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14620316.1985.11515606

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Journal of Horticultural Science
ISSN: 0022-1589 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/thsb19
The in vitro propagation of an endangered species:
Centaurea junoniana Svent. (Compositae)
N. Hammatt & P. K. Evans
To cite this article: N. Hammatt & P. K. Evans (1985) The in vitro propagation of an endangered
species: Centaurea junoniana Svent. (Compositae), Journal of Horticultural Science, 60:1, 93-97,
DOI: 10.1080/14620316.1985.11515606
To link to this article: http://dx.doi.org/10.1080/14620316.1985.11515606
Published online: 27 Nov 2015.
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Date: 26 October 2017, At: 03:22
Journal of Horticultural Science ( 1985) 60 (I) 93-97
The in vitro propagation of an endangered species:
Centaureajunoniana Svent. (Compositae)
By N. HAMMATT• and P. K. EVANS
Department of Biology, Building 44, The University, Southampton S09 SNH, UK
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SUMMARY
Methods are described for the in vitro induction of shoot organogenesis on explants of
cotyledons, hypocotyls, roots and leaves, and for the in vitro propagation through shoot
cultures, of Centaurea junoniana, an endangered plant. Shoots can be rooted and
readily established as plantlets in soil. This work was carried out as a conservation
measure for this species.
Centaurea junoniana Svent. is a woody shrub
indigenous to Gran Canaria, Canary Islands.
The last remaining population of a few
hundred plants is in danger of being engulfed
by lava which would cause 'the extinction of
the species in its native habitat. It is recorded
as existing in two seed banks and in the Jardin
Botanico, Viera y Clavijo, Gran Canaria and
the species is classified as 'Endangered' by the
International Union for the Conservation of
Nature and Natural Resources (I.U.C.N.)
(Lucas and Synge, I978).
In its native habitat it produces terminal,
composite inflorescences, 25-45 mm in
diameter with pale mauve florets. A long
succession of flowers is produced from June to
October. The leaves are pinnately lobed and
often turn red with age (Bramwell and Bramwell, I974). It may thus have potential value
as a horticultural plant, although it is halfhardy in temperate climates and cannot
survive frost.
Many species of plants are now becoming
extinct before possible uses to man are recognized (Synge, 1981) and so these potential
sources of new, renewable resources are lost.
Tissue culture propagation could be a useful
tool in the conservation of rare species,
especially as a method to bulk-up stocks of a
species when its numbers are critically low
(Woods, I982).
In vitro culture of buds has provided a
*Present address: Department of Botany, University of
Nottingham, University Park, Nottingham NG7 2RD.
successful means for propagating woody
species (Abbott, 1978). High levels of
exogenous cytokinins in the medium suppress
apical dominance and stimulate the growth of
axillary buds in the axils of young leaves. Each
axil, in turn, produces its own axillary shoots
and so on (George and Sherrington, 1984).
Such a potentially unlimited system of shoot
production would be of great value in the rapid
propagation of C. junoniana. We report here
the successful establishment and maintenance
of shoot cultures of this species and shoot
organogenesis induced in primary explants of
cotyledons, hypocotyls, roots and leaves.
Rooting of these shoots is also reported.
MATERIALS AND METHODS
Seedling material
Seeds were obtained from the Seed Bank,
Royal Botanic Gardens, Wakehurst Place,
Ardingly, Haywards Heath, West Sussex
RH17 6TN, UK. The seeds were surfacesterilized in 7% v/v 'Domestos' solution for 30
min followed by four 5-min washes in sterile,
distilled water. Seeds were sown in 8 m1 of I%
agar in I5-ml metal-capped Beatson Powder
jars.
Leaf material
The seeds were sown in a mixture of 50%
Levington Universal compost and 50% fine
sand in 5-in plastic pots at 17°C and with a
16 h photoperiod. The seedlings were transferred to John Innes No. I Compost with a
In vitro propagation ofCentaurea junoniana
94
cotyledons and leaves were placed whole on
the medium, and the roots were placed on the
medium in 10 mm-Iong pieces.
