962;Ranal & Santana, 2006), as indicated in Equation 3.
Where Ti represents the time from the beginning of the experiment to the i th interval, Ni is the number of seeds germinated within the i th time interval (not the count, but the count corresponding to the i th interval), and k signifies the total number of time intervals.It is the reciprocal of the mean germination rate ( ̅ ) indicated in Equation 4.
Variance germination time (S 2 T), standard error of germination time (s T ̅), and the coefficient of variation of the germination time (CVT) indicates the germination rate.Speed of accumulated germination (Saccumulated) denotes the rate of cumulative total germinated seeds during the time interval (Santana & Ranal, 2004).Mean germination percentage (G ̅ ) represents the final percentage of germinated seeds per the total time intervals required for final germination time (Czabator, 1962).Geoge's index (GR) quantifies the number of germinated seeds within a specific time interval (George, 1961).The germination index (GI) measures the timing of germinated seeds numbers from the starting day to the interval per the total number of tested seeds (Melville et al., 1980).

SPAD Reading
The chlorophyll concentration was non-destructively measured with a SPAD meter (Konica Minolta SPAD-502 Plus).SPAD reading was used to monitor plant growth and optimize nitrogen fertilization.Furthermore, its mean was immediately calculated before harvesting on day 30.Three mean readings per plant were recorded from the rib of the leaf at the same spot.

Dry Matter and Root-to-Shoot Ratio
Fresh root and shoot samples were washed, air-dried, weighed, and then oven-dried at 45℃ for 60 hours to determine the dry weight.The dry matter was expressed as a percentage using the formula: (fresh weight -dry weight)/dry weight x 100.The root-to-shoot ratio was computed by dividing root dry matter with dry shoot matter.

Statistical Analysis
Recorded data were subjected to the Shapiro-Wilk normality test (p>0.05).Subsequently, a one-way Analysis of Variance (ANOVA) was performed with the "agricolae" package in R statistical software (Mendiburu & Yaseen, 2020).When F values were significant at a p-level of 0.05, treatment means were compared and separated using the Least Significant Difference (LSD) (Mendiburu & Yaseen, 2020).

RESULTS AND DISCUSSION
No significant differences in seeds germination percentage (88.35±1.13%)were observed among the two priming treatments and the control, as presented in Table 2.In this study, seeds treated with bio-nutri-priming and hydropriming exhibited a peak germination time of 2 days, while their counterparts spent 3 days to reach the peak stage.Median germination time was the shortest (1.75 days) for bio-nutri-priming seeds, and the mean time ranged from 2.52 to 2.79 days for priming seeds, (4) Variance germination time (S 2 T ), standard error of germination time ( L of 2% boric acid containing an indicator.The initial purple colour nged to green.The green solution was collected after distilling it 0.01 N HCl.s evaluated with the colourimetric method described by Murphy extracting solution for available P was prepared by combining 60 in a 2000 mL volumetric flask.Reagent A was created by stirring d 148 mL H2SO4, following cooling, 0.2908 g antimony K tartaric by dissolving 1.32 g ascorbic acid and combining it with reagent ocess, 2 g air-dried soil featuring a particle size <2 mm was mixed 0 mL falcon tube, which was then shaken for 45 seconds at a (2 to 5 mL) was added to 8 mL reagent B and made to reach 50 ric flask.The solution was allowed to stand for 15 minutes and .The nutrient contents, including (Ca), sodium (Na), manganese subsequently extracted (Mehlich, 1984;Minca et al., 2013) and sma Optical Emission Spectroscopy (ICP-OES) (Optima 8300 ICP-).
ied when a visible protrusion with at least 0.2 cm length of the seeds were counted daily for seven days.The data obtained were ics" package in R statistical software by comparing germination eters assessed included germination percentage (GP), peak n time (t50) (Coolbear), mean germination time ( ̅ ), variance rror of germination time (s T ̅), coefficient of variation of the umulated germination (Saccumulated), mean germination percentage ation index (GI) (Melville).
as employed to measure the germination capacity (ISTA, 2015), when the highest frequency of germinated seeds is observed rmination time (t50) (Coolbear) signifies the duration required to tion rate (Coolbear, Francis, & Grierson, 1984), as expressed in ), and the coefficient of variation of the germination time (CV T ) indicates the germination rate.Speed of accumulated germination (S accumulated ) denotes the rate of cumulative total germinated seeds during the time interval (Santana & Ranal, 2004).Mean germination percentage ( C. L. Phooi et al. / agriTECH 43 (4) 2023, xxx-xxx notes the median germination time, N is the final number of germinated seeds, or the total numbers of germinated seeds identified during adjacent counts at ctively when Ni< ation time ( ̅ ) is an estimation of the average time needed for the complete h of seeds (Czabator, 1962;Ranal & Santana, 2006), as indicated in Equation 3.
(3) resents the time from the beginning of the experiment to the i th interval, Ni is the minated within the i th time interval (not the count, but the count corresponding and k signifies the total number of time intervals.It is the reciprocal of the mean indicated in Equation 4. (4) mination time (S 2 T), standard error of germination time (s T ̅), and the coefficient of rmination time (CVT) indicates the germination rate.Speed of accumulated ted) denotes the rate of cumulative total germinated seeds during the time interval 2004).Mean germination percentage (G ̅ ) represents the final percentage of r the total time intervals required for final germination time (Czabator, 1962).uantifies the number of germinated seeds within a specific time interval (George, on index (GI) measures the timing of germinated seeds numbers from the starting r the total number of tested seeds (Melville et al., 1980).yll concentration was non-destructively measured with a SPAD meter (Konica lus).SPAD reading was used to monitor plant growth and optimize nitrogen ore, its mean was immediately calculated before harvesting on day 30.Three lant were recorded from the rib of the leaf at the same spot.

