Impaginato 273 Adv. Hort. Sci., 2018 32(2): 273-280 DOI: 10.13128/ahs-20654 Direct and indirect in vitro plant regeneration of two commercial cultivars of perennial ryegrass S. Esmaeili, H. Salehi (*), M. Khosh-Khui Department of Horticultural Science, School of Agriculture, Shiraz University, Shiraz, Iran. Key words: Abscisic acid (ABA), ʻGrasslandʼ, Lolium perenne L., maltose, meristem tip, ʻNumanʼ. Abstract: Experiments were conducted on direct and indirect regeneration from the meristem tip and mature caryopsis explants of Lolium perenne L. ʻNumanʼ and ʻGrasslandʼ. De-husked caryopses were cultured both intact and longitudi- nally sliced on MS media supplemented with 2,4-D alone, and in combinations with BA. The highest percentage of callus induction obtained from intact-sliced caryopses were 71 and 87% for ʻGrasslandʼ on MS basal medium supplemented with 6 mg L-1 2,4-D+ 0.02 mg L-1 BA, and 5 mg L-1 2,4-D. While, for ʻNumanʼ, the highest callus induction was achieved by the same explants as 55% and 72% with 5 mg L-1 2,4-D + 0.02 mg L-1 BA, and 4 mg L-1 2,4-D + 0.02 mg L-1 BA, respec- tively. The best regeneration medium for ʻGrasslandʼ was MS medium supple- mented with 10 g L-1 maltose and 2 mg L-1 ABA. In a separate experiment, meristem tip cultures were incubated on two type combination of plants growth regulators along with control treatment. The best regeneration rate was obtained in both cultivars on MS medium supplemented with 0.1 mg L-1 2,4-D + 0.5 mg L-1 Kin. Plantlets with well-developed roots were transferred to greenhouse condition. Four weeks later, all acclimatized plants were survived. 1. Introduction Turfgrasses control the soil erosion, carpet lawns, cover athletic fields, and beautify the environment. Perennial ryegrass (Lolium perenne L.) is one of the most important turfgrass species for sports fields, golf course fairways, as well as urban landscapes in the areas with temperate climate. That is the fast establishing component of lawn seed mixtures comprising slow growing species such as Kentucky bluegrass (Poa pratensis L.), and is used for winter overseeding on warm-season turfgrasses as well. However, the poor ability to survive in drought regions have limited its distribution. Therefore, improving drought tolerance is an important goal in perennial ryegrass breeding programs via classic or modern (genetic transformation) techniques. Callus production with good quality and effi- cient plant regeneration of various cultivars of perennial ryegrass is a req- uisite to the grass transformation techniques. The formation of embryo- (*) Corresponding author: hsalehi@shirazu.ac.ir Citation: ESMAEILI S., SALEHI H., KHOSH-KHUI M., 2018 - Direct and indirect in vitro plant regeneration of two commercial cultivars of perennial ryegrass. - Adv. Hort. Sci., 32(2): 273-280 Copyright: © 2018 Esmaeili S., Salehi H., Khosh-Khui M. This is an open access, peer reviewed article publi- shed by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 19 October 2017 Accepted for publication 20 March 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(2): 273-280 274 genic calli and plant regeneration were affected by several factors including genotype, explant tissue, culture medium and its supplements (Bhaskaran and Smith, 1990). Many studies have been done on improving tissue culture of perennial ryegrass. In perennial ryegrass, various explants including imma- ture inflorescence, mature seed, leaf base, meristem tip and axillary bud have been used for callus induc- tion, and direct and indirect plant regeneration (Dale, 1977; Dale and Dalton, 1983; Torello and Symington, 1984; Creemers-Molenaar et al., 1989; Altpeter et al., 2000; Bradley et al., 2001; Can et al., 2004; Salehi and Khosh-Khui, 2005; Newell and Gray, 2005; Altpeter, 2006). Among these explants, mature seeds are most commonly used for callus induction and related subsequent studies. Perennial ryegrass is an outcrossing, wind-polli- nated and highly self-incompatible species with abundant genetic variation in each cultivar (Mohr et al., 1998; Smith et al., 2001; Wang et al., 2003; Bolaric et al., 2005). Replacing the seed explants with other vegetative explants would omit this variation. In vitro meristem tip culture is an efficient method for obtaining virus free and identical to mother plant materials. In addition, meristem tip culture is an appropriate method for genetic transformation. In this method, the exposed meristem could