TECHNICAL BULLETIN Y2
MICROPROPAGATION O F AFRICAN VIOLET FROM PETIOLE
AND LEAF BLADE TISSUE
Maria Ioannou
AGRICULTURAL RESEARCH INSTITUTE
MINISTRY O F AGRICULTURE AND NATURAI- RESOURCES
MICROPROPAGATION O F AFRICAN VIOLET FROM PETIOLE
AND LEAF BLADE TISSUE
Maria loannou
SUMMARY
African violet (Sointpai~liaionurziha Wendl.) plants mere produced irom pctiole segment\ and f n ~ mleai hlijde betlions. Both types of tissue were cultured asepticiilly on a Murilsh~geand Skoog (MS) medlum containing <%naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA) and adenine sulfate. The segments produced reisral
shoots in about 8 weeks from culture, which when subcultured on fresh medium dewid of grouth wb\tances, dc"eloped into rooted plantlets which reached trnnspkintable size in 8-10 a r e k r irom transfer. Rooted plantlet, mere
then transplanted in pots containing a sterile >oil mixtuic (2:l v/v of peat m,,% and foreir ,011) a h e r e the) greu tu
flowering within fuur month,. With this technique. more than 51H) plantr could he produced trim ;I s i n ~ l epetiolc or
leaf hlade within a period of 7-8 months.
T h e use of tissue culture as a method of clonal
propagation of plants (in vitro propagation or micropropagation) has been expanding very rapidly in
recent years. It has found wide practical application
in the propagation of herbaceous ornamental plants
and, for some species, like orchids, is presently used
as a standard practice of their culture (Murashige,
1974). Tissue culture allows multiplication of species
difficult t o propagate by conventional methods, and
enables rapid production of a large number of plants
from a Limited number of mother stock plants. Even
with species easily propagated with traditional
methods, tissue culture may be used for rapid clonal
increase of newly selected or rare cultivars. Another
major advantage is that tissue-culture-derived plants
are of superior quality and of better health state
compared to those produced with conventional
methods (Murashige. 1978).
In an effort to introduce the in \,irro propagation
o f ornamental plants in Cyprus. a relevant research
programme was recently undertaken with some
ornamentals such as carnation. African violet.
Kalanchoe. lily and geranium. In this paper the
work with the African violet is reported.
African violet (Soinrpaulia ior~anrhu Wendl.). a
member of the Gesnrriacear, is a popular house
plant with attractive velvety foliage and beautiful
flowers of various colours (purple. pink. white). It is
conventionally propagated with leaf cuttings (Start
and Cumrning. 1976). Each cutting produces three
to five young plants. which normally flower in about
9 months from the time the cuttings are made. Since
this procedure is rather slow and inefficient. micropropagation of African violet would be desirable
for mass and rapid production of superior quality
plants.
1
It1 ~ . i r r opropagation of Sui,rrpuirlin was first reported by Start and Cumming (1976). who regenerated plants by culturing leaf sections on a modified Murashige and Skoog (MS) medium. Cooke
(1977) reported a method for commercial micropropagation of African violet. also from leaf sections. Bilkey et al. (1978) achieved plantlet regeneration from petiole segments cultured on MS
medium. supplemented with a-naphthaleneacetic
acid (NAA) and 6-benzylaminopurine (BA).
In Cyprus. the demand for ornamental house
plants is rapidly increasing. Since local production.
even with conventional propagation methods. is limited. house plants are mainly imported from various European countries. particularly Holland. The
main objectibe of this study was to investigate the
possibility of local propagation of African violet
through the use of suitable tissue culture methods.
MATERIALS AND METHODS
Explant preparation
Healthy mature leaves, with their petioles. were
detached from stock plants grown indoors and used
to prepare explants. either from petiole or from leaf
blade tisue.
Petiole explants. Petioles were rinsed in tap water. washed in mild detergent water and. again.
thoroughl! rinsed in running tap water. They were
then dipped in alcohol. disinfestcd for I5 min in a
15% commercial chlorine solution and. finally.
rinsed three times in sterile distilled water. Follou~ing disinfestation. the damaged ends of each petiole
were removed and the remaining part was sliced
into 10 to 15. 2-3mm thick cross sections using a
double-cdgcd rasor blade. This, and the subsequent
handling of explants. was carried out aseptically in a
Cia% I(HI. laminar flow cabinet (Bassaire 3 MAH).
equipped with UV light and high efficiency particnlate air ( H E P A ) filter.
Leaf blade explants. Detached leaf blades were
thoroufhly uashetl and surface-sterilized with the
procedure described above for petiole tissues. Following disinfestation. the damaged base. the outside
edses and the tip of each leaf blade were removed.
as these areas are considered only marginally productive for root or shoot generation (Start and Cumming. 1976). The rest of the lamina was cut in two
halves along the main central vein and each half was
then sectioned into five 1-1.5 cm2 segments, ensuring that each segment carried a part of the main
vein. This operation was also conducted under
aseptic conditions.
