EPA/600/JA-02/227
2002
Callus Cell Formation and Suspension Culture of Typha latifolia.
Lunique Estime1, Marie O'Shea, Michael Borst
United States Environmental Protection Agency, Water Supply and Water Resources Division,
National Risk Management Research Laboratory 2890 Woodbridge Avenue (MS-104) Edison,
NJ 08837.
Jennifer Gerrity, and Shih-Long Liao
US Infrastructure Inc., 1090 King George's Post Road Suite 407, Edison, New Jersey 08837
Received for publication
The authors gratefully acknowledge the technical assistance of Dr. Chee-Kok Chin from the
Department of Plant Science at Rutgers University. This study was conducted in the Department
of Plant Science, Rutgers University, Cook College, New Brunswick, NJ 08903 through a
contract from the US Environmental Protection Agency's National Risks Management Research
Laboratory, Water Supply and Water Resources Division, Urban Watershed Management
Branch.
1 To whom correspondence should be addressed.
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
-------
Propagation and Tissue Culture
Callus Cell Formation and Suspension Culture of Typha latifolia
Additional Index words. Cattail, tissue culture, growth curve, fresh weight, wetlands
Abstract. This study is the first reported attempt to generate a growth curve from Typha
latifolia L. (broadleaf cattail) callus cells in suspension culture. Several media and hormone
combinations were tested for their capacity to induce callus cell formation from T. latifolia leaf
sections and both male and female inflorescence spikes. A T. latifolia callus cell line was
successfully established from immature female inflorescence spikes. Callus growth on
Gamborgs B5 medium supplemented with 5 mg*!/1 dicamba and 1 mg*!/1 BA was superior to
other media examined. A growth curve in suspension culture was generated on the most
favorable culture medium for callus growth. The mass of the cells increased by 150% by the end
of the growth curve. Chemical names used: dicamba (3,6-dichloro-2-methoxybenzoic acid);
N6-benzyladenine (BA).
Typha latifolia (broadleaf cattail) L. is a native perennial herb that can grow up tolO ft in
height, and forms flowers from June through August. It grows from thick, underground
rhizomes that survive extreme winters and produce shoots in the spring. This plant can produce
more than 2,000 g^m'^year"1 of biomass (Mitsch and Gosselink, 1986). Many consider T.
latifolia to be a nuisance because it grows and reproduces rapidly, however, researchers have
reported that this species can remove pollutants from stormwater wetlands very effectively
-2-
-------
(Breed, 1993; Kadlec and Knight, 1996). T. latifolia grows in freshwater marshes, wet swales,
streams, ponds, and along lake margins (Reddington, 1994). This species is found in all fifty
states and is among the most common aquatic plants. T. latifolia can dominate large areas,
especially where water levels fluctuate (USDA, 1999).
In the early spring, T. latifolia rapidly forms dense colonies that slow down
stormwater-associated flows and allow particles to settle into the sediment (Stockdale, 1991).
T. latifolia is also known to uptake such nutrients and heavy metals from stormwater wetlands as
P, N, Cu, Ni, Zn, and Mg (Prentki et al., 1978; Breed, 1993; Taylor and Crowder, 1983). These
pollutants are then stored in all parts of the plant, including the flower (Taylor and Crowder,
1983). These attributes of T. latifolia make it an excellent candidate for employment in
stormwater constructed wetlands.
In this study we induced callus cell formation from T. latifolia leaf sections, and both
male and female inflorescence spikes, and generated a growth curve for these cells in suspension
culture. We report the initiation of callus cells from 100% of the female explant spikes that
show a faster growth rate on semi-solid medium than reported in earlier studies. Previously,
cattail callus cells were described by Zimmerman and Read (1986) who reported a low
frequency of callus induction; later, Rogers et al., (1998) reported the development of T. latifolia
callus cells that showed a very slow growth rate on Murashige and Skoog (MS) basal medium
(Murashige and Skoog, 1962).
-j-
-------
Material and Methods
T. latifolia leaves and inflorescence segments were collected on June 11, 1999 from
Johnson Park on the Cook Campus of Rutgers University in New Brunswick, NJ. The
developing inflorescence segments were totally enveloped in the foliage leaves. Before
sterilization, the outer foliage leaves were removed and 20- to 30-cm long shoot sections
containing the inflorescence were cut and rinsed with tap water. The shoot sections were rinsed
in ethanol for 1 min then soaked in 0.5% chlorine containing 1% Tween-20® (Carolina
Biological Supply Company) for 10 min followed by three sequential rinses for 1 min in sterile
distilled water. The inflorescence sections were then cut in 5 mm thick cross-section pieces.
The sterilization procedure for the leaves was the same as described for the inflorescence spikes.
