• Open Access

Miscanthus×giganteus plant regeneration: effect of callus types, ages and culture methods on regeneration competence

Authors


Jack M. Widholm, tel: +1 217 333 9462, e-mail: widholm@illinois.edu

Abstract

The perennial rhizomatous grass, Miscanthus×giganteus is an ideal biomass crop due to its rapid vegetative growth and high biomass yield potential. As a naturally occurring sterile hybrid, M. ×giganteus must be propagated vegetatively by mechanically divided rhizomes or from micropropagated plantlets. Plant regeneration through somatic embryogenesis is a viable approach to achieve large-scale production of plantlets in tissue culture. Effect of the callus types, ages and culture methods on the regeneration competence was studied to improve regeneration efficiency and shorten the period of tissue culture in M. ×giganteus. Shoot-forming calli having a yellow or white compact callus with light-green shoot-like structures showed the highest regeneration frequency. Percentage of shoot-forming callus induction from immature inflorescence explants was 41% on callus induction medium containing 13.6 μM 2,4-d and 0.44 μM benzyladenine (BA). The use of a regeneration medium containing 1.3 μM NAA and 22 μM BA was effective at shortening the incubation period required for plantlet regeneration, with 69% of total regenerated plantlets obtained within 1 month of incubation on regeneration medium. Embryogenic-like callus morphotype could maintain regeneration competency for up to 1 year as suspension cultures. Field grown regenerated plants showed normal phenotypic development with DNA content and plant heights comparable to rhizome propagated plants. Winter survival rates of the regenerated plants planted in 2006 and 2007 at the University of Illinois South Farm, Urbana-Champaign, Illinois, were 78% and 56%, respectively.

Introduction

The C4 perennial rhizomatous grass species, Miscanthus×giganteus is a sterile allotriploid (3N=57) hybrid putatively generated from the natural cross between a diploid Miscanthus sinensis (2N=38) and the tetraploid Miscanthus sacchariflorus (4N=76) (Greef et al., 1997). M. ×giganteus is a potential dedicated bioenergy crop due to its high biomass yield (Heaton et al., 2004), conversion of solar radiation to biomass (Beale et al., 1996), and high nitrogen (N) and water used efficiency (Beale & Long, 1997; Beale et al., 1999). The capability of M. ×giganteus to reallocate minerals and other nutrients to underground rhizomes in the fall minimizes need for fertilizer. The rhizomes produce new shoots annually and generate mature stands in 3–4 years providing maximum yields which can be maintained for over 15 years of production (Lewandowski et al., 2000). M. ×giganteus also has a longer growing season in cool climates compared with maize (Zea mays), because of its superior capability to maintain photosynthetically active leaves at a temperature 6 °C below the minimum for maize (Wang et al., 2008). These and other characteristics make M. ×giganteus a viable bioenergy crop which can be used to generate heat, power and fuel, while reducing carbon dioxide emissions (Heaton et al., 2004).

As a naturally occurring sterile hybrid, M. ×giganteus does not form seeds and must be propagated vegetatively by mechanically divided rhizomes or micropropagated plantlets. This requirement makes the establishment of sterile triploid M. ×giganteus costly and labor intensive (Lewandowski et al., 2000). The estimated cost for mechanization of rhizome establishment was €350 ha−1 (US$128 acre−1), and is expected to be reduced to €200 ha−1 about US$74 acre−1 in the future. Micropropagated plants, produced by in vitro culture, require much higher establishment costs of about €3000–6000 (US$2730–5460) per hectare for typical densities of one or two plants per square meter (Lewandowski et al., 2000). The cost of plant propagules and the need to propagate large numbers of plants are key factors constraining widespread planting of M. ×giganteus (Atkinson, 2009). Micropropagation could allow for the rapid multiplication of initial germplasm stocks selected in breeding programs. These materials would then undergo rhizome propagation to fulfill the requirement of large-scale establishment in the field. Micropropagation is also a viable approach for nonseasonal production, production of disease-free plants, germplasm conservation and facilitating international germplasm exchange (Govil & Gupta, 1997). Increasing demands for M. ×giganteus cultivation in various regions and countries requires improvement of current tissue culture micropropagation techniques to reduce propagule cost and increase availability.

