Journal of Babylon University/Pure and Applied Sciences/ No.(3)/ Vol.

(24): 2016

Effect of plant growth regulators BA, 2,4-D, IBA

and Kin on in vitro propagation of white jasmine
(Jasminum azoricum L.)
Siham Abd Alrazzaq Salim
Al Musaib Technical College, Plant Production Department
[email protected]
Abstract
This study was conducted in order to investigate the effect of BA, 2,4-D, IBA and Kin on in vitro
propagation of white jasmine ( Jasminum azoricum L. ) through two ways; in the first way: nodal
explants were cultured on MS medium containing different concentrations of BA ( 0.0 , 0.5 , 1.0 , 2.0 , 4.0
, or 6.0 ) mg/L for direct shoot proliferation. The results showed that BA in concentration 2.0 mg/L gave
the best result in shoot number( 2.5 ), while the control was the best in shoot length ( 2.51 cm ) and number
of nodes per shoot( 4.5 node / shoot ) than other treatments. In the second method: internodal explants
were cultured on MS containing BA( 0.0 , 2.0 , 4.0 , or 6.0 ) mg/L with 2,4-D ( 0.0, 0.01 , 0.05 or 0.1) mg/L
for callus induction and indirect regeneration of shoots. The highest percent of callus induction ( 100% )
was seen in MS supplemented with 4.0 mg/L BA + 0.1 mg/L 2,4-D and 6.0 mg/L BA + 0.1 mg/L 2,4-D.
The proliferated callus was transferred into MS medium supplemented with BA ( 0.0 , 2.0 , 4.0 , or 6.0 )
mg/L with Kin ( 0.0 or 2.0) mg/L for adventitious buds regeneration. The highest number of buds (10.1)
was seen in the combination 4.0 mg/L BA + 2.0 mg/L Kin. Using of IBA (0.0,0.05,0.1,0.5, 1.0 or 2.0)
mg/L in in vitro rooting medium did not give any response of root formation according to the conditions of
experiment, while ex vitro rooting of plantlets on agricultural medium after soaking their bases in a solution
containing 20 mg/L IBA showed that 85% of plantlets were rooted and remained alive. In addition, the ex
vitro rooting resulted in successful hardening of plantlets and reduced time, costs and efforts required for
rooting and hardening.
Key words: Jasminum azoricum, in vitro, regeneration, cytokinin , auxins .
‫الخالصة‬
Kin ‫ و‬IBA ‫ و‬2,4-D ‫ و‬BA :‫تم إجراء ىذه الدراسة بيدف التحري عن تأثير أنواع وتراكيز مختمفة من منظمات النمو النباتية ىي‬
‫ استعممت في الطريقة األولى العقد‬:‫) خارج الجسم الح ي من خالل طريقتين‬Jasminum azoricum L.( ‫في إكثار نبات الياسمين األبيض‬
‫ لتر لتوالد األفرع مباشرة‬/‫ ) ممغم‬0.0 ‫ أو‬0.0 ‫ أو‬2.0 ‫ أو‬1.0 ‫ أو‬0.0 ‫ أو‬0.0 ( BA ‫ مجي از بـ ـ‬MS ‫كأجزاء نباتية بزراعتيا عمى وسط‬
‫ بينما أعطت معاممة السيطرة أفضل استجابة‬, ) 2.0 ( ‫ أعطى أفضل معدل لعدد األفرع‬BA ‫لتر من‬/‫ ممغم‬2.0 ‫ بينت النتائج أن التركيز‬.‫منيا‬
‫ وجرى في الطريقة الثانية زراعة‬.‫فرع) مقارنة بالمعامالت األخرى‬/‫ عقدة‬0.0 ( ‫ سم) ومعدل عدد العقد لكل فرع‬2.01 ( ‫لطول األفرع‬
‫ أو‬0.00 ‫ أو‬0.01 ‫ أو‬0.0 ( 2,4-D ‫لتر مع‬/‫ ) ممغم‬0.0 ‫ أو‬0.0 ‫ أو‬2.0 ‫ أو‬0.0 ( BA ‫ مجي از ب ـ‬MS ‫السالميات عمى وسط‬
MS ‫ في وسط‬%100 ‫ بينت النتائج أن أعمى نسبة الستحثاث الكالس بمغت‬.‫لتر الستحثاث الكالس واخالف األفرع منو‬/‫ ) ممغم‬0.1
‫ وعند نقل الكالس المتوالد‬. 2,4-D ‫لتر‬/‫ ممغم‬0.1 + BA ‫لتر‬/‫ ممغم‬0.0 ‫ و‬2,4-D ‫لتر‬/‫ ممغم‬0.1 + BA ‫لتر‬/‫ ممغم‬0.0 ‫المجيز ب ـ‬
‫لتر إلخالف البراعم العرضية‬/‫ ) ممغم‬2.0 ‫ أو‬0.0 ( Kin ‫لتر مع‬/‫ ) ممغم‬0.0 ‫ أو‬0.0 ‫ أو‬2.0 ‫ أو‬0.0 ( BA ‫ مجي از ب ـ‬MS ‫إلى وسط‬
‫ بالتراكيز‬IBA ‫ أن استعمال‬. Kin ‫لتر‬/‫ ممغم‬2.0 + BA ‫لتر‬/‫ ممغم‬0.0 ‫ في التوليفة‬10.1 ‫ لوحظ أن أعمى معدل لعدد البراعم ىو‬,‫منو‬
‫لتر لتجذير األفرع خارج الجسم الحي لم يعط أية استجابة لمتجذير وفق‬/‫ ) ممغم‬2.0 ‫ أو‬1.0 ‫ أو‬0.0 ‫ أو‬0.1 ‫ أو‬0.00 ‫ أو‬0.0 (
‫ أعطى نسبة‬IBA ‫لتر‬/‫ ممغم‬20 ‫ في حين أن التجذير المباشر لألفرع خارج الزجاج بغمر قواعدىا في محمول يحتوي عمى‬.‫ظروف التجربة‬
‫ فان التجذير المباشر خارج الزجاج ينتج عنو نجاح أقممة‬, ‫ فضال عن ذلك‬, ‫ وبقيت النبيتات عمى قيد الحياة‬%50 ‫استجابة بمغت‬
.‫النبيتات وتقميل الوقت والكمفة والجيد المتطمب لمتجذير واألقممة‬
. ‫ األوكسينات‬, ‫ السايتوكاينينات‬,‫ خارج الجسم الحي‬,‫ الياسمين األبيض‬:‫الكممات المفتاحية‬

