# Influence of environmental factors on seed germination and seedling characteristics of perennial ryegrass (Lolium perenne L.)

### Site description and seed collection

The experiments were carried out at Agronomy Laboratory, Department of Agriculture, Sargodha University, Pakistan (32.0°N and 72.6°E) in 2018. Mature seeds were collected in March 2017 and 2018 in several fallow farms in Layyah and Sargodha. Punjab, Pakistan, and a global sample was prepared. At the time of collection, the seeds were harvested by breaking the stem of the plant about 10 cm in length with a spike each year separately. After that, paper bags were used to transport the seeds to the laboratory, the seeds were detached from the plant spike, cleaned and air-dried at room temperature for 7 days.

Working samples were drawn from the composite sample. After that, the seeds were stored in airtight glass bottles until used in germination experiments.

### General germination test protocol

Prior to the start of subsequent germination or emergence tests, perennial ryegrass seeds were sterilized in 1% sodium hypochlorite (NaClO) for 5 min, then rinsed with distilled water 5 times20. Germination of perennial ryegrass was determined by placing 20 seeds in a 9 cm diameter Petri dish fitted with Whatman No. 10 filter paper, moistened with 3 ml of distilled water or the applicable treatment solution.14. Para-film was used to seal the Petri dishes to prevent water loss. Petri dishes were stored in a germination cabinet (Seedburo Equipment Company, Chicago, IL, USA). Cool white fluorescent bulbs (FL40SBR; National, Tokyo, Japan) were used to produce a photosynthetic photon flux density of 200 µmol m−2 s−1, set to an alternating light/dark cycle of 12 h for all experiments. All experimental tests were carried out at a daytime and nighttime temperature of 25°C, except for the temperature experiment. Seeds were designed to germinate when the radicle reached 2 mm in length. The germinated seeds were counted daily for 30 days. However, in the seed burial depths experiment, when the cotyledon was visible on the soil surface, the seedlings were considered to have emerged. Each experiment was repeated twice using seeds collected in two different years. Each treatment was repeated four times in each experiment.

### Impact of temperature

To study the impact of temperature on the germination of perennial ryegrass, twenty seeds were placed evenly in a Petri dish, lined with filter paper under the seed moistened with 3 mL distilled water and then stored in a incubator at constant temperatures of 20, 25, 30 and 35°C for 15 days.

### Impact of pH

The impact of pH on seed germination of perennial ryegrass was assessed using buffer solutions of pH 5, 6, 7, 8, 9 and 10 which were made according to the method defined by Chachalis and Reddy21. A 2 mM solution of MES [2-(N-morpholino) ethanesulfonic acid] was adjusted to pH 5 or 6 with 1 N hydrochloric acid (HCl) and 2 mM HEPES solution [N-(2-hydroxy-methyl) piperazine-N-(2-ethanesulfonic acid)] was adjusted to pH 7 or 8 with 1 N NaOH. Buffer solutions of pH 9 and 10 were prepared with 2 mM TRICINE [N Tris (hydroxymethyl) methylglycine] and adjusted to each respective pH value with 1 N NaOH. Unbuffered deionized water (pH 6.2) was used as a control.

### Impact of salt stress

To determine the influence of salt stress on perennial ryegrass seed germination, 20 seeds were placed in sealed petri dishes containing various concentrations of sodium chloride (NaCl) at 0, 50, 100, 150, 200 , 250 and 300 mM. However, distilled water was used as a control treatment.

### Impact of osmotic stress

Perennial ryegrass seeds were placed in Petri dishes with an osmotic potential of 0, −0.2, −0.4, −0.6, −0.8, and −1.0 MPa. Osmotic potentials were performed using polyethylene glycol (PEG 8000; Sigma-Aldrich Co., 3050, Spruce St., MO 63130) in distilled water. The equation described by Michel and Kaufmann22 was used for water potential calculation from a known concentration of PEG 6000. Distilled water was used as control treatment.

Water potential = − (1.18 × 10–2) C − (1.18 × 10–4)VS2+ (2.67 × 10–4) 18 CT + (8.39 × 10-seven )VS2T. Where: T represents the temperature in degrees centigrade while C is the PEG concentration.