ot-to-Shoot Ratio
d shoot samples were washed, air-dried, weighed, and then oven-dried at 45℃ rmine the dry weight.The dry matter was expressed as a percentage using the t -dry weight)/dry weight x 100.The root-to-shoot ratio was computed by dividing dry shoot matter.ta were subjected to the Shapiro-Wilk normality test (p>0.05).Subsequently, a Variance (ANOVA) was performed with the "agricolae" package in R statistical & Yaseen, 2020).When F values were significant at a p-level of 0.05, treatment d and separated using the Least Significant Difference (LSD) (Mendiburu & Yaseen, CUSSION t differences in seeds germination percentage (88.35±1.13%)were observed g treatments and the control, as presented in Table 2.In this study, seeds treated g and hydropriming exhibited a peak germination time of 2 days, while their days to reach the peak stage.Median germination time was the shortest (1.75 iming seeds, and the mean time ranged from 2.52 to 2.79 days for priming seeds, ) represents the final percentage of germinated seeds per the total time intervals required for final germination time (Czabator, 1962).Geoge's index (GR) quantifies the number of germinated seeds within a specific time interval (George, 1961).The germination index (GI) measures the timing of germinated seeds numbers from the starting day to the interval per the total number of tested seeds (Melville et al., 1980).

SPAD Reading
The chlorophyll concentration was non-destructively measured with a SPAD meter (Konica Minolta SPAD-502 Plus).SPAD reading was used to monitor plant growth and optimize nitrogen fertilization.Furthermore, its mean was immediately calculated before harvesting on day 30.Three mean readings per plant were recorded from the rib of the leaf at the same spot.

Dry Matter and Root-to-Shoot Ratio
Fresh root and shoot samples were washed, airdried, weighed, and then oven-dried at 45°C for 60 hours to determine the dry weight.The dry matter was expressed as a percentage using the formula: (fresh weight -dry weight)/dry weight x 100.The root-toshoot ratio was computed by dividing root dry matter with dry shoot matter.

Statistical Analysis
Recorded data were subjected to the Shapiro-Wilk normality test (p>0.05).Subsequently, a one-way Analysis of Variance (ANOVA) was performed with the "agricolae" package in R statistical software (Mendiburu & Yaseen, 2020).When F values were significant at a p-level of 0.05, treatment means were compared and separated using the Least Significant Difference (LSD) (Mendiburu & Yaseen, 2020).

RESULTS AND DISCUSSION
No significant differences in seeds germination percentage (88.35±1.13%)were observed among the two priming treatments and the control, as presented in Table 2.In this study, seeds treated with bio-nutripriming and hydropriming exhibited a peak germination time of 2 days, while their counterparts spent 3 days to reach the peak stage.Median germination time was the shortest (1.75 days) for bio-nutri-priming seeds, and the mean time ranged from 2.52 to 2.79 days for priming seeds, which was faster than non-priming seeds.SPAD reading of bio-nutri-priming seeds showed significant differences among treatments in this study, but no substantial variations were found in the dry matter of the plants.