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lower humus content two weeks after germination. Seedling growth in a compost with high
humus content is slow and the native soil is
very low in humus (D. Bramwell, pers.
commun.). Leaf material, two to three weeks
old, was surface sterilized in a 20% v/v
'Domestos' solution for 20 min followed by
four 5-min washes in sterile, distilled water.
Culture media for shoot cultures and shoot
organogenesis
Medium for all cultures consisted of
Murashige and Skoog (M&S) salts and
vitamins (Murashige and Skoog, I962) and
3% w/v sucrose solidified with 0.8% w/v agar.
The medium was dispensed as 8-ml volumes in
I5-ml Beatson Powder jars. Indol-3-yl acetic
acid (IAA), naphthalene acetic acid (NAA)
and benzyl amino purine (BAP) were added at
varying concentrations as growth regulators.
The medium was sterilized by autoclaving at
I2I °C for 20 min.
Medium for rooting the shoots
The medium consisted of half concentration M&S salts and vitamins, I% w/v sucrose
solidified with 0.6% w/v agar. Indol-3-yl butyric
acid (IBA) and NAA were added as growth
regulators. For rooting, shoots were transferred to 60-ml powder jars containing 20 ml
of medium.
Culture conditions
All cultures were maintained on shelves
I30 mm below Sylvania Lifeline Daylight
4300 lighting tubes producing c. 2000 lx with a
I6 h photoperiod. The temperature was.
22°C±2°C.
Transfer ofplantlets to soil
Once rooted, plantlets were extracted from
the medium and any agar still attached to the
roots was washed off with water. The plantlets
were then placed in Levington Universal
compost and were kept for at least one week in
a mist propagator at 29°C. On removal from
the propagator the plantlets were transferred
to John Innes No. I Compost with a low
humus content and were kept in frost-free
conditions.
RESULTS
Explant preparation
Three-week-old seedlings were used as a
source of explants. The cotyledons were
removed about l mm from the cotyledonary
node. The shoot bud was then isolated by
excising the hypocotyl 1 mm below the shoot
tip. The root was then detached from the
hypocotyl. The shoot tips, hypocotyls,
Induction of shoot organogenesis on primary
explants
Shoot organogenesis was induced in all
explants and on a range of different media
(Table I; Figure I). It is clear that the
cotyledons gave the best response for shoot
organogenesis over the range of media tested;
hypocotyls were less responsive and roots only
gave rise to shoots when IAA was the auxin
source. This differential potential of seedling
organs for shoot organogenesis was also found
in another composite, Lactuca sativa L.
(Doerschung and Miller, I967).
Whole leaves placed on a medium
containing 0.5 mg 1-1 BAP and 2 mg l- 1 NAA
thickened and expanded, and within 10 days a
TABLE I
Effects of various combinations of growth regulators on shoot production in seedling primary explants and leaves after
nine weeks in culture. Each value represents the mean of at least five cultures
Growth regulator concentration (mg
NAA
BAP
0.2
0.2
1.0
2.0
2.0
2.0
1.0
1.0
1.0
0.5
0.5
r')
IAA
Mean number of shoots produced per explant
Hypocotyl
0
I
1
1
Cotyledon
0
I
5.2
2.3
Root
Leaf
0
0
0
0
4.7
4.0
2.3
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N. HAMMATT and P. K.
95
EvANS
FIG. I
Leaf explant showing shoot formation three weeks after
culture on medium with 2 mg r• NAA and 0 .5 mg 1- 1
BAP x 10.
hard green callus grew from the edges. By
three weeks most explants underwent shoot
organogenesis, producing as many as seven
shoots in nine weeks.
medium. The presence of a small concentration of auxin in the medium, without BAP,
appeared to maintain some exogenouslygenerated apical dominance effect.
Shoot cultures
Shoot tips placed on various media proliferated and produced different numbers of
axillary shoots (Table II ; Figure 2). In media
containing 2 mg 1- 1 NAA the cut ends of the
hypocotyls and cotyledons produced copious
amounts of callus which tended to engulf the
shoots. By reducing the concentration of NAA
to 0.02 mg 1- 1, it was possible to reduce this
callusing. In cultures where there was no
NAA , growth was poor.
