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The effects of explant type and phytohormones on African violet (Saintpaulia

ionantha) micropropagation efficiency

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BIHAREAN BIOLOGIST 6 (2): pp.73-76 ©Biharean Biologist, Oradea, Romania, 2012
Article No.: 121109 http://biozoojournals.3x.ro/bihbiol/index.html

The effects of explant type and phytohormones

on African violet (Saintpaulia ionantha) micropropagation efficiency

Yousef GHASEMI, Ghorban Ali NEMATZADEH,

Valiollah Ghasemi OMRAN*, Ali DEHESTANI and Soghra HOSSEINI

Agricultural Genetics and Biotechnology Institute of Tabarestan, Sari Agricultural Sciences

and Natural Resources University, Km 9 Darya Road, P.O. Box 578, Sari, Iran.
* Corresponding author, V.G. Omran, Telefax: +981513822715, E-mail: [email protected]

Received: 18. September 2011 / Accepted: 24. April 2012 / Available online: 6. May 2012 / Printed: December 2012

Abstract. This research was conducted to study the effects of two explant types (leaf disc and petiole) and phytohormones including
5 levels of Benzyl Adenine (BA) (0, 0.2, 0.5, 1, 2 mgL-1) and 2 levels of Indole Butyric Acid (IBA) (0 and 0.5 mgL-1) onin vitro
micropropagation of African violet (Saintpaulia ionantha). Based on primary data, experiments were performed with Murashige and
Skoog`s basal media in factorial design was arranged in complete randomized blocks with four replications. Results showed that the
number and length of adventitious shoots were higher in leaf discs compared with leaf petioles. It was also revealed that elimination
of IBA as an auxin source decreased the regeneration efficiency and prolonged the time required for shoot regeneration. Generally,
the highest numbers of adventitious shoots (80 shoots per explant) were observed on a medium containing 0.5 mgL-1 BA along with
0.5 mgL-1 IBA. Among the different hormonal combinations, the medium supplied with 0.2 mgL-1 Gibberellic Acid (GA3) caused the
maximum shoot elongation with an average length of 19.9 mm. The half strength MS medium containing 1 mgL-1 IBA lead to
formation of normal plants with longer roots in micropropagated shoots. The rooted shoots were then acclimatized under high
humidity (80-90% relative humidity) in a mixture of peat moss and perlite (3:1) for three weeks with a survival rate of about 95 %.
Key words: African violet, micropropagation efficiency, phytohormones, explant type.

Introduction plant type. The optimization of in vitro conditions for or-

ganogenesis and somatic embryogenesis decreased the re-
The industry of ornamental plants is growing worldwide lated expenses and improved the commercial production of
and its monetary value has significantly increased over the Saintpaulia ionantha (Rout et al. 2006). Despite the availability
last two decades promising a great potential for continued of the sufficient literature on the micropropagation of Afri-
further growth in both domestic and international markets can violet, most of the successful methodologies have not
(Jain 2002). Major pot plants such as Begonia, Ficus, An- been published due to commercial aspects. This paper de-
thurium, Chrysanthemum, Rosa, Saintpaulia, and Spathiphyllum scribes a highly efficient protocol for shoot induction, plant
are being produced in developed countries (Rout et al. 2006). regeneration and acclimatization of rooted plantlets of Afri-
Many commercial ornamental plants are being propagated can violet.
by in vitro culture on the culture media containing auxins
and cytokinins (Preil 2003, Rout & Jain 2004). Several differ-
ent explants have been used for direct shoot formation from Materials and Methods:

