Switchgrass is one of the important biofuel crops which would contribute to our renewable energy in the future. Research on switchgrass miRNA is still in its infancy and to date, our knowledge has been largely limited to those that are conserved across species. Little is known about many switchgrass-specific miRNAs and their functions. Identification, isolation and functional characterization of switchgrass miRNAs will require more efforts. However, data obtained from many other plant species have clearly demonstrated the importance of these small RNA molecules and their targets in regulating various aspects of plant growth, development and response to environmental stimuli. This points out their great potential for use in plant genetic engineering. It should be noted that although miRNAs could serve as potential tools for genetic manipulation of switchgrass for improvement, altering expression of a miRNA, in many cases, could cause pleiotropic morphological and developmental changes in transgenic plants. Therefore, it is critical to better understand molecular mechanisms underlying miRNA-mediated changes in plant growth and development thereby designing appropriate transgenic strategies to obtain desirable traits with minimum unfavorable side effects.

Acknowledgements

The research in Luo’s lab has been supported by Biotechnology Risk Assessment Grant Program competitive grant no. 2007-33522-18489 and no. 2010-33522-21656 from the USDA National Institute of Food and Agriculture as well as the USDA grant CSREES SC-1700315 and SC-1700450. Technical Contribution No. 6110 of the Clemson University Experiment Station.

Tissue Culture, Genetic
Transformation, and
Improvement of Switchgrass
Through Genetic Engineering

Bingyu Zhao, u* Rongda Qu,2 Ruyu Li,2 Bin Xu1 and
Taylor Frazier1

Tissue Culture

In the early 1990s, the US DOE identified switchgrass (Panicum Virgatum L.) as an herbaceous energy crop and launched research efforts on switchgrass as a biomass energy feedstock (McLaughlin and Kszos 2005). Conger’s laboratory at the University of Tennessee was the pioneer in tissue culture and genetic transformation research on switchgrass. Their first report on callus induction and plantlet regeneration was published in 1994 (Denchev and Conger 1994). Mature caryopses, along with young leaf segments from newly-formed shoots of secondary tillers (lowland cv. Alamo), were used as explants and cultured on MS medium supplemented with auxin,

2,4- D (22.5 pM, or 5 mg/l), and cytokinin, 6-benzylaminopurine (BAP, 45 pM, or 10 mg/l, for mature caryopses and 5 pM, or 1.1 mg/l, for young leaf segments). Mature caryopses cultures were maintained in the dark at 29°C for 4 wk, and callus and "organized structures" were observed. They

department of Horticulture, Virginia Tech, Blacksburg, VA 24061. department of Crop Science, North Carolina State University, Raleigh, NC 27695. ^Corresponding author: bzhao07@vt. edu

were transferred to MS medium without growth regulators and placed under light. Approximately 65 percent of the calluses regenerated into plants. For young leaf segment cultures exposed to the same conditions, embryogenic calluses originated from basal segments of innermost leaf pieces. Non-embryogenic calluses were produced from the remainder of the leaf segments. Histological and scanning electron microscopy (SEM) analyses indicated embryogenesis was the main pathway for regeneration from mature caryopses culture, whereas regeneration from leaf segment cultures was mostly through organogenesis. One thousand regenerated plants were obtained and grown in the field.

In their next publication (Denchev and Conger 1995), the authors evaluated the effects of four concentrations (0, 5, 15, and 45 pM) of BAP in combinations with three concentrations (11.3, 22.5, and 45 pM) of auxins,

2,4- D or picloram, on callus induction and shoot regeneration. With mature caryopses as explants, both embryogenic and non-embryogenic calluses were observed from all 2,4-D-containing media. It was also observed that a supplement of BAP greatly improved the formation of embryogenic calluses and regeneration. In contrast, few embryogenic calluses were observed from picloram-containing media. In both cases, two transfers of calluses to regeneration medium (free of growth regulators) greatly facilitated shoot regeneration. The best regeneration results came from 2,4-D (all three concentrations) in combination with 15 or 45 pM BAP in callus induction media. Young seedling segments were also used as explants but performed very poorly.

Switchgrass is an outcrossing species and thus each plant is an individual genotype. Maintenance of a desirable genotype has to be through vegetative propagation. Correspondingly, Conger’s laboratory developed a node culture procedure for efficient micropropagation of switchgrass (Alexandrova et al. 1996). Tillers of switchgrass (cv. Alamo) with four to six nodes were harvested and individual nodes (below the top node) were excised. The nodes were sliced longitudinally and placed with the cut surface in contact with MS medium supplemented with 30 g/l maltose and four concentrations of BAP (0, 5, 12.5, and 25 pM) as the only growth regulator. A week after culturing nodes under a 16 hr light/8 hr dark photoperiod, shoots began to emerge from the axillary buds at the nodes. Roots were developed 8 wk later when the shoots were transferred to hormone-free medium. The highest shoot numbers came from media that contained 5 or 12.5 pM BAP (six to seven shoots per node) and cultured at 29°C. The number of shoots developed from nodes cultured at 29°C was six-fold higher than those cultured at 22°C, most likely owing to the fact that switchgrass is a warm-season grass.

