Effects of rootzone CO₂ and O₂ levels on seed germination and stolon growth of Cynodon dactylon

Abstract

The role of rootzone CO₂ and O₂ levels on seed germination and plant growth is not fully defined. The aim of the present study was to investigate the effects of supraoptimal CO₂ in combination with different O₂ concentrations on seed germination and the growth of pre-rooted stolon segments of Cynodon dactylon. Seeds of “Princess 77” were germinated in specialized airtight tubes for 21 days. Gas mixtures containing 40–20, 40–10, 40–2.5% CO₂–O₂, or air were injected every 12 h. The establishment of bermudagrass stolons took place in micro-lysimeters for 28 days, while the rootzone was injected with either ambient air (control) in natural conditions or with the above-mentioned gas mixtures. For the determination of the effects of soil gases on stolon growth, the cumulative clippings dry weight, the root dry weight, the root total area and total length were recorded. The leaf chlorophyll, the relative water content, and the soluble sugars of both leaves and roots were also measured. The presence of 40–20, 40–10 and 40–2.5% CO₂–O₂ increased the final germination compared with the control. The gas mixtures as rootzone gases, affected the plant growth rate differently. The root growth was suppressed in all three gas treatments, but fructose and glucose root concentrations of the stolons treated with 40–2.5% CO₂–O₂ were significantly higher 28 days after the initiation of the gas treatments (DGT). Plants under the 40–20% CO₂–O₂ treatment showed initially a high sward growth. The Chlb concentration showed significant reduction under 40–10 and 40–2.5% CO₂–O₂, while the Chla/b of the leaves increased in the treatment with 40–2.5 CO₂–O₂ 28 DGT.

Keywords:

• Bermudagrass
• chlorophyll content
• growth rate
• landfills
• relative water content
• root system

Introduction

The effect of soil atmosphere composition on the establishment and growth of turfgrass species is an intriguing issue that challenges turfgrass managers. It is documented that the concentration of underground gases changes in turfgrass stands, such as golf course greens (Bunnell and McCarty 1999) under various abiotic stresses. The concentration of CO₂ is elevated whilst depletion of soil O₂ occurs during hot weather, through enhanced root and microbial respiration (Waddington and Baker 1964, Williamson 1964). Moreover for the rehabilitation of landfills and unsanitary landfills coverage by turfgrasses is recommended by the Environmental Protection Agency (2001) due to the shallow root system of the plants and their adaptability to several stresses. Basri (1998) and Morgan et al. (1993) reported advantages of using waste landfills as parks and recreational areas, such as cheap land use, proximity to urban centers, and flat topography suitable for park and golf course construction. They also reported an increase in the real estate value of lands after restoration. A major problem for the restoration, however, is biogas migration by diffusion through the landfill cap, especially in older or unsanitary landfill sites that are usually unlined. Methane and carbon dioxide represent about 90% of the total volume of biogas in a ratio of 1:1 (Arthur et al. 1981). In a case study, Christophersen et al. (2001) recorded a high CO₂ flux during the occurrence of high soil temperatures and moisture, and lateral gas migration to adjacent soils. In addition Scheutz and Kjeldsen (2006) reported a possible elevation of CO₂ concentration up to 35% in the first 10 cm of soil columns flushed with gas mixtures of 50% CH₄–50% CO₂.

The effects of gas concentration on seed germination of turfgrass species are not yet defined, whilst even though there are studies on other species, the results are often controversial. Baskin and Baskin (2001) reported that Oryza sativa and Echinochloa crus-galli are the only two species whose seeds are capable of germinating in almost anoxic conditions, while Leather et al. (1992) attributed the reduced germinability of dormant caryopses of Echinochloa crus-galli under 75% or 100% CO₂ as being likely due to the inhibition of root protrusion. In addition, Marchiol et al. (2000) reported that simulated landfill gas concentration of 16% O₂, 8% CO_2, 3% CH₄, and 73% N₂ resulted in delayed seed germination of Vicia villosa and Lotus corniculatus, while no significant result was observed in seeds of Trifolium pratense and T. repens.

The negative effects of high CO₂ rootzone concentration levels on Agrostis stolonifera have been reported in earlier studies. A very poor visual quality under 10% soil CO₂ concentration along with stunted root growth was recorded (Bunnell et al. 2002), while Rodriguez et al. (2005) reported an imbalance between respiration and photosynthesis at high CO₂ concentrations. Furthermore, the decline of turf growth, quality, chlorophyll content, and net photosynthetic rate under low soil aeration of A. stolonifera was reported from Huang et al. (1998).

