Final report to the Australian Flora Foundation
Micropropagation of threatened species of Persoonia
J.R. Gorst, Department of Plant Science, University of Tasmania
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Abbreviations used in the text : IAA = indole-3-acetic acid: IBA = indole-3-butyric
acid; 2.4-D = 2.4- dichloropnenoxyacetic acid; 2iP = N6-iso pentenyl amino
i) Preliminary work
Work on the project commenced in November 1991 with collections of fruits
and stem lengths of P. juniperina from the Tasman Peninsula for
the purpose of conducting germination trials and preliminary micropropagation
and cuttings trials on a species which is widespread and not endangered.
It was hoped from this to be able to identify problems that might be characteristic
of the family as a. whole before any work was undertaken on rare members
of the family. Of 40 fruits. 31 were found to contain a grub - later identified
as a member of the Cecidomyiidae; this left few viable fruits for experimentation
but these remaining fruits were surface sterilized, half were treated
in 32% HC1 at 60° C for 2 min. and they were all then placed on an
agar medium and left at 24° C. To date none of the fruits has germinated.
For the tissue culture experiments 30 nodal explants and 30 shoot tips
were surface sterilized in 2% (av. chlorine) sodium hypochlorite and placed
on a high nutrient - MS - (Murashige & Skoog. 1962) and low nutrient
– B5 – (Gamborg et al) medium. Half of all of these cultures
showed fungal contamination within 2 weeks and of the non-contaminated
explants,. all had turned black and died within 8 weeks. For glasshouse
experiments, a total of 30 cuttings were taken and were treated in the
standard way used by the Royal Tasmanian Botanical Gardens for cutting
material (i.e. no hormone treatment; a “Clonex” dip supplying
3 g/L IBA; a "Clonex" dip supplying 8 g/L IBA) - the cuttings
were kept in the mist propagator at the Botanical Gardens. Most of the
cuttings turned black and died without developing adventitious roots,
within 3 months; cuttings still alive have not produced roots.
This preliminary work thus identified three main problem areas :
i) germination / viable seed
ii) phenolic production in tissue culture and cutting material
iii) contamination in explants taken from field-grown material for tissue
In subsequent collections of P. juniperina and another species,
P. gunnii, in January 1992 these problems were confirmed.
At the end of January 1992, we were fortunate enough to be included on
a National Parks flight down to Melaleuca in the South-west of the state
and used the opportunity to search for the rarest Tasmanian persoonia
P. moscalii which is thought to occur only in the vicinity of
Bathurst Harbour. In habit and habitat, this species is very similar to
P. gunnii and, in fact, had been included with P.gunnii
until a recent revision of the Tasmanian Persoonias (Orchard, 1983). The
plant proved to be in a fairly inaccesible and remote area and given this,
plus the fact that the likelihood of being able to achieve any success
with propagation was very low (because of results with P. juniperina
and P.gunnii), we decided that we would focus all efforts on
P. gunnii until such time as we felt confident about the conditions
necessary for successfully handling this species.
ii) Experiments with P. gunnii
a} Embryo culture : Because of widespread reports of the difficulty
of germinating Personia (see e.g. Wrigley & Fagg, 1989) embryo culture
offers a possible means of bypassing dormancy problems; the technique
involves removal of the intact embryo from the seed and culturing it on
a suitable nutrient medium. P. gunnii (which occurs at higher
sub-alpine altitudes in Tasmania e.g. Mt Field, Hartz Mountains and Lake
St Clair) flowers in late March and produces most fruit in mid-April although
fruits can still be found on plants almost all year round. The fruit is
a fleshy (turning from green to dark purple as it matures) drupe and the
seeds are found inside a stony endocarp. Our observations indicated that
two seeds are usually produced but that one invariably aborts when the
embryos are still very immature (heart-shaped stage). We collected mature
fruits throughout the year (January, March, April, May, July, September)
from the locations mentioned above and found that the best method of removing
embryos was to crack surface-sterilized fruits open with a nut-cracker
and aseptically dissect out the embryo; the procedure was not entirely
satisfactory because of the difficulty in removing the embryo from the
closely adhering testa without damaging it. Embryos not removed from the
testa did not respond well; damaged embryos showed marked reddening (phenolic
production ?) and death; immature embryos died. The best time of year
for taking fruits was April/May - outside this, fruits showed high abortion
rates and insect damage.
The embryos were placed on a variety of culture media both liquid and
solid testing the effect of growth regulators such as cytokinin, auxin
and gibberellic acid (GA3). Nutrient levels, sucrose concentration and
dark/light incubation were also tested. In the end, none of the treatments
tried was satisfactory; the embryos in all cases were extremely slow to
grow and tended to be highly distorted, producing a large single root
(members of the Persoonia genus are not thought to produce proteoid
roots [Wrigley & Fagg, 1989] and anyway, such roots require a soil
bacterium for initiation and therefore do not develop in sterile conditions,
so this finding cannot be explained in these terms) and very little or
no shoot development. However, in a few instances (embryos cultured on
a medium containing 2 µM 2,4-D + 0.2 µM kinetin for 28 days
and then transferred to a medium containing 0.75 µM IAA + 5 µM
2iP. Shoot growth began after 4 months; also, embryos grown in a very
small volume of liquid medium containing 0.1 µM 2iP grew quite well
although they were vitrified (a physiological condition, essentially brought
on by "waterlogging" of tissue) and hence would present problems
in planting out to soil. GA3 was used at various levels (0.1 - 10 µM)
and there was some indication that the higher level may be beneficial
but unfortunately the results for this experiment were confounded by contamination
problems; other workers have suggested that GA3 treatments may help in
germination of intact seeds (Wrigley & Fagg, 1989). These results
suggests that embryo culture may have promise but further experimentation
would have to be done at optimal collection time.
b) Micropropagation : The first problem to overcome with this
method was disinfestation of field- grown material. Various treatments
were tried (e.g. sodium hypochlorite, benzalkonium chloride, mercuric
chloride and hydrogen peroxide) in combination with pretreatments in benlate
and running water. It was found that a (1h - overnight) treatment in running
water was very successful. The problem of blackening of explants (phenolic
production) proved to be insurmountable in the time frame of the project.