Begonia, Cyclamen, Anthurium, Dracaena and Saintpaulia (Rout Plant material and explant sterilization
et al. 2006). Saintpaulia ionantha, commonly known as African The plant materials were obtained from commercial providers and
violet, is one of the most important commercial flowers that propagated at GABIT (Genetics and Agricultural Biotechnology In-
belong to the family Gesneriaceae. Some characteristics such stitute of Tabarestan) greenhouse. The plants were washed under
as variation in color and shape and tolerance to shade, made running tap water and then sterilized by 1% sodium hypochlorite so-
lution containing 0.5 % Tween-20 for 15 minutes. After that, the ster-
it a popular household ornamental flower in the world
ilized plants were rinsed with distilled water for three times, then
(Grout 1990). Leaf cutting has been the traditional method leaf discs (about 1 cm2) and petioles (1 cm) were used as explants for
for African violet propagation and propagated new plantlets in vitro micropropagation on different MS media (Murashige &
have been produced with many difficulties like emergence Skoog 1962) supplemented with different phytohormones.
of asymmetrical plants, high growing space requirements
Adventitious shoot induction
and disease development (Torres 1988). Species belonging to
Shoot induction media (SIM) contained MS salts fortified with B5 vi-
Gesneriaceae are mainly micropropagated from buds or ad- tamins, 30 grL-1 sucrose, 0.7% agar and 6-benzylaminopurine (BA) as
ventitious shoots which are directly regenerated on leaf sec- cytokinin source at different concentrations (0, 0.2, 0.5, 1 and 2 mgL-
tions (Sameera et al. 2006). African violet has been propa- 1) and indole-3-butyric acid (IBA) as auxin source (0 and 0.5 mgL-1).

gated successfully on tissue culture media by many re- The pH of media was adjusted to 5.8 before autoclaving. All cultures
searchers e.g., from vegetative parts, i.e., leaf segments were maintained in glass jars for 4 weeks in a growth room at 25 ± 1
(Redway 1991, Lo 1997) and from floral parts (Molgaard et ºC with 16 h light daily (Approximately 2500 Lux).
al. 1991). Different factors such as growth regulators, explant Shoot elongation
type, explant orientation and growth conditions (light and The shoots from the most appropriate SIM were subcultured for fur-
temperature) affect the regeneration frequency. Jain (1997) ther growth in shoot elongation media (SEM). These media consisted
micropropagated Saintpaulia ionantha by culturing leaf disks of MS basal salts with 5 hormonal treatments (control (hormone
on MS medium containing 0.22– 0.50 μM BA. They sug- free), 0.2 mgL-1 BA, 0.2 mgL-1 Kin, GA3 at 0.2 and 0.5 mgL-1). Shoots
were cultured and maintained at the same growth conditions men-
gested that Addition of auxins with cytokinins is essential
tioned for shoot induction, and were subcultured after 4 weeks.
for shoot induction and multiplication depending on the ex-
74 Ghasemi, Y. et al.