### Impact of seed burial depth

The impact of seed burial depth on seed emergence was studied in the greenhouse of the College of Agriculture, Sargodha University, Pakistan. Twenty perennial ryegrass seeds were planted on the soil surface or covered with soil (30% silt, 30% clay and 40% sand) at sowing depths of 0, 1, 2, 3, 4, 5, 6 and 7 in 15 cm diameter plastic pots. Throughout the experiment, the greenhouse temperature was maintained at 25 ± 2°C during the day and night. The pots were watered as needed to maintain sufficient soil moisture. Seedlings were considered to have emerged when the cotyledons were visible on the soil surface.

Percentage germination or emergence data of perennial ryegrass obtained from experiments regarding osmotic stress, NaCl concentration and seed burial depth were subjected to nonlinear regression analysis. Germination percentage data at various concentrations of osmotic potential and NaCl were fitted to a 3-parameter logistic model using Sigma Plot 2008 software (version 11.0, SyStat Software GmbH, Schimmelbuschstrasse 25 D-40699 Erkrath Germany). The fitted model was:

$$G(% ) = frac{{mathop Gnolimits_{max } }}{1 + mathop {left( {frac{x}{{mathop xnolimits_{50} } }} right)}nolimits^{g} }}$$

(1)

where g is the total percentage of germination at the concentration X, X50 is the osmotic potential or NaCl concentration for 50% suppression of maximum sprouting and g denotes the slope and gmaximum is the maximum germination percentage.

A logistic model with three parameters:

$$Eleft( % right) = frac{{E_{max} }}{{1 + left( {frac{x}{{x_{50} }}} right)^{e } }}$$

(2)

was set at the percentage of emergence of seedlings obtained at different burial depths from 0 to 7 cm, where E is the total percentage of seedling emergence at the burial depth X, X50 is the planting depth for 50% suppression of maximum seedling emergence and e denotes the slope, Emaximum is the maximum percentage of seedling emergence.

The time to germination or initial emergence (Ti) was recorded when the first seed germinated or emerged. The time to 50% germination or emergence (J50 Where E50) was estimated using a formula described by Coolbear et al.23:

$$T_{50} ;ou;E_{50} = t_{i} frac{{left( {frac{N}{2} – n_{i} } right)(t_{j} – t_{i} )}}{{(n_{j} – n_{i} )}}$$

(3)

where NOT is the final number of seeds germinated or emerged, and notI and notI are the cumulative number of seeds germinated by adjacent counts at times youI (day) and youI(day), respectively, when notINOT/2notI .

Average time to germination or emergence ( GMTWhere MEET ), which is a measure of germination or emergence rate, was calculated from Ellis and Roberts24 :

$$MGT;or;MET = frac{{sum {Dn} }}{sum n }$$

(4)

where not is the number of seeds that germinated during the dayD and not is the number of days counted from the start of the germination experiment.

The germination or emergence index (GIWhereIE), which is a measure of percentage and germination rate was calculated as described by the Association of Official Seed Analysis25using the following formula:

$$GI;{text{or}};EI = frac{{Number;of;germinated;{text{or}};emerged;plants}}{Days;of ;first;count} + cdots + { }frac{{No;of;gerimated;{text{or}};emerged;seeds}}{Days;of;final; count}$$

(5)

Data for root length (cm), shoot length (cm), root fresh weight (g), root dry weight (g), shoot fresh weight (g) and root dry weight (g) Perennial rye shoots were collected throughout the study using standard procedures.

### statistical analyzes

All experiments were performed in a completely randomized design (CRD) with four repetitions and each experiment was repeated twice. The data collected was subjected to a one-way ANOVA. The significance of the treatment means was practiced using the least significant difference (LSD) test at a probability level of 5%.26.

### Research Ethics

Experimental research and field studies on plants (cultivated or wild), including collection of plant material, complied with relevant institutional, national and international guidelines and legislation. Prior permission was obtained from the farm owner and the research, innovation and commercialization offices of Sargodha University, Pakistan.