In vitro propagation of M. ×giganteus has been conducted by shoot regeneration from axillary nodes and apical meristems followed by in vitro tillering (Nielsen et al., 1993; Nielsen et al., 1995; Lewandowski, 1997), and by plantlet regeneration through somatic embryogenesis of callus induced from shoot apices, leaf sections and immature inflorescence tissues (Holme & Petersen, 1996; Lewandowski, 1997). Previous investigations have focused on optimization of tissue culture conditions to improve the induction rate of the embryogenic callus type, described as a compact opaque white callus, by addition of proline and different carbon sources, and the use of suspension culture methods in M. ×giganteus (Holme et al., 1997; Petersen et al., 1999). Combination of auxin (2,4-dichlorophenoxyacetic acid, 2,4-d) and cytokinin (benzyladenine, BA) in callus induction medium increased the frequency of both embryogenic and shoot-forming callus induced from leaf sections and shoot apices compared with the medium containing auxin alone (Petersen, 1997). Relatively high regeneration rates of M. ×giganteus callus was also achieved by the use of 2,4-d and BA when callus is induced from immature inflorescence tissues (Lewandowski, 1997).

The aim of this study was to improve regeneration efficiency through shoot-forming callus culture and shorten the period of tissue culture required for M.giganteus plant regeneration. Different ages and types of callus maintained on either solidified medium or suspension cultures were also tested to elucidate the possible maintenance period of regenerable callus for long-term subculture. Regenerated plants were transplanted to field plots to evaluate winter survival, biomass productivity and somaclonal variation.

Materials and methods

Callus induction and maintenance culture

Immature inflorescence tissues, approximately 5–20 mm in length, were harvested from M. ×giganteus plants grown in the greenhouse, and sterilized by immersion in 0.5% NaOCl solution for 3 min. Sterilized explants were cut into 5–7 mm sections and placed on callus induction medium (M1BA) consisting of MS basal salts and MS vitamins (Murashige & Skoog, 1962) with 2,4-d (13.6 μM), BA (0.44 μM), 2.88 g L−1l-proline, 30 g L−1 sucrose, and 750 mg L−1 MgCl2·6H2O as described by Petersen (1997). All media were supplemented with 2 g L−1 Phytagel (Sigma-Aldrich, St. Louis, MO, USA), and adjusted to pH 5.5 before autoclaving. Fifteen explants were placed on each callus induction medium (M1BA) with 15 replications. Cultures were incubated in darkness at 27 ± 2 °C, and subcultured at 1-week intervals for the first 2 weeks and then at 2-week intervals for 4 weeks. Six weeks after culture initiation, the total number of calli induced was recorded to calculate callus induction percentage. The different callus types were separated by their visual appearance and the number of each callus type was counted. Callus classification was as described by Lewandowski (1997), Holme & Petersen (1996) and Petersen (1997) for M. ×giganteus callus evaluation. One callus unit was defined as 2 mm diameter calli. The percentage of each callus type was calculated as: callus type percentage=(number of each callus type/total number of callus pieces) × 100.

Two types of calli, compact and white callus (embryogenic-like callus) and a yellow or white compact callus with translucent and light-green shoot-like structures (shoot-forming callus), were maintained separately in solid or liquid callus maintenance medium for 1 year. Callus maintenance medium (M1) has the same composition as callus induction medium without BA. Ten pieces of calli (1–2 mm diameter) were transferred into each solid callus maintenance medium or 50 mL of liquid callus maintenance medium in 250 mL Erlenmeyer flasks, and the proliferated callus masses were subcultured by continuous selection for original callus type at 2-week intervals. The flasks with liquid medium were shaken at 120 rpm at 27 ± 2 °C in darkness.