In 1998, the same laboratory reported multiple shoot clump formations when mature caryopses were cultured in media with a combination of various concentrations of 2,4-D and thidiazuron (TDZ) (Gupta and Conger 1998). Caryopses germinated in the medium and multiple shoot meristems were formed at the shoot apex (Fig. 1). The phenomenon was only observed when both 2,4-D and TDZ were present in the medium. The optimal combination of the growth regulators for producing the highest number of shoots was determined to be 4.5 pM 2,4-D and 18.2 pM TDZ. Both lowland (cv. Alamo) and upland cultivars (cv. Trailblazer, Blackwell) had similar responses, though at different frequencies. SEM and histological analyses revealed that the multiple shoot formation was caused by activation of axillary buds and de novo formation of adventitious buds. The shoot clusters were successfully transferred to soil after rooting in hormone-free medium.

In 1999, Conger’s laboratory reported the development of suspension cultures of switchgrass in MS liquid medium containing 2,4-D (9 pM) and BAP (4.4 pM). Initiated from embryogenic calluses from young inflorescence cultures, the suspensions contained various developmental stages of somatic embryos, which regenerated or germinated into plantlets after being transferred to solid medium (Gupta and Conger 1999). In a related report, Odjakova and Conger (1999) studied the effect of callus age and osmotic pretreatment on embryogenic cell formation and the regeneration ability of the suspension cultures (cv. Alamo). It was observed that 10-day-old calluses performed better than 20- and 30-day-old calluses, and that 0.3 M each of sorbitol and mannitol was superior over 0.1 and 0.2 M each.

Based on experience and previously established methods in switchgrass tissue culture, Conger’s laboratory went on to successfully obtain transgenic switchgrass plants (see below). In a report from Somleva et al. (2002), the authors identified several genotypes from cv. Alamo and induced embryogenic calluses from various explants of these genotypes for transformation. Details on how to identify such genotypes and how to maintain them were not described.

A similar approach was recently reported by Xu et al. (2011). These authors first identified certain lines, such as HR8, whose mature caryopses- derived calluses had high regeneration ability in tissue culture experiments. HR8 plants were cross-pollinated with another high regeneration line, HR7, for seed production. Seeds collected from HR8 have a higher germination rate than the unselected Alamo seeds (82 vs. 72 percent). Eighty-five percent of the calluses induced from germinated HR8 seeds were embryogenic, and 84 percent of those regenerated into plants, whereas the corresponding rates of unselected Alamo seeds were only 36 and 21 percent, respectively. The response of HR8 to ABA supplementation to the medium was also different from the unselected Alamo seeds. The addition of ABA to callus induction medium reduced seed germination and increased embryogenic callus formation from HR8 seeds. For example, at 10 pM of ABA, the seed germination rate was reduced to 51 percent but embryogenic callus formation increased to 99 percent for HR8. However, unselected seeds, placed at the same concentration of ABA, exhibited a decline in both germination rate and embryogenic callus formation. Further analysis revealed that the endogenous ABA levels in HR8 seeds were about 3-fold higher than the ABA concentration in unselected Alamo seeds. Additionally, fungal endophytes were observed from some switchgrass callus culture, which may negatively affect callus growth and regeneration.

Burris et al. (2009) employed a new culture medium, LP9, in switchgrass tissue culture, which was modified from culture medium described by Lu et al. (2006). The medium by Lu et al. contains macroelements of N6 medium (Chu 1975), and microelements and vitamins of B5 medium (Gamborg et al. 1968). It is also supplemented with casein hydrolysate, proline, and glutamine. In the LP9 medium by Burris et al. (2009), dicamba was replaced by 2,4-D (5 mg/l), and both BAP and myo-inositol were removed. Proline was also reduced from 0.5 g/l to 0.1 g/l. Inflorescences from tillers of E2 to E4 stages (Moore et al. 1991) were first cultured on MS+BAP medium for 10 days and then transferred to the LP9 medium. Approximately one third of the calluses induced were "brittle and white", similar to the Type II callus described in maize culture (Armstrong and Green 1985). The regeneration ability of the friable, embryogenic, type II callus lines could last more than six months. Agrobacterium-mediated transgenic plants were obtained from the type II calluses.

Calluses similar to the reported type II callus were also observed by Li and Qu (2011) in lowland cultivars of Alamo, Performer, and Colony. However, different medium ingredients were used. Mature caryopses were first cultured on MS-based medium containing 5 mg/l 2,4-D and 1 mg/l BAP for 6-8 wk. Approximately 15 percent of the induced calluses were white, compact, embryogenic calluses. They were transferred to the same medium supplemented with 2 g/l L-proline. Approximately 50 percent of the subcultured calluses became white and friable for cv. Alamo and Colony. Although both types of calluses were highly regenerable, the friable calluses were more competent for transformation. However, this kind of callus tends to regenerate albino plants after a long term culture (100 percent albino after 14 mon of culture). Another kind of callus, one that is yellow and friable, was observed mainly from cv. Performer (Fig. 2). This kind of callus was also highly regenerable, but maintained green plant regeneration ability much longer. In an experiment, 100 percent of such calluses still regenerated into green plants after 14 mon of culture without any albinos. Supplementation of proline to the culture medium not only promoted type II callus formation, but also enhanced callus growth.

Color image of this figure appears in the color plate section at the end of the book.