directly be used for transformation; then after, will multiplied in vitro, or can form multiple shoot clumps that can be bombarded with the gene(s) of interest, and then each meristem will regenerated the mature plants. Therefore, development of an optimized and short time tissue culture protocol plays a critical role in successfully transformation of the grass species. To the best of our knowledge, there is no report on meristem tip culture of ʻGrasslandʼ and ʻNumanʼ cul- tivars of perennial ryegrass. In addition to, it has been shown stimulating effect of ABA on somatic embryogenesis of some grass species calli such as wheat (Qureshi et al., 1989), maize (Close and Ludeman, 1987), Kentucky bluegrass (Van Ark et al., 1991) and bermudagrass (Li and Qu, 2002). The objectives of the present investigation were comparison of the callus induction on intact and lon- gitudinally sliced caryopses, and evaluation of the effects of ABA and maltose combinations on plant regeneration of two Lolium sp. cultivars to optimize their tissue culture condition. Furthermore, a sepa- rate experiment was conducted to find a direct regeneration and fast tissue culture method for these cultivars. 2. Materials and Methods Seed surface sterilization Seeds of the two commercial cultivars of perenni- al ryegrass, ʻGrasslandʼ and ʻNumanʼ, were pur- chased from Iran Bazr Seed Company (Karaj, Iran). Seeds were socked in 50% H2SO4 for 15-20 min to remove the husks (Salehi and Khosh-Khui, 2003). The de-husked seeds were surface sterilized with 70% ethanol for 1 min and immediately were treated with a house-hold bleach (5.25% available chlorine) for 20 min, then rinsed 6 times with sterile distilled water. Callus induction The surface sterilized seeds were used both intact and longitudinally sliced. For callus induction, MS basal medium (Murashige and Skoog, 1962) plus 3% sucrose supplemented with 4, 5, 6, 7, 8, 9 and 10 mg L-1 2,4-D alone and with 0.02 mg L-1 BA. Intact and longitudinally sliced seeds were cultured on 80 mm petri dishes containing callus induction media. After 4 weeks, the calli were separated, transferred to the same fresh media and maintained for 3 weeks. Then, the calli were transferred to optimum MS media, supplemented with 4 mg L-1 2,4-D + 0.02 mg L-1 BA and 5 mg L-1 for ʻNumanʼ and ʻGrasslandʼ, respec- tively. The number of seeds that induced callus was recorded according to seed viability. The callus induc- tion rate was calculated as the number of caryopses which induced callus over the total number of explants plated × 100. All cultures were kept at 24±1°C in dark. Plant regeneration To determine the best regeneration media for ʻNumanʼ and ʻGrassland ,̓ the embryogenic calli were transferred to MS media containing 3% sucrose and 0, 10, 15 and 20 g L-1 maltose with 0, 1.7, 2 and 2.3 mg L-1 ABA. Filter-sterilized ABA was added after autoclaving when the media tempera- ture dropped to 50°C. Plant regeneration was scored 7-10 days after transferring the calli to the regeneration medium. The criterion used to determine regeneration was the formation of a distinguishable shoot at least 1 cm in length. Plant regeneration rate was defined as the percentage of callus that had regenerated shoots. The regeneration cultures were maintained at 24±1°C with a 16 h light/8 h dark cycle under a light intensity of 20 µmol m-2 s-1. Meristem tip culture In a second experiment, surface sterilized intact caryopses were incubated on 80 mm diameter petri dishes containing MS media, 3% sucrose, solidified Esmaeli et al. - In vitro plant regeneration of perennial ryegrass cultivars 275 with 0.8% plant agar and were kept at 24±1°C in dark. After emerging radicles, the petri dishes were transferred to 16h/8h light/dark photoperiodic con- dition. Meristem tips of 7-10 days old seedlings were excised by a Stereo microscope (C121506 Zeiss Stemi 1000 Binocular Stereo Zoom 0.7-3.5x Microscope 10x Eyepieces, Germany) and cultured on three different MS media including hormone-free MS, MS supple- mented with 0.01 mg L-1 2,4-D + 0.2 mg L-1 Kinetin, MS supplemented with 0.1 mg L-1 2,4-D + 0.5 mg L-1 Kinetin. All media were supplemented with 3% sucrose and pH was adjusted to 5.8 prior to autoclav- ing. The length of the meristem tips cultured ranged from 0.1-1 mm and consisted of the meristem dome plus one or several leaf primordia. After 2 weeks, the number of direct regenerated plantlets was recorded in each treatment based on green and healthy