Several researchers have also reported an effect of the soil gas atmosphere on sugar concentrations of the upper plant parts and the root system (Huang and Johnson, 1995, Albrecht et al. 2004), and their probable role in a mechanism of plant protection.

Bermudagrass (Cynodon dactylon L. Pers.) is a warm season turfgrass species that exhibits tolerance mechanisms under submergence (Tan et al. 2010), although Ashraf and Yasmin (1991) reported sensitivity of an ecotype of the species, under continuous waterlogging. Due to the absence of essential information and contradicting results of the current literature, we studied the effects of supraoptimal CO₂ concentrations combined with varied O₂ levels on bermudagrass seed germination and vegetative propagation.

Materials and methods

Seed germination

Seeds of Cynodon dactylon L. Pers. “Princess 77” (Desert Sun, Phoenix AZ, USA) coated with Penokoted Locked-On^TM were germinated at 30/25 °C day/night temperatures, for 21 days as is indicated by ISTA (1999). For each gas treatment 200 seeds were placed for germination in four transparent plexiglass tubes (50 seeds/tube) having a length of 250 mm and an internal diameter of 14 mm, on sterilized filter paper soaked with 0.5 mL sterilized distilled water per tube. During the germination period water was added as needed in order to keep the filter paper moist. Both ends of the tubes were closed airtight with silicon rubbers connected to on-off valves, while for the gas flushing a system of silicon pipes was used. For the gas treatments standardized mixtures were applied, while the control was supplied with ambient air. The concentrations of the gas mixtures were the following: 40% CO₂ either with 20%, 10% or 2.5% O₂ in 40%, 50%, or 57.5% N₂ respectively. The application of gas mixtures or ambient air was performed manually every 12 hours and the flux and final concentration were measured at the outlet valve with a detector for landfill gas GA94 (Geotechnical Instruments, Ltd., UK) via an infrared detector for CO₂ and an electrochemical detector for O₂ concentration measurement.

For the determination of the effects of the gas environment, the number of germinating seeds was recorded daily and the time to reach 50% of cumulative germination of viable seeds (T₅₀) was evaluated.

Stolon establishment and growth

For the determination of the effects of gas concentration in the rootzone on the vegetative establishment of bermudagrass, three node stolon segments were obtained from a plantation of C. dactylon “Princess 77.” The maternal plantation was growing in a heated greenhouse during the winter months, under long days. The long day conditions were achieved with supplemental photoperiod light from 17:00 h to 21:00 h, provided by 100 W incandescent bulbs, having light intensity 40 µmol m⁻² s⁻¹, at plant level. The cultivation techniques were as needed for the optimal growth of the turf, and the cutting height was 25 mm. Stolons of the same age and similar thickness were cut on May 2007 and three node segments having 2 to 3 leaves were put for a day in water for root induction. The rooted segments were rinsed with tap water and placed in the micro-lysimeters for further rooting.

Micro-lysimeters

The establishment and growth of stolons took place in airtight PVC micro-lysimeters sized 100 mm length×100 mm width×150 mm height. At the bottom and the two alternative vertical sides, on-off valves were adjacent, each having an attached PVC tube of 0.95 cm. The bottom valve was the inlet of irrigation water and fertilizer solution or drainage of the excess of liquids. For irrigation and fertilization the tube system was connected with a peristaltic pump (Newport Electronics, Inc., USA). The other two valves served as inlet (the lower one) and outlet (the upper one) of the gas mixtures. On the top of each micro-lysimeter a 5 mm thick opaque cap was placed, having 81 holes with 5 mm diameter.

