8 Kaviani et al.

Int. J. Biosci. 2015

RESEARCH PAPER OPEN ACCESS

Micropropagation of Begonia rex Putz. by 6-benzyladenine and

α-naphthalene acetic acid

Behzad Kaviani1*, Davood Hashemabadi1, Hossein Khodabakhsh1, Rasoul Onsinejad1,

Mohammad Hossain Ansari2, Naghi Haghighat1

1Department of Horticultural Science, Rasht Branch, Islamic Azad University, Rasht, Iran

2Department of Agronomy, Rasht Branch, Islamic Azad University, Rasht, Iran

Key words: In vitro culture, Plant growth regulators, Ornamental plants, Tissue culture.

http://dx.doi.org/10.12692/ijb/6.5.8-15 Article published on March 09, 2015

Abstract

Begonia (Begonia rex Putz.), an ornamental plant, is used for landscape and pot purposes. For efficient

regeneration of this species, a simple protocol is presented. Leaf explants dissected from 7-month-old mother

plants grown in a greenhouse were cultured on Murashige and Skoog (MS) medium supplemented with 16

combinations of 6-benzylaminopurine (BA; 0.00, 0.50, 0.60 and 1.00 mg l-1) and α-naphthaleneacetic acid

(NAA; 0.00, 0.10, 0.25 and 0.50 mg l-1). Here, we present a protocol includes single medium for maximum

induction and growth of all traits related to micropropagation of begonia. The optimum plant growth regulators

(PGRs) for maximal shoot number (44.33 per plant), node number (12.31 per plant), leaf number (76.25 per

plant), root number (11.42 per plant), and root length (6.00 cm per plant) were 0.50 mg l-1 BA + 0.10 mg l-1 NAA.

The plantlets regenerated in vitro with well-developed shoots and roots were successfully established in pots

containing humus, sand and garden soil (2:1:1) and grown in a greenhouse with a 90% survival rate. The

regenerated plants were morphologically identical to the mother plants. The described protocol offers a simple

and fast protocol for micropropagation of begonia for large-scale multiplication.

* Corresponding Author: Behzad Kaviani b.kaviani@yahoo.com

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print), 2222-5234 (Online)

http://www.innspub.net

Vol. 6, No. 5, p. 8-15, 2015

9 Kaviani et al.

Int. J. Biosci. 2015

Introduction

Begonia is a genus of perennial flowering plants in

the family Begoniaceae. The genus contains about

1,400 different plant species (the sixth-largest

angiosperm genus) and the most famous species of

begonia is rex (David and Frodin, 2004). The

begonias are native to moist subtropical and tropical

climates in South and Central America, Africa, and

southern Asia. Some species are commonly grown

indoors as ornamental houseplants in cooler climates.

In cooler climates some species are cultivated outside

in summertime for their bright colorful flowers, which

have sepals but no petals. The leaves, which are often

large and variously marked or variegated, are usually

asymmetric (unequal-sided) (David and Frodin,

2004).

The techniques for tissue culture of ornamental plants

are being continuously improved to meet the demand

of the floriculture breeding and industry (Rout et al.,

2006). Tissue culture has become a routine technique

in agricultural and horticultural development which

has revolutionized the ornamental industry and most

popular application of this technique is

micropropagation (Maira et al., 2010; Bhattacharya

and Bhattacharyya, 2010). In the field of ornamental

plants, in vitro culture has allowed mass propagation

of superior genotypes and plant improvement, thus

enabling the commercialization of healthy and

uniform planting material (Winkelmann et al., 2006;

Nhut et al., 2006). Among all factors affected on plant

in vitro micropropagation, plant growth regulators

(PGRs) have the most effect. Some studies connection

with in vitro micropropagation of begonia were done

by some researchers (Bouman and Clerk, 2001;

Kishimoto et al., 2002; Espino et al., 2004; Mendi et

al., 2009; Kabirnataj et al., 2012). Begonias are easy

to propagate vegetatively by stem, leaf or top cuttings,

or by splitting. The use of plant growth regulators,

especially cytokinins, enhance the regeneration of

buds on leaves explants of Begonia rex. Shoot

regeneration was also obtained from petioles on MS

medium containing NAA and BA via organogenesis

(Jain and Ochatt, 2010). Sometimes, there is

heterogeneity in Begonia plants obtained by ex vitro

propagation. Thus, the objective of the present study

was to evaluate the effects of different concentrations

of BA and NAA on regeneration of shoot and root in

Begonia rex Putz.

