PLANT TISSUE CULTURE

Plant regeneration from cell suspension-derived protoplastsof Populus × beijingensis

Xiao Cai & Xiang-Yang Kang

Received: 5 January 2013 /Accepted: 3 June 2013 / Editor: J. Forster# The Society for In Vitro Biology 2013

Abstract A protocol for plant regeneration from cellsuspension-derived protoplasts of Populus × beijingensis isdescribed. Protoplasts were isolated from cell suspension cul-tures 6 d after subculture and further cultured in liquidNH4NO3-free Murashige and Skoog (MS) medium supplemented with0.6 M glucose, 9.05 μM 2,4-dichlorophenoxyacetic acid, and0.89 μM 6-benzyladenine at a density of 2×105 protoplasts permilliliter. The initial plating efficiency and final plating efficien-cy recorded after 10 and 30 d reached 33.7 and 1.07%, respec-tively. The proliferated calli transferred to regeneration mediumsupplemented with 2.22 μM 6-benzyladenine and 0.54 μM α-naphthaleneacetic acid gave the highest rate of shoot formation(44.4%). All protoplast-derived shoots were able to form rootson half-strength MS medium supplemented with 2.46 μMindole-3-butyric acid.

Keywords Plant regeneration . Cell suspension . Protoplastculture . Populus × beijingensis

Introduction

Populus species and their hybrids are sources of woody bio-mass for the forest products industry and are widely cultivatedglobally. However, progress in classical breeding and selection

is limited because of long generation times and the presence ofseasonal dormancy (Confalonieri et al. 2003). Somatic hybrid-ization and genetic transformation allow the exploration of newpathways for the genetic improvement of poplars, which de-pends upon the establishment of a fully developed protoplast-to-plant system for Populus species.

Russell and McCown (1986) reported plant regenerationfrom leaf protoplasts of Populus alba × Populus grandidentata.Protocols for recovery of plants from protoplasts of Populustremula as well as Populus nigra × Populus trichocarpa (Rus-sell and McCown 1988), P. nigra × Populus maximowiczii(Park and Son 1992), P. tremula × P. alba (Chupeau et al.1993), Populus simonii (Wang et al. 1995), and P. alba (Qiaoet al. 1998) have been developed. However, protoplastculture of poplar is still far from being used in breeding pro-grams because of the low reproducibility and the limitedefficiency of plant regeneration. Additional research is neededin order to establish an efficient and reproducible protoplast-to-plant system for most cultivars of poplar.

Populus × beijingensis was selected and cultivated from theartificial crossing of female P. nigra var. pyramidalis and malePopulus cathayanaKehd. As a valuable germplasm, it is widelyplanted in northern China because of its fast growth, ease ofvegetative propagation, and cold-resistant traits. However,in vitro culture and genetic transformation of P. × beijingensishas received little attention (Li et al. 2013). In this study, wereport an efficient and reproducible protocol for plant regenera-tion from cell suspension-derived protoplasts ofP.× beijingensis.

Materials and Methods

Plant materials. Fruiting branches from a female and a maletree of P. × beijingensis were collected from the campus ofBeijing Forestry University. Branches were kept in a green-house in water-filled containers at 25°C. Flowers were thencontrol-pollinated and seeds were collected at maturity.

X. Cai :X.<Y. Kang (*)Key National Engineering Laboratory for Tree Breeding,Key Laboratory for Genetics and Breeding of Forest Treesand Ornamental Plants, Ministry of Education,Beijing Forestry University, Beijing 100083, Chinae-mail: kangxy@bjfu.edu.cn

X. CaiInstitute of Cotton, Beijing Forestry University Key NationalEngineering Laboratory for Tree Breeding, Key Laboratory forGenetics and Breeding of Forest Trees and Ornamental Plants,Ministry of Education Hebei Academy of Agriculture and ForestrySciences, Shijiazhuang 050051, China

In Vitro Cell.Dev.Biol.—PlantDOI 10.1007/s11627-013-9540-x

Surface disinfection was accomplished by immersing seedsin 70% (v/v) ethanol for 1 min, then 1% (v/v) sodium hypo-chlorite for 20 min, followed by rinsing at least three timeswith sterile distilled water. Seeds were placed horizontally onMurashige and Skoog (MS) medium (Murashige and Skoog1962) containing 3% (w/v) sucrose (Amresco, Solon, OH)and 0.6% (w/v) agar (Wako Co, Osaka, Japan). Hypocotylswere excised from 15-d-old seedlings and cut into 10 mmsections before placing on callus induction medium (MSmedium supplemented with 3% (w/v) sucrose, 0.6% (w/v)agar, 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and0.44 μM 6-benzyladenine (BA)). All media were adjusted topH 5.8 and autoclaved at 121°C for 20 min. Ten explantswere cultured in each Petri dish (90×18 mm) containing50 mL medium. All the cultures were maintained in the darkat 25±1°C for 4 wk.

