Crop Science Journal of Natural Resources and Life Sciences Education
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Published online 6 May 2005
Published in Crop Sci 45:1069-1072 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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NOTES

EMBRYOGENIC CALLUS INDUCTION AND REGENERATION IN A PENTAPLOID HYBRID BERMUDAGRASS CV. TIFTON 85

Mukesh Jaina, Kudithipudi Chengalrayana, Maria Gallo-Meagherb,* and Paul Mislevyc

a Agronomy Dep., Univ. of Florida, Gainesville, FL 32611-0300
b Agronomy Dep., Plant Molecular and Cellular Biology Program, and Genetics Institute, Univ. of Florida, Gainesville, FL 32611-0300
c Range Cattle Research and Education Center, Univ. of Florida, 3401 Experiment Station, Ona, FL 33865-9706

* Corresponding author (mgmea{at}mail.ifas.ufl.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Materials and Methods
 Results and Discussion
 REFERENCES
 
Hybrid bermudagrass [Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt Davy] cv. Tifton 85 is a major forage grass in the southeastern USA that suffers severe yield losses because of insect damage. A facile, cultivar-specific regeneration protocol is required to pursue genetic improvement of Tifton 85 for incorporating insect resistance into this valuable forage crop. An efficient regeneration system that uses somatic embryogenesis from immature inflorescences of Tifton 85 has been established. Young, immature inflorescence explants proved to be an excellent source for obtaining embryogenic callus, as opposed to apical meristems and nodal segments. Embryogenic callus with demonstrable morphogenetic competence was obtained on MS basal medium containing 30 g L–1 sucrose, 4 mg L–1 ; 2,4-dichlorophenoxyacetic acid (2,4-D), 0.01 mg L–1 6-benzylaminopurine (BAP) and 200 mg L–1 casein hydrolysate. The regenerants were rooted on hormone free MS basal medium supplemented with 30 g L–1 sucrose, successfully established in soil under greenhouse conditions, and did not show any phenotypic differences compared with wild-type Tifton 85 plants.

Abbreviations: ABA, (±)-abscisic acid • BAP, 6-benzylaminopurine • MS, Murashige and Skoog medium • N6, Chu's N6 medium (Chu, 1981)


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Materials and Methods
 Results and Discussion
 REFERENCES
 
BERMUDAGRASS is widely used as a warm-season turf and forage grass in the temperate and tropical regions of the world. Triploid bermudagrass cultivars (2n = 3x = 27) are sterile F1 hybrids of Cynodon dactylon (L.) Pers. (common bermudagrass, 2n = 4x = 36) x C. transvaalensis Burtt-Davy (African bermudagrass, 2n = 2x = 18), and are exclusively vegetatively propagated. The bermudagrass cv. Tifton 85 is a sterile, pentaploid F1 hybrid (2n = 5x = 45) between a South African plant introduction (PI 290884 or Tifton 296, 2n = 2x = 18) and Tifton 68, an F1 hybrid between PI 255450 and PI 293606, both from Kenya (2n = 6x = 54) (Burton and Monson, 1984; Burton, 1993). Compared with its parents, Tifton 85 has improved persistence because of above ground stolon spreading as well as subterranean rhizomes. Because of its high yields, good digestibility and protein content, along with its ability to sustain overgrazing and dry weather conditions (Hill et al., 1993), Tifton 85 has become a major forage grass in the southeastern USA. However, its high susceptibility to the insect pest fall armyworm [Spodoptera frugiperda (J.E. Smith)] warrants genetic engineering approaches to produce pest resistant germplasm. Traditional breeding methods for Tifton 85 are impossible because of the sterile nature of all hybrid bermudagrass cultivars. A facile and efficient regeneration protocol is, therefore, an essential prerequisite for any efforts aimed at genetic improvement of Tifton 85 in vitro.