Materials and methods

Plant materials and surface sterilization

Adult plants of begonia (Begonia rex Putz.) were

brought from a greenhouse in Abbasabad city,

Mazandaran province, northern part of Iran. Leaves

dissected from mother plants were put in washing

liquid, immediately and transferred to the tissue

culture laboratory. In laboratory, leaves were washed

and then immersed in 2 mg l-1 fungicide

carboxytyram for 2 min. Then, leaves were immersed

in distilled water and one or two drops of Tween-20

for 20 min. then rinsed with distilled water. Under

laminar air flow cabinet, leaves were disinfected by

immersion in 70% ethanol for 4 sec. followed by 1%

HgCl2 for 7 min. then 20% sodium hypochlorite

(NaClO) for 10 min. Finally, leaves were thoroughly

rinsed by sterile distilled water for 1, 3 and 5 min.

Leaves were divided to the pieces with dimensions of

1.5 cm × 1.5 cm and were used as explants.

Culture medium and growth conditions for leaves

Surface sterilized leaves were inoculated in Murashige

and Skoog (MS) medium (Murashige and Skoog,

1962) with 3% (w/v) sucrose. The medium was

solidified with 0.70% Agar-agar. The pH of the

medium was adjusted to 5.6–5.8. The 30 ml medium

was dispensed into each 250 ml glass dish. The

culture glass dishes containing the media were

autoclaved at 121°C for 20 min. Two leaves were

inoculated in each glass dish and plugged firmly. All

the cultures were maintained at 24 ± 2°C, RH 70-80%

under a 12 h photoperiod at a photosynthetic flux of

50 µmol m-2 s-1, provided by cool daylight fluorescent

lamps. Subculture was done fortnightly.

Maintenance of shoot cultures and multiplication

The medium was divided to several media containing

0.00, 0.50, 0.60 and 1.00 mg l-1 BA and 0.00, 0.10,

0.25 and 0.50 mg l-1 NAA, individually and/or in

combination (16 treatments). First morphological

10 Kaviani et al.

Int. J. Biosci. 2015

changes were revealed after two weeks on MS

medium and the first shoots were produced after 4

weeks. Shoots were directly produced on leaves

completely after five weeks. Experiments were carried

out in three replications and two explants for each

replication.

Rooting in vitro and plantlets acclimatization

Rooting was done in MS medium supplemented with

0.00, 0.50, 0.60 and 1.00 mg l-1 BA and 0.00, 0.10,

0.25 and 0.50 mg l-1 NAA, individually and/or in

combination (16 treatments) (as the same as shoot

multiplication media). The pH of the media, autoclave

time and environmental conditions were similar with

shoot production media. For easy acclimatization, the

glass dishes with plantlets (fully expanded leaflets)

were kept open for 2–3 h after removing the plugs in

the culture room itself. Then, the plantlets were

removed from the culture media and washed with

distilled water; then transferred to plastic cups (15-cm

in diameter) containing a mixture of humus, sand and

garden soil (2:1:1). Plantlets were kept in a

greenhouse at 27 ± 1°C, 4000–5000 lux light and 75–

80% RH with periodic irrigation.

Measured characters

Characters including shoot number, node number,

leaf number, root length and root number of begonia

(Begonia rex Putz.). were calculated after 42 days.

Experimental design and data analysis

The experimental design was R.C.B.D. Each

experiment was carried out in three replicates and

each replicate includes two explants. Analysis of

variance (ANOVA) was done using SAS and SPSS

statistical software and means were compared using

the Least Significant Difference Test (LSD) at 5%

probability level.

Results

Effect of BA and NAA on shoot number

The largest number of multiplication rate (46.24

shoots per explant) was recorded with 0.10 mg l-1

NAA + 0.50 mg l-1 BA (Table 1). Media

supplementation with NAA was a little better than BA

for shoot induction, singularly (Table 1). This result

revealed that NAA was comparable with BA for shoot

induction. The concentration of 0.25 mg l-1 NAA +

0.60 mg l-1 BA was relatively suitable for induction of

shoot per plantlet (43.86) (Table 1).

Table 1. Mean comparison of the effect of different concentrations of BA and NAA on shoot number, node

number, leaf number, root length and root number of begonia (Begonia rex putz).