At the bottom of each micro-lysimeter two pieces of Enkamat (Colbond, the Netherlands) totalling a height of 40 mm with cheesecloth on top were placed to prevent downwards substrate particle migration (Figure 1). The substrate was composed of screened perlite with particle distribution of 2.5–5.0 mm, with a 10-mm top layer comprised of sandyloam soil to support the stolons’ establishment. Before planting the micro-lysimeters were saturated with water and allowed to drain freely for 24 h. Preliminary tests were performed to determine the irrigation needs in order to maintain field capacity during the experimental period. Plants were fertilized weekly by the application of full-strength Hoagland's solution via the irrigation system. The pre-rooted stolon segments having roots less than 5 mm long, were placed manually in the holes, and left for 5 days to acclimatize in the growth chamber. After this period each one of the 81 holes that included a single stolon was sealed – with the help of a syringe – using aquarium silicone, in order to completely separate the rootzone with the injected gases from the aerial plant parts. The treatments were gas mixtures consisting of 40% CO₂ either with 20%, or 10% or 2.5% O₂ in 40%, 50% or 57.5% N₂ respectively. Injection of the root zone with ambient air served as the control. The concentration of the gases at the outlet was measured manually three times per day with a detector for landfill gas GA94 (Geotechnical Instruments, Ltd., UK). The plants were kept in a controlled environment under 30/25 °C day/night temperatures with light intensity 450 µmol m⁻² s⁻¹, 16/8 h daylength photoperiod and 45% RH for 28 days after the initiation of the gas treatments (DGT).

Figure 1.  Representation of the micro-lysimeter used for the stolon establishment experiment.

Growth parameters

For the evaluation of supraoptimal rootzone CO₂ combined with different O₂ levels, on sward growth, the cumulative clipping yield was measured. Mowing was performed with a motorized hand-held trimmer at 25 mm height at weekly intervals. The clippings were oven dried at 75 °C for 48 h and weighed. For the determination of root growth four destructive measurements were performed at 7, 14, 21, and 28 DGT, and the total area, the total length, and the dry weight were measured according to Nektarios et al. (2004). More specifically after thorough washing, the roots were stained with 0.1% trypan blue FAA. The stained roots were scanned with a flat-bed scanner, and analysis of the black and white pictures was performed with Delta-T SCAN Image Analysis (Delta-T Devices Ltd., UK) of the total area and total length. For the determination of the physiological response of bermudagrass under altered rootzone gas concentration, the relative water content (RWC) of the leaves was measured as described by Jiang and Huang (2001). Fully expanded leaves were weighed immediately after sampling, and were placed in deionized water for 4 h, and the turgid weight was obtained. The leaves were oven dried afterwards for 24 h and the dry weight was recorded.

Leaf chlorophyll (Chl) content was determined as referred by Arnon (1949). Fresh leaf tissue weighing 0.5 g was lyophilized in a mortar with 5 mL of 80% acetone aqueous solution. The extract was dissolved up to volume of 100 mL with the same solution, and measured in 663 and 645 nm with a spectrophotometer (Lamda1, Perkin Elmer, USA).

The content of sucrose, glucose, and fructose, of the leaf blades, and the roots were also determined. The tissues were put immediately after sampling for freeze drying in a Heto LyoLab 3000 (Heto-Holten A/S, Allerød, Denmark) freeze drier for 3 days and reweighed. The dry material (40–50 mg) was washed with 5 mL petroleum ether and the sugars extracted twice with 80% ethanol. The ethanol was removed by a stream of nitrogen and the residue dissolved in HPLC-grade water (1.0–2.0 mL) to dissolve the sugars, and charcoal (10–12 mg) added to decolorize the solution. After centrifugation at 2950×g for 5 min the supernatant was removed for HPLC analysis (Piccaglia and Galleti 1988). Samples (20 µL) were injected into a 305×7.8 mm HC-75 Ca^{+ +} column (Hamilton BonaduzAG, Bonaduz, Switzerland) at 65–75 °C, with the mobile phase of HPLC grade water supplied by a Hewlett-Packard 1050 isocratic pump (Hewlett-Packard GmbH, Boblingen, Germany) at 0.8 mL min⁻¹. After leaving the column, the sugars were detected using a refractive index detector (Hewlett-Packard HP 1047A, Hewlett-Packard GmbH, Boblingen, Germany).

Experimental setup and statistical analysis

The experimental setup was a completely randomized design, with four gas treatments (including control) and four replicates per treatment for the seed germination trial, while the stolon growth and physiological trials had six replicates per treatment. All the experiments were replicated twice, with similar results in both replications. Analysis of variance was performed to all data with STATGRAPHICS Centurion XV and the means were compared using Fisher's least significance difference at 0.05 probability level. For the comparison of means of the same treatments on different dates t-test was used at 0.05 probability level.

