Inducing Adventitious Rooting in Cycad Leaves

by Robert Buckley
32005 Pleasant Glen Road, Trabuco Canyon, California, U.S.A. 92679-3228
[email protected]

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old leaf bases on the sides of the trunk (Pant, 1990).
ycads are exotic, ancient and rare, hence their
In South Africa, observations by Roy Osborne (1988)
popularity as collectibles. They also tend to
reproduce very slowly. The time from the that “whole leaves removed from the parent plant
pollination of the female cone to the emergence of and placed in a warm, porous medium . . . (produce)
callogenesis and occasional rhizogenesis . . . .”
the first leaf in a seedling can be measured in years.
Faced with a steady decline in habitat and pressure Supporting this, a happy accident planting dislodged
from both poachers and collectors, many species are leaves of Encephalartos lehmannii, one of which
rooted and produced a new plant after several years,
at risk of extinction. To quote Chamberlain, the living
cycads “are the last of their race, restricted in prompted Nat Grobbelaar to experiment with excised
leaves from ten different species of Encephalartos.
geographic distribution, and struggling for their very
existence.” If cycads are to continue, discovering He was able to show that Encephalartos leaf bases
faster, more efficient means of reproduction is rooted easily (even without being treated with rooting
paramount. These should be simple techniques that hormones) and that these rooted leaves were capable
can be used effectively by almost anyone having of surviving for long periods (7.5 years). Of these
survivors a small percentage regenerated the terminal
basic horticultural skills. Fortunately, the “primitive”
heritage of the cycad has bestowed upon it a unique bud (apical meristem) that gives rise to leaves,
survival advantage. Almost every part of a cycad, cataphylls, sporophylls, and internal stem and root
even individual pinnae (those that have a midrib), systems, and produced new leaves (Grobbelaar,
are capable of regenerating roots. Tom Broom, in a 1993). In 1994, Osborne and Dalzell performed a
recent article in the Cycad Newsletter, described similar experiment with excised Encephalartos
numerous techniques for deriving new plants from woodii leaves treated with “Seradix” rooting powder
pieces of trunk, stem, and roots. and placed in pots of clean sharp river sand. These
were maintained with bottom heat and intermittent
Leaf cuttings can also an excellent source of new spray mist for six years and of these, several of these
plants. Cycad leaves are remarkably resistant to explants also produced new leaves.
desiccation, staying fresh for days after being cut.
Cycads also show a tendency to develop roots from

Figures 1 and 2. Callus and roots on leaf tip cutting of Z. furfuracea (note nitrogen fixing nodules).

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In 1972, I excised the pinnae from a Cycas circinallis
leaf, sliced the rachis into 3 inch segments, dipped
the segment ends into “Rootone” rooting powder and
placed the cuttings in a mist tank under fluorescent
GRO-lights. After 3 months, a few of the pinnae had
produced small callus buds, and of these, two
developed roots. Several rachis cuttings also swelled
and split at the leaf base and rooted. However, the
roots ceased growing when the pinnae withered.
Cuttings of this size possessed the ability, but lacked
the starch reserves necessary for long term
regeneration.

I began to experiment with larger leaf cuttings, using

orchid bark as the support medium. I found that some
leaves would root readily (see Figures 2, 7 & 8), but
that once the leaf itself died, the new roots and callus Figure 4. A rooted Microcycas leaf cutting potted
ceased growing. The photosynthic support of the leaf in a mixture of pumice and sand
seemed to be the driving impetus for regeneration.
from the failures and near successes of past years. A
It appeared that for leaf cuttings to live long enough
to regenerate into a new plant the candidate had to mist tank was used to keep the leaf hydrated long
be a young, newly hardened leaf supplied with enough to generate callus tissue and roots. I also
proper humidity, light, and abundant nutrients. switched from using orchid bark as the potting
medium to sterilized sharp sand and pumice. Once
In the Fall of 1997, I began to work with leaves of the leaf cuttings had formed roots, I moved them
Microcycas calocoma applying what I had learned into an Aerojet hydrophonic unit that sprayed the
suspended cuttings with nutrient solution every 5
minutes for 40 seconds. This caused more rapid root
formation than would soil culture. This is important
because the cutting has to develop a large callus mass
and an extensive root system before the leaf reaches
it natural senescence and began to die.