Root length (cm) Root number Leaf number Node number Shoot number NAA + BA (mg L-1)

2.00 ± 1.18h 3.48 ± 2.76n 24.65 ± 13.24m 3.43 ± 2.51o 29.33 ± 9.15i 0.00 + 0.00

4.33 ± 1.15b 6.39 ± 0.14f 51.42 ± 13.53c 5.96 ± 0.02h 34.09 ± 4.38gh 0.00 + 0.50

3.66 ± 0.48d 5.61 ± 0.64i 44.41 ± 6.52f 8.26 ± 2.32c 35.89 ± 2.59fg 0.00 + 0.60

2.66 ± 0.52f 4.68 ± 1.57j 33.04 ± 4.85j 6.55 ± 0.61f 31.77 ± 6.70hi 0.00 + 1.00

2.00 ± 1.18h 4.33 ± 1.91l 24.06 ± 13.82n 5.06 ± 0.88k 42.00 ± 3.52bc 0.10 + 0.00

6.00 ± 2.12a 11.42 ± 5.17a 76.25 ± 38.36a 12.31 ± 2.37a 46.24 ± 7.76a 0.10 + 0.50

4.33 ± 1.15b 9.60 ± 3.35d 52.45 ± 14.56b 5.80 ± 0.14i 40.26 ± 1.78bcde 0.10 + 0.60

3.00 ± 0.18e 6.18 ± 0.07g 36.77 ± 1.12h 6.86 ± 0.92e 36.47 ± 2.01efg 0.10 + 1.00

3.66 ± 0.48d 9.77 ± 3.52c 42.90 ± 5.01g 4.70 ± 1.25l 41.66 ± 3.18bcd 0.25 + 0.00

4.33 ± 1.15b 8.16 ± 1.91e 49.46 ± 11.57d 9.06 ± 3.12b 42.00 ± 3.52bc 0.25 + 0.50

4.00 ± 0.82c 11.27 ± 5.02b 46.11 ± 8.22e 8.20 ± 2.26d 43.86 ± 5.38ab 0.25 + 0.60

2.33 ± 0.85g 6.03 ± 0.22h 26.44 ± 11.44l 5.62 ± 0.32j 40.23 ± 1.75bcde 0.25 + 1.00

1.66 ± 1.52i 2.25 ± 4.00p 19.03 ± 18.86o 3.47 ± 2.45n 38.00 ± 0.48def 0.50 + 0.00

3.00 ± 0.18e 4.50 ± 1.75k 33.91 ± 3.98i 5.98 ± 0.04g 39.22 ± 0.74cdef 0.50 + 0.50

2.33 ± 0.85g 3.55 ± 2.70m 26.72 ± 11.17k 4.45 ± 1.49m 38.25 ± 0.22cdef 0.50 + 0.60

1.66 ± 1.52i 2.80 ± 3.45o 18.60 ± 19.29p 3.45 ± 2.55p 36.33 ± 2.15fg 0.50 + 1.00

In each column, means with the similar letters are not significantly different at 5% level of probability using LSD

test.

11 Kaviani et al.

Int. J. Biosci. 2015

Our findings demonstrated that the smallest number

of shoot (29.33 per explant) was produced in medium

without PGRs (control) (Table 1). Concerning the

shoot number per plantlets, all concentrations of NAA

were suitable and induced the relatively good number

of shoot as compared to the all concentrations of BA.

There was significant differences between BA

(p≤0.05), NAA (p≤0.01), and interaction effects of BA

and NAA (p≤0.05) on shoot number (Table 2).

Table 2. Analysis of variance (ANOVA) for the effect of different concentrations of BA and NAA, singularly or in

combination with each other, on the shoot number, node number, leaf number, root length and root number of

begonia (Begonia rex Putz.).

M.S. df Source of variations

Root number Root length Leaf number Node number Shoot number

27.55** 11.98** 1721.00** 22.91** 42.18* 3 BA

77.98** 6.48** 1116.00** 15.39** 2.90** 3 NAA

8.70** 1.25** 229.00** 4.10** 44.13* 9 BA × NAA

0.19 0.05 18.74 0.10 14.52 32 Error

7.03 7.22 11.42 5.38 9.90 c.v.

* And **: Significant at α = 5% and 1%, respectively.

Effect of BA and NAA on node number

Differences of node number in explants grown under

BA, NAA and combination of BA and NAA were

significant (p≤0.01) (Table 2). Node number was

varied with BA and NAA concentrations (Tables 1).