Plant regeneration

Effect of the callus types, ages and culture methods on the regeneration competence was studied with two different types of callus cultured on either solid or in liquid callus maintenance medium on two different regeneration media (MR1-1 and MR1-2) 1, 2, 4, 6, 8, 10 and 12 months after callus initiation. Each type and age of calli was transferred into the regeneration medium consisting of MS basal salts and MS vitamins (Murashige & Skoog, 1962), 20 g L−1 sucrose and 750 mg L−1 MgCl2·6H2O with the addition of á-naphthaleneacetic acid (NAA) (1.3 μM) and BA (22 μM) for MR1-1 medium and 2,4-d (4.5 μM) and BA (22 μM) for MR1-2 medium. Regeneration media was solidified with 3 g L−1 Phytagel, and adjusted to pH 5.5 before autoclaving. Four Petri dishes with 10 calli (3–4 mm diameter) each were plated out for each treatment, and subcultured at 3-week intervals. Growth conditions were 16 h of cool white fluorescent light (40 μmol m−2 s−1 of photosynthetically active radiation) at 27 ± 2 °C. Shoots regenerating from callus were transferred into MS basal medium to induce roots, and rooted plantlets were later transferred to plastic pots (700 cm3) containing a 1 : 1 : 1 (v/v/v) mixture of peat moss, vermiculite and perlite in the greenhouse. The number of plantlets regenerated in each treatment was recorded when they were transferred from regeneration to rooting medium during a 4-month-period of incubation on regeneration medium. Multiple shoot clumps that could not be divided were considered as one plantlet. The regeneration frequency was calculated as the number of regenerated plantlets divided by total number of calli.

The incubation period required for callus differentiation into plantlets on regeneration medium was measured by counting the number of plantlets regenerated from shoot-forming calli after 1 month culture on MR1-1 and MR1-2. In this experiment, shoot-forming calli were induced from immature inflorescence tissues harvested from field grown M.×giganteus materials in July at the University of Illinois South Farm. Twenty calli per treatment were used for regeneration in five replications. To estimate the regeneration potential of calli remaining after removing the regenerated plantlets, the calli were subcultured on the same regeneration medium keeping the same replications for one additional month. The regeneration frequency of second set of calli was also calculated by same procedure.

Phenotypic evaluation and DNA content analysis of field-grown regenerated plants

Field plantings and growing of regenerated plants were conducted at the University of Illinois South Farm, Urbana-Champaign, Illinois, from 2006 to 2009. A total of 54 and 25 regenerated plants hardened in greenhouse for 2 month and hardened off outside for about 1 month were planted in a single block at low density (0.9 m spacing) in the spring of 2006 and in high density (0.3 m spacing) in the spring of 2007, respectively. Two morphological characteristics, plant height and stem diameter, were measured in fall 2009 at the mature flowering stage of the regenerated plants. Plant height was measured from the soil surface to the apex of the inflorescence, and stem diameter was measured as the width of stem at the 1 m height of the stem from the soil surface with a slide caliper. Winter survival rate was calculated as the percentage of plants that survived the first winter season. Temperatures in Illinois during 2006 and 2007 winter seasons (December through February of the next year) were near average and below average, respectively (National Weather Service Regional Office, http://www.crh.noaa.gov/crh). Precipitation was above average during the same periods in Illinois.

Leaves from five individual plants were selected from each of the 2006 and 2007 regenerated field plants based on their phenotypic variation in plant height, and DNA content of these plants compared with leaves from five plants randomly selected from rhizome propagated M. ×giganteus plants. Approximately 500 mg of young and healthy leaf tissue of each regenerated plant were collected and ploidy levels determined by flow cytometry (Model LSRII, BD Biosciences at the University of Illinois-Keck Biotechnology Flow Cytometry Facility) as described by Rayburn et al. (1989, 2009) using sorghum nuclei as an internal standard.

Statistical analysis

Plant regeneration frequency of shoot-forming calli incubated on regeneration medium for 1 or 2 months, and phenotypic evaluation data obtained from field grown regenerated plants with replications were analyzed by the GLM procedure and LSD tests using sas statistical analysis package (sas version 8.0). DNA content collected from the field-grown regenerated plants was subjected to statistical analysis using the LSD test to evaluate for significant differences between regenerated plants and rhizome propagated plants.

