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A xylem mutant (vse) was isolated from a Bambusa edulis (Odashima) Keng plantlet following vegetative micropropagation and subculture for 7 consecutive years and induced to proliferate in medium supplemented with 0.1 mg·L–1 (0.5 μM) thidiazuron (TDZ) and to develop roots in medium supplemented with 5 mg·L–1 (26.9 μM) α-naphthaleneacetic acid (NAA). Subsequent investigations comparing the growth habits of mutant plantlets with those of the wild type indicated that the growth of the former was retarded in a greenhouse. Several morphological abnormalities were observed in the vse mutant: it had thinner stems with fewer trichromes on the surface; the xylem vessels were smaller in diameter and contained crystal-like structures in the pith; the leaves were shorter and narrower with a sharp leaf blade angle; the roots were thinner and contained fewer xylem cells. The cation concentrations of both the mutant and wild type were similar in the in vitro analysis, except for those of iron and potassium, which were lower in mutant leaves in vivo. In 2-month-old mutant plants, iron chlorosis was observed on young leaves and a potassium defi ciency was observed on older leaves. After 1 year of growth in the greenhouse, all of the wild-type plants had survived, but only 27% (16/60) of the mutant vse plants were alive. Received for publication 20 Nov. 2005. Accepted for publication 29 Apr. 2006. 1To whom correspondence and reprint requests should be addressed; e-mail address: cslin99@hotmail.com (CSL); wcchang2@gate.sinica.edu.tw (WCC). Bambusa edulis is a semitropical evergreen species known locally in Taiwan as “gray feet green bamboo.” It is a mediumsized clumping bamboo that is cultivated extensively for the quality of its edible young shoots, which are considered to be a culinary delicacy in Taiwan, as well as for its wood products. It is also commonly used as an ornamental, both in the garden, where it can reach a height of 19.8 m, and in pots, with a more restricted height of 3 m. Although various propagation techniques are available for Bambusoideae in general and B. edulis in particular, including plant division, vegetative propagation, and seed propagation, these methods are severely limited when utilized for largeor mass-scale propagation. It has been proposed that only micropropagation—propagation by tissue culture—will meet the estimated demand for the mass propagation of 500,000 bamboo plants per year (Gielis et al., 2001). In B. edulis, protocols for micropropagation (Lin and Chang, 1998) and somatic embryogenesis (Lin et al., 2003, 2004) have been successfully developed and used to maintain multiple shoots in vitro for 7 years. Rooted plantlets derived from the shoots maintained in these in vitro tissue culture systems were recently transferred to the greenhouse. We found that the vegetative tissuederived somatic embryos (Lin et al., 2004) were retarded with respect to their growth characteristics and displayed chlorosis of the young leaves, indicating a nutrient imbalance. We denoted this aberrant plant type as the vse mutant. Since xylem is the pathway through which the plant transports its water and nutrients and given the economic potential of successful micropropagation techniques for bamboo, it is important to understand the relation between nutrient transport and vascular structure of the wild type and somaclonal variation in B. edulis. Consequently, the aim of the investigation reported here was to compare the wild type and the micropropagated vse mutant in terms of morphology, anatomy, and nutrient analysis in an attempt to clarify the relation between xylem structure and cation content in both types of bamboo. In addition, we examined the specifi c morphological changes that have occurred in the mutant relative to the wild type. 4-751, p445-451 445 6/30/06 12:43:27 PM 446 J. AMER. SOC. HORT. SCI. 131(4):445–451. 2006. Materials and Methods MEDIUM PREPARATION. The basal medium comprised MS salts (Murashige and Skoog, 1962), 100 mg·L–1 myoinositol (Sigma, St. Louis), 0.5 mg·L–1 nicotinic acid (Sigma), 0.5 mg·L–1 pyridoxineHCl (Sigma), 1 mg·L–1 thiamine-HCl (Sigma), 2 mg·L–1 glycine (Sigma), 2 g·L–1 Gelrite (Kelco, San Diego), and 30 g·L–1 sucrose (Taiwan Sugar Co., Taipei, Taiwan). Growth regulators were added prior to the adjustment of the medium to pH 5.7. The media were autoclaved at 121 °C for 15 min. PLANT MATERIALS. Bambusa edulis multiple shoots were used as explants and incubated on MS medium supplemented with 0.1 mg·L–1 (0.5 μM) TDZ (Sigma) (Lin et al., 2004) to induce in vitro shoot proliferation. Each explant (wild type and vse) contained approximately fi ve shoots. Root development was induced in the developing shoots following a 1-month culture on MS medium supplemented with 5 mg·L–1 (26.9 μM) NAA (Sigma). The explants were maintained at 26 °C under a 16/8 h (light/dark) photoperiod with light supplied by fl uorescent tubes (FL-30D/29, 40 W; China Electric, Taipei, Taiwan) at an intensity of 54 mol·m–2·s–1. The rooted