The explants were then placed into separate petri dishes containing either MS or Gamborgs B5
basal media supplemented with plant growth regulators (PGRs) dicamba or 2,4-D (2,4-Dichloro-
phenoxy acetic acid) at 1, 2.5, or 5 mg*!/1 (Murashige and Skoog 1962; Gamborg et al., 1976).
All media contained 0.8% agar, 3% sucrose, and were adjusted to a pH of 5.6 with IN HCL or
IN KOH. The petri dishes were placed in the dark at 25 ± 1 'C. Once callus cells were
established, they were subcultured onto their original basal media with the addition of 0, 0.5, 1,
2.5, or 5 mg*!/1 BA (N6-benzyladenine). The cells were subcultured every 4 weeks onto the
same medium from which they were derived. Callus cells on petri dishes placed in 12 L/D
-4-
-------
photoperiod formed shoots and were not used in this study.
Suspension cultures were generated from actively-growing undifferentiated callus cells
isolated from 3-week-old stock cultures maintained on B5 basal medium supplemented with 5
mg*!/1 dicamba and 1 mg*!/1 BA. Callus tissue weighing 5 gm were placed into 125 mL
Erlenmeyer flasks containing 60 mL of this basal medium without agar. Thirty-six flasks were
set up for this experiment and placed on a rotary shaker operated at 110 rpm in the dark at
25 ± 1 'C.
Once removed from the shaker, the flask contents were vacuum filtered to separate the
callus cells from the medium. The cells were harvested, and their fresh weights were obtained to
determine the net weight increase. Initially, the growth curve was intended to last 24 days, with
6 flasks removed from the shaker every 4 days. At day 16 of the growth curve, it was apparent
that the cells would not reach the end of their exponential growth phase by day 24. Therefore, at
day 20 adjustments were made in the number of flasks removed from the shaker. During this
study, six flasks were harvested on days 4, 8, 12, and 16, while three flasks were harvested on
days 20, 24, and 32.
-5-
-------
Results
All three explant sources produced callus cells after 3 to 4 weeks in culture (Table 1),
however, the immature female spikes were the superior explant source. Callus initiation from
the female spike typically occurred between 7 and 10 days. They initially produced white, off-
white, or beige callus cells on both MS and B5 media. After the third subculture the callus cells
began to darken. The callus cells formed a hard, compact-to-loose mass. Callus initiation was
from the inflorescence not from the stem. The callus mass formed only on the outer ring of the
explant and not in the center (Fig. 1). This is similar to what was observed in Gramineae species
(Straub et al., 1992). The callus cells did not form a complete spherical mass until separated
from the explant and sub cultured.
Callus cells were initiated from female spikes on both B5 and MS basal medium at all
PGR concentrations tested (Table 1). Abundant callus growth from the female spike was
obtained from MS basal medium supplemented with 5 mg*!/1 dicamba, which produced hard
white, loose cells; B5 basal medium containing 2.5 mg*!/1 2,4-D produced soft white, loose
cells; and, B5 basal medium containing 5 mg*!/1 dicamba produced hard beige callus cells.
Callus initiation from immature male spikes was low (33%), and these callus cells grew
very slowly. Only MS basal media induced callus formation from the male inflorescence spikes.
-6-
-------
MS basal media supplemented with 5 mg*!/1 2,4-D and MS basal media supplemented with 1
mg*!/1 dicamba induced hard, loose beige cells. The callus cells initiated from the male spikes
were produced after 12 to!4 days in culture. These cells were subcultured once, but eventually
died.
In addition to the female and male inflorescence spikes, leaf tissue was the final explant
source used in this study. Callus initiation from the leaf was slightly higher than the immature
male spikes. The leaf produced callus cells at a rate of 66% and 33% on MS and B5 basal
media,
respectively. Most of these cells were hard and white and grew at a very slow rate. A period of
14 to 21 days was required for callus initiation from the leaf tissue. Even after these cells
formed, several months were required for them to reach a size sufficient for subculturing, we
therefore determined that these cells were not suitable to establish a cell line.
Only callus cells derived from female spikes were suitable for subculturing. They were
subcultured 4 weeks after initiation. Callus cells initiated on B5 basal media were bigger and
had a more uniform growth pattern and color than the cells derived from MS basal media. All of
the callus cells derived from the immature female spikes were subcultured onto B5 basal
medium containing various PGR concentrations. After the third subculture, cells on B5
containing 1 and 5 mg*!/1 dicamba began to form roots (Fig. 2). To prevent root formation and
stimulate callus growth, the cells were subcultured onto the same basal medium from which they
-7-
-------
were derived with the addition of BA (Table 2). BA prevented root formation and accelerated
the rate of callus growth. B5 basal medium supplemented with 5 mg*!/1 dicamba and 1 mg*!/1
BA was found to produce the largest mass of cells at the fastest rate (Fig. 3). This media was
selected as the optimum growth medium.