Many treatments were tried including -
* addition of anti-phenolic agents to culture media - polyvinyl-pyrrolidone,
serum albumin, dithiothreitol, ascorbic acid, benzyladenine, indole-butyric
acid, activated charcoal, aminooxyacetic acid
* pretreatment of explants in anti-phenolics prior to transfer to media
* low temperature incubation
* dark incubation
* various basal media - MS, B5, Woody Plant Medium (Lloyd & McCown,
calcium medium, addition of growth regulators
* liquid and solid media
* orientation of explants in media
* type of explant taken i.e. shoot tip, nodal
* time of year at which explants were taken
* moving explants to fresh medium every day
None of these treatments in any way inhibited blackening - some increased
it. This phenolic problem in the Proteaceae is not unique to Persoonia
with other laboratories reporting severe blackening in Banksia
(Dixon, pers. com. - Kings Park & Botanic Garden, Perth) and my own
laboratory (in a study this year designed to complement the Persoonia
work) having similar results with Bellendena montana and to a
lesser but still significant degree with Agastachys odorata ,
Cenarrhenes nitida, Orites diversifolia and Lomatia
tinctoria. In fact, very few members of the family have been successfully
tissue cultured because of the phenolic problem. The genus which responds
best in tissue culture is Grevillea (see e.g. Gorst et al., 1978;
Bunn & Dixon, 1992a); there also appears to be some success with Hakea
and Telopea speciosissima (see Barlass, 1991) and Stirlingia
latifolia (Bunn & Dixon, 1992b).
iii) Experiments with other members of the Proteaceae
This year, as indicated above, an Honours student - Mr Patrick Ball -
has surveyed a number of other members of the Tasmanian Proteaceae. There
has been varying success with these. Bellendena montana and Telopea
truncata respond well to embryo culture ; Cennarrhenes nitida
and Orites diversifolia show a marked seasonal response to micropropagation
techniques and it is only in recent collections that explants have remained
green and showed signs of growth - it remains to be seen whether these
explants can now respond to media designed to induce rapid proliferation;
Lomatia tinctoria explants taken at any time throughout the year
initially responded well to culturing but phenolic problems arose as soon
as material was subcultured.
Like most other members of the Proteaceae, Persoonia seems to
present problems in its response to micropropagation techniques. The most
significant of these is production of phenolics which leads to rapid blackening
and death of explants and which cannot be inhibited by standard anti-phenolic
treatments. Until the problem can be overcome, there is no chance of being
able to tissue culture Persoonia or to achieve any repeatable
success with taking cuttings (some amateur botanists have reported successes
with cuttings but the success rates are extremely low and cannot be linked
to any particular treatment - Wrigley & Fagg, 1989). The next stage
is probably to collaborate with a chemist and attempt to identify the
major phenolic(s) produced, in the hope that this might then suggest a
means to inhibit synthesis.
Embryo culture of Persoonia is much less successful than for
other members of the Proteaceae but, with further experimentation, may
prove to be a feasible way of overcoming seed germination difficulties.
The experiments have, at least, demonstrated that dormancy is probably
linked with inhibitors produced within the seed (since the embryos do
show precocious germination when removed from the seed) although there
are obviously inherent physiological factors acting, as well, to account
for the slow development of seedlings.
Barlass, M. (1991). Commercial production in Australia and New Zealand.
In : Micropropagation - Technology & Application, eds. P.C. deBergh
& R.H. Zimmerman. Kluwer Academic Publishers pp. 191 - 198.
Bunn, E. & Dixon, K.W. (1992 a). In vitro propagation of the rare
and endangered Grevillea scapigera (Proteaceae) HortScience 27
: 261 - 262.
Bunn, E. & Dixon, K.W. (1992 b). Micropropagation of Stirlingia
latifolia (Proteaceae). an important cut flower from Western Australia.
HortScience 27 ; 368.
Gamborg, O.L.; Miller, R.A. & Ojima, K. (1968). Nutrient requirements
of suspension cultures of soybean root cells. Exp. Cell Res. 50 : 151
Gorst, J.R.; Bourne, R.A.; Hardaker, S.E.; Richards, A.E.; Dircks, S.
& deFossard, R.A. (1978). Tissue culture propagation of two Grevillea
hybrids. Proc. Int. plant Prop. Soc. 28 : 435 - 446.
Lloyd, G. & McCown, B. (1980) Commercially-feasible micropropagation
of mountain laurel (Kalmia latifolia) by use of shoot-tip culture.
Proc. Int. Plant Prop. Soc. 30 : 421 - 427.
Murashige, T. & Skoog, F. (1962). A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol. Plant. 15 : 473 -
Orchard, A. E. (1983) The Tasmanian species of Persoonia Smith (Proteaceae).
Brunonia 6:217- 240.
Wrigley, J.W. & Fagg, M. (1989). Banksias, Waratahs and Grevilleas.
Angus & Robertson.