Establish of cell suspension culture. To establish cell sus-pension cultures, 1 g friable calli induced from hypocotylswas transferred to a 150-mL flask containing 50 mL of MSliquid medium (MS medium supplemented with 3% (w/v)sucrose, 0.15% (w/v) glutamine, 0.05% (w/v) malt extract,9.05 μM 2,4-D, and 0.44 μM BA) at pH 5.8. The cultureswere incubated on a rotary shaker (100 rpm) at 25°C in thedark, and 1 mL cells was subcultured to 50 mL of freshmedium every 12 d. The cell suspension cultures used inthis study were obtained within 2 mo.

Protoplast isolation. Cell suspension cultures, 6 d after thelast transfer to fresh medium, were used for protoplast isola-tion. The cells were collected on a 150-μm mesh and about1 mL packed cell volume of the suspension culture wastransferred to a Petri dish (60×15 mm) containing 5 mLenzyme solution. The enzyme solution consisted of 1%(w/v) cellulase Onozaka RS (Yakult, Tokyo, Japan), 1%(w/v) macerozyme R-10 (Yakult, Japan), 5 mM 2-(N-morpholino)ethanesulfonic acid (MES), and 11% mannitolin cell protoplast washing medium (CPW; Frearson et al.1973) salt solution at pH 5.8, and was filter-sterilized using a0.22-μm pore size Millipore filter. The cell–enzyme mixturewas incubated in the dark on a rotary shaker (80 rpm) for 4–6 h at 28°C. After digestion, protoplasts were separated fromthe debris by filtrating through a 45-μm pore size stainlesssteel mesh, and then transferred to a 10-mL glass centrifugetube. The filtrate was centrifuged at 100×g for 3 min. Thepellet of protoplasts was resuspended in 10 mL washingsolution (CPW salts supplemented with 11% mannitol and5 mM MES) and centrifuged at 100×g for 3 min, and thewash was repeated three times.

The protoplast yield was determined by counting the num-ber of protoplasts using a hemocytometer. The viability of theprotoplasts was assessed with 0.01% (w/v) fluorescein diacetate(FDA) under UV-light microscope (Olympus BX51).

Protoplast culture. Isolated protoplasts were cultured at den-sities of 1.0×105 or 2.0×105 protoplasts per mL in NH4NO3-free MS medium (MS basal medium minus NH4NO3) usingtwo different culture protocols: thin liquid culture andagarose-embedded culture. For thin liquid culture, 2 mL ofnewly isolated protoplasts was spread as a thin layer on thebase of a Petri dish (60×10 mm). Seven days after cultureinitiation, 0.2 mL of fresh culture medium was added to eachPetri dish. Then, the osmotic pressure was gradually reducedby adding 0.5 mL fresh medium lacking osmoticum andplant growth regulators (PGR) every 7 d. For agarose-embedded culture, aliquots of protoplasts suspended at twicethe final culture concentration were mixed with an equalamount of protoplast culture medium containing 1.2% lowmelting temperature agarose (Promega®, Madison, WI) at30°C and 2 mL of the mixture was plated in each Petri dish(60×10 mm). Protoplasts were cultured at a density of1×105 and 2×105 protoplasts per mL. In addition, the effectof carbon sources (0.6 M glucose, 0.6 M sucrose, 0.6 Mmannitol, and 0.2 M mannitol + 0.4 M glucose in combina-tion) on protoplast culture was also evaluated (Table 2).Protoplasts were cultured in liquid NH4NO3-free MS medi-um supplemented with 9.05 μM 2,4-D and 0.89 μM BA at adensity of 2×105 protoplasts per mL. To evaluate the effectof different PGR (4.52, 9.05, or 13.57 μM 2,4-D; 0.22, 0.44,or 0.89 μM BA; 0, 1.08, or 2.69 μM α-naphthaleneaceticacid (NAA)) combinations on colony formation, protoplastswere cultured at a density of 2×105 protoplasts per mL inliquid NH4NO3-free MS medium supplemented with 0.6 Mglucose.