plantlets produced and then the percentage of direct regeneration of meristem tips was calculated. One hundred meristem tips were cultured in each petri dish. All media were placed in dark for 3 days at 24±1°C and then were transferred to 16h/8h light/dark photoperiodic condition. Light intensity was provided by cool-white fluorescent lamps at a photon flux density of 20 µmol m-2 s-1. Statistical analysis Complete randomized design with factorial arrangements was used for all experiments. For cal- lus induction, each treatment had four replications, each replicate consisted of 100 seed. Experiments on direct and indirect regeneration were done with three replications. All data were analyzed with SAS 9.1 software and means were compared using LSD test at 5% level. 3. Results and Discussion Effects of cultivars, explants and hormonal treat- ments and their interaction on callus induction Plant growth regulators (PGRs) are specific mole- cules used in plants and supplemented at relatively low concentrations to work as signaling compounds for plant growth and development (Sauer et al., 2013). The most extensively used and studied class of PGRs in plant tissue cultures are auxin and cytokinin (Ikeuchi et al., 2013). Among auxin sources, 2,4-D is known as the most effective PGR to induce callus for- mation with stimulating cell elongation and enlarge- ment in many plant species especially turfgrasses. In addition, low levels of cytokinins such as BA and BAP enhanced callus regeneration ability in several grass species (Zhong et al., 1991; Van der Valk et al., 1995; Chaudhury and Qu, 2000; Bradley et al., 2001). Calli were observed in 7 to 15 days after placing the explants on callus induction media. Their mor- phological appearance was hyperhydrated and white. After 4 weeks, three types of calli were observed including: i) white and hyperhydrated; ii) yellowish and friable; iii) compact, nodular, yellowish to opaque (Fig. 1 B-D). This variability is probably due to different tissues origin. Results showed that callus induction was notably increased by 28% in sliced caryopses (61.37) compared to intact caryopses (48.04). In addition, there was a significant difference Fig. 1 - Steps of caryopses culture of two perennial ryegrass cultivars. A) The de-husked caryopses and surface sterilization of caryopses, right ‘Grassland’, left ‘Numan’. B) Calli derived from longitudinally sliced caryopsis tissue, C) Embryogenic callus type ii. D) Embryogenic callus type iii. E) Green shoots produced on embryogenic callus. F) Shoot proliferation on the regeneration medium after two weeks. G) Plants acclimatized in soil mixture after one week. H) Healthy and green plants maintained in greenhouse after one month. Adv. Hort. Sci., 2018 32(2): 273-280 276 between two cultivars. The callus induction raised by 24% in ʻGrasslandʼ (60.57) compared to ʻNumanʼ (48.83) (Data not shown). In ʻGrasslandʼ, the highest good quality callus induction was obtained at 5 mg L-1 2,4-D in sliced caryopses, while ʻNumanʼ had the highest callus induction at 4 mg L-1 2,4-D with 0.02 mg L-1 BA treat- ment in sliced caryopses (Table 1). In ʻGrasslandʼ, higher concentrations of 2,4-D declined callus induction with explants sliced and entire caryopses. However, more calli showed white color with increasing 2,4-D concentration in men- tioned media. The effectiveness of 2,4-D in inducing the formation of callus is attributed to its main char- acteristic which can stimulate cell division of plant tissues and strongly suppress their organogenesis. In a similar study, Liu et al. (2006) used 2,4-D and BAP for callus induction in perennial ryegrass. They found that the highest callus induction rate with the best callus quality was obtained on MS medium con- taining 5 mg L-1 2,4-D and 0.05 mg L-1 BAP. Preliminary studies on the effectiveness of BAP on somatic embryogenesis in callus culture medium in several grass species were reported (Zhong et al., 1991; Chaudhury and Qu, 2000; Bradley et al., 2001). According to previous reports, the optimal BAP con- centration in the induction medium should be 0.02- 0.05 mg L-1 for perennial ryegrass. In this experiment, no significant difference was found on media supplemented with 0.0 or 0.02 mg L-1 BA. However, this amount of BA had positive effect on morphology and cellular structure of calli and their capacity to regenerate plantlets. As previously stated, faster and more callus induc- tion of longitudinally sliced caryopses