Results and discussion

The presence of supraoptimal CO₂ promoted the total germination percentage of bermudagrass seeds, regardless of the O₂ concentration (Table I). No differences were observed in total germination percentage of seeds (approximately 71%) in 40% CO₂–20% O₂ or 40% CO₂–10% O₂, but both were significantly higher compared with control. However, the T₅₀ increased by 2 days when O₂ decreased to 10% (Table I). The precise mode of action of elevated CO₂ levels on bermudagrass seed germination cannot be easily explained, but it can be hypothesized that elevated CO₂ could trigger the carbonic anhydrase, phosphoenolpyruvate carboxylase system present in seeds (Khayat 1991, Aivalakis et al. 2004) for assimilation of CO₂. In such a case, carbonic anhydrase could provide bicarbonates for phosphoenolpyruvate carboxylase to produce oxaloacetate, a dicarboxylic acid of Kreb's cycle. Similar results were obtained by Bibbey (1948) who recorded a positive effect of 4.8–7.2% CO₂ on the germination of dormant seeds of Avena fatua, and Yoshioka et al. (1995) reported a 10% increase of Setaria faberi seeds under 300 mmol mol⁻¹ CO₂ concentration.

Table I. Total seed germination (%) and T₅₀ (days) of bermudagrass seeds being treated with: 40–20 % [CO₂–O₂], 40–10 % [CO₂–O₂], 40–2.5 % [CO₂–O₂] and [Control]: air.

	Total germination (%)
Gas treatment	21d	T50 (days)
Control	45.5 c	−^†
40–20% [CO2–O2]	70.8 a	3.6 b
40–10% [CO2–O2]	70.6 a	5.8 ab
40–2.5% [CO2–O2]	57.0 b	7.8 a
LSD	10.4	3.6
Means followed by different letters within a column are significantly different at p = 0.05.
†T50 was not calculated due to germination lower than 50%.

The observed low germination percentage of control (Table I) could be attributed to the absence of cold storage of the seeds, since they were stored at room temperature (20±2 °C) during the experimental period. The final germination percentage of seeds of “Princess 77” that were stored at 6 °C with 33% RH was approximately 70% (Hacisalihoglu 2007).

The reduction of O₂ concentration to hypoxic levels (2.5%) resulted in an almost 14% suppression of the total germination along with a further increase of the T₅₀, compared to 40–20%, or 40–10% CO₂–O₂ treatments. Seed germination nevertheless, remained 12% higher compared with the control (Table I). The lower total germination of bermudagrass seeds under 40–2.5% CO₂–O₂ could be attributed to the combination of high CO₂ and hypoxia to root protrusion, as Leather et al. (1992) suggested for dormant caryopses of E. cruss-galligerminating under 75% or 100% CO_2, and Grable and Danielson (1965) for Z. mays germinating under 30% CO₂ and 20% O₂.

The effects of the supraoptimal CO₂ in the rootzone, on pre-rooted stolons, varied with the O₂ concentration. The mean cumulative clippings yield was suppressed when O₂ concentration was 10% or 2.5%. Significant differences however were evident in 40% CO₂–2.5% O₂ on the second and the last sampling date compared with the control (Figure 2). Huang et al. (1998) reported a decrease of the shoot dry weight of A. stolonifera when the oxygen diffusion rate was reduced, but Rodriguez et al. (2005) did not record differences in shoot dry weight of the same species even when the rootzone was subjected to 10% CO₂–10% O₂.

Figure 2.  Cumulative clippings dry weight for plants treated with gas mixtures of 40–20%, 40–10%, 40–2.5% [CO₂–O₂], and [Control]: air. Bars represent Fisher's least significance difference (LSD) at p = 0.05.

Bermudagrass plants treated with 40–20% CO₂–O₂ exhibited slightly higher shoot growth during the experimental period compared with the control, though significant differences were evident only at the first sampling date (Figure 2). The normal O₂ concentration may have sustained the aerobic metabolism, turning the carbon translocation to the growth of the sward, according to a model described by Mendelssohn et al. (1981). High shoot dry weight has also been reported by Huang et al. (1997) in Triticum aestivum plants under high rootzone CO₂ and ambient O₂ concentration. Similar shoot growth was observed in potato plants when the rootzone was aerated with a gas stream of 45% CO₂ and 21% O₂ (Arteca et al. 1979).