All culture dishes were sealed with Parafilm® and incu-bated at 27°C in the dark. The initial plating efficiency (IPE)was defined as the percentage of protoplasts that divided atleast once after 10-d culture. Final plating efficiency (FPE)was the percentage of protoplasts producing colonies (morethan 0.1 mm in diameter) after 30-d culture.

Plant regeneration. After 1–2 mo of culture, visible micro-calli were transferred to callus proliferation medium(NH4NO3-free MS medium supplemented with 3% (w/v)sucrose, 0.6% agar (w/v), 4.52 μM 2,4-D, and 0.89 μMBA, pH 5.8). The cultures were maintained in the dark forfurther development. The proliferated calli were then trans-ferred to MS medium supplemented with BA (1.33, 2.22,4.44, or 8.89 μM) and NAA (0, 0.54, 1.08, or 1.61 μM) forshoot regeneration. The shoot formation rate represented thepercentage of differentiated calli after 6-wk culture on dif-ferentiation medium. When differentiated shoots were ap-proximately 1–2 cm in length, shoots were excised and cul-tured for rooting on half-strength MS medium supplementedwith 2.46 μM indole-3-butyric acid (IBA), 3% (w/v) sucrose,and 0.6% (w/v) agar at pH 5.8 (Cai and Kang 2011). Thecultures, for differentiation and rooting, were kept in 100-mL

CAI AND KANG

Erlenmeyer flasksunder an illuminationat 30–40μmolm−2 s−1

for 16 h photoperiod at 27°C. Rooted plantlets were trans-ferred to the plastic pots (9×8 cm) containing a 1:1:2 mixtureof peat, perlite, and sand, and maintained in a greenhouse.

Data analysis. All experiments were repeated three times.The data collected were analyzed using the analysis of var-iance (Gomez and Gomez 1984). The arcsine-transformationwas applied to percentage data to stabilize variances, and thelog-transformation was used when data were not normallydistributed. Significance of differences was determined byDuncan’s multiple range test at a 0.05 level of probability.

Results

Protoplast isolation and culture. Cell suspensions, 6 d aftersubculture, at the logarithmic growth stage were used for proto-plast isolation. Freshly isolated protoplasts from cell suspensions

ranged from 15 to 40 μm in size (Fig. 1a). Protoplasts appearedspherical in shape and their viability was assessed with FDA(Fig. 1b), revealing an average viability of 90–95%.

Cell wall formation started within 3 d for thin liquid culture.After 4–5-d culture, cells became oval in shape and several cellsshowed the first cell division (Fig. 1c), and second divisionswere observed after 7-d culture (Fig. 1d, e). Small coloniesconsisting of four to ten cells were observed after 10–14-dculture (Fig. 1f). After 5 wk of culture, the cell colonies (1–2 mm in diameter, Fig. 1g) were visible to the naked eye.

Protoplast-derived calli were obtained from both thinliquid culture as well as agarose-embedded culture. A highercell density (2×105 protoplasts per mL) yielded a highplating efficiency for thin liquid culture, while a lower den-sity (1×105 protoplasts per mL) was beneficial for agarose-embedded culture. However, the highest division frequencyand plating efficiency were observed using thin liquid cul-ture at a density of 2×105 protoplasts per mL (Table 1).Therefore, the thin liquid system was employed for furtherexperiments.

Figure 1. Plant regenerationfrom embryogenic cellsuspension-derived protoplastsof P. × beijingensis. (a) Freshlyisolated protoplasts. Bar,20 μm. (b) Viability ofprotoplasts stained byfluorescein diacetate. Bar,50 μm. (c) First cell division.Bar, 50 μm. (d–e) Second celldivision. Bar, 50 μm. (f)Protoplast-derived smallcolony. Bar, 50 μm. (g) Micro-calli formation. Bar, 1 cm. (h)Protoplast-derived micro-calliproliferation. Bar, 1 cm. (i)Adventitious shoot budformation. Bar, 1 cm. (j) Shootsdeveloped from protoplast-derived calli. Bar, 1 cm. (k)Regenerated plantlet Bar, 1 cm.(l) Plantlet growing in soil, 2 wkafter transfer. Bar, 1 cm.