Rooting medium (RM) shoots and in the lack of IBA, the shoot induction was post-
After 1 month, elongated shoots were transferred to 1⁄2 strength of poned. Number of induced shoots was higher in medium
Murashige and Skoog`s basal medium (BM) supplemented with 3 with 0.5 mgL-1 IBA compared with hormone free media (Fig.
different concentrations (0.1, 0.5 and 1 mgL-1) of indole-3-butyric
1).
acid (IBA) and Naphthalene Acetic Acid (NAA) phytohormones.
As indicated in Fig. 2, the length of produced shoots in
Acclimatization IBA was longer than those generated on hormone free me-
The rooted plantlets were transferred to pots containing peat moss dia. Based on reports from Rout et al. (2006), the shoot num-
and perlite with a ratio of 2:1 and covered with transparent plastic ber significantly increased by auxin hormone treatment in
cover. The pots were incubated under high humidity at 25°C with 16
African violet. Assessment of BA effect on shoot induction
h of light per day.
(without considering the IBA level) revealed considerable
Experiment design and Data analysis changes in shoot numbers and their lengths with variations
All investigations were performed as factorial experiments in a com- in BA concentrations. The highest number of shoots was ob-
pletely randomized design with four replications (glass jars) per served on media containing 1 mgL-1 BA, while the longest
treatment and 4 explants per replication. Data of each experiment
shoots were developed on media supplemented with 0.2
were subjected to analysis of variance (ANOVA) by the General Lin-
ear Models procedure using SAS software. Means were compared
mgL-1 BA (Fig 3). Kushikawa et al. (2001), reported that the
using the Least Significant Difference (LSD) method at P≤ 0.05. highest shoot regeneration from hypocotyls after inoculation
with Agrobacterium was achieved in a medium supple-
mented with 1 mgL-1 BA. Daud and Taha (2008), reported
Results and Discussion the highest shoot regeneration from floral explants in a me-
dium fortified with 1 mgL-1 BA and 2 mgL-1 NAA.
Shoot induction As indicated in Table 1, there are significant reciprocal
The results have shown the significant effect of explant type effects between phytohormones` levels and explants type on
and hormonal combinations on shoot primordia induction days to shoot formation, number of shoots and shoot length.
after 2-3 weeks, in other words, compared to the petiole ex- Observation of shoot induction in all media revealed the
plants; the leaf explants are more efficient for shoot induc- high potential of the Saintpaulia for adventitious shoot for-
tion (Table 1). mation. The media containing 0.5 or 1 mgL-1 BA along with
Both explants produced green shoot primordia after 2-3 0.5 mgL-1 IBA was more capable for shoots formation rather
weeks of culture which were developed to adventitious than the other media (17-18 days after culture). As seen in
shoots after 4-5 weeks. Similar results were reported by Table 1, addition of IBA in media caused rapid shoot induc-
Sunpui and Kanchanapoom (2002). They reported a higher tion.
rate of shoot induction from petioles compared to leaf discs, Mithila et al., (2008) reported that application of Thidia-
while the regenerated shoots from leaf discs were longer zuron at concentrations below 0.5 mgL-1 increased shoot in-
than the petiole derived shoots. Sharifi et al. (2010) investi- duction from Saintpaulia leaf and petiole explants, while
gated different solidifying agents (potato starch, agar and higher concentrations promoted somatic embryogenesis.
liquid media) on in vitro regeneration of African violet and The highest number of shoot was induced from petiole
concluded that agar is the best agent for media preparation explants on media supplemented with 0.5 mgL-1 BA and 0.5
for Saintpaulia micropropagation. mgL-1 IBA, with an average of 80 shoots per explant. The
Compared to explants type, hormonal combinations had longest shoots belonged to media containing 0.2 or 0.5 mgL-1
a more remarkable effect on shoot induction. Primary inves- BA along with 0.5 mgL-1 IBA from leaf disc (with an average
tigations showed that IBA is essential for rapid induction of of 7.6 mm), while shorter belong to the media containing

Table 1. Effects of combination of explant type, IBA and BA on shoot number, length
and time required for shoot regeneration in MS medium after 4 weeks.
IBA BAP Days to shoot Num of shoot Length of shoot
Explant
(mg/l) (mg/l) induction /explant /explants (mm)
Leaf disc 0 0.2 24-25 50c 6.3bc
Leaf disc 0 0.5 24-25 34.3ef 4.6ef
Leaf disc 0 1 20-22 45cd 4.3efg
Leaf disc 0 2 20-22 28.3fg 7abc
petiole 0 0.2 24-25 28.3fg 3.6fgh
petiole 0 0.5 24-25 24.6g 2.8h
petiole 0 1 20-22 33.3ef 3.3gh
petiole 0 2 20-22 30fg 5df
Leaf disc 0.5 0.2 19-20 43.3cd 7.6a
Leaf disc 0.5 0.5 17-18 80a 7.3ab
Leaf disc 0.5 1 17-18 63.3b 7.6a
Leaf disc 0.5 2 22-23 58.3b 4.3efg
petiole 0.5 0.2 19-20 33.3ef 6cd
petiole 0.5 0.5 17-18 46.6c 4.6ef
petiole 0.5 1 17-18 46.6c 4efg
petiole 0.5 2 22-23 38.3de 4efg
LSD 8.1 1.1
‫٭‬Means with the same letter do not have significant differences at P≤ 0.05.
The effects of explant type and phytohormones on African violet (Saintpaulia ionantha) micropropagation efficiency 75

only 0.5 mgL-1 BA, (with an average of 2.8 mm). These re-
sults indicated the crucial role of auxin (IBA) in promoting
both number and length of the regenerated shoots.

