African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12271

Full Length Research Paper

Improved embryogenic callus induction and plant regeneration in big bluestem (Andropogon gerardii Vitman), a potential bioenergy feedstock

Pramod Pantha
  • Pramod Pantha
  • Department of Agriculture, University of Arkansas at Pine Bluff, Pine Bluff, AR 71601, USA.
  • Google Scholar
Sathish Kumar Ponniah
  • Sathish Kumar Ponniah
  • Department of Agriculture, University of Arkansas at Pine Bluff, Pine Bluff, AR 71601, USA.
  • Google Scholar
Sixte Ntamatungiro
  • Sixte Ntamatungiro
  • Department of Agriculture, University of Arkansas at Pine Bluff, Pine Bluff, AR 71601, USA.
  • Google Scholar
Muthusamy Manoharan
  • Muthusamy Manoharan
  • Department of Agriculture, University of Arkansas at Pine Bluff, Pine Bluff, AR 71601, USA.
  • Google Scholar


  •  Received: 25 June 2016
  •  Accepted: 07 September 2016
  •  Published: 28 September 2016

 ABSTRACT

The objective of this study was to develop an efficient regeneration protocol in big bluestem, a potential feedstock that produces huge biomass. Embryogenic calli were induced from the seeds of cultivars, Kaw and Earl, on Murashige and Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0.2, 0.5, 1.0, 2.0, 3.0 and 5.0 mg l-1) alone or in combination with 6-benzylaminopurine (BA) (0.5 mg l-1) or L-proline (2.0 g l-1). In Kaw, the highest number of embryogenic calli (39.1%) was induced on MS + 0.5 mg l-1 2,4-D and L-proline, whereas in Earl, the highest number of embryogenic calli (39.8%) was obtained on MS medium containing 1.0 mg l-1 2,4-D and L-proline. The embryogenic calli were then transferred to regeneration media (MS medium supplemented with kinetin, 0.2, 0.5, 1.0, 3.0 and 5.0 mg l-1 or BA, 0.2, 0.5, 1.0, 3.0 and 5.0 mg l-1). Shoots were regenerated on all of the concentrations tested and the regeneration percentage and number of shoots per calli increased with the increase in BA or kinetin concentration. Regenerated shoots were transferred to half strength MS medium for rooting. The fully developed plantlets were established in the greenhouse. The regeneration protocol established in this study may be used for the application of genetic engineering technologies in big bluestem.

 

Key words: Big bluestem, biomass, embryogenic calli, L-proline, regeneration.


 INTRODUCTION

Big bluestem (Andropogon gerardii Vitman) is a warm-season (C4) perennial grass native to North America (Boe et al., 2004). It is adapted to most native prairie ecosystems and comprises as much as 80% of plant biomass in prairies in the North American Mid-western grassland (Gould and Shaw, 1983; Knapp et al., 1998). Compared  to  switchgrass  (Panicum  virgatum   L.)   and Indiangrass (Sorghastrum nutans (L.) Nash), big bluestem produces twice the biomass due to its efficient nutrient utilization (Johnson and Matchett, 2001). Currently, big bluestem is considered a potential biomass feedstock for lignocellulosic ethanol production (Zhang et al., 2012). However, the biggest limiting factor in the efficient utilization of  plant  biomass for  cellulosic  bio-fuel  is  the presence of the ligno-hemicellulose complex that prevent the enzyme cellulase from effective conversion of biomass to biofuel (Sticklen, 2008). While approaches such as acid or heat pre-treatment are being used to break the ligno-hemicellulosic complex, genetic engineering technology holds great promise for reducing lignin content in biomass through down-regulation of lignin pathway genes (Hisano et al., 2009). Such an approach requires an efficient plant regeneration system, which is not available for a number of important cultivars in big bluestem.
 