Results

Callus induction from immature inflorescence tissue

Immature inflorescence explants initiated callus about 2 weeks after culture on M1BA medium containing 13.6 μM 2,4-d and 0.44 μM BA at a frequency of 78 ± 7.8% when measured 6 weeks after initiation. Small brown calli not distinguishable from the swollen explants were not counted. Three different types of callus were identified after 6 weeks of culture (Fig. 1a–c). The dominant type of callus (41 ± 4% of the total) was a yellow or white compact callus with light-green shoot-like structures (shoot-forming callus) (Fig. 1a). The percentages of compact, white callus (embryogenic-like callus) (Fig. 1b) and soft, friable callus (Fig. 1c) were 22 ± 2.1 and 37 ± 3.7%, respectively. We found that continuous subculture of shoot-forming calli on M1BA solid callus induction medium caused browning of most of calli. Maintenance of shoot-forming calli on solid medium containing only 2,4-d mostly produced yellowish and semi-soft callus type with a small portion of embryogenic-like callus type. Shoot-forming callus maintained in liquid medium also turned into the yellowish and semisoft callus type, and most then formed roots on regeneration medium (Fig. 1d).

Figure 1.

 Plant regeneration from callus cultures of Miscanthus×giganteus. (a) Shoot-forming callus induced from immature inflorescence tissue. (b) Embryogenic-like callus. (c) Soft, friable callus. (d) Yellow, semi-soft, root-forming callus (bar=1 mm). (e) Callus induction from immature inflorescence explants after 3 weeks incubation on M1BA callus induction medium. Callus differentiation into plantlets after 3 weeks incubation on MR1-1 medium (f) and MR1-2 medium (g). Plates are 10 cm in diameter. (h) Regenerated plants in the field in 2009. Person in the figure is 1.7 m tall.

Influence of callus type, age and culture maintenance method

The responses of the shoot-forming and embryogenic-like calli for plant regeneration are presented in Table 1. One-month-old shoot-forming calli showed the best regeneration performance compared with embryogenic-like calli. Dramatic reduction in regeneration potential with increasing age of shoot-forming callus culture was observed. After 1 month of subculture on solid callus maintenance medium, the estimated regeneration frequency of shoot-forming calli dropped to 0.3 and 0.1 plantlet per calli on MR1-1 and MR1-2 regeneration medium, respectively. No plants could be regenerated from the friable callus. Optimum concentration of NAA and BA used in MR1-1 medium was obtained from our preliminary regeneration experiments conducted by treatment of eight combinations of NAA (1.3 and 5 μM) with BA (5, 10, 15 and 22 μM). Highest regeneration efficiency was found at the combination of 1.3 μM NAA and 22 μM BA (data not shown). Most of previous regeneration conditions employed with M. ×giganteus were based on 22 μM BA (Holme & Petersen, 1996) or combination of 4.5 μM 2,4-d and 22 μM BA (MR1-2 medium) (Holme et al., 1997).

Table 1.   Frequency of plant regeneration from shoot-forming and embryogenic-like calli on regeneration media MR1-1 and MR1-2
Callus
age
(month)*
Regeneration frequency of calli maintained on
solid callus maintenance medium
MR1-1 medium
(1.3 μM NAA and
22 μM BA)
MR1-2 medium
(4.5 μM 2,4-D and
22 μM BA)
Shoot-
forming
calli
Embryogenic-
like calli
Shoot-
forming
calli
Embryogenic-
like calli
  • *

    Callus age was time after placement on initiation medium.

  • †Regeneration frequency was estimated as the average number of regenerated plantlets per calli after 4 months incubation on regeneration medium with 3 weeks interval subculture (40 calli were tested for each treatment).

10.930.301.330.25
20.300.000.100.50
40.000.000.100.90
60.000.000.000.00
80.000.000.000.00
100.000.000.000.00
120.000.000.000.00

Regeneration of embryogenic-like callus type was less dependent on callus age compared with shoot-forming callus type. Continuous selection of the embryogenic-like callus type for 2 and 4 months of subcultures on solid medium increased the regeneration frequency, but after over 5 months of subculture no plant regeneration resulted regardless of callus types and regeneration medium. Suspension culture of embryogenic-like callus type in liquid medium did maintain regeneration competence for up to 12 months (Table 2). The highest plantlet regeneration was obtained from 6-month-old embryogenic-like calli. Regeneration frequency of suspension aggregates decreased after 6 months, but plantlets were still being obtained after 1 year of culture. Twenty percent of total regenerated plants obtained from 8-month-old suspension aggregates were albinos, whereas all other regenerated plants from different ages and types were normal green.