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Introduction
Plants of Jasminum genus are evergreen climbing or erect shrubs with thin woody
stems of older growth belonging to the family Oleaceae, they are distributed in the
warmer parts of Asia, Europe and Africa (Ramdas et al., 1993). The flowers are very
fragrant used for production of perfumes, soap, and cosmetic industry ( Alikhan et al.,
1989 ). Pharmacology researches revealed anticancer activity of the extracts of
jasmine on human epidermoid carcinoma of nasopharynx and anti-inflammatory
effect against acute and chronic inflammation, while the oil is used externally to
soothe dry or sensitive skin. The extracts of flowers and leaves of jasmine species
showed antibacterial (Kumar et al., 2007) and as herbicides ( Poonpaiboonpipat et al.,
2011). Several studies have been done on aroma and oil of jasmine, but reports about
the propagation are few. The plant tissue culture technique permits regeneration of
uniform plants . Plantlets were regenerated from leaf callus of J. grandiflorum
after sub cultured on MS provided with BAP (2 ppm) with sucrose at
concentration 1.5% ( Gomathi et al., 2007 ) . Multiple shoots of J. officinale were
produced from axillary buds cultured on MS containing 45% sucrose + 4mg/L
BA + 0.1mg/L NAA (Bhattacharya and Bhattacharya, 2010).
Jasminum azoricum is a climbing shrub ( 2 to 3 m in height) , leaves are trifoliate,
flowers are fragrant formed in groups of 1-5 at the end of branches, with a small green
calyx and a white tubular corolla expanding into 6 petal lobes. The present study aimed
to determine the optimal combinations of different plant growth regulators for micro
propagation of J. azoricum using plant tissue technique.
Materials and Methods
Preparation of plant materials:
Nodes and internodes were used as explants which collected from a garden grow adult
shrub of J. azoricum. After removing off leaves; explants were washed with water and
liquid soap to remove dirt, and then they were washed under running tap water for
1 hr. Surface sterilization was achieved in the laminar – air flow cabinet with 2%
(v/v) Clorox (6% NaOCl) solution containing 2 drops of tween 20 for 18 min., then
they were rinsed for two times with sterile distilled water (two min. in each). Nodal
and intermodal explants (1.5 – 2.0 cm) were prepared for culture.
Culture initiation and shoot multiplication:
The nodal explants were cultured on MS medium (Murashige and Skoog, 1962)
containing 30% sucrose, 0.7% agar, and supplemented with various concentrations of
benzyl adenine ( BA) ( 0.0, 0.5, 1.0, 2.0, 4.0, or 6.0 ) mg/L for shoot initiation. The pH
of medium was 5.7 ± 0.1 before autoclaving at 121ºC and 1.04 kg/cm² for 15 min. 10
replicates were used for each concentration. Cultures were incubated under 16 h
photoperiod with light intensity of 1000 lux at 25 ± 2ºC. Results were taken after five
weeks of culture. The shoots that were proliferated in vitro were cut into nodal explants
and recultured on the same basal medium with the best results of BA concentration from
the previous experiment above in order to produce multiple shoots.
Callus induction and shoot regeneration:
For callus induction , internodal explants were cultured on MS medium containing
30% sucrose, 0.7% agar, and supplemented with 2,4-Dichlorophenoxy acetic acid (2,4-
D) at concentrations of (0.0, 0.01, 0.05, or 0.1) mg/L in combination with BA at
concentrations of (0.0, 2.0, 4.0, or 6.0 ) mg/L. After four weeks, the induced

