The ability of auxin to induce callus was associated with the relative strength of the four auxins tested, with 20 or 50 µM 2,4-dichlorophenoxyacetic acid giving ...
In Vitro Cell. Dev. Biol.--Plant 33:20-25, January 1997 © 1997 Societyfor In Vitro Biology 1071-2690/97 $05.00+0.00
EFFECTS O F M E D I U M C O M P O N E N T S A N D L I G H T O N CALLUS I N D U C T I O N , G R O W T H , A N D F R O N D R E G E N E R A T I O N IN LEMNA GIBBA (DUCKWEED) H. K. MOON ANDA. M. STOMP1 Institute of Forest Genetics, Suwon Kyonggido, Republic of Korea (H. K. M.); ForestryDepartment, North Carolina State University, Box 8002, Raleigh, North Carolina27695-8002 (A. M. S.)
(Received 2 April 1996; accepted 23 September 1996; editor G. C. Phillips)
SUMMARY
Basal media, plant growth regulator type and concentration, sucrose, and light were examined for their effects on duckweed (Lemna gibba) frond proliferation, callus induction and growth, and frond regeneration. Murashige and Skoog medium proved best for callus induction and growth, while Schenk and Hildebrandt medium proved best for frond proliferation. The ability of auxin to induce callus was associated with the relative strength of the four auxins tested, with 20 or 50 ~tM 2,4-dichlorophenoxyacetic aci¢l giving the highest frequency (10%) of fronds producing callus. Auxin combinations did not improve callus induction frequency. Auxin in combination with other plant growth regulators was needed for long-term callus growth; the two superior plant growth regulator combinations were 10 ~tM naphthaleneacetic acid, 10 ~tM gibberellic acid, and 2 p.M benzyladenine with either 1 or 20 ~tM 2,4-dichlorophenoxyacetic acid. Three percent sucrose was best for callus induction and growth. Callus induction and growth required light. Callus that proliferated from each frond's meristematic zone contained a mixture of dedifferentiated and somewhat organized cell masses. Continual callus selection was required to produce mostly dedifferentiated, slow-growing callus cell lines. Frond regeneration occurred on Schenk and Hildebrandt medium without plant growth regulators but was promoted by 1 pM benzyladenine. Callus maintained its ability to regenerate fronds for at least 10 mo. Regenerated fronds showed a slower growth rate than normal fronds and a low percentage of abnormal morphologies that reverted to normal after one or two subcultures. Key words: tissue culture; plant growth regulators; media; light; sucrose.
lection of over 900 strains across all species of Lemnaceae by Landolt (1986). Individual strains within this collection have been used for a variety of duckweed studies by a number of researchers over the last several decades. The collection has recently been transferred to our laboratory. Commercial use of duckweeds in a variety of applications is increasing. A recent patent search has identified 78 patents. Applications include the use of duckweed and its proteins for livestock feed and food additives (Dewanji, 1993; Rokonuddin et al., 1993; Haustein et al., 1994), the use of duckweed for wastewater cleanup and for integrated wastewater-aquacuhure systems (Oron et al., 1988; Cui et al., 1994), and isolation of useful secondary metabolites synthesized by duckweeds, including a steroid that induces flowering (Kaihara and Takimoto, 1991), an algicide (Crombie and Heavers, 1992), and compounds with pharmacological activity (Mesmar and Abussaud, 1991). Duckweeds are quite sensitive to environmental pollutants and are used as bioindicator species (Hoist and Ellwanger, 1982; ASTM, 1991; Jenner and Janssen-Mommen, 1993). Fresh and processed duckweed are marketed for human consumption. Further development of commercial uses of duckweeds could be done by exploiting the genetic diversity within the Lemnaceae or by enhancing the genetic diversity through genetic engineering. Despite increasing interest in both basic science and technological applications, cell and molecular technology for duckweed has not been developed to any great extent. Methods for mutagenesis are available (Slovin and Cohen, 1988), and limited gene cloning has been done
INTRODUCTION