Seeds Emergence Performance in Bio-Nutri-Priming Seeds
The significantly elevated germination rate observed in priming seeds, compared to none-priming seeds, could be attributed to metabolic repairing and increased production of metabolites needed for germination during the imbibition process (Afzal et al., 2016).Hydropriming and bio-nutri-priming facilitated the dehydration of seeds post-priming, followed by reinitiation under favorable conditions for cell elongation and radicle protrusion (Lutts et al., 2016).Moreover, protein expression post-priming improved seed emergence (Afzal et al., 2016).
Bio-nutri-priming outperformed hydropriming, probably due to the leachate nutrient storage within the seeds aided by microbes during seed germination phase III, namely cell elongation, leading to radicle protrusion (Afzal et al., 2016).Similarly, previous studies showed that Agnihotra ash, compost tea, and hot water priming significantly enhanced germination rates in treated rosemary seeds compared to dry seeds (Sharma et al., 2019).The examined seeds stored a substantial amount of carbohydrates to endure low oxygen stress conditions, conferring improved germination in those subjected to bio-nutri-priming (Deshmukh et al., 2020;Paparella et al., 2015).
s identified when a visible protrusion with at least 0.2 cm length of the rminated seeds were counted daily for seven days.The data obtained were tionmetrics" package in R statistical software by comparing germination ).Parameters assessed included germination percentage (GP), peak ermination time (t50) (Coolbear), mean germination time ( ̅ ), variance ndard error of germination time (s T ̅), coefficient of variation of the d of accumulated germination (Saccumulated), mean germination percentage d germination index (GI) (Melville).
centage was employed to measure the germination capacity (ISTA, 2015), presents when the highest frequency of germinated seeds is observed edian germination time (t50) (Coolbear) signifies the duration required to germination rate (Coolbear, Francis, & Grierson, 1984), as expressed in (2) was diluted with distilled water up to 100 mL.A mixture of 10 mL of 30% NaOH and 10 mL extractant was subjected to distillation with 10 mL of 2% boric acid containing an indicator.The initial purple colour of the boric acid-indicator mixture changed to green.The green solution was collected after distilling it to a volume of 50 mL for titration with 0.01 N HCl.Available phosphorous (P) was evaluated with the colourimetric method described by Murphy and Riley (Murphy & Riley, 1962).The extracting solution for available P was prepared by combining 60 mL of 1 N H4F and 200 mL of 1 N HCl in a 2000 mL volumetric flask.Reagent A was created by stirring with 12 g of ammonium molybdate and 148 mL H2SO4, following cooling, 0.2908 g antimony K tartaric was added.Reagent B was prepared by dissolving 1.32 g ascorbic acid and combining it with reagent A up to a volume of 250 mL.In this process, 2 g air-dried soil featuring a particle size <2 mm was mixed with 14 mL extracting solution in a 50 mL falcon tube, which was then shaken for 45 seconds at a constant speed.The resulting filtrate (2 to 5 mL) was added to 8 mL reagent B and made to reach 50 mL using distilled water in a volumetric flask.The solution was allowed to stand for 15 minutes and read spectrophotometrically at 885 nm.The nutrient contents, including (Ca), sodium (Na), manganese (Mn), iron (Fe), and zinc (Zn), were subsequently extracted (Mehlich, 1984;Minca et al., 2013) and analyzed with Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) (Optima 8300 ICP-OES, Perkin Elmer, Waltman, MA, USA).

Seeds Germination Test
Seed germination was identified when a visible protrusion with at least 0.2 cm length of the seedling radicle emerged.Germinated seeds were counted daily for seven days.The data obtained were analyzed using the "germinationmetrics" package in R statistical software by comparing germination traits (Aravind et al., 2019).Parameters assessed included germination percentage (GP), peak germination time, median germination time (t50) (Coolbear), mean germination time ( ̅ ), variance germination time (S 2 T), standard error of germination time (s T ̅), coefficient of variation of the germination time (CVT), speed of accumulated germination (Saccumulated), mean germination percentage (%), Geoge's index (GR), and germination index (GI) (Melville).
The germination percentage was employed to measure the germination capacity (ISTA, 2015), as shown in Equation 1.
The seed germination index of Brassica napus L. was amplified by biopriming with bacteria (Bacillus subtilis) and fungus (Macrophomina phaseolina) (Mousavi & Omidi, 2019).Bacteria priming has been observed to increase seed germination percentage in saline conditions (Chu et al., 2019;Ghorbanpour & Hatami, 2014) and shorten the mean germination time to 1.28-4.25 days (Ghorbanpour & Hatami, 2014;Mousavi & Omidi, 2019).This impact was assumed to be triggered by the completion of pre-germination metabolite activity and reorganization of membrane structure (Muhammad et al., 2015).Dry seeds coated with Pseudomonas fluorescens AB254 facilitated germination through improved water imbibition, leading to a moisture content reaching 35-40% (Callan, 1990;Sisodia et al., 2018).Furthermore, biopriming with bacterial antagonists boosted the antagonist population, protecting the rhizosphere from plant pathogen infection (Mahmood & Kataoka, 2018).The effectiveness of biopriming using plant growth-leachate as a bio-nutri-priming solution held promising potential for addressing food waste challenges while concurrently promoting plant growth.The substantial improvements in germination index and median germination time observed in bio-nutri-priming seeds were attributed to improved nutrient storage within seeds facilitated by microbial assistance.Furthermore, increased SPAD reading could reduce fertilizer requirements due to augmented nutrient use efficiency post bio-nutri-priming.This observation indicated the advantageous role of Sandwich compost leachate in priming, such as the enhancement of seed germination and leaf greenness.The use of this approach aligned with the demand for leafy vegetables in Malaysia, contributing to food and nutritional security promotion.