The suspension cultures were established on B5 medium supplemented with 5 mg*!/1
dicamba and 1 mg*!/1 BA (Fig. 4). The lag phase lasted 4 days during which the cell mass
increased by 2%. Subsequently, the cells entered their exponential growth phase, which lasted
20 days. During this time, the cell mass increased 150%. The doubling time for the cells in
suspension culture was 16 days. Compared with other wetland plants grown in suspension
culture, the growth rate of these cells was very slow. For example, Distichilis spicata (seashore
saltgrass) exhibits a doubling time of approximately 40 hours, and Catharanthus roseus
(madagascar periwinkle) doubles in 2 days (MacCarthy et al., 1980; Warren and Gould, 1982).
The rapidly dividing D. spicata has a lag period of 2 days and an exponential growth phase of 7
days (Warren and Gould, 1982); C. roseus has a lag period of 4 days and an exponential growth
phase of 14 days (MacCarthy et al., 1980).
Discussion
Using several tissue sources as explants, we have identified several new media
formulations that can initiate T. latifolia callus cell formation. However, only the cells initiated
from immature female spikes were suitable to establish a cell line because the cells formed a
single layer that spread evenly across the petri plates, and the general color and appearance of
the cells remained consistent after many subcultures, also, after adding BA to the media, no
-------
apparent signs of embryogensis or organogenesis were visible. These characteristics presented a
very stable cell line that can remain in culture indefinitely.
Zimmerman and Read (1986) concluded the type and concentration of PGR were critical
for callus formation from T. latifolia. They reported callus initiation from male and female
spikes used as explants. As the concentration of PGR changed, the frequency of callus formation
varied. Zimmerman and Read (1986) reported callus initiation from 65% and 49% of the T.
latifolia immature male and female inflorescence spikes respectively. The optimum basal
medium developed in this present study induced callus formation from 100% of the female
spikes used as explants (Table 1). In contrast, this present study indicates that explant source is
the most important factor in callus formation; immature female spikes produced callus cells that
could be used to establish a cell line when grown on several different media, while male
inflorescence spikes and leaf sections did not produce viable cell lines on any of the media
tested. In addition, the T. latifolia cells developed by Rogers et al., (1998) appeared to be about
7.5 mm in diameter at 9 weeks. In comparison, the T. latifolia cells developed in this study were
more than 20 mm in diameter at 4 weeks. Therefore, to produce an abundant mass of cells
relatively quickly, the culture media presented in this report is more suitable, because it
generates callus cells at a higher frequency which had a faster growth rate than the callus cells
reported by either Zimmerman and Read (1986), or Rogers et al., (1998).
It is unclear why the cells in suspension culture had an extended doubling time compared
with other wetland plants, especially since the cells had a rapid growth rate on semisolid medium
-9-
-------
when compared with the cattail cells formed by Rogers et al., (1998). In suspension culture, the
cells developed in this study exhibited clearly distinguishable lag, exponential, and stationary
phases of growth (Fig 4). However, because the cells had a normal growth cycle, the slow rate
of growth was not a major concern.
Literature Cited
Breed, C. 1993. Constructed wetlands R&D facility at TVA's national fertilizer and
environmental research center, p. 369-372. In: Moshiri, G.A (ed.). Constructed wetlands
for water quality improvement. Lewis Publishers. Boca Raton, FL.
Gamborg, O.L., T. Murashige, T.A. Thrope, and I.K. Vasil. 1976. Plant tissue culture media. In
Vitro. 12:473-478.
Kadlec, R.H, and R.L. Knight. 1996. Treatment wetlands. CRC Lewis Publishers. Boca Raton,
FL.
MacCarthy, J.J., D. Ratcliff, and H.E. Street. 1980. The effect of nutrient medium composition
on the growth cycle of Catharanthus roseus G. Don cells grown in batch culture. J.
Exper. Botany. 31:1315-1325.
Mitsch, W.J., J.G. Gosselink. 1986. Wetlands. Van Nostrand Reinhold Company. NY.
Murashige, T., F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiologia Plantarum. 15:473-497.
Prentki, R.T., T.D. Gustafson, and M.S. Adams. 1978. Nutrient movements in lakeshore
marshes, p. 169-194. In: Good, R. E., D.F. Whigham, and R.L. Simpson (eds.)
Freshwater wetlands, ecological processes and management potential. Academic Press.
New York.
-10-
-------
Reddington, C.B. 1994. Plants in wetlands. Kendal/Hunt Publishing Company. Dubuque, IA.
Rogers, S.D., J. Beech, and K.S. Sarma. 1998. Shoot regeneration and plant acclimatization of
the wetland monocot cattail (Typha latifolia). Plant Cell Rpt. 18:71-75.