Culturing procedures and nutrient media
Micropropagation of Sainrpauliu proceeded in
three major steps, which included culture establishment and shoot proliferation (stage I), shoot development and rooting (stage 11) and potting and
plant establishment (stage 111).
Culture estaMishment and shoot proliferation
(stage I). The explants were transferred aseptically
into 100 ml glass jars (10-15 explants per jar), each
containing 40 ml of solidified sterile medium. The
cultures were then placed in an incubator (GCA
and 16h
Precision Stientific, Model 818) at 25-27
photoperiod (fluorescent illumination) for shoot
proliferation.
Various modifications of MS (Murashige and
Skoog, 1962) nutrient medium were tested for the
shoot proliferation stage. All contained MS mineral
salts (macro and micronutrients), supplemented
with different combinations of organic ingredients,
which included a solidifying agent (agar), a source
of carbon, vitamins and growth regulators. Two
types of agar were tested, 1.e. Difco noble agar
(highly purified) and Merck microbiological grade
agar (less pure). Glucose and sucrose were tested as
carbon sources. Vitamins and growth regulators
tested in various combinations were myoinositol,
thiamine HCI, adenine sulfate, nicotinic acid,
pyridoxin HCI, glycine, 6-benzylaminopurine (BA)
and a-naphthaleneacetic acid (NAA).
For medium preparation, separate stock solutions
of macronutrients and micronutrients (Table 1) were
used. Iron was added to the medium as separate
stock solution of ferric sodium salt EDTA. BA and
NAA were disolved in 1N HCI and 1N NaOH, respectively. The pH of the medium was adjusted to
5.7 with 0.1 N NaOH before autoclaving, an.d prior
to adding agar. Following addition of 0.7% agar, the
medium was autoclaved in a conventional pressure
cooker for 5 min to dissolve the gelling agent, and
was then dispensed in 100 ml glass jars (Beech Nut),
capped with aluminum foil, and was autoclaved
once more in a pressure cooker for 10 min for
sterilization.
Table 1. Composition of eulhlre medium for shoot polieration of
African violeta.
Ingredient
Conccntr;ltion
mgIL
A. Mineral salts
I . Macrooutr~enls
NHqN03
KNO?
moved progressively, allowing the plants to adjust to
lower humidity. Half-strength Hoagland solution
was used for watering and fertilizing the plants. Eollowing an acclimation and establishment period of 2
weeks, the potted plants were transferred to the
greenhouse, where they were treated as normal
plants.
RESULTS AND DISCUSSION
Culture establishment and shoot proliferation
The MS nutrient medium, supplemented with
adenine sulfate 30 rng/L, BA 0.4 mgIL and NAA
0.1 mg/L (Table I ) , was found to be the most effective for induction of shoots from both petiole and
leaf blade explants. After incubation for 2 weeks on
this medium, tissues of explants enlarged and thickened; bud regeneration became evident during the
fourth week of culture and continued for several
weeks, resulting in a large number of adventitious
shoots, ranging from 20 to 130 on each segment.
2. Micronutrients
H3B03
MnS044H20
ZnS04.4H20
KI
Nd2MoO42H2O
CuSOjSH20
CuC12,6H20
R. Organic compounds
Sucrose
Mjoinositol
Thi;unine. H C I
6-hcnzylaminopurine ( R A )
cr-nitphthalrneacetic acid ( N A A )
Adenine sulkits
AS"'
3WK)
I(XJ
n.4
11.4
0. I
30
7000
" I'hc
rame medium. but devold of B A . N A A snd adenine
solf;irc. was also uscrl ;is routing substrate.
Shoot development and rooting (stage 11). Regenerated shoots, 2-5 mm long, were excised 8 weeks
from culture and transferred to freshly prepared
rooting medium, which was identical to the shoot
proliferation medium but was devoid of growth substances. Shoots were either separated and transferred individually to the rooting medium or transferred a5 clusters of shoots, in an effort to avoid the
time consuming separation procedure. Subsequent
development and performance of shoots on the
rooting medium were assessed and the above two
methods were evaluated.
Potting and plant establishment (stage In). In this
final stage of micropropagation, rooted plantlets 1-2
cm long, were taken from the rooting medium,
rinsed gently to remove agar, and planted in 6.5 cm
pots containing a sterile soil mixture (2:l viv of peat
moss and forest soil). The potted plants were placed
in a growth room at 20-25 C and 12h photoperiod.
