Proc. Fla. State Hart. Soc. 96: 266-269. 1983.
MICROPROPAGATION OF LEATHERLEAF FERN
(RUMOHRA ADIANTIFORMIS)1
Shirmy Y. Chen and Paul E. Read
Department of Horticultural Science and
Landscape Architecture,
University of Minnesota,
1970 Folwell Ave., St. Paul, MN 55108
words, tissue culture, preconditioning,
Additional index
cytokinin.
Abstract. Large-scale micropropagation of Rumohra adian-
tiformis (G. Forst.) Ching was achieved. Excised rhizome tips
developed into plantlets on modified Prague's medium. Pre
conditioning of the intact rhizome tips affected both the
contamination rate during stage I and the plant growth
during stage II in tissue culture. With 10 mg/Biter isopentenyl
adenine (2iP), 10-15 mg/liter kinetin or 1 mg/liter zeatfn in
the medium, 20 to 25 fronds were obtained after 9 wk in
light (12 hr at 30 fxe or 70 fie/m~2/tec). Cytokinin was also
essential for the development of new rhizome masses, which
facilitate subculturing. Fronds were subcu!tured and resulted
in 30-40 fronds/flask from solid (regardless of kinetin level)
and liquid (with 0.5 mg/liter kinetin) cultures. Explant length
interacted with solid vs. liquid media, with >3 cm best for
solid and <1 cm best for liquid. Potential annual propagation
rate from a single rhizome tip can be as high as 16,000,000
plantlets ready for potting and transfer to the greenhouse.
The cut fronds of leatherleaf fern are commonly used
as background greenery by florists. Commercial propagation
is by rhizome division, but because frequent replanting is
necessary, there is a demand for large scale propagation.
Although ferns have proven to be amenable to culture
in vitro (5, 6), there are neither reports specifically on large
scale micropropagation directly from underground rhizome
tips nor of leatherleaf fern.
Since preliminary investigations illustrated an undesir
able propensity for severe explant contamination, an ob
jective of this study was to discover methods requisite for
rapid large scale micropropagation.
Materials and Methods
Greenhouse grown rhizome tips were used as the source
of explants. Intact rhizome tips were preconditioned by ex
posing them to the air in the greenhouse for 1 wk prior to
the excision of the explants. For surface disinfestation
studies, collected rhizome tips were first soaked in a 10%
diluted commercial bleach (0.5% NaOCl) for 20 min. 95%
ethanol for 5 min. and 0.5% NaOCl for 20 min. The
wetting agent, Tween 20, was also added at 0.03% to the
0.5% NaOCl. Following each of the 3 soakings, the rhizome
tips were rinsed 3 times with autoclaved distilled water for
5 min. Rhizome tips were then transferred to a petri dish
and cut into 6-mm lengths. A new blade was used for each
cutting process in order to minimize possible tissue dam
age and each blade was heat sterilized and cooled prior to
use. Each tip was transferred aseptically onto 25 ml of
medium in 125-ml Erlenmeyer flasks. The basal medium
used was modified Prague's medium (2), amended with 2%
sucrose, organic substances (1, 4), and cytokinins and/or
2,4-dichlorophenoxyacetic acid (2,4-D). The medium was
iScientific Journal Series Paper 13,699, Minnesota Agriculture
Experiment Station.
266
adjusted to pH 5.7 before solidifying with 8% agar. The
medium was then sterilized by autoclaving for 20 min. at
121 °C at 1.06 kg/cm2. All treatments contained 3 to 5 repli
cates. Cultures were kept at 25°C under 12 hr of light
supplied by cool white fluorescent tubes.
To investigate the importance of the preconditioning
procedure, as well as the factors affecting the rate of con
tamination, a 2x4x3 factorial experiment with a completely
randomized design was then conducted. The factorial com
binations were the rhizome tips with and without outer cell
layers removed, the length of time for which intact rhizome
tips were exposed to the greenhouse air (1, 4, 7 and 10 days),
and Tween 20 at levels of 0.03%, 2.5% and 5%. At 2i/2 wk
after initial culture, percentage contamination rate was
recorded. At the end of 20 wk, plantlets surviving con
tamination were measured and number of fronds, frond
lengths, number of roots, and root lengths were recorded
in order to study the performance related to length of pre
conditioning time.
For the cytokinin effect on organogenesis, 2 concentra
tions (1 or 10 mg/liter) of each of the cytokinins (benzyla-
denine BA), 2 iP, kinetin and zeatin) were tested. Data col
lected were number and lengths of fronds and roots after
9 wk of culture in light (12 hr, 70 /xE/m2/sec). A further
experiment followed to test for other factors affecting
plantlet development. In a 2x2x5 factorial experiment with
a completely randomized design, combinations were the
light intensities at 30 vs. 70 ^E/m2/sec, explants with and
without cell layers removed, and kinetin levels at 0, 5, 10, 15
and 30 mg/liter.
Fronds collected from in vitro cultures were recultured
on solid and liquid media. Each was a 42 factorial experi
ment with a completely randomized design. The factorial
combinations were kinetin at 0, 0.2, 2 and 20 mg/liter and
2,4-D at 0, 0.05, 0.1 and 0.2 mg/liter. After 6 wk they were
all transferred to fresh liquid medium without growth
regulators for another 6 wk. Data were then collected at
the end of the total 12 wk.
Some fronds collected from in vitro culture were con
ditioned in agitated liquid medium without growth regu
lators. At the end of every 6 wk, old liquid medium was
discarded and fresh liquid medium was added. Fronds
conditioned for 28 wk were collected for a 3x7x6 factorial,
randomized block design experiment, with liquid and solid
media. There were 3 blocks with fronds of 3 different
lengths, < 1 cm; 1 to 2.9 cm; and > 3 cm, in initial
cultures. Kinetin at 0, 0.1, 0.5, 1, 2.5, and 10 mg/liter and
2,4-D at 0, 0.1, 1, 2, and 5 mg/liter were other treatments. At
the end of 12 wk in culture, number of fronds and roots;
lengths of fronds and roots; and weights of fronds, rhizome
mass, callus, roots, and gametophytes were recorded. When
fronds were collected for rooting, treatments of liquid and
solid media with 0.01 mg/liter of 2,4-D were tested.
Results and Discussion
The rhizome tips preconditioned in the air for 7 days
when combined with 2.5% Tween 20 in NaOCl during the
surface disinfestation procedure had the least contamination
(Fig. 1). Those which were preconditioned for 7 days had
better form and growth (Fig. 2 8c 3) and data collected from
those which were free of contamination showed that
number of days which rhizome tips were preconditioned
affected the number of fronds (Fig. 4). Also, those rhizome
Proc. Fla. State Hort. Soc. 96: 1983.