in comparison with intact caryopses were detected. Increase in con- tact surface of tissue could be an important reason for callus induction. These findings are similar to other studies which have been conducted by Altpeter et al. (2000), Altpeter and Xu (2000), Bai and Qu (2001) and Bradley et al. (2001) on Lolium sp. and Festuca sp. Bai and Qu (2001) improved both callus production and regeneration rate of tall fescue, Festuca arundinacea Schreb. with slicing seeds in cal- lus culture medium. Also, similar inclusion on chop- ping the mature seeds of Lolium sp. and Festuca sp. to suppress germination and stimulation of callus production were reported (Altpeter et al., 2000; Altpeter and Xu, 2000). Although, Salehi and Khosh- Khui (2005) obtained contrary results by using hori- zontally sliced caryopses of Lolium sp. cultured on callus induction medium. These results might be related to genotypic differences and callus origin. These results might be used as a general application for other grass species. Interaction effect of ABA and maltose on plant regen- eration The highest regeneration rates were recorded for both cultivars on MS medium supplemented with 30 g L-1 sucrose, 10 g L-1 maltose and 2 mg L-1 ABA. As shown in figure 2, ʻGrasslandʼ has higher regenera- tion rate (~13%) than ʻNumanʼ. Incidentally, maltose had not notably effect on plant regeneration percent- age in both cultivars lonely but ABA concentrations showed significant effects on plant regeneration rate (Fig. 2). This result can be referred to the role of ABA in maturation of embryos (Finkelstein et al., 1985). The most important source of energy for plant growth in the culture medium is carbon. Several Table 1 - Interaction of different concentrations of 2,4-D and BA on callus induction of intact and sliced caryopses in two cultivars of Lolium perenne L. 2,4-D (mg L-1) BA (mg L-1) ‘Grassland’ ‘Numan’ Intact seeds Sliced seeds Intact seeds Sliced seeds 4 0 49± 3.9 de 69± 6.0 bc 49±2.4 de 54±3.5d 5 0 61±2.7 c 84±3.2 a 46±1.9 de 61±3.8 c 0.02 59±5.2 c 73±2.0 ab 55±1.8 d 41±2.9 e 6 0 51±3.4 de 77±3.8 a 30±2.9 g 62±3.6 c 0.02 62±5.4 c 71±2.3 b 53±6.7 d 54±4.7 d 7 0 71±0.6 ab 60±4.8 c 41±3.2 e 58±1.7 cd 0.02 55±3.5 d 75±5.5 ab 53±2.7 d 65±4.9 bc 8 0 53±1.1 d 73±6.3 ab 54±1.2 d 67±3.5 b 0.02 46±5.0 e 69±3.4 bc 45±6.6 e 53±1.7 d 9 0 46±5.0 e 53±6.4 d 35±1.6 f 38±1.9 f 0.02 38±6.6 f 57±2.5 c 29±2.4 g 53±1.9 de 10 0 43±7.9 e 54±4.1 de 38±2.8 f 41±3.2 e 0.02 54±7.4 d 56±4.1 d 34±4.9 f 54±2.5 d Values represent mean ± SE. Means with the same letter are not significantly different at p ≤ 0.05. Fig. 2 - Regeneration rates of two cultivars of Lolium perenne L. in combination concentrations of ABA (0, 1.7, 2 and 2.3 mg L-1), and maltose (0, 10, 15 and 20 g L-1 Mal) under same concentration of sucrose (30 g-1 Suc). Esmaeli et al. - In vitro plant regeneration of perennial ryegrass cultivars 277 studies have reported the effects of carbohydrates such as sucrose and maltose on different plant species like wild cherry, rubber tree, oilseed rape and sugarcane (Reidiboym-Talleux et al., 1998; Blanc et al., 1999; Slesak and Przywara 2003; Gill et al., 2004). Maltose is the best carbon source for callus induction and regeneration medium. Furthermore, maltose, as an osmoticum, plays a major role in the optimization of callus cellular environment and protect calli by reducing ethylene (Darachai et al., 2004; Zaidi et al., 2006). The regeneration rate was affected by using mal- tose, to almost five-fold increase at 20 g L-1 maltose compared to 30 g L-1 sucrose in both cultivars studied (Fig. 2). In addition, ABA increased the regeneration rate ~25 and 16 fold at 2 mg L-1 in comparison with 0 mg L-1 in ʻGrasslandʼ and ʻNumanʼ, respectively. The combination of maltose and sucrose and ABA at high concentrations had no promoting effect on plant regeneration in both cultivars. In ʻGrasslandʼ, significant declines was observed at 2.3 mg L-1 ABA + 20 g L-1 maltose (71.3%) and 1.7 mg L-1 ABA + 10 g L-1 maltose (100%). According to the results, both culti- vars showed different responses at high concentra- tions of maltose and ABA combination. Generally, the regeneration rate dramatically reduced by 100% at 2.3 mg L-1 ABA + 20 g L-1 maltose in ʻNumanʼ compared to ʻGrasslandʼ (Fig. 2). A probable reason for reducing the regeneration percentage is related to sucrose in regeneration media that stimulates the ethylene production in plant tissues which can cause browning of