Table 2.   Effect of callus suspension culture on maintenance of regeneration competence for long-term subculture
Callus
age*
(month)
Regeneration frequency of calli maintained by
suspension culture
MR1-1 medium
(1.3 μM NAA and
22 μM BA)
MR1-2 medium
(4.5 μM 2,4-D and
22 μM BA)
Shoot-
forming
calli
Embryogenic-
like calli
Shoot-
forming
calli
Embryogenic-
like
calli
20.050.030.100.90
40.000.030.000.78
60.000.000.001.00
80.000.000.000.50
100.000.000.000.55
120.000.000.000.45

Rapid regeneration of shoot-forming callus

The effect of two auxin-like growth regulators, NAA or 2,4-d, on the regeneration efficiency and speed of plantlet regeneration from shoot-forming callus were further evaluated. Combination of NAA and BA in regeneration medium (MR1-1 medium) stimulated rapid shoot regeneration and growth from shoot-forming calli compared with 2,4-d and BA in combination (MR1-2 medium) (Table 3). Regeneration frequency of shoot-forming calli on MR-1 medium was significantly higher than the frequency obtained from MR1-2 medium during the first month of incubation, and total number of regenerated plants obtained after 2 months of incubation on regeneration medium was also significantly higher on MR1-1 medium. Most of regenerated plantlets (69% of total regenerated plantlets) appeared within 1 month of incubation in MR1-1 medium, while only 30% of total regenerated plantlets were produced in the same culture period on MR1-2 medium. Fig. 1f and g taken after 3 weeks incubation of shoot-forming calli on regeneration medium showed the distinct differences in shoot regeneration and growth. Shoot-forming calli placed on MR1-2 medium formed many green spots indicating initiation of callus differentiation with few tiny multiple shoots on the surface of calli during 3 weeks (Fig. 1g), but the growth of differentiated shoots were relatively slow compared with the regenerated shoots on MR1-1 medium (Fig. 1f).

Table 3.   Effect of two different regeneration media (MR1-1 and MR1-2) on rapid plant regeneration from 6 weeks old shoot-forming calli
Regeneration mediumRegeneration frequency
After 1 month incubation
on regeneration
medium*,§
After 2 months incubation
on regeneration
medium†,§
Total regeneration
frequency‡,§
  • *

    Regeneration frequency was estimated as the number of regenerated plantlets divided by total number of calli after 1 month incubation on regeneration medium without subculture (20 calli were tested for each treatment with five replications).

  • †After taking out the regenerated plantlets from first month incubated calli materials, calli were subcultured and incubated on regeneration medium again for one more month, and then the number of regenerated plantlets was counted.

  • ‡Total number of regenerated plants obtained for 2 month of calli incubation on regeneration medium was used to estimate total regeneration frequency.

  • §

    Means within each column with different letters are significantly different at Pφ0.05, using Fisher's LSD.

MR1-10.84 ± 0.08a0.35 ± 0.08a1.19 ± 0.15a
(1.3 μM NAA and 22 μM BA)
MR1-20.29 ± 0.05b0.54 ± 0.07a0.83 ± 0.08b
(4.5 μM 2,4-D and 22 μM BA)

Phenotypic and cytogenetic variation of field grown regenerated plants

To determine if there were any phenotypic differences between field grown regenerated plants and rhizome propagated plants, two morphological characters, plant height and stem diameter were measured (Table 4). Plant height of regenerated plants planted in 2007 at high density (0.3 m spacing) was significantly higher than rhizome propagated plants planted in 2004 and regenerated plants planted in 2006 at low density (0.9 m spacing). Regenerated plants planted in low density were statistically larger in stem diameter but lower in plant height compared with the rhizome propagated plants and regenerated plants planted at high density. The average DNA content of regenerated plants selected for short plant height showed no significant differences with rhizome propagated plants and normal phenotype regenerated plants with means of 7.19 ± 0.13 and 7.24 ± 0.05 pg, respectively (Table 4). Winter survival rates of field planted regenerated plants in 2006 and 2007 were 78% and 56%, respectively.