TW20
% TW20
5°/* TW20
10
DAYS
Fig. 1. Contamination rate (%) of leatherlcaf fern 2j/2 wk after
initial culture in vitro. TW 20 = Tween 20.
Fig. 2. Plantlet formation from a leatherleaf fern rhizome tip pre
conditioned for 10 days.
Fig. 3. Plantlet formation from a leatherleaf fern rhizome tip pre
conditioned for 7 days.
tips with outer cell layers intact resulted in more fronds
in both 4 day and 7 day treatments (Fig. 5).
With different cytokinins in the medium, leatherleaf
fern responded differently (Table 1). BA retarded the
growth at both levels. With 10 mg/liter of 2 iP or 10 mg/
liter of kinetin in the medium, 20 to 25 fronds were obtained
after 9 wk culture in light. Zeatin at 1 mg/liter gave the
same effect as 10 mg/liter of kinetin or 2iP.
Within the range of 5 mg/liter to 15 mg/liter of
kinetin, removal of rhizome tip outer cell layers did not de
crease the number of fronds. However, at 30 mg/liter
kinetin, removal of outer cell layers did benefit frond pro
duction, and with no kinetin in the medium, removal of
Proc. Fla. State Hort. Soc. 96: 1983.
S. E. = 1.95
6.037s TW20——
1
DAYS
Fig. 4. Mean number of fronds/flask produced from leatherleaf fern
rhizome tips preconditioned at various lengths of time (1, 4, 7, and
10 days) and Tween 20 at levels of 0.03, 2.5, and 5%.
S. E. = 1.95
1 h 7 10
DAYS RHIZOME TIPS EXPOSED
Fig. 5. Mean number of fronds/flask produced from leatherleaf
fern rhizome tips preconditioned at various lengths of time (1, 4, 7,
and 10 days) with outer cell layers intact (T) or with outer cell layers
removed (C).
outer cell layers caused decreased frond production (Fig. 6).
Kinetin at both 10 mg/liter and 15 mg/liter resulted in a
similar amount of fronds (20 to 25) after 9 wk in culture at
either 70 ^ or 30 ^e/m^sec (Fig. 7).
The fronds derived from tissue culture and recultured
on either solid or liquid medium did not respond further
to 2,4-D concentrations. Kinetin at 0 to 0.2 mg/liter in
solid or liquid medium resulted in best frond development,
but only 5 to 6 fronds were formed. At 20 mg/liter kinetin
in the medium, new frond development was completely in
hibited. Fronds recultured on solid medium increased the
frond number 2 fold (9 to 11 fronds) after being transferred
to a liquid medium without growth regulators. Those
fronds recultured on liquid medium initially did not in
crease in number of fronds produced after being transferred
to a liquid medium without growth regulators.
The results from the conditioned fronds showed that
the fronds conditioned (in liquid medium without growth
regulators and fresh liquid medium replaced every 6 wk)
for 28 wk were sensitive to 2,4-D concentrations only at 5
mg/liter level, with frond number decreasing in solid as
well as in liquid cultures. Increasing kinetin levels to 5
to 10 mg/liter decreased the number of fronds in liquid
culture. Conditioned fronds were not sensitive to kinetin
267
Table 1. Effect of cytokinins on plantlet development.

Treatments (ing/ liter)

BA 2iP kinetin zeatin 95%
Control 1 10 1 10 1 10 1 10 C.I.