Duckweeds are the sole members of the Lemnaceae. All 32 species within the family's 4 genera are free-floating, aquatic monocots, found on still or slow-moving fresh water. The duckweeds have long been an object of botanical fascination in the field, in the laboratory, and most recently for their potential in wastewater remediation (Landolt and Kandeler, 1987). They can be conveniently grown in the laboratory on simple mineral nutrient solutions, proliferating asexually, through budding of new daughter fronds. Although morphologically reduced, duckweeds are complete plants, with flowers, seeds, roots, leaves, stems, and vascular tissue. The genome size of duckweeds is small, ranging from 0.15 to 1.63 pg DNA/1 C, with chromosome numbers varying widely, from 20 to well over 100 (Urbanska-Worytkiewicz, 1980). Studies to estimate the genetic diversity within the family, either using secondary metabolites (McClure and Alston, 1966) or isozymes (Crawford and Landoh, 1993), revealed considerable variation across genera, species, and geographical isolates within species (strains). The ease of propagation, clonal growth habit, morphological organization, and small genome size make the duckweeds excellent subjects for developmental and molecular studies with the laboratory convenience of yeast. The ease of asexual propagation and long-term stability of clones has allowed the establishment of an extensive col1To whom correspondenceshould be addressed. 20
21
DUCKWEED CALLUS AND REGENERATION (Silverthorne and Tobin, 1990), but convenient, highly efficient methods for gene transfer remain elusive. Tissue culture has only been explored to a limited extent. Chang and co-workers demonstrated that callus formation and frond regeneration are possible in Lemna gibba (Chang and Chiu, 1976, 1978) and Lemna perpusilla (Chang and Hsing, 1978); however, no attempt was made to optimize culture procedures. Our studies expand previous work on tissue culture methodology to better define optimal basal medium formulation, plant growth regulator type and concentration, light, and sucrose concentration for frond proliferation and callus induction and growth in Lemna gibba. Our ultimate goal is the development of an efficient callus culture and frond regeneration system to use in gene transfer methodology. MATERIALS AND METHODS Duckweed plants used in these experiments were produced from a L. gibba G3 culture provided by J. Slovin, USDA (Beltsville, MD). Frond stock cultures were maintained on modified Hoagland's solution containing 0.08 mg/ 1 (16 riM) CuSO4.5H20 and 10 g/1 sucrose. All media components were combined, the media pH adjusted to 5.8, and then autoclaved at 121 ° C for 20 rain, with the exception of indoleacetic acid (IAA) and gibberellic acid 3 (GAs), which were filter-sterilized and then added to cooled, autoclaved media. Unless stated otherwise, incubation of stock fronds and experimental cultures was at 23 ° C, under a 16-h light photoperiod of approximately 40 ttmol m-2s -~ illumination provided by Gro-Lux fluorescent lights. Stock fronds taken from 2-wk-old cultures were used to test the effects of (a) basal media, (b) plant growth regulator type and concentration, and (c) sucrose on frond proliferation, frond survival, and callus induction. Callus proliferated in these experiments was then used to test the effects of plant growth regulator type and concentration, sucrose concentration, and light on callus proliferation and frond regeneration. Frond proliferation and callus induction. The differing abilities of basal media to support frond proliferation and callus induction were tested using: MS (Murashige and Skoog, 1962), half-strength MS, NN (Nitsch and Nitsch, 1969), SH (Schenk and Hildebrandt, 1972), and B5 (Gamborg et at., 1968). Each medium contained 30 g/1 sucrose, 50 I.tM 2,4- dichlorophenoxyacetic acid (2,4-D), and 10 g/1 Difco Bacto-Agar. Four fronds per medium treatment were placed ventral side down on 25 ml of medium in a 100 X 15 mm petri dish, then sealed with parafilm. The experiment was replicated 5 times (5 plates), giving a total of 20 observations per treatment. After 4 wk in culture each frond was scored for frond proliferation, frond survival, and callus induction. Callus derived from this initial experiment was transferred to MS medium with either 50 p21,/2,4-D and 2 I.tM benzyladenine (BA) or 1 I.tM 2,4-D, 10 /aM naphthaleneacetic acid (NAA), 10 ~M GA3, and 2 ~tM BA to maintain growth. This callus was used in subsequent experiments to test the effects of plant growth regulators, sucrose, and light on callus growth and frond regeneration. Two experiments were conducted to determine the effects of (a) auxin type and concentration and (b) sucrose concentration on frond proliferation, frond survival, and callus induction. The auxin experiment had 