Minimum node number per explant (3.43 and 3.45)

was recorded in the plantlets grown on the medium

without PGRs and medium containing the highest

concentrations of NAA and BA (0.50 mg l-1 + 1.00 mg

l-1), respectively (Table 1). The medium with 0.50 mg

l-1 NAA without BA was produced a few number of

node per plantlet (3.47), too. Largest number of node

per plantlet (12.31) was achieved on MS medium

supplemented with 0.10 mg l-1 NAA + 0.50 mg l-1 BA

(Table 1). Among all concentrations of NAA, 0.10 mg

l-1 induced largest number of node per plantlets (5.06)

(Table 1). Also, among all concentrations of BA, 0.60

mg l-1 induced largest number of node per plantlets

(8.26) (Table 1).

Effect of BA and NAA on leaf number

Leaf number was varied with BA and NAA

concentrations (Tables 1). Differences of leaf number

in explants grown under BA, NAA and combination of

BA and NAA were significant (p≤0.01) (Table 2).

Maximum number of leaf per plantlet (76.25) was

obtained on MS medium supplemented with 0.10 mg

l-1 NAA + 0.50 mg l-1 BA (Table 1). Minimum leaf

number per explant (18.60) was calculated in the

plantlets treated with the highest concentrations of

NAA and BA (0.50 mg l-1 NAA + 1.10 mg l-1 BA) (Table

1). The media containing 0.50 and 0.10 mg l-1 NAA

without BA and control with induction of 19.03, 24.06

and 24.65 leaves per explant were not proper

treatments. Among all concentrations of NAA, 0.25

mg l-1 induced maximum leaf number per plantlets

(42.90). On the other hand, among all concentrations

of BA, 0.50 mg l-1 induced maximum leaf number per

plantlets (51.42) (Table 1).

Effect of BA and NAA on root length

In the case of root length, BA, NAA and interaction

effect of these two PGRs had significant effect on

rooting capacity and the best results (6.00 cm per

plantlet) was obtained with shoots treated with 0.10

mg l-1 NAA + 0.50 mg l-1 BA (Fig. 1, Tables 1 and 2).

However, 0.50 mg l-1 BA without NAA, 0.10 mg l-1

NAA + 0.60 mg l-1 BA and 0.25 mg l-1 NAA + 0.50 mg

l-1 BA media all with 4.33 cm long induced proper

root length (Table 1). Least root length (1.66 cm per

explants) was observed in shoots grown on media

containing 0.50 mg l-1 NAA without BA and 0.50 mg l-

1 NAA + 1.00 mg l-1 BA (Table 1). Control medium and

medium supplemented with 0.10 mg l-1 NAA without

BA induced less root length (both with 2.00 cm long)

(Table 1).

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Int. J. Biosci. 2015

Fig. 1. Stages of micropropagation of begonia (Begonia rex Putz.). (A) Mother plants, (B) Preparation of explants

from leaf, (C) Explants on MS medium containing PGRs for proliferation, (D) Shoot proliferation on medium

enriched with 0.10 mg l-1 NAA + 0.50 mg l-1 BA in the beginning of differentiation, (E) Fully shoot proliferation on

medium supplemented with 0.10 mg l-1 NAA + 0.50 mg l-1 BA ready for rooting, (F) A plantlets with roots

produced on medium containing 0.10 mg l-1 NAA + 0.50 mg l-1 BA.

Effect of BA and NAA on root number

The data clearly show that root number is strongly

affected by BA, NAA and interaction effect of BA and

NAA (p≤0.01) (Table 2). Best results in terms of

rooting frequency and mean number of roots (11.42)

was obtained in medium enriched with 0.10 mg l-1

NAA + 0.50 mg l-1 BA (Fig. 1, Table 1). However, 0.25

mg l-1 NAA + 0.60 mg l-1 BA (with 11.27 roots), 0.25

mg l-1 NAA without BA (with 9.77 roots) and 0.10 mg

l-1 NAA + 0.60 mg l-1 BA (with 9.60 roots) induced

good mean number of roots (Table 1). With regard to

BA concentration, 0.50 mg l-1 induced suitable root

number (6.39 per plantlet). Also, among all

concentrations of NAA, 0.25 mg l-1 induced maximum

root number per plantlets (9.77). Smallest number of

root per shoot (2.25, 2.80 and 3.48) was observed in

plantlets treated with 0.50 mg l-1 NAA without BA,

the highest concentrations of NAA and BA and

control, respectively (Table 1). The in vitro

regenerated plantlets with well-developed roots were

acclimatized ex vitro and eventually established in a

greenhouse with high relative humidity (75–80%).