9 23 21 1.19**z

O
No. of fronds (cm) 7 1 0 25 10
Length of fronds 4.8 0.4 0 3.1 0.8 2.2 0.8 1.4 0.24**
No. of roots 13 0 0 15 0 13 0 0 1.80**
Length of roots (cm) 1.3 0 0 2.4 0 2 0 0 0.54**

z**Significantly different at the 0.01 level of probability.

KINETIN mg/liter

Fig. 6. Mean number of fronds/flask produced from leatherleaf fern

cultured in vitro at various kinetin levels (0, 5, 10, 15, and 30 mg/liter)
Fig. 8. A leatherleaf fern frond derived from tissue culture contain
with rhizome tip outer cell layers removed (C) or remaining intact (T).
ing a single frond and a small amount of rhizome mass.

S.E. = 1.95 X 3.2

25
2k

\\
23
22

'/ \\
21

\\
20

■ 19
18

/ / \ \
\\
17
16
15
. Ik / J \
13 \
12
IS \
. 11 \
10 SI \
9
/ > 30 AH
7

10 15 30

KINETIN mg/liter

Fig. 7 Mean number of fronds/flask produced from leatherleaf fern
cultured in vitro at various kinetin levels (0, 5, 10, 15, and 30 mg/liter)
at different light intensities (30 /jE/m2/sec and 70 E//)
applied in solid culture. However, without kinetin present
in the medium, fronds produced from reculturing did not
produce rhizome masses, which is important for further sub-
culturing of fronds (Fig. 8). Also, explant length interacted
with solid vs. liquid media, with > 3 cm best for solid and
< 1 cm best for liquid. Therefore, in liquid culture, 0.5
mg/liter of kinetin with 0.01 to 0.1 mg/liter of 2,4-D was
the best combination, and in solid culture, 0.1 mg/liter of
kinetin with no 2,4-D was best. In liquid and solid cultures,
30 to 40 fronds were produced (Fig. 9).
At stage III, solid medium used for rooting resulted in
better survival rate of 92% vs. 58% when liquid medium
was used.
In commercial operations, the most economical cytokinin
to use for producing crops with equivalent quality is
Fig. 9. Leatherleaf fern fronds produced from reculturing.
kinetin. The preconditioning of the intact rhizome tips
(Fig. 10) in the air in the greenhouse condition is very im
portant. Except for the difference in light intensity, which
was much higher when they were preconditioned, the results
of this experiment were similar to those from Knauss's (3)
work with Boston fern and diffenbachias. He concluded that
the preconditioning of explant source plants not only de
creased the rate of contamination during stage I but also
affected the multiplication of the propagules in tissue
culture.
The system for production of leatherleaf fern from tissue
culture can be best described as shown in Fig. 11, a method
which enables the production of leatherleaf fern from rhi
zome tips and gave a potential annual propagation rate of
up to 16,000,000 (Table 2) plantlets for supply of new stock
plants (Fig. 12).

268 Proc. Fla. State Hort. Soc. 96: 1983.

 Table 2. Potential annual propagation rate of leatherleaf fern from
tissue culture system.

a. Production of fronds

No. of weeks No. rhizome tips No. of fronds

12 20 to 22
99 600 to 660
32 18,000 to 19,800
42 540,000 to 594,000
52 16,200,000 to 17,820,000

b. Plantlets ready for pot planting

56 16,200,000 to 17,820,000

Fig. 10. Intact rhizome tips of leatherleaf fern being preconditioned

in the greenhouse.

Fig. 12. Leatherleaf fern stock plants produced from the micro-
propagation system in this study.

Fig. 11. The system for production of leatherleaf fern from tissue
culture.
Acknowledgment
The authors wish to acknowledge Leben's Florist, Rose-
ville, Minnesota for furnishing plant material.
Literature Cited
1. Chen, S. Y. and Paul E. Read. 1980. In vitro propagation of leather
leaf ferns (Rumohra adiantiformis). Hort Science 15:417.
2. Hirsch, A. M. 1975. The effect of sucrose on the differentiation of
excised fern leaf tissue into either gametophytes or sporophytes.
Plant Physiol. 56:390-393.
3. Knauss, J. F. 1979. Contamination in plant tissue cultures. Proc.
Fla. State Hort. Soc. 92:341-343.
4. Mehra, P. N. and D. S. Sulklyan. 1969. In vitro studies on apogamy,
apospory, and controlled differentiation of rhizome segments of
the fern Ampelopteris prolifera (Retz.) Copel. J. Linn. Soc. Lond.
Bot. 62:431-443.
5. Kshirsagar, M. K. and A. R. Mehta. 1978. In vitro studies in ferns:
growth and differentiation in rhizome callus of pteris vittata.
Phytomorphology. 28(l):50-59.
6. Pedhya, M. A. and A. R. Mehta. 1982. Propagation of fern (Nephro-
lepis) through tissue culture. Plant Cell Rpt. 1:261-263.

Proc. Fla. State Hort. Soc. 96: 1983. 269