Despite its importance as a potential bioenergy grass, very few studies have been carried out on in vitro regeneration of big bluestem (Chen et al., 1977; Chen and Boe, 1988; Li et al., 2009a). Chen et al. (1977) and Chen and Boe (1988) regenerated plants from calli that were induced from young inflorescence on Linsmaier and Skoog (LS) medium supplemented with 5.0 mg l-1 of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l-1 kinetin. Li et al. (2009a) obtained embryogenic calli from mature seeds on LS medium containing 2.0 to 4.0 mg l-1 2,4-D for big bluestem cultivars Bison and Bonilla. Cultivars such as Bison and Bonilla are well adapted to US Midwest and Western states; however, a regeneration system is not available for cultivars that are adapted to US Southern states such as Arkansas and Texas.
 
The objective of this study was, therefore, to establish an efficient regeneration system from the calli of mature seeds in big bluestem cultivars, Kaw and Earl, that are well adapted to the southern US. Mature seeds are considered an important source of explants, as they are readily available and used frequently for in vitro regeneration (Li et al., 2009a; Lee et al., 2006, 2008).


 MATERIALS AND METHODS

Plant materials
 
Mature seeds of big bluestem grass cultivars, Kaw and Earl, were obtained from Turner Seeds, Breckenridge, TX, USA. The seeds were sterilized with 70% ethanol for 30 s, followed by 20% commercial bleach (sodium hypochlorite, 5.25%) containing two drops of Tween 20 (MP Biomedicals, LLC, Aurora, Ohio, USA) for 45 min, and then washed three times with sterile distilled water for 5 min each.
 
Callus induction
 
Sterilized seeds were cultured for callus induction on petri plates containing MS (Murashige and Skoog, 1962) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.2, 0.5, 1.0, 2.0, 3.0 and 5.0 mg l-1) alone or in combination with 6-benzylaminopurine (BA) (0.5 mg l-1) or L-proline (2.0 g l-1). The components of MS medium as well as growth regulators, 2,4-D and BA were obtained from Sigma-Aldrich (St. Louis, MO, USA). L-Proline was obtained from Caisson Labs (Logan, UT, USA). Media were supplemented with 30 g l-1 of sucrose (PhytoTechnology Laboratory, Shawnee Mission, KS, USA); pH was adjusted to 5.7 with 1 N NaOH before sterilizing at 121°C (120 kPa for 20 min) and the media were solidified  with   4.0 g l-1   Gelzan   (Phyto  Technology   Laboratory).  
 
Callus induction was performed under dark at 28°C (Isotemp Incubator, Fisher Scientific, Hanover Park, IL, USA). Primary shoots from seed were removed 10 days after inoculation and subculture was done 3 weeks after inoculation on the same medium. Overall 18 treatments were tested for embryogenic callus induction in each cultivar. The frequency of embryogenic calli was calculated by: [the number of seeds producing embryogenic calli/the number of seeds germinated on callus induction medium] × 100.
 
Shoot regeneration, rooting, and plant establishment
 
Embryogenic calli were transferred to regeneration medium for shoot regeneration. Regeneration media contained MS medium supplemented with kinetin (0.2, 0.5, 1.0, 3.0 and 5.0 mg l-1; Sigma-Aldrich) or BA (0.2, 0.5, 1.0, 3.0 and 5.0 mg l-1) and 30 g l-1 of sucrose); pH was adjusted to 5.7 with 1 N NaOH before sterilization and the media were solidified with 4.0 g l-1 Gelzan. The cultures were incubated in a plant tissue culture chamber at 25°C under 16 h photoperiod (Percival Scientific, Perry, IA, USA).
 
After 4 weeks, the regenerated plants were transferred to rooting medium. Rooting medium contained half-strength MS medium with 30 g l-1 of sucrose; pH was adjusted to 5.7 with 1 N NaOH before sterilizing and the medium was solidified with 4.0 g l-1 Gelzan. The rooting was carried out in a plant tissue culture chamber under a 16 h photoperiod at 25°C (Percival Scientific, Perry, IA, USA). Rooted plants, after 2 to 3 weeks, were transferred to peat pellets for 1 week for hardening and then to the greenhouse for further growth.
 