22 auxin treatments: 4 auxins, 2,4-D, 1AA, NAA, and indolebutyric acid (IBA) were each tested at 4 concentrations: O, 2, 20, and 50 p.M (16 treatments), and 20 taM IAA, IBA, or NAA were tested in combination with either 20 or 50 p.M 2,4-D (6 treatments). MS containing 30 g/1 sucrose and 10 g/l agar was used as the basal medium. In the sucrose experiment, concentrations of 0, I0, 30, and 80 g/l were tested using MS basal medium supplemented with 50 IxM 2,4D. In both experiments, 4 fronds per medium treatment were placed ventral side down on 25 ml of medium in a 100 × 15 mm petri dish, sealed with parafilm. After 4 wk in culture, each frond was scored for frond proliferation, frond survival, and callus induction. Each experiment was replicated 5 times giving a total of 20 observations per treatment. Callus proliferation. The type and concentration of plant growth regulator, basal medium strength, sucrose concentration, and light intensity were tested in separate experiments for their effects on callus proliferation. Nine combinations of plant growth regulators were tested: (1-3) 2, 20, and 50/aM of 2,4-D alone; (4-5) 20 taM 2,4-D combined with 10 taM NAA or 10 I.tM GA3; (6) 20 ~ 2,4-D combined with 10 p.M each of NAA and GA3, (7~3) 20 taM
2,4-D, 10 IxM NAA, and 10/aM GA3 combined with either 2 taM BA or 2 N6-[2-isopentenyl]adenine (2iP); and (9) 1/aM 2,4-D, 10 I.tM NAA, and 10 IxM GAs combined with 2 ~ BA. The basal medium was MS containing 30 g/1 sucrose and solidified with 4 g/1 Difco Bacto-Agar and 1.5 g/1 Gelrite. Callus used for this experiment had been maintained for 12 wk on MS medium supplemented with 50 IxM 2,4-D and 2/aM BA, 30 g/1 sucrose, and solidified with 10 g/1 agar. For each of the 9 treatment combinations, 4 callus pieces of 0.5-0.7 cm in diameter were plated on 25 ml of medium and the experiment was replicated 5 times giving a total of 20 observations per treatment. After 4 wk in culture, fresh weights of each callus piece were taken to evaluate each treatment for their ability to support callus growth. Medium type and strength, sucrose concentration, and light were tested for their effects on callus proliferation in a nested experiment consisting of 12 treatments. Full- and half-strength MS media, each supplemented with either 10, 30, or 80 g/1 sucrose were placed at 23 ° C under the standard 16-h light photoperiod or in darkness, by wrapping the plates in aluminum foil. All media were solidified with 4 g/l Difco Bacto-Agar and 1.5 g/1 Gelrite, and supplemented with 20 laM 2,4-D, 10 laM NAA, 10 taM GAs, and 2 taM BA. Callus used for this experiment had been maintained for 16 wk on MS supplemented with 50 taM 2,4-D and 2 p.M BA. For each of the 12 treatment combinations, 5 callus pieces of 0.5 cm in diameter were plated on 25 ml of medium and the experiment was replicated 3 times giving a total of 15 observations per treatment. After 4 wk of culture, the fresh weight of each callus piece was measured to evaluate treatment effects on callus proliferation. Frond regenerationfrom callus. Both SH and MS basal media were used to test the ability of callus to regenerate fronds. Each medium was supplemented with BA or 2iP at 1 or 10 I.tM. All media were solidified with 4 g/1 Difco Bacto-Agar and 1.5 g/1 Gelrite. Callus used for this experiment was maintained on MS medium supplemented with I p.M 2,4-D, 10 p.M NAA, 10 ~ / G A s , and 2/aM BA for 16 wk. For each of the 8 basal medium/cytokinin treatment combinations, 5 callus pieces, each weighing approximately 0.03 g, were plated on 25 ml of medium and the experiment was replicated 3 times giving a total of 15 observations per treatment. After 4 wk in culture, the ability of each callus piece to regenerate fronds (frequency of frond regeneration) and the number of fronds regenerated per callus piece was determined. Fronds regenerated in this experiment were used to test the relative growth rates between regenerated fronds and stock fronds maintained on modified Hoagland's solution. Both regenerated and stock fronds were cultured on SH or MS medium without plant growth regulators, or with either BA or 2iP at 1 taM. For each of the 12 treatment combinations, 4 fronds were plated on 25 ml of medium and the experiment was replicated 5 times giving a total of 20 observations per treatment. After 4 wk of culture, frond proliferation was determined by final frond count and by total frond fresh weight. All experiments were completely randomized designs. Statistical analyses were done using SAS (SAS Institute Inc., Cary, NC) analysis of variance procedures. Duncan's Multiple Range test was used to establish main effects of medium type and auxin type in Table 1 and Figure 2; contrast analyses were performed on the data from experiments with factorial structure. Specific analyses and the results are noted in the appropriate figure and table footnotes and text. All tests for significance were conducted at the c~ = 0.05 level. RESULTS AND DISCUSSION