For the common bermudagrass Cynodon dactylon, Ahn et al. (1985) demonstrated induction of embryogenic callus from immature inflorescence explants on N6 medium supplemented with 1 mg L–1 2,4-D. Germination of the somatic embryos was achieved on hormone-free N6 medium. Further, these embryogenic cultures were maintained for more than 80 wk without losing their morphogenetic potential. The results were further extended to an array of various forage and turf type bermudagrass cultivars (Ahn et al., 1987). A similar mode of regeneration from embryogenic callus cultures for bermudagrass cultivars A-10978b, A-12164 and Brazos, was achieved for immature floral tissue explants cultured on Murashige and Skoog medium (MS, Murashige Skoog; Murashige and Skoog, 1962) containing 1 to 3 mg L–1 2,4-D (Artunduaga et al., 1988). Casein hydrolysate (200 mg L–1) in addition to 3 mg L–1 2,4-D was found to be stimulatory for induction of embryogenic callus for the bermudagrass cv. Zebra (Artunduaga et al., 1989). Congruous to the reports published earlier for other bermudagrass cultivars, only the young inflorescence explants of cv. Tifgreen and Savannah yielded embryogenic callus cultures on MS medium supplemented with 1 to 3 mg L–1 2,4-D and 0.01 mg L–1 BAP. Scanning electron microscopic studies confirmed somatic embryogenesis to be the major route for plant regeneration (Chaudhury and Qu, 2000). A stimulatory effect of abscisic acid (ABA) on repetitive somatic embryogenesis and improved germination of embryos on supplementation with gibberellic acid (GA3) in the regeneration medium was observed for hybrid bermudagrass cv. Tifgreen (Li and Qu, 2002). Repetitive somatic embryogenesis (rarely reported for monocots) was also observed for immature inflorescence explants of bermudagrass cv. Savannah cultured on ABA supplemented medium (Li and Qu, 2002). Establishment of highly regenerable callus lines from young inflorescence cultures of common bermudagrass cv. J1224, and recovery of transgenic plants expressing the gusA and bar genes has recently been reported (Li and Qu, 2003). Likewise, Zhang et al. (2003) and Goldman et al. (2004) have reported biolistic transformation of triploid bermudagrass cv. TifEagle with the hpt and bar genes, respectively. The suspension and embryogenic callus cultures were, however, obtained from nodal explants obtained from stolons.

In this paper, an optimized protocol for in vitro culture of immature inflorescence explants of hybrid bermudagrass Tifton 85 is reported followed by regeneration and establishment of chlorophyllous, true-to-type Tifton 85 plants in the greenhouse.


   Materials and Methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results and Discussion
 REFERENCES
 
Unemerged immature inflorescences, after the appearance of their flag leaves, were randomly collected from pastures located at the Range Cattle Research and Education Center, Ona, FL. After the removal of leaf blades, the intact inflorescences surrounded in layers of leaf sheath were rinsed briefly in 70% (v/v) ethanol, and sterilized in 150 mL L–1 commercial bleach (containing 6 mL L–1 sodium hypochlorite, v/v) for 20 min, followed by five washes, 8 to 10 min each, in sterile distilled water. The young inflorescences, approximately 1 to 3 cm in length, were sliced into segments of approximately 5 mm and cultured on callus induction medium. Aseptic shoot apices and nodal segments from the stolons were isolated similarly as 3 to 5 mm explants that were sliced in half longitudinally and plated with their cut sides in contact with the culture medium.

The callus initiation medium consisted of MS basal salts and vitamins (M5524 and M3900, respectively, Sigma, St. Louis, MO), 30 g L–1 sucrose, pH 5.8, and gelled with 3 g L–1 phytagel (Sigma, St. Louis, MO). Various growth hormones were added as indicated, to determine the callus initiation and regeneration potential. Regeneration and rooting of embryogenic calli were obtained on hormone-free MS basal medium containing 30 g L–1 sucrose. Screening of growth hormones for optimizing a successful regeneration protocol was done following a completely randomized experimental design (10 explants cultured per 100- x 15-mm Petri plate containing 30 mL medium each, five replications per treatment). The rooting of regenerants was obtained in Magenta GA-7 vessels (Sigma, St. Louis, MO) (50 mL medium per vessel). For all in vitro cultures, Plant Preservative Mixture (PPM, 1 mL L–1; Plant Cell Technologies, Washington, DC.) was included in the medium as a precautionary measure to prevent potential contamination. All the cultures were maintained at 26°C, under cool white fluorescent light at a photon flux density of 60 µmol m–2 s–1 with a 16-h light regimen, unless otherwise stated.