Experimental design and statistical analysis
 
The experiment was conducted in six replicates with 25 seeds per treatment and data presented here represent an average of six experiments. The experiment was performed as a completely randomized design. To assess the treatment effects, the percentages of primary calli, embryogenic calli and shoot regeneration were subjected to Analysis of Variance (ANOVA) and mean separation was performed using least significant difference (LSD) with SAS software, version 9.2 (SAS Institute Inc., 2008).


 RESULTS AND DISCUSSION

Seed germination
 
Germination of mature seeds started 3 days after inoculation in both Kaw and Earl. Germination percentage ranged from 80.0 to 82.7 for Kaw and 60.3 to 75.3 for Earl (Table 1). Germination data were recorded 10 days after seed inoculation.
 
 
Effect of 2,4-D on embryogenic calli induction
 
Callus was initiated 7 days after inoculation of Kaw and 10 days after inoculation of Earl on MS medium containing different concentrations of 2,4-D. The callus thus formed was white and non-nodular (Figure 1a). After 3 weeks, callus was sub-cultured onto the same medium. After 1 week of subculture, nodular structures, which were smaller in size, light yellow, friable and fast-growing, started  appearing  on  the  outer  surface of the calli. The fast growing nodular calli were recognized as embryo-genic (Figure 1b), while the larger, white, slow growing non-nodular calli were identified as non-embryogenic (Figure 1a). The formation of two distinct calli, embryogenic and non-embryogenic, with different regeneration potential is an important characteristic of cereals and grasses (Pola et al., 2008). The auxin 2,4-D has been successfully used to induce embryogenic calli in grasses such as big bluestem (Chen et al., 1977; Chen and Boe, 1988; Li et al., 2009a), Indiangrass (Chen et al., 1979; Li et al., 2009b), Brachypodium (Zombori et al., 2011) and little bluestem (Schizachyrium scoparium (Michs.) Nash) (Songstad et al., 1986; Li et al., 2009a). In our study, 23.8% of germinated seed produced embryogenic calli on MS medium containing 3.0 mg l-1 2,4-D in Kaw and 24.5% of germinated seed produced embryogenic calli on MS medium with 2.0 mg l-1 2,4-D in Earl (Table 1). The percentage of embryogenic calli induced in our study is comparable to the percentage of embryogenic calli (23.1%) obtained with the supplementation of 2,4-D in other big bluestem cultivars, Bison and Bonilla (Li et al., 2009a).
 
 
Effect of L-proline on embryogenic calli induction
 
The use of L-proline in the medium has  a  positive  effect on the frequency of callus induction in rice (Oryza sativa L.), a member of grass family (Chowdhry et al., 1993; Ge et al., 2006). In this study, we have tested the effectiveness of L-proline (2.0 g l-1) on embryogenic calli induction in Kaw and Earl (Table 1). With the addition of L-proline, the frequency of embryogenic calli was significantly increased to 39.1% on MS medium supplemented with 0.5 mg l-1 2,4-D in Kaw and 39.8% on MS medium containing 1.0 mg l-1 2,4-D in Earl. The percentage of germinated seed that produced embryogenic calli in this study (39.1% in Kaw and 39.8% in Earl) was significantly higher (69 to 72%) than the percentage of embryogenic calli obtained in big bluestem cultivars, Bison and Bonila (23.1%) (Li et al., 2009a). Clearly, the increase in embryogenic calli frequency obtained in this study can be attributed to the supplementation of L-proline in callus induction medium. In switchgrass cv. Alamo, addition of L-proline increased the embryogenic calli formation efficiency by 30% (Li and Qu, 2010). The effectiveness of L-proline for the initiation and maintenance of embryogenic calli has also been reported in rice (Datta et al., 1992; Kishor et al., 1999) and alfalfa (Medicago sativa L.) (Shetty and McKersie, 1993). L-Proline also promoted embryogenesis in somatic tissue cultures of Zea mays (Armstrong and Green, 1985; Vasil and Vasil, 1986) and wheat (Triticum aestivum L.) (Gill and Gosal, 2015).
 