The surviving frequency was 90% months after

acclimatization. Micropropagated plants showed good

growth and uniformity ex vitro and exhibited normal

development.

13 Kaviani et al.

Int. J. Biosci. 2015

Discussion

Effect of BA and NAA on aerial parts

The combination of 0.10 mg l-1 NAA + 0.50 mg l-1 BA

was found to be the most suitable growth regulators

for obtaining the maximum shoot number, node

number, leaf number, root length and root number in

micropropagation of begonia (Begonia rex Putz.).

Full plantlets were produced at the same medium.

This approach saves money, because root induction is

most expensive step during micropropagation

process. Also, contamination percentage reduces

because of reduction of subculture. The present study

showed important role of suitable concentrations of

an auxin and cytokinin on formation of shoots, leave,

nodes and roots. The positive role of BA among all

cytokinins for shoot induction and growth have been

shown (Kaviani, data not published). Study of

Kabirnataj et al. (2012) on micropropagation of

Begonia rex showed that the highest adventitious

shoot regeneration with an average number of 41.6

was calculated from leaf disc explants after 5 weeks

culture on MS medium supplemented with 1 mg l-1 BA

and 0.5 mg l-1 IBA. The lowest adventitious shoots

were observed on petioles which were cultured in the

medium containing 0.2 mg l-1 BA and 0.2 mg l-1 NAA.

Mendi et al. (2009) demonstrated that in begonia

regeneration, 1 mg l-1 NAA with 2 mg l-1 BA were more

effective on regeneration. Combination of auxin with

cytokinin in regeneration medium increased

regeneration efficiency in begonia (Mendi et al.,

2009). These workers reported that at high

concentration of cytokinin, auxin had positive effect

on regeneration but at low concentrations of these

PGRs the rate of regeneration decreased. Espino et al.

(2004) reported that BA was more effective cytokinin

on shoot production than kinetin. Nhut et al. (2005)

investigated the effect of TDZ, BA and NAA on shoot

bud formation of begonia (Begonia spp.) using

transverse thin cell layer of petiole. These researchers

showed that the maximum shoot bud formation was

obtained on medium enriched with 0.2 mg l-1 TDZ +

0.2 mg l-1 NAA. Cytokinins are usually applied to

stimulate shoot multiplication and node production

(van Staden et al., 2008; Chawla, 2009; Gomes et al.,

2010). The combination of BA and NAA is applied

more than the other combinations for

micropropagation of ornamental plants (Paek and

Hahn, 2000; Ghaffari Esizad et al., 2012; Kaviani et

al., 2014; Kaviani, data not published).

Effect of BA and NAA on root

Most roots were produced on media containing both

of BA and NAA. The production of 9.77 roots per

explants at the base of shoots on medium enriched

with 0.25 mg l-1 showed the important role of auxins

for root induction. Some studies on other ornamental

plants showed the positive effect of auxins on rooting

(Jain and Ochatt, 2010; Kaviani et al., 2011; Ghaffari

Esizad et al., 2012; Kaviani et al., 2014; Kaviani, data

not published). Some investigations revealed the

positive effect of cytokinins on root induction (Gomes

et al., 2010; Ghaffari Esizad et al., 2012; Kaviani et

al., 2014). In the present study, maximum root

induction and root growth was obtained in medium

supplemented with 0.10 mg l-1 NAA + 0.50 mg l-1 BA.

In some study on micropropagation of ornamental

plants, root formation was inhibited or reduced in the

culture medium enriched with BA (Jain and Ochatt,

2010; Han et al., 2004). Investigation of Kabirnataj et

al. (2012) on shoots rooting of Begonia rex showed

that the largest number of root was obtained in

control medium with the average of 4.1 roots per

plantlet and the lowest one was in presence of NAA 1

mg l-1 with the average of 1.5 roots per plant. Based on

these researchers, presence of auxin is not essential

for root induction in begonia and rooting was

occurred in auxin-free medium. Concentrations of 0.5

mg l-1 NAA and 0.5 mg l-1 IBA caused the highest root

length and concentration higher than 0.5 mg l-1 had

inhibitory effect on it (Kabirnataj et al., 2012).

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