Over a period of two and a half years eight excised

Microcycas leaves were cultured using this process,
a small sample, admittedly, but all my sole specimen
of Microcycas calocoma could support without
sustaining harm.

Of these eight leaves, three have died: one from

accidental dehydration while in the mist tank, one
never produced callus or root tissue and died after
six months in the mist tank, and a healthy, fully rooted
leaf died due to being “pushed” to produce a new
foliage leaf before it was ready. Three long term
survivors have now developed large root systems,
and two are in an early callus stage (not all leaves
Figure 3. The donor Microcycas plant were started at the same time).
remains in good health.
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Typically, a callus will form at base of the petiole regeneration and new leaf formation, based on
after roughly three months. The size of callus will documented development in Encephalartos leaf
vary. Roots appear simultaneously, or soon after cuttings, requires another 3 to 4 years. It remains to
callus formation. Root growth accelerates in the be seen if hydrophonic culture will speed up this
hydrophonic solution. Roots that develop in the process.
hydrophonic unit are long and straight, but once
potted in fast draining soil, the roots begin to As long as proper light, warmth and humidity are
resemble normal cycad roots. Apical meristem provided, cycad leaves readily produce both callus

Preparation and Culture of Excised Cycad Leaves:

1. Remove healthy leaf from donor plant (leaf should be at least 4 months old, well hardened). This can be the tip of
a large frond, or the base. 2’ long cuttings work best. Soak entire cutting in 10% sodium hypochlorite
for 10 minutes to discourage bacteria and fungal spores.

2. Rinse cutting in tap water for several minutes and shake dry. Soak cut base in B1 rooting solution for 10 minutes,
then dip base in a commercial rooting & fungicide powder such as Rootone, or equivalent.

3. Boil enough sharp sand to fill a small, clean plastic pot (about 20 minutes). Drain and allow to cool.

4. Fill small plastic pot with sand and place on a petri dish. Push a hole into the sand with a sterile spoon.
Place the base of the cutting in the sand and position so that it leans at a low angle. Tamp down the sand to hold
the cutting in place.

5. Place pot in mist tank. For new cuttings, spray briefly with distilled water once a day for two months.
Keep temperature at 90˚F during day, no lower than 75˚F at night. Maintain humidity between 80 to 90%.
Use a “fogger” set to come on 4 times a day. Put mist tank lighting on a timer (16 hours ON, 8 hours OFF).

6. Once a callus and roots have formed, transfer cutting to Aerojet net pot with its bottom cut out. Place the leaf
stem through the hole in the neoprene plug and replace pot in Aerojet unit under plant lights. Mix per 1 gallon
H2O: (2.8 grams Base 5-11-26, 3.5 grams 15.5-0-0 Cal. Nitrate, 1.12 grams Magnesium Sulfate, pH= 6.0/6.5)

7. Monitor root growth. When roots become too large for the Aerojet spray tray, remove the cutting from the net pot
and transfer to a deep pot filled with pumice, sand, and loam. Place in bright shade, keep moist and fertilize.

Figure 5. A rooted leaf cutting is placed

through a hole in the pot’s
neoprene plug. This cutting is Figure 6. An Aerojet hydrophonic unit available from
now ready for the Aerojet unit seaofgreen.com, Phoenix, Arizona, U.S.A.
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material and roots. I have observed callus and root
growth in the genus Cycas, Encephalartos, Zamia,
Dioon, and Microcycas. Not all cuttings respond as
well as those pictured.

Leaf cuttings of Stangeria, Bowenia, Lepidozamia,

Ceratozamia, and Macrozamia have so far shown
negative results, but this may have been due to the
cuttings not being good candidates; root cuttings of
Stangeria produce new plants readily - Stangeria
was one of the first cycads to be successfully tissue
cultured (Osborne & van Staden, 1987).
Figure 7. This root of this cutting has broken away
In the early stages of callus development, it’s from base of the leaf. Callus tissue was
important to monitor hydration (turgor) of the cutting. unusually small in this cutting. This 2
In Microcycas, the rachis will show a distinct ridge year old leaf of D. spinulosum was
between leaflets when it is beginning to dry out. Soon cultured for 7 months.
after, leaflets will begin to turn yellow and drop off does not regenerate within the callus, the rooted leaf
unless water is added to the potting sand and the plant will never be more than a clonal “monster”. An
is misted more frequently. A rounded rachis without extensive study was made of the structure and growth
indentations is the sign of a fully hydrated cutting. of the apical meristem (shoot apex) in Cycas (Foster,
1939). Sixty 2-year old Cycas revoluta seedlings
Experimental cuttings of leaf tip material are also were sectioned and examined microscopically to
being tested to determine their growth potential. determine if there was a single “permanent” specific
When severed at mid-leaf and cultured with several apical cell responsible for shoot growth. (A rooted
rows of pinnae removed, callus material has been
observed swelling the rachis and splitting the area
where the pinnae were formerly attached.