 
 
Effect of BA on embryogenic calli induction
 
In general, the addition of 0.5 mg l-1 BA on MS medium supplemented with 2,4-D did not significantly increase embryogenic calli formation in Kaw and Earl (Table 1). In Kaw, the maximum of 26.6% embryogenic calli was obtained on MS medium containing 3.0 mg l-1 2,4-D and 0.5 mg l-1 BA, while in Earl, the maximum of 27.7% embryogenic calli was formed on MS medium containing 1.0 mg l-1 2,4-D and 0.5 mg l-1 BA. It has been reported that BA in combination with auxins such as 2,4-D has a positive effect on callus induction in grasses (Altpeter and Posselt, 2000; Chaudhury and Qu, 2000; Bai and Qu, 2001). However, BA in combination with 2,4-D did not have a positive effect on callus induction in seashore paspalum turfgrass (Paspalum vaginatum Swartz) (Neibaur et al., 2008). The BA requirement may be species-specific (Chaudhury and Qu, 2000) and depends on the specific endogenous  hormone  levels  (Bhaskaran
and Smith, 1990).
 
Shoot regeneration
 
The actively growing embryogenic calli were transferred to MS medium with different concentrations of kinetin or BA for shoot induction. Addition of cytokinin in the regeneration medium can significantly increase plant regeneration in grasses (Songstad et al., 1986; Fei et al., 1997). In the present study, the percentage of the shoot regeneration and number of shoots per calli increased with the increasing concentrations of kinetin or BA in both Kaw and Earl (Table 2). However, compared to BA, kinetin was better with 100% regeneration achieved in both Kaw and Earl on MS medium containing 5.0 mg l-1 kinetin (Table 2) Similar high percentage of regeneration was observed with the addition of 5.0 mg l-1 kinetin in the regeneration  medium  for  big  bluestem  cultivars,  Bison and Bonila (Li et al., 2009a). Interestingly, only 70.0 and 83.4% shoot regeneration was observed in Kaw and Earl, respectively, on MS medium containing 5.0 mg l-1 BA. An average of 7.4 shoots in Kaw and 8.2 shoots in Earl were regenerated on regeneration medium containing 5.0 mg l-1 kinetin, while an average of 5.7 shoots in Kaw and 6.6 shoots in Earl were regenerated on MS with 5.0 mg l-1 BA. 
 
 
Rooting and plant establishment in greenhouse
 
More than 90% of the regenerated shoots were success-fully rooted on half strength MS without the addition of growth hormones. A similar percentage of rooting was achieved in big bluestem cultivars, Bison and Bonila (Li et al., 2009a). Fully rooted plants were transferred initially to peat pellets for hardening for one week and then to the greenhouse for further growth and development.
 
In conclusion, we have successfully established an improved regeneration system in two cultivars of big bluestem, Kaw and Earl, which are grown in the southern US. Both the cultivars showed enhanced embryogenic calli formation (39.1% in Kaw and 39.8% in Earl) on MS medium containing 2,4-D and L-proline. In addition, shoots were successfully regenerated (100% in Kaw and Earl) on MS medium containing kinetin (5.0 mg l-1). Because of high-frequency embryogenic calli formation and efficient plant regeneration, this protocol may be used for the application of genetic engineering technologies in big bluestem cultivars, Kaw and Earl.
 
 
 

 


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.


 ACKNOWLEDGEMENTS

The author, Muthusamy Manoharan thank Dr. Lynn Dahleen, L&Js Homegrown Exchange LLC, Albany, Wisconsin, for critical review of the manuscript and the Plant Powered Production (P3) Center, which was funded wholly or in part by the National Science Foundation (NSF) EPSCoR Program and the Arkansas Science and Technology Authority (ASTA) for providing funding support for this study. The NSF EPSCoR award number is: EPS-1003970.