Supraoptimal CO₂ concentration in the root system resulted also in differences of the leaf chlorophyll content (Table II). Significant differences were observed 28 DGT in leaf Chlb content of the plants treated with 40–2.5% CO₂–O_2, while the Chla content remained unaffected. The Chla/b increased significantly 28 DGT in the treatment with hypoxic O₂, as a result of the reduced Chlb content. Differences were also observed between the two sampling dates both in Chlb and Chla/b, in all treatments except for the 40–2.5% CO₂–O₂. The decrease of Chlb content of T. aestivum under waterlogging has been reported from Olgun et al. (2008), while Jiang and Wang (2006) recorded a reduction in total leaf chlorophyll content of A. stolonifera, under waterlogged conditions. A higher susceptibility of Chlb content compared with Chla and an increased ratio was recorded by Zaidi et al. (2003), in different genotypes of Z. mays under waterlogging.

Table II. Leaf chlorophyll content and relative water content of C. dactylon, when the root system was treated with 40–20% [CO₂–O₂], 40–10% [CO₂–O₂], 40–2.5% [CO₂–O₂], and [Control]: air at 7 DGT and 28 DGT.

	Chla (mg g^−1 FW)		Chlb (mg g^−1 FW)		Chla/b		RWC (%)
Gas Treatment	7d	28d	7d	28d	7d	28d	7d	28d
Control	2.6	2.7	1.1	2.0a^*	2.4	1.4b^*	62.3a	65.9a
40–20% [CO2–O2]	2.7	2.5	1.1	1.8a^*	2.6	1.4b^*	58.9ab	58.3ab
40–10% [CO2–O2]	2.6	2.5	1.0	1.7a^*	2.5	1.5b^*	55.9b	57.8ab
40–2.5% [CO2–O2]	2.6	2.7	1.1	1.1b	2.4	2.4a	57.8b	50.9b
	NS	NS	NS	*	NS	*	*	*
Means followed by different letters within a column are significantly different at p = 0.05. Means of the same line between the two different sampling dates followed by * are significantly different at p = 0.05.
*: Significance at p = 0.05, NS: non-significant at p = 0.05.

A reduction in RWC of the leaf blades was recorded at 7 DGT, when bermudagrass root system was subjected to all gas treatments. Significant differences though were observed in the treatments with 40% CO₂ and 10% or 2.5% O₂ compared with control (Table II). The RWC of plants treated with the hypoxic O₂ concentration remained lower than control at 28 DGT. A similar reduction of leaf water potential was reported by Huang et al. (1997) in T. aestivum leaves, when the root system of the plants was treated with 10% CO₂ and 5% O₂. The decrease in Chlb content along with the lower RWC might have imposed photosynthesis impedance, especially in plants treated with 40–2.5% CO₂–O₂.

At 7 DGT the sucrose content of leaves in the treatments with 40% CO₂–20% and 2.5% O₂ was significantly reduced compared with the control (Table III). The glucose content of leaves decreased in all supraoptimal CO₂ treatments, both at 7 and 28 DGT. The fructose content was significantly lower in the leaves of plants treated with 2.5% O₂ compared with the control at 7 DGT, while at 28 DGT a significant reduction occurred when rootzone O₂ was either 20% or 2.5% (Table III). The variable response of the leaf sugar content could be the result of the imposed stress and the different sward growth among the treatments. Huang and Johnson (1995) also reported variable responses of the sugars in T. aestivum leaves after 21 d under hypoxic conditions that were attributed to a decline in photosynthesis, due to the stress conditions. By contrast Albrecht et al. (2004) recorded an increase in carbohydrate levels of the shoots of wheat plants exposed to hypoxia, suggesting that an adequate photosynthesis rate was preserved during the 6 days of hypoxia.

Table III. Concentration of soluble sugars in C. dactylon leaves, when the root system was treated with 40–20% [CO₂–O₂], 40–10% [CO₂–O₂], 40–2.5% [CO₂–O₂], and [Control]: air at 7 DGT and 28 DGT.

	Sucrose (mg g^−1 DW)		Glucose (mg g^−1 DW)		Fructose (mg g^−1 DW)
Gas treatment	7d	28d	7d	28d	7d	28d
Control	67.4a	27.2	20.3a	14.3a	11.4a	6.2ab
40–20% [CO2–O2]	52.9b	27.1	16.5b	11.3b	10.3ab	5.6b
40–10% [CO2–O2]	70.9a	26.4	12.4b	11.1b	9.5ab	8.2a
40–2.5% [CO2–O2]	33.3c	25.1	11.7b	10.9b	8.1b	4.5b
	***	NS	***	*	*	*
Means followed by different letters within a column are significantly different at p = 0.05.
*: Significance at p = 0.05, ***: Significance at p = 0.0001, NS: non-significant at p = 0.05.