Frond proliferation and callus induction. Frond proliferation preceded callus formation on all basal media tested supplemented with 50 ~M 2,4-D, beginning 3 - 5 d after culture establishment and continuing for the first 2 wk of culture. Fronds curled irregularly in all treatments and white or yellow senescent fronds were observed after 2 wk. By Week 4, frond proliferation on MS or half-strength MS media was significantly retarded over that seen on SH, NN, or B5 media (Table 1). Frond senescence was low and uniformly distributed across all media, except SH medium, which showed significantly greater senescence (Table 1). The frequency of callus induction from fronds varied significantly among basal media. Media that promoted frond proliferation did not favor callus induction @able 1). Callus induction was best on MS and half-strength MS media but the frequency of fronds giving rise
22
MOON AND STOMP
FIG. 1. (a) Callus proliferation at the frond base. (b) Callus proliferation at the root tip. (c) Callus proliferationfrom meristematic area. White arrows indicate areas of callus proliferation. (d) Established callus after 16 wk in culture. (e) Frond regeneration from 16-wk, established callus culture. F = frond; CA = callus area. Magnification = X 12.
to callus was less than 10% (Table 1). Nitsch and Nitsch (1969) medium proved the least successful for callus induction. The superiority of MS medium for callus induction and growth has been seen with other monocotyledonous species (Sharp et al., 1984; Ahloowalia, 1984). Chang and Chiu (1976) found that MS supported callus growth in L. perpusiUa. The timing of callus induction, location of responsive tissue, and type of callus did not vary among basal media. Callus induction was slow and first visible after 3 wk in culture. Callus arose occasionally from the frond base (Fig. 1 a), the root tip (Fig. 1 b), and most often from the meristematic region on the ventral surface of the frond (Fig. 1 c). The callus from the meristematic region was a compact but watery mixture of pale green, yellow, and white areas (Fig. 1 c). This callus could be established in long-term culture, whereas that arising
from the frond base or root tip grew slowly and did not persist after 10 wk in culture. In an attempt to increase callus induction frequency, frond developmental stage, cutting the frond, and placement of the frond, ventral surface up or down, were tested but these treatments were without effect. Auxin type, concentration, and combinations of auxins all affected frond proliferation, frond senescence, and callus induction (Fig. 2). In general, auxin was found to stimulate frond proliferation at low concentrations and inhibit it at higher concentrations. Within each auxin concentration, the ranking of auxins relative to their ability to promote frond proliferation was consistent and the auxins differed significantly from each other, with IAA the best, followed by IBA, NAA, and 2,4-D. With the weakest auxins, IAA and IBA, inhibition of frond proliferation was seen only at the highest concentration (50
DUCKWEED CALLUS AND REGENERATION
23 TABLE 1
3OO
i
THE EFFECT OF BASAL MEDIUM TYPE ON FROND PROLIFERATION, CALLUS INDUCTION, AND FROND SENESCENCE AFTER 4 WK OF CULTURE
200
Medium Type~,
Conb'ol
2.4-D
NAA
IAA
IBA
MS ½MS NN SH B5
% Fronds
Proliferation Rate ~J
% Fronds Forming callus~
Senescing ~r
22.8 b 21.5 b 27.8 a 29.5 a 27.6 a
9.6 a 9.0 a 2.5 c 4.7 b 3.9 bc
27.0 b 26.5 b 27.4 b 34.6 a 26.6 b
lO 9 8
g
r
~
5
N
3 2 1 0 Control
2,4-D
NAg,
IAA
IBA
2O
aEach medium was supplemented with 50 uM 2,4-dichlorophenoxyacetic acid (2,4-D). ~Proliferation rate was calculated as the ratio of the final number of fronds to the initial number of fronds planted. cFronds were scored positively for callus induction when three criteria were met: (a) cell multiplication and tissue swelling was obvious in the ventral meristematic zone; (b) tissue color changed from dark green to pale green, yellow-green, yellow, or white; and (c) the texture of the tissue was unorganized and without epidermis. "Fronds were scored as senescing when they had turned yellow or white. °MS = Murashige and Skoog (1962); ½ MS = half-strength MS; NN = Nitsch and Nitsch (1969); SH = Shenck and Hildebrandt (1972); B5 = Gamborg et al. (1968). '~4eans within the column followed by the same letter were not found significantly different using Duncan's critical range test at the 0.05 level or less (~ < 0.05).