 REFERENCES

Altpeter F, Posselt UK (2000). Improved plant regeneration from cell suspensions of commercial cultivars, breeding and inbred lines of perennial ryegrass (Lolium perenne L.). J. Plant Physiol. 156:790-796.
Crossref

 

Armstrong CL, Green CE (1985). Establishment and maintenance of friable embryogenic maize callus and the involvement of L-proline. Planta 164:207-214.
Crossref

 
 

Bai Y, Qu R (2001). Factors influencing tissue culture responses of mature seeds and immature embryos in turf-type tall fescue (Festuca arundinacea Schreb.). Plant Breed. 120:239-242.
Crossref

 
 

Bhaskaran S, Smith RH (1990). Regeneration in cereal tissue culture: a review. Crop Sci. 30:1328-1336.
Crossref

 
 

Boe A, Keeler K, Norrmann GA, Hatch SL (2004). The indigenous bluestems (Bothriochloa, Andropogon and Schizachyrium) of the western hemisphere and gamba grass Andropogon gayanus) In. Moser L, Byron B, Sollenberger L (eds) Warm-season (C4) grasses ASA, Madison, WI, Pp. 873-908.

 
 

Chaudhury A, Qu R (2000). Somatic embryogenesis and plant regeneration of turf-type bermudagrass: effect of 6-benzyladenine in callus induction medium. Plant Cell Tissue Organ Cult. 60:113-120.
Crossref

 
 

Chen CH, Boe AA (1988). Big bluestem (Andropogon gerardii Vitman), little bluestem [Schizachyrium scoparium (Michs.) Nash] and indiangrass [Sorghastrum nutans (L.) Nash] In. Bajaj YPS (ed) Biotechnology in Agriculture and Forestry 6 Crops II, Springer-Verlag.

 
 

Chen CH, Lo PF, Ross JG (1979). Regeneration of plantlets from callus cultures of Indiangrass. Crop Sci. 19:117-118.
Crossref

 
 

Chen CH, Stenberg NE, Ross JG (1977). Clonal propagation of big bluestem by tissue culture. Crop Sci. 17:847-850.
Crossref

 
 

Chowdhry CN, Tyagi AK, Maheshwari N, Maheshwari SC (1993). Effect of L-proline and L-tryptophan on somatic embryogenesis and plantlet regeneration of rice (Oryza sativa L. cv. Pusa 169) Plant Cell Tissue Organ Cult. 32:357-361.
Crossref

 
 

Datta SK, Datta K, Soltanifar N, Donn G, Potrykus I (1992). Herbicide resistant indica rice plants from IRRI breeding line IRRI after PEG mediated transformation of protoplast. Plant Mol. Biol. 20:619-629.
Crossref

 
 

Fei S, Read PE, Riordan T (1997). In Vitro regeneration of buffalograss through immature inflorescence culture. Int. Turfgrass Res. J. 8:283-289.

 
 

Ge XJ, Chu ZH, Lin YJ, Wang SP (2006). A tissue culture system for different germplasms of indica rice. Plant Cell Rep. 25:392-402.
Crossref

 
 

Gill AK, Gosal SS (2015). Improved somatic embryogenesis in Indian bread wheat (Triticum aestivum L.) cultivar HD 2967 through media manipulations. J. Cell Tissue Res. 15:4855-4860.

 
 

Gould FW, Shaw RB (1983). Grass systematics. 2nd (ed) College Station: Texas A&M University Press.

 
 

Hisano H, Nandakumar R, Wang ZY (2009). Genetic modification of lignin biosynthesis for improved biofuel production. In Vitro Cell Dev. Biol. Plant 45:306-313.
Crossref

 
 

Johnson LC, Matchett JR (2001). Fire and grazing regulate below ground processes in tallgrass prairie. Ecology 82:3377-3388.
Crossref

 
 

Kishor PBK, Sangam S, Naidu KP (1999). Sodium, potassium, sugar, alcohol and proline mediated somatic embryogenesis and plant regeneration in recalcitrant rice callus. Plant Tissue Cult. Biotech.: Emerging Trends, Proc. Symposium, Hyderabad, India, Pp. 78-85.