Shoots produced in this way were small and
difficult to manipulate. Small shoots could be
elongated by placing the shoot cultures on
M&S medium lacking growth regulators with
2% w/v sucrose. In the presence of a small
concentration of NAA (0.02 mg 1- 1) fewer
shoots elongated and these shoots tended to be
shorter than when there was no NAA in the
Rooting of shoots
Shoots obtained from shoot cultures or by
induction on primary explants could be
successfully rooted in vitro. Two growth
regulators, IBA and NAA, both used in media
for the in vitro rooting of various woody
TABLE II
Effects of various concentrations of BAP and NAA upon
the number of shoots produced per shoot tip explant in
culture after jive weeks in culture. Each value represents
the mean of at least jive cultures
Growth regul ator concentration
(mg r•)
NAA
BAP
2
0.1
2
2
0 .2
0.5
2.5
5.0
Mean number of
shoots per
culture
2.5
2.3
12.4
14.1
In vitro propagation ofCentaurea junoniana
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96
FIG .
2
Shoot culture produced from a seedlin g s hoot tip after
five weeks on medium with 0.2 mg 1- 1 NAA and 5 mg 1- 1
BAP x 4.
species (James, 1983) were tested. Initially
either NAA or IBA at 0.1 mg J- I was used.
After five weeks NAA mainly caused callusing
of the cut ends of the excised shoots although
a few fragile roots were produced. With IBA,
root production was enhanced but at
0.1 mg 1- 1 small emerging roots immediately
callused. All 25 shoots tested produced roots
over 20 mm long within three weeks when the
I BA concentrtion was reduced to 0.0 I mg J- I
(Figure 3).
These rooted shoots could all be successfully transferred to soil.
DISCUSSION
The methods described show that it is
possible rapidly to produce large numbers of
FIG. 3
Plantl et of Ce/1/aurea j unoniana produced by micropro pagation.
plantlets of Centaurea junoniana from a very
small amount of initial explant material. This
will prove useful in the conservation of this
species and also as a method to bulk up stocks
of this species once its uses to man have been
assessed. The shoot culture method will be
especially useful in cloning particular genotypes which may have horticultural value.
We wish to acknowledge the assistance of
the Royal Botanic Gardens, Kew, for supplying seeds of C. junoniana. The photographic
assistance of Mr B. Lockyer and Mr B. V.
Case is much appreciated.
REFERENCES
ABBOTT, A. J. ( 1978). Practise and promise of micropropagation of woody species. Acta
Horticu/turae, 79, 113- 27.
N. HAMMATI and P. K. EvANS
97
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DoERSCHUNG, M. R. and MILLER, C. 0. (1976). Chemical control of adventitious organ
formation in Lactuca sativa L. American Journal of Botany, 54, 410-13.
BRAMWELL, D. and BRAMWELL, Z. (1974). Wildflowers of the Canary Islands, Stanley Thomas.
GEORGE, E. F. and SHERRINGTON, P. D. (1984). Plant propagation by tissue culture, Exegetics
Ltd.
JAMES, D. J. (1983). Adventitious root formation in vitro in apple rootstocks (Malus pumila).
I. Factors affecting the auxin sensitive phase in M.9. Physiologia Plantarum, 51, 149-53.
LucAs, G. and SYNGE, H. (1978). IUCN Red Data Book, International Union for the
Conservation of Nature and Natural Resources.
MuRASHIGE, T. and SKOOG, F. (1962). A revised medium for rapid growth and bioassays with
tobacco tissue cultures. Physiologia Plantarum, 15,473-97.
SYNGE, H. (Ed.) (1981). Biological aspects of rare plant conservation, Wiley.
Wooos, A. (1982). The propagation and distribution of rare and endangered plants. IUCN
Threatened Plants Committee Newsletter No.9, 14-15.
(Accepted 3 July 1984)
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