PLANT REGENERATION FROM CELL SUSPENSION-DERIVED PROTOPLASTS

In this study, four types of carbon sources and osmoticstabilizers were tested. Significant differences in IPE (thepercentage of protoplasts that divided at least once after 10-dculture) and FPE (the percentage of protoplasts producingcolonies more than 0.1 mm in diameter after 30-d culture)were observed among the different carbon sources (Table 2).Glucose, supplemented medium at 0.6 M, resulted in thehighest IPE (33.7%) and FPE (1.07%). Protoplasts culturedin medium supplemented with 0.4 M glucose and 0.2 Mmannitol showed almost the same response to those culturedin 0.6 M glucose-containing medium but with FPE reducedto 0.81%. Few protoplasts sustained cell division with 0.6 Mmannitol in the culture medium. Therefore, our resultsshowed that glucose was the most efficient carbon sourcefor further development of colonies.

Different PGR combinations supplemented in protoplastculture medium had different effects (Table 3). The highestFPE was obtained on medium containing 9.05 μM2,4-D and0.44 μM BA. However, NAA with BA in combinationresulted in a significant lower FPE than 2,4-D with BA in

combination. Results indicated that 2,4-D might have had abeneficial effect on cell division. The IPE and FPE were notsignificantly affected by the concentrations of BA.

After visible protoplasts-derived calli were subcultured tocallus proliferation medium, the colonies grew to 5–10 mmwithin 3 wk (Fig. 1h).

Plant regeneration. The calli transferred to the regenerationmedium started to form shoots after ∼4-wk culture (Fig. 1i).The shoot formation efficiency was significantly affected bythe concentration of BA and NAA in the regeneration medi-um (Table 4). The combination of 2.22 μMBA and 0.54 μMNAA gave the highest shoot formation efficiency (44.4%).No shoots were obtained when 8.89 μM BA and 0.54 μM

Table 1. Effect of culture method along with plating density on proto-plast culture

Culture method Plating density(protoplasts/mL)

IPE (%) FPE (%)

Thin liquid culture 1×105 24.3±5.9 b 0.78±0.08 c

2×105 33.7±1.2 a 1.07±0.11 a

Agarose-embeddedculture

1×105 16.7±3.2 c 0.87±0.04 b

2×105 15.8±2.0 c 0.03±0.01 d

Data represent the mean ± SE of three replicates. Protoplasts werecultured in NH4NO3-free MS medium supplemented with 0.6 M glu-cose, 9.05 μM 2,4-D, and 0.89 μM BA. Different letters indicate thesignificant differences by Duncan’s multiple range test at the 0.05 levelof probability

IPE initial plating efficiency, FPE final plating efficiency, MSMurashige and Skoog, 2,4-D 2,4-dichlorophenoxyacetic acid, BA 6-benzyladenine

Table 2. Effect of carbon sources on protoplast culture

Carbon source IPE (%) FPE (%)

0.6 M glucose 33.7±1.2 a 1.07±0.12 a

0.6 M sucrose 24.4±3.0 bc 0.12±0.04 c

0.6 M mannitol 21.7±3.5 c 0.04±0.02 d

0.2 M mannitol and 0.4 M glucose 28.7±6.1 ac 0.81±0.13 b

Data represent the mean ± SE of three replicates. Protoplasts werecultured in liquid NH4NO3-free MS medium supplemented with9.05 μM 2, 4-D and 0.89 μM BA at a density of 2×105 protoplastsper mL. Different letters indicate the significant differences byDuncan’s multiple range test at the 0.05 level of probability

IPE initial plating efficiency, FPE final plating efficiency, MSMurashige and Skoog, 2,4-D 2,4-dichlorophenoxyacetic acid, BA 6-benzyladenine

Table 3. Effect of PGR combinations on protoplast culture

2,4-D (μM) BA (μM) NAA (μM) IPE (%) FPE (%)

4.52 0.89 0 20.6±2.5 b 0.57±0.10 c

9.05 0.89 0 32.2±1.0 a 1.10±0.06 a

13.57 0.89 0 21.7±1.7 b 0.46±0.08 c

9.05 0.44 0 30.6±3.5 a 1.19±0.14 a

4.52 0.89 1.08 22.2±3.5 b 0.88±0.09 b

0 0.89 2.69 13.9±1.0 c 0.15±0.03 d

0 2.22 1.08 8.3±2.9 d 0.04±0.01 d

Data represent the mean ± SE of three replicates. Protoplasts werecultured in liquid NH4NO3-free MS medium supplemented with0.6 M glucose at a density of 2×105 protoplasts per mL. Differentletters indicate the significant differences by Duncan’s multiple rangetest at the 0.05 level of probability