Enhancement of SPAD Reading through Bio-Nutri-Priming
Biopriming has been found to elevate SPAD values significantly (Roslan et al., 2020), corroborating the results of this study.Based on previous investigations, SPAD reading showed a high correlation (r 2 =0.94) with chlorophyll content (Chu et al., 2019;James et al., 2002).High chlorophyll content was observed in a plant grown from biopriming seeds (Singh et al., 2020).The improved nutrient uptake in bio-nutri-priming plants supported root development (Sarkar, Sankar, et al., 2021).Bio-nutri-priming also enhanced soil microbe activity, mineralising organic nutrients into inorganic forms that could be retained as soil solution (Sarkar, Sankar, et al., 2021).Superior assimilation in plants contributed to higher SPAD readings (Sarkar, Sankar, et al., 2021).Therefore, bio-nutri-priming enhanced nitrogen use efficiency (Sarkar, Rakshit, et al., 2021).Another contributing factor might be the noninvolvement of indirect mechanisms in the synthesis and activity of antioxidant enzymes such as peroxidase, catalase, and superoxide dismutase in host tissues capable of harming the plant (Singh et al., 2020).These observations aligned with previous studies indicating significant improvements in chlorophyll biosynthesis and photosynthetic activity (Chitra & Jijeesh, 2021;Zulueta-Rodríguez et al., 2015).Further nutrient analysis could improve the priming method, specifically regarding nutrient content, as SPAD reading correlated with ascorbic acid content (Yaseen & Takacs-Hajos, 2022).

Shoot and Root Dry Matter
Biopriming has been shown to augment plant biomass (Sivakumar et al., 2017).The lack of significant influence from biopriming on plant dry matter in this study might be due to the plants being in a similar growth phase.Therefore, further exploration should be conducted on aspects including plant growth performance, omics (e.g., metabolomics and proteomics), and responses to biotic and abiotic stressors (e.g., pest infection, saline, and drought).Investigations related to destructive sampling could enhance the understanding of dry matter accumulation across different growth cycle stages.

CONCLUSION
In conclusion, bio-nutri-priming using Sandwich compost leachate significantly enhanced mean germination time and SPAD reading.The addition of this C. L.Phooi et al. / agriTECH 43 (4) 2023, xxx-xxx    Where t50 denotes the median germination time, N is the final number and Ni and Nj stand for the total numbers of germinated seeds identified durin times Ti and Tj, respectively when Ni< +1 2 <Nj.
C. L.Phooi et al. / agriTECH 43 (4) 2023, xxx-xxx    Where t50 denotes the median germination time, N is the final number of germinated seeds, and Ni and Nj stand for the total numbers of germinated seeds identified during adjacent counts at times Ti and Tj, respectively when Ni< germination time (S2T), standard error of germination time (ST), coefficient of variation of the germination time (CVT), speed of accumulated germination (Saccumulated), mean germination percentage ( C. L. Phooi et al. / agriTECH 43 (4) 2023, xxx-xxx

Table 1 .
The physicochemical properties of sandwich compost leachate and tap water *mean ± standard error.

Table 2 .
Germination performance, root-to-shoot ratio, and root and shoot dry matter based on seeds treatments including bio-nutri-priming, hydropriming, and no priming Means with the same letter were not significantly different between treatments using LSD.*** p<0.00, ** p<0.001, * p<0.01, and p>0.05: not significant.

Table 2 .
Germination performance, root-to-shoot ratio, and root and shoot dry matter based on seeds treatments including bio-nutri-priming, hydrop and no priming