10
5
ContrOl
2,4-D
NAA
IAA
IBA
FIG. 2. The effects of auxin type and concentration on frond proliferation, percent callus induction, and percent frond senescence after 4 wk in culture. Frond proliferation was the ratio of final frond number to original number of fronds plated. Percent callus induction was the ratio of the number of fronds that had proliferated callus to the number of fronds proliferated in 4 wk. Percent frond senescence was the ratio of the number of white or yellow fronds to the total number of fronds proliferated in 4 wk. Significant main effects of auxin type were separated via Duncan's Multiple Range test. Differences in auxin concentrations within auxin were separated via a general linear model (PROC GLM) contrast analysis. 2,4-D = 2,4-dichlorophenoxyacetic acid; NAA = naphthaleneacetic acid; IAA = indoleacetic acid; IBA = indolebutyric acid.
laM). Frond proliferation was inhibited at all levels of 2,4-D tested. Frond senescence followed an inverse pattern relative to frond proliferation with senescence lowest on treatments showing the greatest proliferation. There was no significant difference among treatment means for frond proliferation or senescence using combinations of IAA, IBA, or NAA with 2,4-D from that seen with 2,4-D alone (data not shown). High levels of auxin proved necessary for callus induction, with 2,4-D significantly better than the other three auxins in the frequency of fronds producing callus (Fig. 2). Significantly greater callus induction was obtained using 2,4-D at 20 or 50 laM, relative to 2 I.tM 2,4-D; however, the higher concentrations did not differ from each other in their ability to promote callus production, with 9.5 and 9.2% of fronds showing callus production, respectively. The highest concentration (50 pM) of the next strongest auxin, NAA, was significantly less effective for callus induction relative to 2,4-D and was
the only treatment without 2,4-D in which callus induction was observed. Callus induction was observed with all combinations of 2,4-D with other auxins; however, all of these treatments were significantly less effective than 2,4-D alone. Our results differ significantly from those of Chang and co- workers (Chang and Chiu, 1976, 1978) who reported that 50% of their L. gibba G3 cultures produced callus when cultured on MS medium with 10 mg/1 2,4-D (45 IxM) and 1 mg/l 2iP (4.9 lttM). Our efforts to repeat their system resulted in only 6% of fronds giving rise to callus. We hypothesize that the difference in callus induction frequencies results from a difference in the criteria used to score cellular proliferation as "callus." In our experiments, most cellular proliferation consisted of domes sheathed in a surface layer of epidermal cells. If we had scored this cellular proliferation as "callus," our callus induction frequency would have been much higher. We scored only undifferentiated cellular proliferation as "callus." This callus was difficult to maintain requiring reselection at each subculture, a characteristic observed for a number of other species (Mehta et al., 1993; Rogers, 1993; Adda et al., 1994). Within a given treatment, not all fronds responded in an identical manner. This results implies that some epigenetic factor(s) has a main effect on callus induction, as all fronds in any treatment are presumably identical because of the clonal nature of duckweed proliferation. We are currently examining the extent to which the state of frond expansion (from primordia to fully expanded fronds) might regulate callus induction. Budding order could also regulate frond response, as Ashby et al. (1948) showed that daughter fronds arising from one mother frond are not identical. Both frond proliferation and callus induction showed a strong requirement for sucrose. Sucrose-free medium showed very low levels of frond proliferation, no callus induction, and almost half (49.2%) of all fronds had senesced by 4 wk in cultures. Frond proliferation and the percentage of fronds forming callus was greatest at 30 g/1 sucrose (25-fold frond proliferation in 4 wk and 9.1% of fronds form-