IPE initial plating efficiency, FPE final plating efficiency,MSMurashigeand Skoog, 2,4-D 2,4-dichlorophenoxyacetic acid, BA 6-benzyladenine,NAA α-naphthaleneacetic acid

Table 4. Effect of PGR combinations on plant regeneration fromprotoplast-derived calli

BA (μM) NAA (μM) Shoot formationefficiency (%)

1.33 0.54 13.3±6.7 c

2.22 0.54 44.4±3.8 a

4.44 0.54 28.9±3.8 b

8.89 0.54 0.0±0.0 d

4.44 1.08 11.1±3.8 c

2.22 1.08 40.0±6.7 ab

2.22 1.61 2.2±3.8 d

2.22 0 0.0±0.0 d

Data represent the mean±SE of three replicates. The shoot formationrate represents the percentage of calli differentiated after 6 wk of cultureon differentiation medium. Different letters indicate the significantdifferences by Duncan’s multiple range test at the 0.05 level ofprobability

PGR plant growth regulator,BA 6-benzyladenine,NAAα-naphthaleneaceticacid

CAI AND KANG

NAA were used. When 2.22 μM BA was used, the shootformation efficiency was strongly influenced by the concen-tration of NAA. The absence of NAA or the addition of1.61 μM NAA greatly inhibited shoot formation. Lowerconcentrations of NAA (0.54 μM or 1.08 μM) were pre-ferred for shoot regeneration (Fig. 1j).

All regenerated shoots were able to form roots on half-strength MS medium containing 2.46 μM IBA (Fig. 1k).Plantlets were transferred to the soil, with a survival rate ofmore than 95% (Fig. 1l).

Discussion

The most critical step in a protoplast-to-plant regenerationsystem is to obtain protoplast-derived micro-calli efficientlyand reliably. In this study, we investigated the parametersthat affected the formation and differentiation of protoplast-derived micro-calli and report an efficient and reproducibleregeneration protocol from cell suspension-derived proto-plasts of P. × beijingensis. Cell suspension cultures forprotoplast systems have been also described for other plantspecies such as Cyclamen persicum (Winkelmann et al.2006), mango (Ara et al. 2000), and Phalaenopsis (Shresthaet al. 2007).

Various protoplast culture systems have been developed fordifferent Populus species. A floating polyester screen diskmethod was described using poplar leaf protoplasts (Russelland McCown 1986, 1988). Park and Son (1992) comparedfour culture methods (hanging drop, liquid drop, liquid plat-ing, and fabric-supported semi-solid agar plating) and foundthat the fabric-supported semi-solid agar plating method wassuperior for colony formation for P. nigra × P. maximowiczii.In the present study, protoplasts of P. × beijingensis weresuccessfully cultured in both the thin liquid system as wellas the agarose-embedded system. However, colonies grewfaster and higher plating efficiencies were achieved using thethin liquid culture than the agarose-embedded system. Theresults imply that liquid culture was more suitable for proto-plast culture of P. × beijingensis. This result resembles thatreported by Qiao et al. (1998), who cultured P. alba L. pro-toplasts using a liquid system. Liquid culture provides an easyway to add fresh medium and to transfer protoplast-derivedmicro-calli. Adding fresh medium may enhance the cell divi-sion frequency through reduction of medium osmolarity andat the same time supply new nutrients for cells to sustaindivision. Also, other studies have shown that a decrease inthe concentration of osmoticum was essential for protoplastdevelopment (Nakano et al. 1995; Mizuhiro et al. 2001).

The carbon source supplied in the culture medium greatlyimpacts protoplast cell division and colony formation. Car-bon sources used in several studies for protoplast culture

have included glucose (Nakano et al. 2003), sucrose (Panet al. 2004; Prange et al. 2010), mannitol (Chen et al. 2004;Lian et al. 2012), maltose (Ma et al. 2003), and myo-inositol(Kim et al. 2003). A synergistic effect was also observedwhen different carbon sources were used in combination(Park and Son 1992; Wang et al. 1995; Castelblanque et al.2010). Of the four carbon sources tested in this study, glu-cose alone gave the highest plating efficiencies while man-nitol alone obtained the lowest plating efficiencies. Our re-sults showed that glucose was the most appropriateosmoticum and carbon source for protoplast culture of P. ×beijingensis. Similar results have also been demonstrated forcell culture of P. alba (Qiao et al. 1998). Perhaps, as glucoseis a monosaccharide, it can easily be consumed by thegrowing cells and thus results in a decrease of osmoticpressure in the medium.