Shoot elongation
Adventitious shoots produced after one month on media
containing 0.5 mgL-1 BA and 0.5 mgL-1 IBA, were subcul-
tured on SEM media for further growth. Results showed that
the highest elongation rates related to the media supple-
mented with 0.2 mgL-1 GA3 (Fig4), with an average shoots
length of 19.9 mm after 3 weeks.
Shoots cultured on media with 0.2 mgL-1 BA and 0.2
mgL-1 Kinetin, produced new short shoots with proliferated
small leaves (Picture 1). It appears that use of kinetin and
benzyladenine decreased apical dominance resulting en-
hanced shoot proliferation with less growth. On the other
Figure 1. Effect of explant type on shoot number and shoot
length of African violet on MS medium. hand, shoots cultured in media lacking phytohormones, had
green broad leaves with roots emerging on some shoots (Fig.
4). The shoots on GA3 containing medium had narrow leaf
blades and longer petioles. Application of GA3 produced
longer distinct shoots which facilitated dividing and subse-
quent subculture of shoots for rooting. Jia-Tao (2006) re-
ported that employing GA3 promoted the number of nor-
mally regenerated shoots in African violet.

Figure 2. Effect of different levels of IBA on shoot number

and shoot length of African violet on MS medium.

Figure 4. Effect of hormonal treatment on elongation of

adventitious shoots of African violet produced on MS medium.

Rooting
The results showed that different treatments had significant
effects on roots number, length and the morphology of the
regenerated plantlets. Maximum and minimum root num-
bers were obtained in media containing 1 and 0.1 mgL-1
NAA, respectively (Fig. 5).
The primary results indicated that there is a positive cor-
relation between NAA concentration and rooting percent-
age. Root length was also affected by hormonal alterations
and maximum root length was obtained in media containing
1mgL-1 IBA and minimum root length was observed in me-
dia supplemented with 0.5 mgL-1 NAA (Fig. 6).
Auxin type and concentration affected the morphology
of the regenerated plantlets. The use of 0.1 mgL-1 NAA pro-
Figure 3. Effect of different levels of BA on the length of the duced normal plants, while increasing NAA concentration to
adventitious shoots produced from African violet 0.5 and 1mgL-1, caused some abnormalities such as, callus
explants on MS medium. formation at the shoot base and appearance of abnormal
shoots with small leaves. In contrast, most of the plants
 76 Ghasemi, Y. et al.

rooted in media supplemented with different levels of IBA thermore, it was showed that addition of IBA at a concentra-
showed normal morphology. tion of 0.5 mgL-1 in the shoot induction media enhanced the
Therefore, despite of the higher root number in media regeneration efficiency. The highest numbers of adventitious
containing NAA, the media with IBA were indicated as the shoots were produced on media containing 0.5 mgL-1 BA
most appropriate media for rooting of Saintpaulia. Similar re- and 0.5 mgL-1 IBA (80 shoots per explant). Addition of GA3
sults were also reported for some ornamental plants by dif- in elongation media caused a significant increase in shoot
ferent researchers as well (Hutchinson 1981, Lo 1997). growth. It was also revealed that compared to NAA, IBA
was more appropriate for rooting process and produced
more normal plantlets. The present study provides a com-
prehensive methodology for high efficiency micropropaga-
tion of African violet for commercial production.

Acknowledgements. This research was conducted with the financial

support of the Genetics and Agricultural Biotechnology Institute of
Tabarestan (GABIT), Sari, Iran.

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Conclusions

According to the results of the present study, it was revealed

that the regeneration efficiency of the leaf blades is higher
than that of petiole explants, which can be attributed to the
higher nutritional and hormonal content of the tissue. Fur-

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