calli. On the contrary, maltose might protect the calli from browning. However, beneficial effect of maltose was observed on embryogenesis and regeneration of cereals such as rice, wheat and perennial ryegrass, but the mechanisms of maltose role in tissue and cell culture media is not yet completely known. There were significant differences between two cultivars according to plant regeneration rates. Our results are in accordance with previous studies on Kentucky bluegrass (Van Ark et al., 1991), Zoysiagrass (Dhandapani et al., 2008), and bermudagrass (Li and Qu, 2002). According to the results of Van Ark et al. (1991), by adding ABA to regeneration medium, the percentage of calli with somatic embryos or embryo- like structures increased (up to 29.6%) as compared to the control (16.4%). In addition, Dhandapani et al. (2008) found that ABA significantly increased embryo- genic callus formation from stem nodes, but not from young inflorescences. ABA not only promotes the transition of somatic embryos from the proliferation to the maturation phase (Langhansova et al., 2004), but it also enhances embryo quality by increasing des- iccation tolerance and preventing precocious germi- nation (Li et al., 1997; Robichaud et al., 2004; Vahdati et al., 2006; Rai et al., 2008). Ultimately, based on mentioned authors results, the role of ABA on increasing somatic embryogenesis and regeneration were affected by several factors including origin of explant, physiological status and environmental con- dition. Direct regeneration in meristem tip culture An efficient and rapid method was developed for direct regeneration of two cultivars of perennial rye- grass. The results of meristem tip culture provided good information regarding changings in PGRs and their effects on plantlets growth and development. There was significant difference between two culti- vars in plantlet regeneration rate. In ʻGrasslandʼ, the overall regeneration rate of meristem tips was about 63.5%, while this parameter was approximately 55.7% in ʻNumanʼ. Meristem elongation and development as a single plantlet on MS medium without PGRs was observed (Fig. 3C). More plantlets were obtained from ʻNumanʼ meristem tips in comparison to ʻGrasslandʼ on hormone-free MS medium (Fig. 3D). The same results were previously reported on Curculigo latifo- lia (Babaei et al., 2014). In the present study, overall regeneration percentage of both cultivars on MS media was obtained approximately 41%, while in Fig. 3 - Steps of meristem tip culture. A) Ten days old seedling. B) Isolated meristem tips. C) Meristem tip cultured on MS medium. D) Meristem tips cultured on MS medium supplemented with 0.01 mg L-1 2,4-D + 0.2 mg L-1 Kin. E) Meristem tips on MS medium supplemented with 0.1 mg L-1 2,4-D +0.5 mg L-1 Kin. Adv. Hort. Sci., 2018 32(2): 273-280 278 L-1 BA was selected. In sliced caryopses more callus induction was observed in both cultivars. The best regeneration medium for them was also recom- mended. Generally, low concentrations of maltose and ABA was more effective on regeneration rate of two cultivars. In addition, an efficient and rapid pro- cedure for direct in vitro regeneration of two peren- nial ryegrass cultivars has been established. Despites, MS hormone-free medium was an acceptable medi- um for regeneration of meristem tips but the highest regeneration rate of meristem tips was observed with 0.1 mg L-1 2, 4-D + 0.5 mg L-1 Kin in both culti- vars. There was a positive relation between length of meristem tips and their survival percentage. It is now possible to test the effectiveness of this technique to produce virus-free plants. Moreover, the results can definitely improve the transformation efficiency of these cultivars. References ALTPETER F., 2006 - Perennial ryegrass (Lolium perenne L.). Agrobacterium protocols. Volume 2. - Methods Mol. Biol., 344: 55-64. ALTPETER F., XU J., 2000 - Rapid production of transgenic turfgrass (Festuca rubra L.) plants. - J. Plant Physiol., 157: 441-448. ALTPETER F., XU J., AHMED S., 2000 - Generation of large numbers of independently transformed fertile perennial ryegrass (Lolium perenne L.) plants of forage-and turf- type cultivars. - Mol. Breed., 6: 519-528. BABAEI N., PSYQUAY ABDULLAH N.A., SALEH G., LEE ABDULLAH T., 2014 - An efficient in vitro plantlet regeneration from shoot tip cultures of Curculigo latifo- lia, a medicinal plant. - The Sci. World J., 2014: 1-9. 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