shoots were transferred to a greenhouse without hardening. HISTOLOGICAL STUDIES. Plant tissues were fi rst fi xed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) for 4 h at room temperature, then post-fi xed in 2% (v/v) OsO4 for 2 h, washed in phosphate buffer, dehydrated through an acetone series, infi ltrated with Spurr’s resin, and polymerized at 70 °C (Spurr, 1969). For light microscopy, the embedded tissues were cut into 0.7-μm-thick sections on a Rechert Ultracut S ultrathin microtome (Rechert, Vienna, Austria), stained with 0.1% toluidine blue, and viewed under a light microscope. The photographs of the sections were printed out and used for determining the number and diameters of the cells of the roots and shoots. The longest cross-section that did not cross the xylem was considered to be the diameter. The number of cells falling on this line was taken to be the cell number. For viewing in the scanning electron microscope (model DSMS 950; Carl Zeiss, Oberkochen, Germany), the tissues were fi xed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) for 4 h at room temperature, dehydrated through an ethanol series (Dawns, 1971), dried in a critical point dryer (HCP-2; Hitachi, Tokyo), and coated with gold in an ion coater (IB-2; Giko Engineering, Tokyo). MEASUREMENT OF CATION CONTENT. Leaves were collected from the wild-type and vse mutant plants cultured under different growth conditions (in vitro or different lengths of time in the greenhouse). The samples were fi rst oven-dried at 65 °C for 3 d. The dried sample (0.1 g) was weighed into a crucible and placed in a muffl e furnace at 200 °C for 2 h, followed by 400 °C for 1 h, and fi nally 550 °C for 1 h. The ashed samples were dissolved in 5 mL 2 N HCl and fi ltered through Whatman #42 fi lter paper. To measure the potassium and magnesium content of the ashed samples, 0.1 mL of fi ltered solution was diluted with 4.9 mL of deionized water and potassium and magnesium content determined by means of a Varian 20 Techtron atomic absorption spectrophotometer (Techtron, Melbourne, Australia). For calcium content measurement, 0.1 mL of the fi ltered solution, 3.9 mL deionized water, and 1 mL 5% lanthanum oxide were mixed for atomic absorption analysis (Martin-Prevel et al., 1987). STATISTICAL ANALYSIS. The analysis of variance was conducted using Costat (CoHort Software, Monterey, Calif.). Duncan’s multiple range test was used for mean separation when signifi cance treatment effects existed (Duncan, 1955). Results MORPHOLOGY OF THE VSE MUTANT PLANT. The leaves of vse plantlets derived from multiple shoots maintained in continuous subculture for 7 years on MS medium supplemented with 0.1 mg·L–1 TDZ were slightly smaller than those of the wild type at the 21-d stage (Fig. 1A), but there was no signifi cant difference in shoot proliferation (Fig. 1B). Following the transfer of both the wild-type and vse rooted plantlets to the greenhouse, differences in growth characteristics became more visible (Fig. 1C–F). We measured stem diameter (Fig. 2A), leaf length (Fig. 2B), and leaf width (Fig. 2C) during in vitro culture and at the 3and 6month intervals, respectively. Our measurements showed that the difference in leaf size between the wild-type and vse mutant was Fig. 1. Comparison of the Bambusa edulis vse mutant and wild-type phenotypes. (A) Leaves from a 21-d-old wild-type (upper) and vse (lower) mutant plant grown on MS medium (Murashige and Skoog, 1962) supplemented with 0.1 mg·L–1 thidiazuron (bar = 1.5 cm). (B) Multiple shoots from a 21-d-old wild-type (right) and vse (left) mutant plant grown on MS medium supplemented with 0.1 mg·L–1 thidiazuron (bar = 1.5 cm). (C) Three-month-old wild-type plantlet grown in the greenhouse (bar = 3 cm). (D) Three-month-old vse plantlet grown in the greenhouse (bar = 3 cm). (E) Six-month-old wild-type plantlet grown in the greenhouse (bar = 12 cm). (F) Six-month-old vse plantlet grown in the greenhouse (bar = 3 cm). 4-751, p445-451 446 6/30/06 12:43:44 PM 447 J. AMER. SOC. HORT. SCI. 131(4):445–451. 2006. proportional to the duration of the growth period in the greenhouse, with the leaves of the wild type becoming signifi cantly longer and wider than those of the vse mutant over time (Fig. 2B–C). The stem diameter of the wild type was signifi cantly larger than that of the vse mutant by the 3and 6-month stage (Fig. 2A, Table 1). There was a 100% survival rate of the wild-type plants (16/16) after 1 year, whereas only 27% (16/60) of the vse plants were alive after the same length of time. STEM STRUCTURE. The structure of the stems of wild-type and vse mutant plantlets after 3 months of growth in the greenhouse was examined and compared (Fig. 3, Table 1). Horizontal sections of the stem revealed that the vse mutant had a reduced mean total number of cells, a reduced mean number of pith cells, and smaller xylem and pith cells relative to the wild-type plantlets (Table 1, Fig. 3A–D)

Changes in the Morphology and Cation Content of a Bambusa edulis Xylem Mutant, vse, Derived from Somaclonal Variation