24
MOON AND STOMP
ing callus; both means separable by Duncan's Multiple Range test). Frond proliferation was significantly inhibited by 80 g/l sucrose (17fold) but the percentage of callus induction was not significantly different from that obtained on 30 g/1 sucrose (9.1% versus 8.5%). Frond senescence showed a strictly inverse relationship to sucrose with 80 g/1 sucrose giving significantly less frond senescence than 30 g/1 (9.5% versus 26.7%, respectively). Frond curling was observed in the 80 g/1 sucrose treatment. Callus proliferation. Callus proliferation was quite slow on MS medium supplemented with 50 I.tM 2,4-D and 2 ~M BA. Because increasing the 2,4-D concentration alone did not enhance callus growth (data not shown), nine combinations of 2,4-D with NAA, GAz, BA, and 2iP were tested to determine if they would increase the rate of callus growth. Two combinations were found best, although not significantly different from each other: either 1 or 20 ~tM 2,4-D with 10 laM NAA, 10 ttM GA3, and 2 laM BA. This combination of NAA, GAz, and BA with 1 or 20 laM 2,4-D resulted in significantly increased callus fresh weight gain, 0.43 and 0.39 g, respectively, in 4 wk over that seen with 50 laM 2,4-D alone (0.16 g) or with 20 IxM 2,4-D and 10 ttM NAA (0.30 g). BA promoted callus proliferation better than 2iE Although tested in combination with 20 ~tM 2,4-D alone or with 20 laM 2,4-D and 10 I.tM NAA, a beneficial effect of GA3 on fresh weight gain was not statistically verified. Sucrose concentration and the presence of light had strong, statistically separable main effects (via contrast analysis) on callus growth. Mean callus fresh weights after 4 wk in culture in the presence or absence of light were 0.16 g versus 0.04 g, respectively. Callus cultured without light did not grow, regardless of basal medium formulation or plant growth regulator levels. During 4 wk of culture in the absence of light, the callus turned yellow or white and gradually died. Our results differed from those of Chang and Chiu (1978) who observed callus growth in the absence of light. This discrepancy could have resulted from incomplete darkness in the experiments of Chang and Chiu (1978). We have observed slow callus growth under light levels of 5 lamol m-2s -1 (unpublished data). No significant difference was observed between mean callus fresh weights after 4 wk on media containing 10 or 30 g/l sucrose (0.121 g versus 0.120 g, respectively), however, callus fresh weight was significantly reduced on medium containing 80 g/1 sucrose (mean fresh weight of 0.058 g in 4 wk). Some cultures reddened on their surface when callus was grown on medium with 80 g/1 sucrose. Proliferating callus could be separated into two types based on color and the degree of organization. A relatively dedifferentiated callus was identified that was a pale green to greenish yellow color. This relatively unorganized callus had a slower growth rate than the second, more organized callus type, which had a darker green color, patches of epidermal cells, and a faster growth rate. As these mixed cultures were maintained, sectors would brown and die, allowing careful selection of one callus type from the other. Repeated selection produced cultures that were primarily undifferentiated; however, organized patches of callus continued to arise. Frond regeneration. Both SH and MS basal media supplemented with cytokinin were tested for their ability to regenerate fronds using callus that had been established in vitro for at least 16 wk (Table 2). All treatments, including cytokinin-free media, supported frond regeneration by Week 3. A significant main effect of basal medium type was observed when measured as the percentage of calli regenerating fronds or the number of fronds regenerated per callus piece. SH medium was superior to MS medium independent of cytokinin
TABLE 2 THE EFFECTS OF BASALMEDIATYPE AND CYTOKININTYPE AND CONCENTRATIONON THE FREQUENCYAND EFFICIENCYOF FROND REGENERATIONFROM CALLUS AFTER 4 WK IN CULTURE Basal Medium°
Cytokinin uM b
% Callus Regenerating Fronds
Number of Fronds Regenerated/Callus Piece
SH
control, 0 BA, 1 BA, 10 2ip, 1 2iP, 10 control, 0 BA, 1 BA, 10 2iP, 1 2iP, 10
66.7 100.0 66.7 80.0 86.7 46.7 53.3 33.3 53.3 60.0
12.2 25.4 9.9 12.8 15.0 2.1 1.9 2.2 3.3 4.8
MS
a SH=Schenk and Hildebrandt (1972); MS=Murashige and Skoog (1962). b BA = Benzyladenine;2iP = N6-[2-isopentenyl]adenine.