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embryogenic callus was transferred into MS free- hormone medium for two weeks,
then cultured on medium containing BA at concentrations of (2.0, 4.0, or 6.0) mg/L
with (0.0 or 2.0 mg/L) of kinetin . All cultures were incubated under the same
conditions that mentioned previously. Results were evaluated after five weeks of
culture.
Rooting of plantlets and acclimatization:
The proliferated plantlets (1.5 – 5.0 cm height ) were cultured in half strength MS
medium supplemented with different concentrations of IBA (0.0, 0.05, 0.1, 0.5, 1.0, 1.5,
or 2.0) mg/L for the in vitro rooting process, while ex vitro rooting process was
achieved by soaking the bases of plantlets in a solution containing 20 mg/L IBA and
cultured in pots containing agricultural medium of river soil and peat moss (2:1), then
covered with beakers to prevent the loss of water in order to achieve rooting and
acclimatization in one process. Results were taken after four weeks of culture .
Statistical analysis:
Data were statistically analyzed in a Completely Randomized Design (CRD). Mean
values were compared using Least Significant Difference (LSD) test at 0.05 (SAS, 2001).
Results and Discussion:
Shoot proliferation from nodes
Results in table-1 show the effect of different concentration of BA on shoot
proliferation from axillary buds. The majority of explants produced either short
multiple shoots or only single long shoots. There was shoot formation (1.2) in basal MS
medium without any addition of plant growth regulators (control). This average was
appeared to be significant with the graduate increasing in cytokinin concentration until
it was reached to the concentration 2.0 mg/L BA, which gave the highest shoot
number gave (2.5) followed by the concentration 4.0 mg/L BA gave (2.3). However,
there was a significant decreasing in shoot number with concentration of 6.0 mg/L BA
gave (1.9), but it was remained significant as compared with control.

Table 1: Effect of different concentration of BA in MS medium on shoot number, shoot

length, and average number of nodes of Jasminum azoricum L.
BA(mg/L) Shoot number Shoot length (cm) Number of nodes

0.0 1.2 2.51 4.50

0.5 1.9 1.78 3.19

1.0 2.0 1.82 3.10

2.0 2.5 1.62 2.60

4.0 2.3 1.55 2.20

6.0 1.9 1.32 2.20

L.S.D.(0.05) 0.41 0.13 0.10

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Data also showed that there was a significant and best elongation of shoots of the
control gave ( 2.51 cm) followed by the concentrations of 0.5 and 1.0 mg/L BA
which gave ( 1.78 and 1.82 cm respectively) as compared with other treatments . The
lowest shoot length was observed with 6.0 mg/L BA , which was 1.32 cm.
Significant difference was found in the average number of nodes per plant
(Table-1 ) in which the control gave the maximum number of nodes (4.50 ) than other
treatments. The Figure-1 shows the shoot proliferation and multiplication in vitro .
Cytokinins induced multiple shoot formation, but the proper type and concentration of
these hormones are different for each plant species ( Luo et al., 2009 ; Gantait et al.,
2011 ; Wangren, 2011; Shen et al. , 2013 ).