   Results and Discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
REFERENCES
 
Explants obtained from young inflorescences, shoot apices, and nodal segments were tested for their callus initiation and regeneration response on various media. Given the extremely limited seasonal availability of the reproductive plant material, the auxin treatment matrices were designed with 1 mg L–1 increments. An extensive screening of various auxins such as 2,4-D (1–6 mg L–1), dicamba (1–5 mg L–1), and indole acetic acid (IAA, 1–5 mg L–1) either alone or in combination with kinetin (1, 2, and 3 mg L–1) or BAP (0.01, 0.1, and 1 mg L–1), identified only 2,4-D and BAP to be useful for callus induction, growth and establishment of regenerating callus cultures (data not shown). Callus induction from immature inflorescence (less than 3 cm) explants was achieved after 4 wk of culture on medium containing MS salts and vitamins, supplemented with 30 g L–1 sucrose, 1-6 mg L–1 2,4-D, and 0.01 mg L–1 BAP, incubated at 26°C under dark conditions (Table 1). Four milligrams per liter 2,4-D was found to yield maximum callus induction with 88 (± 13)% of the explants responding, followed by 66 (±11)% on 3 mg L–1 2,4-D. Callusing response was lower by 73, 34, and 39% on 1, 2, and 6 mg L–1 2,4-D, respectively, as opposed to 4 mg L–1 2,4-D. Gain in callus fresh weight (observed visually) was also substantially higher in medium containing 4 mg L–1 2,4-D. Following 4 wk of dark incubation, the cultures were transferred under 16-h photoperiod for 3 wk, to achieve shoot differentiation on the same medium (Fig. 1a and b). On average, 6 to 10 regenerating shoots were scored per callus after the three-week culture cycle. More than 60% of the total calli (obtained on 4 and 6 mg L–1 2,4-D) regenerated shoots when transferred to the second culture cycle, even though the incidence of callus induction was significantly higher (1.6-fold) in the presence of 4 mg L–1 2,4-D, when compared with 6 mg L–1 2,4-D. Notably, the incidence of callus induction was significantly higher in the presence of 3 mg L–1 2,4-D as opposed to 6 mg L–1 2,4-D, but the regenerative potential on the former auxin concentration was severely restricted by as much as 24%. Occasionally, root differentiation was also observed under dark incubation conditions, but such cultures failed to regenerate shoots on transfer to light conditions. Callus obtained from the inflorescence explants was initially watery, pale to translucent white, and on transfer to light conditions, gave rise to opaque, grayish white sectors, which were compact and dry in texture. The compact nature of the callus and its regeneration potential were positively correlated. 6-Benzylaminopurine (0.01 mg L–1) was found to be essential for obtaining morphogenetic differentiation of the callus, as complete absence of BAP in the callus induction and maintenance medium, resulted in poor regeneration of callus cultures, with occasional differentiation of one or two shoots. Regeneration in the absence of BAP was highly inconsistent, and therefore could not be evaluated statistically. Higher concentrations of BAP (0.1 and 1 mg L–1) resulted in excessive browning and compromised the growth of callus cultures, adversely affecting the establishment of regenerating callus.


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  		Table 1. Effect of 2,4-D concentration on the regeneration of callus obtained from the immature inflorescence (less than 3 cm) explants of hybrid bermudagrass Tifton 85.