Table 4.   Comparison of the phenotypic characters and DNA contents between plants derived from in vitro plant regeneration and rhizome propagation
Plant originNumber
of lines
Plant height
(meter)*
Stem
width (cm)*
Winter
survival
rate (%)
DNA content
(picogram/
nuclei)
  • *

    Two morphological characteristics, plant height and stem diameter, were measured in fall 2009 at the same mature flowering stage. Plant height was measured from the soil surface to the apex of the inflorescence, and stem diameter was measured as the width of stem at the 1 m height of the stem from the soil surface with a slide caliper of plants from the interior rows of the plots. Means within each column with different letters are significantly different at P φ0.05, using Fisher's LSD.

  • Spacing of low-density planting and high-density planting were 0.9 and 0.3 m, respectively.

  • ND, not determined.

Regenerated plants planted in 2006 in low density planting183.17 ± 0.16b1.03 ± 0.07a787.19 ± 0.13a
Regenerated plants planted in 2007 in high density planting143.68 ± 0.13a0.83 ± 0.05b567.25 ± 0.01a
Rhizome propagated plants planted in 2004 in high density planting143.37 ± 0.10b0.79 ± 0.03bND7.24 ± 0.05a

Discussion

Our experimental data demonstrated that the types and ages of callus induced from immature inflorescence tissues are critical factors determining the efficiency of plantlet regeneration in M. ×giganteus. Shoot-forming calli induced from immature inflorescence tissues showed the greatest regeneration frequencies in our experiments. Plant regeneration was reported to be improved by using shoot-forming callus induced from leaf sections and shoot apices of M. ×giganteus (Petersen, 1997). Combination of 2,4-d and BA in callus induction medium was effective to improve plant regeneration as well as induction frequency of shoot-forming callus in M. ×giganteus (Lewandowski, 1997; Petersen, 1997). Our preliminary experiments conducted by regeneration of calli induced from immature inflorescence tissues with or without BA also showed higher regeneration frequencies from callus induced by 2,4-d and BA than the callus induced by 2,4-d alone (data not shown). Maintenance of shoot-forming callus was not possible under our current culture conditions as was observed by Petersen (1997).

Loss of regeneration capability with increasing age of callus is a critical problem for callus maintenance and regeneration in M. ×giganteus, which has been observed for other grass species (Cai & Butler, 1990; Brisibe et al., 1994). Holme et al (1997) established a suspension culture system to maintain embryogenic callus cultures in M. ×giganteus, and high regeneration efficiency was obtained from the 18 months old suspension aggregates. Our experiments confirmed that the regeneration competence of embryogenic-like callus culture could be maintained for long-term culture periods (up to 1 year) by suspension culture. Fine selection and maintenance of a compact, white nodular callus type also seems to be an important factor affecting regeneration efficiency of embryogenic-like callus cultures. Regeneration frequency of embryogenic-like calli was increased after 1 or 2 months of subculture on solid callus maintenance medium. Although no regeneration was recorded from the relatively old callus materials in our first experiment, later experiments generated a few regenerated plants (data not shown). This kind of variation in plant regeneration could be due to callus types selected and maintained by continuous selection of embryogenic or proembryogenic callus type during subculture procedures. The embryogenic potential of wheat callus cultures could be maintained for 20 months by continuous selection of embryogenic clumps at subculture (Yang et al., 1991).

Regeneration response of embryogenic-like calli was relatively slow compared with shoot-forming calli. Although most of embryogenic-like calli initiated callus differentiation through the formation of green spots on the surface of calli, only a few regenerated plantlets appeared within 1 month of incubation on MR1-2 regeneration medium, with short multiple shoots developing from the differentiated callus later. Embryogenic-like calli continued to proliferate on MR1-2 regeneration medium.

Rapid and more synchronized regeneration from shoot-forming calli occurred on MR1-1 medium containing NAA and BA. Combination of NAA and BA has previously been employed to induce shoots from nodal segments of M. ×giganteus (Nielsen et al., 1993), but this has not been tested for regeneration via somatic embryogenesis in M. ×giganteus. Addition of NAA into the regeneration medium may stimulate shoot elongation of calli already differentiated or undergoing cell differentiation. The lack of effect of NAA and BA in combination on regeneration of embryogenic-like callus type supports this possibility.