type or concentration (number of fronds regenerated 13.04 versus 1.47, respectively; for the percentage of callus pieces regenerating fronds 80.0% versus 49.33%, respectively, both sets of values separable by contrast analysis). Cytokinin type, BA versus 2-iP, within medium was not associated with a significant effect on either the number of fronds regenerated per callus piece or the percentage of callus pieces regenerating fronds. However, in SH medium, 10 IxM BA significantly promoted a greater number of fronds regenerated per callus piece when compared to the 1 ttM BA treatment or the control (0 ~tM BA). The calli derived from the initial cultures were transferred to fresh medium at 3-wk intervals and their ability to regenerate fronds was maintained for more than 10 mo., although callus growth rate slowed as the cultures aged. Frond regeneration from established callus cultures (Fig. 1 d) started on the callus upper surface and was visible by the 2nd wk after transfer to medium supplemented with cytokinin (Fig. 1 e). Chang and Chiu (1978) reported frond regeneration after 8 wk using MS medium. Differences in regeneration media between our work and that of Chang and co- workers is the most likely cause of the differing results. Variation in frond size, color, degree of flatness, multiple frond fusions, and vitrification occurred in regenerating fronds during the first 3 wk of regeneration. By the end of the first subculture period (4 wk), frond morphology appeared indistinguishable from stock fronds. Fronds regenerated from callus, when returned to liquid medium, proliferated normally and were comparable to our stock frond cultures. ACKNOWLEDGMENTS Our thanks to Dr. Ben Bergmann for help with statistical analyses. REFERENCES Adda, S.; Reddy, T. P.; Kavi Kishor, P. B. Somatic embryogenesisand organogenesis in Guitzotia abyssinica. In Vitro Cell. Dev. Biol. 30P:104-107; 1994. Ahloowalia, B. S. Forage grasses. In: Ammirato, E V.; Evans, D. A.; Sharp, W. R., et al. eds. Handbook of plant cell culture. Volume3. New York: Macmillan Publishing Co.; 1984:91-125.