 


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  		Fig. 1. Callus induction and regeneration from immature inflorescence explants of hybrid bermudagrass Tifton 85. (a) Callus initiated from immature inflorescence (less than 3 cm) explants of Tifton 85 cultured on MS medium containing 30 g L–1 sucrose, 4 mg L–1 2,4-D and 0.01 mg L–1 BAP for 4 wk in the dark. (b) Initial shoot regeneration on the same medium after 3 wk under a 16-h light regimen. (c) Regenerated plants of Tifton 85 rooted on hormone-free MS medium. (d) Regenerated plants of Tifton 85 after acclimatization in the greenhouse.

 
All attempts to regenerate callus obtained from vegetative tissue explants met with limited success, even though the callus initiation from such material was always more than 80 to 90%. The maturity of the grass inflorescence used as the explant source was critical in obtaining high callus regeneration. Only immature small inflorescences (less than 3 cm) with yellowish to pale green racemes were useful for initiation of callus and its subsequent regeneration. Mature or nearly mature floral tissue either gave rise to callus with severely restricted regeneration potential or failed to initiate callus altogether. Casein hydrolysate (200 mg L–1) supplementation showed marginal improvement in the morphogenetic response of the callus. The data are summarized in Table 2.


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  		Table 2. Effect of explant source and casein hydrolysate on induction and regeneration of callus of hybrid bermudagrass Tifton 85.

 
Other supplements like coconut water (5–20%, v/v) and ABA (1–5 mg L–1) were also tested on the basis of the results from preliminary experiments or previously published reports, but showed no stimulatory effects on the callus regeneration process. Similarly, N6 (Chu, 1981) and B5 (Gamborg et al., 1968) basal salt media and vitamin formulations, used in various combinations, had little effect on increasing the regeneration competence of the embryogenic callus (data not shown).

A comparative assessment of regeneration protocols published for various bermudagrass cultivars necessitates the need for in vitro culture procedures optimized specifically for a given cultivar. For example, while nodal explants were used to establish embryogenic callus and suspension cultures of triploid bermudagrass cv. TifEagle (Zhang et al., 2003), mainly only inflorescence explants have proved useful for obtaining regeneration for other bermudagrass varieties (Li and Qu, 2003, Chaudhury and Qu, 2000, Artunduaga et al., 1988, Ahn et al., 1985). Likewise, regeneration of TifEagle was obtained on medium containing 30 µM dicamba and 20 µM BAP. Notably, an absolute requirement for substantially lower BAP concentration (0.01 mg L–1 or 0.044 µM) in combination with 2,4-D was observed for regeneration of bermudagrass cvs. Savannah, Tifgreen (Chaudhury and Qu, 2000) and Tifton 85 (present study), with higher concentrations (0.1 and 1 mg L–1) proving detrimental for morphogenetic competence. Similarly, shoot regeneration from inflorescence explants of Tifton 85 cultured on medium containing dicamba was severely inhibited, as opposed to the shoot regeneration in presence of 2,4-D. Other parameters such as the basal salt and vitamin formulations and supplements such as ABA, gibberellic acid, and casein hydrolysate may also have a bearing on the establishment of embryogenic cultures of a given bermudagrass genotype, with adequate regeneration potential.

The regenerating callus cultures were easily and efficiently rooted on hormone-free MS basal medium containing 30 g L–1 sucrose (Fig. 1c). All the regenerants thrived well following transplantation to soil under greenhouse conditions (Fig. 1d). The regenerated plantlets were always green and morphologically indistinguishable from the plants propagated vegetatively ex vitro. To our knowledge, this is the first report of in vitro regeneration of hybrid bermudagrass cv. Tifton 85. Further efforts are being directed toward optimization of biolistic transformation of somatic embryogenic cultures of this important forage grass.


   ACKNOWLEDGMENTS
 
The authors wish to thank Drs. F. Altpeter and K. H. Quesenberry for a critical review of the manuscript.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 REFERENCES
 
This work was approved for publication as Journal Series No. R-10490 by the Florida Agricultural Experiment Station.

Received for publication June 1, 2004.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 REFERENCES
 


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