 
 

Knapp AK, Briggs JM, Blair JM, Turner CL (1998). Patterns and controls of aboveground net primary production in tallgrass prairie. In. Knapp AK, Briggs JM, Harnett DC, Collins SL (eds) Grassland dynamics: Long-term ecological research in tallgrass prairie. Oxford University Press, New York pp. 193-221.

 
 

Lee KW, Ahsan N, Lee SH, Lee DG, Kim KH, Alam I, Kwon SY, Kim JS, Back K, Lee SS, Lee BH (2008). Responses of MxPPO overexpressing transgenic tall fescue plants to two diphenyl-ether herbicides, oxyfluorfen and acifluorfen. Acta Physiol. Plant. 30:745-754.
Crossref

 
 

Lee SH, Lee DG, Woo HS, Lee KW, Kim DH, Kwak SS, Kim JS, Kim, HG, Ahsan N, Choi MS, Yang JK, Lee BH (2006). Production of transgenic orchardgrass via Agrobacterium-mediated transformation of seed-derived callus tissues. Plant Sci. 171:408-414.
Crossref

 
 

Li R, Qu R (2010). High throughput Agrobacterium-mediated switchgrass transformation. Biomass Bioenergy 35:1046-1054.
Crossref

 
 

Li Y, Gao J, Fei SZ (2009a). High frequency embryogenic callus induction and plant regeneration from mature caryopsis of big bluestem and little bluestem. Sci. Hortic. 121:348-352.
Crossref

 
 

Li Y, Gao J, Fei SZ (2009b). High frequency in vitro embryogenic callus induction and plant regeneration from Indiangrass mature caryopsis. Sci. Hortic. 119:306-309.
Crossref

 
 

Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-497.
Crossref

 
 

Neibaur I, Gallo M, Altpeter F (2008). The effect of auxin type and cytokinin concentration on callus induction and plant regeneration frequency from immature inflorescence segments of seashore paspalum (Paspalum vaginatum Swartz). In Vitro Cell Dev. Biol. Plant 44:480-486.
Crossref

 
 

Pola S, Mani NS, Ramana T (2008). Plant tissue culture studies in sorghum bicolor: immature embryo explants as the source material. Intl. J. Plant Prod. 2:1-14.

 
 

SAS Institute Inc. (2008). SAS Software version 9.2. SAS Institute, Cary, NC.

 
 

Shetty K, McKersie BD (1993). Proline, thioproline and potassium mediated stimulation of somatic embryogenesis in alfalfa (Medicago sativa L.). Plant Sci. 88:185-193.
Crossref

 
 

Songstad DD, Chen CH, Boe AA (1986). Plant regeneration in callus cultures derived from young inflorescences of little bluestem. Crop Sci. 26:827-829.
Crossref

 
 

Sticklen MB (2008). Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nat. Rev. Genet. 9:433-443.
Crossref

 
 

Vasil V, Vasil IK (1986). Plant regeneration from friable embryogenic callus and cell suspension cultures of Zea mays L. J. Plant Physiol. 124:399-408.
Crossref

 
 

Zhang K, Johnson L, Nelson R, Yuan W, Pei Z, Wang D (2012). Chemical and elemental composition of big bluestem as affected by ecotype and planting location along the precipitation gradient of the great plains. Ind. Crop Prod. 40:210-218.
Crossref

 
 

Zombori Z, Szécsényi M, Györgyey J (2011). Different approaches for Agrobacterium-mediated genetic transformation of Brachypodium distachyon, a new model plant for temperate grasses. Acta Biol. Szeged. 55:193-195.

 

 




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