The root system was negatively affected from the presence of 40% CO₂. At the last two sampling dates, the root dry weight increased in control plants, but remained reduced in all the other gas treatments (Figure 3). Total root length, and total root area exhibited a similar pattern as the root dry weight, though the detrimental effect of 40% CO₂ concentration on the root length was evident from the first sampling date (Figure 3). The negative influence of the three gas mixtures with supraoptimal CO₂ was apparent at 28 DGT when the total root length of the control was about 47% higher than in 40–10%, 40–2.5% CO₂–O₂, and about 61% higher than in 40–20% CO₂–O₂. The most profound inhibitory effect of supraoptimal CO₂ concentration on total root area was at 20% O₂, probably due to the higher shoot growth rate (Figure 3). The findings are in accordance with Bunnell et al. (2002) who recorded the severe reduction of root dry mass and length of A. stolonifera, in the presence of 10% CO₂, and Geisler (1967) who observed interactive effects of CO₂ and O₂ concentrations on dry mass and length of barley roots. Huang et al. (1997) attributed the reduction of root elongation under high CO₂ to the inhibition of root respiration and cell viability.

Figure 3.  Root growth parameters for plants treated with gas mixtures of 40–20%, 40–10%, 40–2.5% [CO₂–O₂], and [Control]: air. Bars represent Fisher's least significance difference at p = 0.05. A: Root Dry Weight, B: Root total length and C: Root total area.

The concentration of soluble sugars in roots was different among the gas treatments (Table IV). The sucrose content was significantly higher in roots treated with 40% CO₂ content and 20% O_2, both at 7 and 28 DGT, probably as a result of the reduced root growth. The glucose contents increased 7 DGT in roots of plants treated with 40% CO₂ and 10% or 2.5% O_2, while the fructose and glucose concentration was significantly higher in the treatment with hypoxic O₂ at 28 DGT. The results are in accordance with the findings of Huang and Johnson (1995) and Barrett-Lennard et al. (1988) for soluble sugars of root apices and whole roots of flooded T. aestivum plants. Both researchers suggested that highly soluble sugars in hypoxic roots can be a part of a protective or a tolerance mechanism, for plant survival under unfavorable conditions. Furthermore, Albrecht et al. (2004) have attributed the higher concentration of soluble sugars in wheat roots under hypoxia, both to a supply of the root system with an amount of photosynthetically derived carbohydrates, and the reduced metabolite demand of strongly retarded roots.

Table IV. Concentration of soluble sugars in C. dactylon roots, when the root system was treated with 40–20% [CO₂–O₂], 40–10% [CO₂–O₂], 40–2.5% [CO₂–O₂], and [Control]: air at 7 DGT and 28 DGT.

	Sucrose (mg g^−1 DW)		Glucose (mg g^−1 DW)		Fructose (mg g^−1 DW)
Gas treatment	7d	28d	7d	28d	7d	28d
Control	9.7b	9.8b	9.2b	9.5b	11.3a	10.2b
40–20% [CO2–O2]	23.5a	58.0a	9.4b	11.7b	11.0a	14.9b
40–10% [CO2–O2]	9.2b	24.7b	18.2a	11.1b	11.0a	9.7b
40–2.5% [CO2–O2]	9.2b	23.9b	20.9a	40.3a	14.1a	24.8a
	***	***	***	***	NS	*
Means followed by different letters within a column are significantly different at p = 0.05.
*: Significance at p = 0.05, ***: Significance at p = 0.0001, NS: non-significant at p = 0.05.

In conclusion, the supraoptimal CO₂ levels promoted bermudagrass seed germination; however, the O₂ concentration influenced the germination rate. The presence of 40% CO₂ concentration in the rootzone of bermudagrass stolon segments during establishment prevented the root growth independently of the O₂ concentration. The overall reduced root growth along with a higher sward growth of plants treated with 40–20% CO₂–O₂ indicated a more severe stress compared with the other treatments with lower O₂ concentrations. The presence of 40% CO₂ and hypoxic rootzone O₂ concentrations for 28DGT affected negatively the sward growth rate, the Chlb content, and the RWC, without being lethal to the plants.