Stockdale, B.C. 1991. Freshwater water wetlands, urban stormwater, and nonpoint pollution
control: A literature review and annotated bibliography. Resource planning section King
county department of parks planning and resources. Seattle, Washington.
Straub, P.P., D.M. Decker, and J.L. Gallagher. 1992. Characterization of tissue culture
initiation and plant regeneration in Sporobolus virginicus (Gramineae). Amer. J. Bot.
79:1119-1125.
Taylor, G.J., A.A. Crowder. 1983. Uptake and accumulation of heavy metals by Typha
latifolia in wetlands of Sudbury, Ontario region. Can J. Bot. 61:63-73.
USDA, NRCS. 1999. The plants database (http://plants.usda.gov/plantsy National Data Center.
Boca Rouge, LA 70874-4490. USA.
Warren, R.S., A.R. Gould. 1982. Salt tolerance expressed as a cellular trait in suspension
cultures developed from the halophytic grass Distichilis spicata. Z. Pflanzenphysiol. Bd.
107:347-356.
Zimmerman, E.S., P.E. Read. 1986. Micropropagation of Typha species. HortScience. 21:1214-
1216.
-11-
-------
Table 1: Callus production from T. latifolia using various organs as sources of explants.
Plant Growth
Regulator
(PGR)
1 mg« L1 2,4-D
2.5 mg« L1 2,4-D
5 mg« L1 2,4-D
Plant Growth
Regulator
(PGR)
1 mg« L1
Dicamba
Callus Growth
Texture
Color
N
Callus Growth
Texture
Color
N
Callus Growth
Texture
Color
N
Callus Growth
Texture
Color
N
B5 Basal Medium
Male
None
0
None
0
None
0
Female
Moderate
Hard
White
60/60
Abundant
Soft-Loose
White
60/60
Moderate
Hard-Loose
White
60/60
Leaf
Very little
Hard
Brown
12/60
Very Little
Hard
White
12/60
None
0
B5 Basal Medium
Male
None
0
Female
Moderate
Hard-Loose
White
36/60
Leaf
None
0
MS Basal Medium
Male
None
0
None
0
Very Little
Hard
Beige
60/60
Female
Moderate
Hard/Loose
White
60/60
Moderate
Hard/Loose
White
60/60
Moderate
Hard/Loose
Off-White
60/60
Leaf
Slow
Hard
White
12/60
None
0
Very/Little
Hard
White
48/60
MS Basal Medium
Male
Very Little
Hard/Loose
White
48/60
Female
Moderate
Hard
Off-White
48/60
Leaf
None
0
-12-
-------
2.5 mg« L1
Dicamba
mg« L
Dicamba
Callus Growth
C*t\lt\r
N
Callus Growth
Pnlnr
N
None
0
None
0
Moderate
HarH
48/60
Abundant
Hard
60/60
None
0
None
0
None
0
None
0
Moderate
HarH
Whitp
60/60
Abundant
White
60/60
Very Little
HarH
24/60
Very Little
HarH
White
12/60
N=Number of explants forming callus cells/Number of explants placed in culture
Table 2: Maintenance of T. latifolia callus cells on B5 basal media supplemented with various
hormone concentrations after 3 weeks in culture.
BA Concentration
0 mg-L'1
0.5 mg-L/1
1 mg-L/1
2.5 mg-L/1
5 mg-L/1
Auxin Concentration
2.5mg-L/1 2,4-D
5 mg'L"1 Dicamba
2.5mg-L/1 2,4-D
5 mg'L"1 Dicamba
2.5mg-L-1 2,4-D
5 mg'L"1 Dicamba
2.5mg-L-1 2,4-D
5 mg'L"1 Dicamba
2.5mg-L-1 2,4-D
5 mg'L"1 Dicamba
Callus Growth2
++
+++
+++
+++
+++
++++
+++
+++
+
+++
z+ = No callus growth; ++ = 1 - 10 mm callus; +++ = 11 - 20 mm callus; ++++ = > 20 mm callus
-13-
-------
Figure 1: Callus initiation from female spikes.
Figure 2: Root formation of from callus cells grown on B5 supplemented with 5 mg'L"1
dicamba and 1 mg«L BA"1
-14-
-------
Figure 3: Callus cell formation on optimum basal medium.
-15-
-------
14
1?
cs m
•S
1:
O)
'CD
5 «
CO
CD
LL
c
>1
,
§
L
n
1
1 f
o
^ 1
I
? 1
n
L
] I
R ?
n
0 ?
n
J
4 ?
-
L
8
r=T
[3
3
T=,
&1
?
~T ±Std. Dev.
1 — 1 ±Std. Err.
D Mean
Incubation period (days)
Figure 4: Suspension culture of T. latifolia cells grown in B5 medium supplemented with 5 mg
•L"1 dicamba and 1 mgrL"1 B A.
-16-
------- |