The deleterious effects of ammonium have been notedpreviously for in vitro studies on poplars (Russell andMcCown 1986, 1988; Park and Son 1992; Qiao et al. 1998),Citrus (Grosser 1994), Robinia pseudoacacia (Kanwar et al.2009), and mulberry (Umate et al. 2005). In this study,NH4NO3-free MS medium successfully supported sustainedcell division and colony formation.

The plating density is crucial for maximizing cell wallregeneration and concomitant daughter cell formation, ascells are known to stimulate mitotic division of adjacent cellsby releasing growth factors (Davey et al. 2005). For Dian-thus acicularis, the optimum plating density was 1×105

protoplasts per mL in liquid medium (Shiba and Mii 2005).However, a plating density of 1×106 protoplasts per mL wasapplied inMusa spp. using the nurse culture method (Assaniet al. 2006). In our study, we found that a plating density of2×105 protoplasts per mL was suitable for liquid culture,while agarose-embedded culture required a plating densityof 1×105 protoplasts per mL.

The combination of BA and 2,4-D is considered to be themost effective for callus proliferation (compared to cytokininor auxin alone) for in vitro culture of some Populus species(Park and Son 1988; Michler and Bauer 1991). Our studyrevealed that a higher concentration of 2,4-D (9.05–13.57 μM) combined with a lower concentration of BA(0.44–0.89 μM), which was similar to the PGRs used duringcell suspension culture, was the most effective combinationof PGRs for colony formation and protoplast-derived calliproliferation.

Different requirements of PGRs for shoot formation mayresult from different levels of endogenous hormones withinthe species being cultured. Shoot formation in poplar can bemanipulated in vitro by altering the ratio of auxin to cytoki-nin. Adventitious shoot formation usually requires the pres-ence of exogenous cytokinin. The cytokinins used in studieson poplar protoplast culture for shoot formation have includ-ed zeatin (Park and Son 1992), thidiazuron (Russell and

PLANT REGENERATION FROM CELL SUSPENSION-DERIVED PROTOPLASTS

McCown 1986; 1988; Chupeau et al. 1993), and kinetin(Qiao et al. 1998). Auxins such as NAA and indoleaceticacid were often used in combination with cytokinins forshoot regeneration (Park and Son 1988; Perales and Schieder1993; Umate et al. 2005; Thomas 2009). In protoplast cultureof P. simonii, multiple shoots were regenerated on the MSmedium containing 4.44 μMBA, 2.32 μM kinetin, 2.28 μMzeatin, and 0.54 μM NAA. In this study, 2.22 μM BA plus0.54 μM NAA generated the highest rate of shoot formationfrom protoplast-derived calli. A ratio reflecting a high levelof cytokinin and a low level of auxin also proved to beefficient for shoot formation in several earlier studies (Azadet al. 2006; Guo et al. 2006; Borgato et al. 2007).

Prior to this study, there had been no systematic and com-prehensive study on the parameters critical for successfulprotoplast culture and plant regeneration for poplars. Howev-er, the problem of low reproducibility for regeneration fromprotoplasts still imposes restrictions on its potential commer-cial applicability. Parameters investigated in this study includ-ed the culture method, plating density, carbon source, PGRcombinations for micro-colony formation, and concentrationPGRs for plant regeneration. The results obtained provide thebasis for an efficient and reproducible protoplast-to-plant pro-tocol for P. × beijingensis. The application of this protocol toanother Populus species (i.e., Populus pseudo-simonii Kitag.)has been carried out and has resulted in successful plantregeneration from protoplasts (X. Cai, personal communica-tion), indicating a more broad applicability of this system. Cellfusion, which is based on an efficient protoplast-to-plant sys-tem, offers a potential approach for genetic improvement. ForPopulus species, studies on cell fusion have been limited(Sasamoto et al. 2000) but are currently a focus in our labo-ratory. In summary, application of this promising protoplast-to-plant protocol is expected to boost the genetic improvementof Populus species.

Acknowledgments The authors thank Dr. Zhenghai Zhang fromChinese Academy of Agricultural Sciences for revising the manuscript.This work was financially supported by the Forestry Public BenefitResearch Foundation (201004009).

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PLANT REGENERATION FROM CELL SUSPENSION-DERIVED PROTOPLASTS