Apical Meristem Regeneration - This is crucial for

the creation of a new plant from a leaf cutting. The
cells that form new leaves (leaf primordium)
surround and overlap this area. If an apical meristem

Figure 8. Two views of root development on an E. lebomboensis cutting after eight months.
This cutting came from the tip of the leaf, rather than its base.
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leaf would be incapable of producing new foliage capable of regenerating an initiation zone (and new
unless some portion of this meristem tissue had been leaves) from cuttings of root tissue alone (of course,
captured along with the leaf base as it was cut from being subterranean, Stangeria lacks a true stem).
the stem. For this reason, Osborne recommends that Time will show if leaf base cuttings and leaf tip
at least 1 mm of stem tissue be included in the leaf cuttings both have the ability to give rise to new
cutting.) However, Foster’s, and other studies have plants. Further experimentation in cloning offsets
shown that there is no single apical cell, but rather from leaves may reveal that the basic structure of
several initial cells (an initiation zone) that lie above the apical meristem in cycads is more generic and
a mound of cells known as the central mother zone adaptable than we currently believe possible.
and these give rise to all other types of zones in the
apex by cyclic episodes of division. Despite having limited culture material available for
Microcycas, the cuttings produced seem promising
So here we have an interesting mystery. Is it possible for the artificial propagation of this rare genus.
for a new initiation zone to spontaneously appear in Hopefully, experiments similar to this one and those
the mass of undifferentiated regenerative tissue that performed by Grobbelaar and Osborne will inspire
forms in a cultured leaf base? This is currently others to experiment on their own and help ensure
unclear. Two early rooted cuttings were from rooted that the “Living Cycads” remain just that.
leaf tips, not leaf bases. There is also the incidence
of Cycas pinnae rooting. Additionally, Stangeria is

Figure 9. Root development (left) in a Microcycas cutting after 4 months in mist tank and (right)
a cutting after 4 months in a mist tank and 3 months in the Aeroject unit (shown below).

Figure 10. A Microcycas cutting

being moved into the
Aerojet unit.

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 Figure 11. Two views of the roots of the Microcycas cutting shown in Figure 4. Photo at left
shows the still expanding callus area (note cracks). This cutting is 18 months old.
Acknowledgements:
Roy Osbornewas kind enough to review the first draft of this article and provide helpful suggestions for
improvement. Thanks also to Roy Osborne, Libby Besse and Severn Doughty for providing copies of
hard to find reference material, which proved to be invaluable.
References and additional reading:

Broome, T., Never Throw Away a Cycad, The Cycad Newsletter, 21(3), pp. 8-10, 1998.

Foster, A.S., Structure and Growth of the Shoot Apex of Cycas Revoluta, American Journal of Botany, 26, pp. 372-386, 1939.

Grobbelaar, N., Vegative Propagation of Encephalartos species using Excised Leaves, Proceedings of the Third International
Conference on Cycad Biology, pp. 85-89, 1995

Norstag, K.J., and Nicholls, T.J., The Biology of the Cycads, Cornell University Press, pp. 38-39, 1997

Osborne, R., Hopes Fade for a Wild Encephalartos woodii, Encephalartos , 40, pp. 22-23, 1994.

Osborne, R., and Dalzell, C., Potential Propagation of Encephalartos Woodii from Excised Leaves, Encephalartos 45,
pp 16-17, 1996.

Osborne, R., and van Staden, J., In Vitro Regeneration of Stangeria eriopus, HortScience 22(6): 1326, 1987.

Pant, D. D., and Das, K., Occurance of Non-Coralloid Aerial Roots in Cycas. Memoirs of the New York Botanical Garden.
57: 56-62. 1990.
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