Field grown regenerated plants showed normal phenotypic development with plant heights and stem diameters comparable to rhizome propagated plants in our experiments. Planting of the mature rhizomes grown for about 3 months in the greenhouse or outside without subdivision may accelerate the growth and development of regenerated plants of M. ×giganteus. Higher planting density did affect the development of field planted regenerated plants as these plants were significantly taller with reduced stem diameters than in the low-density planting (Table 4). Christian & Haase (2001) reported that high-density planting of Miscanthus results in improved competition with weeds and the achievement of high yields more rapidly. The small phenotypic differences noted are most likely due to microclimate differences and not somaclonal variation since the DNA content of the regenerated plants and the rhizome propagated plants were not significantly different. It has been shown that regenerated plants produced through somatic embryogenesis are competitive with rhizome propagated plants in terms of good morphological development and yield potential in M. ×giganteus (Lewandowski, 1998; Clifton-Brown et al., 2007). Higher shoot length and number of shoots per plant was observed in micropropagated plants over rhizome propagated plants in the first and second ratoon by Lewandowski (1998). Micropropagated plants have proven to be more susceptible to winter losses than rhizome propagated materials (Lewandowski, 1998). Poor over-wintering of the rhizomes in the first year after planting was a problem in Northern Europe (Clifton-Brown & Lewandowski, 2000). Field tests of M. ×giganteus in southern Ireland showed 60% and 53% of winter survival rate from rhizome propagated plants and micropropagated plants, respectively (Clifton-Brown et al., 2007). Our field experiments showed 78% and 56% of winter survival rates from the regenerated plants transplanted in 2006 and 2007, respectively. Field trials of rhizome propagated M. ×giganteus previously planted in 2002 in Illinois demonstrated 100% of over-winter survival in Central (Urbana), Southern (Dixon Springs) Illinois, and 86% in Northern Illinois (Dekalb) (Heaton et al., 2008). Monthly average soil temperatures measured 4 inches below the soil surface at Urbana, Illinois during the 2006 and 2007 winter seasons (December through February of the next year) ranged from −2.5 to 2.7 °C, and −2.0 to 0.8 °C, respectively, whereas they ranged from −0.3 to 2.4 °C during 2002 winter season (Illinois & Water Climate Summary, http://www.sws.uiuc.edu) when 100% survival was observed. This result suggests that soil temperature during the first winter season may be an important factor affecting winter survival of field-grown regenerated plants. Further study is required to elucidate the proper size of rhizomes and transplanting time for improvement of winter survival rate in field planted regenerated plants.

Our regeneration methods show a high frequency of plantlet regeneration with a reduced tissue culture period that is applicable for large scale production of regenerated plants in M. ×giganteus. Immature inflorescences are abundant materials which can be easily obtained from field-grown M. ×giganteus during the summer or from greenhouse grown plants throughout the year. Enough shoot-forming calli can be produced 6–8 weeks after culture of immature inflorescence tissues on M1BA medium, and most plantlet regeneration is achieved within 2 months on MR1-1 regeneration medium. Regenerated plantlets need to hardened off and allowed to form appropriate sized of rhizomes during the winter season in the greenhouse or winter nurseries, and then these propagules can be ready for large-scale field planting in the spring. Maintenance of shoot-forming callus and their regeneration competence for long-term tissue culture periods need to be improved to save the cost and time required for continued immature inflorescence harvesting and cultivation of explant sources. Various combinations of auxin and cytokinin concentrations in callus maintenance medium will be tested in the future in attempts to improve long-term maintenance of shoot-forming callus.

Acknowledgements

This work was supported by funds from the Illinois Council on Food and Agricultural Research, the Energy Biosciences Institute (EBI), the Illinois Agricultural Experiment Station and the Consortium for Plant Biotechnology Research (CPBR) by DOE Prime Agreement No. DE-FG36-02GO12026. This support does not constitute an endorsement by DOE or by The Consortium for Plant Biotechnology Research, Inc. of the views expressed in this publication. We also thank Wei Q. Zhong for technical support.

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