DUCKWEED CALLUS AND REGENERATION Ashby, E.; Wangermann, E.; Winter, E. J. Studies in the morphogenesis of leaves. III. Preliminary observations on vegetative growth in Lemna minor. New Phytol. 49:374-381; 1948. ASTM, American Society for Testing Materials. Standard guide for conducting static toxicitl~ tests with Lemna gibba G3.E1415- 91. ASTM Annual Book of Standards. Volume 11.04. Philadelphia, PA. 1991. Chang, W. C.; Chiu, R L. Induction of callus from fronds of duckweed (Lemna gibba L.). Bot. Bull. Academia Sinica 17:106-109; 1976. Chang, W. C.; Chiu, P. L. Regeneration of Lemna gibba G3 through callus culture. Z. Pflanzenphysiol. Bd. 89.S:91-94; 1978. Chang, W. C.; Hsing, Y. I. Callus formation and regeneration of frond-like structures in Lemnaperpusilla 6746 on a defined medium. Plant Sci. Lett. 13:133-136; 1978. Crawford, D. J.; Landolt, E. Allozymic studies in Spirodela (Lemnaceae): variation among conspecific clones and divergence among species. Syst. Bot. 10:389-394; 1993. Crombie, L.; Hearers, A. D. Synthesis of an allelopathic cyclopentenone from Lemna trisulca. J. Chem. Soc. Perkin Trans. 1:2685-2687; 1992. Cui, Y. B.; Chen, S. L.; Wang, S. M. Effect of ration size on the growth and energy budget of the grass carp, Ctenopharyngodon idella Val. Aquaculture 123:95-107; 1994. Dewanji, A. Amino acid composition of leaf proteins extracted from some aquatic weeds. J. Agric. Food Chem. 41:1232-1236; 1993. Gamborg, O. L.; Miller, R. A.; Ojima, K. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50:151-158; 1968. Haustein, A. T.; Gilman, R. H.; Skillicorn, R W., et al. Performance of broiler chickens fed diets containing duckweed (Lemna gibba). J. Agric. Sci. 122:285-289; 1994. Hoist, R. W.; Ellwanger, T. C. Pesticide assessment guidelines, subdivision J hazard evaluation: nontarget plants. EPA-540/9-82- 020; Washington, DC: Government Printing Office; 1982. Jenner, H. A.; Janssen-Mommen, J. P. M. Duckweed Lemna minor as a tool for testing toxicity of coal residues and polluted sediments. Archives Environ. Contam. Toxicol. 25:3-11; 1993. Kaihara, S.; Takimoto, A. A flower-inducing substance derived from norepinephrine upon contact with intact Lemna plants. Plant Cell Physiol. 32:1107-1109; 1991.
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Landolt, E.; Kandeler, R. The family of Lemnaceae, a monographic study. Volume 2 of the monograph: phytochemistry; physiology; application; bibliography. Zurich, Switzerland: Veroffentlichungen des Geobotanischen Institutes ETH, Stiftung Ruebel; 1987. McClure, J. W.; Alston, R. E. A chemotaxonomic study of Lemnaceae. Am. J. Bot. 53:849~360; 1966. Mehta, U. J.; Hazra, S.; Mascarenhas, A. F. Somatic embryogenesis and in vitro flowering in Brassica nigra. In Vitro Cell. Dev. Biol. 29P:14; 1993. Mesmar, M. N.; Abussaud, M. The antibiotic activity of some aquatic plants and algal extracts from Jordan. Qatar Univ. Sci. J. 11:155-160; 1991. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473496; 1962. Nitsch, J. R; Nitsch, C. Haploid plants from pollen grains. Science 163:8587; 1969. Oron, G.; De-Vegt, A.; Porath, D. Nitrogen removal and conversion by duckweed grown on wastewater. Water Res. 22:179-184; 1988. Rogers, S. M. D. Culture phenotype effects on regeneration capacity in the monocot Haworthia comptoniana. In Vitro Cell. Dev. Biol. 29P:9-12; 1993. Rokonuddin, A.; Kabirullah, M.; Khan, S. A., et al. Preparation of poultry feed for starters using duckweeds and mixed pulses. Bangladesh J. Sci. Ind. Res. 28:33-37; 1993. Schenk, R. U.; Hildebrandt, A. C. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50:199-204; 1972. Sharpe, W. R.; Evans, D. A.; Ammirato, P. V., et al., eds. Handbook of plant cell culture. Volume 2. Crop species. New York: Macmillan Publishing Co.; 1984:69-136. Silverthome, J. Post-transcriptional regulation of organ-specific expression of individual rbcS mRNAs in Lemna gibba. Plant Cell 2:1181-1190; 1990. Slovin, J. R; Cohen, J. D. Levels of indole-3-acetic acid in Lemna gibba G3 and in a large Lemna mutant regenerated from tissue culture. Plant Physiol. 86:522-526; 1988. Urbanska-Worytkiewicz, K. Cytological variation within the family of Lemnaceae. Veroff. Geobot. Inst. Eidg. Tech. Hochsch. Stift. Ruebel Zuer. 70:30-101; 1980.