Figure(1): Shoots proliferation from nodes of Jasminum azoricum. A: nodes cultured on free
MS medium(right) and on MS+ 1.0mg/L BA ( left). B: shoots multiplication on MS + 2.0
mg/L BA.

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Callus induction and buds regeneration:

Results in table ( 2 ) revealed that there are significant differences in the effect of the
different concentrations of BA and 2,4-D on the callus induction. The best concentrations
of BA that gave the best percentage of callus induction were 4.0 and 6.0 mg/L, which
gave 62.5 and 72.5 %, respectively, whereas 0.1 mg/L 2,4-D gave a significant difference
( 82.5 % ) than other combinations. The highest percent of callus induction ( 100 % ) was
seen in explants grown in MS medium containing 4.0 mg/L BA+ 0.1 mg/L 2,4-D and 6.0
mg/L BA+ 0.1 mg/L 2,4-D , whereas the control did not give any response for callus
formation.

Table 2: Effect of different concentrations of BA and 2,4-D on the percentage of callus

induction from internodes of Jasminum azoricum .
2,4-D(mg/L)
BA(mg/L) 0.0 0.01 0.05 0.1 Mean effect of BA

0.0 0.0 10.0 30.0 60.0 25.0

2.0 10.0 20.0 60.0 70.0 40.0

4.0 30.0 40.0 80.0 100.0 62.5

6.0 30.0 70.0 90.0 100.0 72.5

Mean effect of 2,4-D 17.5 35.0 65.0 82.5

BA = 17.260
L.S.D.( 0.05 ) 2,4-D= 17.260
BA × 2,4-D = 24.720

Many researchers showed that cytokinins and auxins induced callus formation
in many plants (Darion et al., 2010; Hesar et al., 2011). It was known that the cytokinins
and auxins are used to promote the formation of callus in many excited and in vitro
cultured explants or organs (Ibrahim et al., 2013) .
The induced callus was transferred into MS medium without any addition of
plant growth regulators for two weeks, then it was transferred into fresh MS
medium containing different concentrations of BA (0.0, 2.0, 4.0, or 6.0) mg/L in
combination with Kin (0.0 or 2.0 mg/L) for adventitious buds regeneration. Data in
Table (3) showed that there were significant differences of BA concentrations on the
regenerated buds from callus (Figure- 2) . The highest number of buds was found in
the concentration of 4.0 mg/L BA, which gave 16.9 buds, other concentrations of BA
were comparatively better than the control. Results also showed that the effect of
Kin in concentration 2.0 mg/L (7.05 buds) was significant than control (3.4 buds). The

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interaction between BA and Kin showed that the highest number of buds was
(10.1) seen in the combination of 4.0 mg/L BA + 2.0 mg/L Kin .

Table 3: Effect of different concentrations of BA and Kin on the buds regeneration from
callus of Jasminum azoricum .
Kin ( mg/L )
BA ( mg/L ) Mean effect of BA
0.0 2.0

0.0 0.0 0.4 0.20

2.0 0.7 9.0 4.85

4.0 6.8 10.1 16.90

6.0 6.1 8.7 7.40

Mean effect of Kin 3.4 7.05

BA = 1.603
L.S.D. ( 0.05 ) Kin = 1.134
BA × Kin = 2.268

Organogenesis in explants during micro propagation takes place either directly

or after callus formation. Studies on many ornamental plants showed both kinds of
organogenesis . Cytokinins are known to promote in vitro regeneration of organs or
buds from callus tissues of many ornamental plants ( Gomathi et al., 2007) .

A B

Figure ( 2 ): A- Callus induction on internodes cultured on MS medium

supplemented with BA and 2,4-D.
B- Regeneration of buds from callus cultured on MS medium supplemented with BA and
Kin.

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Rooting and acclimatization:

According to the conditions of the experiment, all of plantlets failed to root in vitro
on all IBA concentrations, but they continued in their growth, while the ex vitro rooting
process showed that 85% (Figure- 3) of plantlets were successfully rooted and
hardened. Most of recent studies focused on the rooting and hardening of in vitro micro
propagated plantlets in one process by treated them with different types and
concentrations of auxins and cultured directly in agricultural media (Gantait et al., 2011).
It could be concluded that this method of propagation lead to efficient acclimatization
procedure and save the resources of time, labor and reduce the cost of production.

Figure(3): A,, direct rooting and acclimatization of in vitro micro propagated plantlets.
B,, ex vitro formation of roots on the base of jasmine plantlet.

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