T o prevent dehydration, the young plants were covered with inverted glass heakers. which were re-
Bilkey et al. (1978) reported shoot regeneration
from petiole sections of African violet on MS
medium supplemented with 0.1 mgiL NAA and 0.01
mg/L BA. In the present study, however, trials with
the above formulation failed to induce shoot production, although the cultures were incuhated for
several months; instead. plenty of adventitious roots
developed.
Start and Curnming (1976), working with leaf sections of African violet, obtained maximum shoot induction on a modified MS medium containing 0.1
mg/L NAA, 5.0 mg/L BA and 125 mglL adenine
sulfate. Our results indicate that much lower concentrations of BA (0.4 mglL) and adenine sulfate
(30 mg/L) (Table 1) were adequate for the production of a large number of shoots from both petiole
and leaf blade sections.
Trials with different sources of carbon in the nutrient medium showed that there was no difference
between glucose and sucrose. Similar results were
obtained when Difco noble agar was compared to
Merck microbiological grade agar.
Shoot development and rooting
Best results were obtained when regenerated
shoots, 3 mm or longer. were excised and transferred individually to the rooting medium. Such shoots
developed into rooted plantlets. which reached
transplantable size ( I - ? cm) within 8-10 weeks from
transfer.
Explants kept on the initial medium for a long
period of time continued to proliferate but a few
shoots grew and supprcsed the development of the
rest. A similar response was obsen-ed when the explants u e r c suhcultured into the rooting medium as
clusters. rather than as individual shoots. in an effort to omit the separation procedure. Thin and
crowded rootcd plants developed which could not
be separated easil! for transplanting.
Potting and plant establishment
Using the acclimation procedure already described. essentially 100% of the ported plantlets
u e r e succcs5fully estahlishsd in soil. After transfer
to thc greenhouse. rhs plants grew rapidly and
reached flouering stage within 4 months from potting. T h e surlival rate was near 100%. plants and
flowers appeared true-to-type and no abnormalities
in'plant developement were observed. More than
500 marketable African violet plants could be produced from a single petiole or leaf bli~dewithin 7-8
months from culture initiation. compared to only
three to five plants per leaf cutting produced over a
period of nine months with the conventional propagation method.
Qualify of tissue -culture -derived plmts
Plants produced with tissue culture were of
supcrior quality compared to those produced with
the con\cntional leaf cutting method. The latter
plants ere ver) often poorly formed, asymmetrical.
with elongated sidew;i).s-displaced petioles and small
leaves (Start and Cumming. 1976). In contrast, all
o u r plants conformed to the esthetically and com-
mercially desirable type of African violet, consisting
of a flat rosette whorl of large leaves on short
petioles. T h e unusual upright growth habit, encountered in some cases by St;~rtand Cumming (1976).
was not observed.
Both. petioles and leaf blades were suitable
sources of tissue for micropropagation. In contrast
t o results reported by Kukulczanka and Suszynska
(1972) (cited by Bilkey et al., 1978), petiole tissue
possessed a high regenerative capacity, comparable
t o that of leaf blade tissue. In addition, the shape of
the petiole made sterilization and aseptic handling
easier, and allowed greater uniformity in the size of
tissue explants. Petiole tissue was also less prone to
damage during sterilization and handling than leaf
blade tissue. Therefore, in agreement with the conclusion of Bilkey e t al. (1978). it appears that there
a r e several advantages in using petiole over leaf
blade tisslle for commercial micropropagation of
African violet.
LITERATURE CITED
Bilkey, P.C., B.H. Mc Cown, and A.C. Hildebrandf. 1978. Microprop;tg;llton of African violet from petiole cn,ss\ections. tiorrSci<~,zc<,1337.38.
Cooke. R.C. 1977. Tissue culture propagation of
African violets.
HoriSrrcrzc~, 12:579.
Mur;l\hlpc. T 1071. P1;int prop;igation through tissue cullure.
A,i,leoi Rrb,ii.%r~of Plmr Physioiug? 2 5 : 135-166.
hlul;~\hipr.T. 19711. Thr irnpilct of plant tlssue culture on
;~griculfurc.I n : Fronric.r.7 of Pbnr Tissue Cuirure 1978.
( A . Thorpe. ed.). pp. 15-26 Proceedings of the 4th International Congrcss of Planl Tissue and Cell Culture,
University of Calgary. Alherta. Canada.
hlur;t,hige. T.. and F. Skoog. I962 A revised medium for rapid
growth and biaass;iys with tobaccu tissue cultures.
Physic~iogiii Pl~,nr,nmn~m15:473-497.
Stilrt. E D . .; i n i l B.G. Cumming. 1'176.
I n virro propag;iIion of
.krinrpni<lirr i<,,rii,,rlrii Wcndl. HonSci?,rrr ll:?01-206.
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