INTERNATIONAL JOURNAL OF BUSINESS, SOCIAL AND SCIENTIFIC RESEARCH

ISSN: 2309-7892, Volume: 3, Issue: 1, Page: 35-38, January-February, 2015

Short Communication
IN VITRO REGENERATION AND RAPID MULTIPLICATION OF TUBEROSE

M. R. Ali1, M. H. Akand1 , M. E. Hoque2 , Homayra Huq2 , H. Mehraj1 and *A.F.M. Jamal Uddin1

M. R. Ali, M. H. Akand, M. E. Hoque, Homayra Huq, H. Mehraj and AFM Jamal Uddin (2015) In Vitro
Regeneration and Rapid Multiplication of Tuberose. Int. J. Bus. Soc. Sci. Res. 3(1): 35-38. Retrieve from
http://www.ijbssr.com/currentissueview/14013078

Received Date: 02/01/2015 Acceptance Date: 25/01/2015 Published Date: 26/01/2015

Abstract
An experiment was conducted at Tissue Culture Laboratory, Department of Biotechnology,
Sher-e-Bangla Agricultural University, Dhaka, Bangladesh from July 2011 to March 2012 for In
Vitro regeneration of tuberose. Experiment consisted ten combinations of different concentrated
KIN and IAA as supplemented with MS medium. Early shoot (10.5 days), leaf (14.6 days) and
root (16.0 days) initiation were found from MS+0.5 mg/L IAA +1.0 mg/L KIN. Maximum
number of leaves (2.99/plantlet), length of leaf (5.48 cm) , number of roots (5.69/plantlet) and
length of root (3.49 cm) were also found from MS+0.5 mg/L IAA +1.0 mg/L KIN.
Key words: Tuberose, regeneration and multiplication.
Introduction
Tuberose (Polianthes tuberosa L.) is a cut flower belongs to the Amaryllidaceae family. The bulbous
plants are commercially perpetuated by using their underground storage organs such as rhizomes o f
tuberose, corms of gladiolus and bulbs of lilies. Production of tuberose is greatly constrained by the bulb
quality, physiological decline and accumulation of pathogen. The effects of di fferent phytohormonal
combinations and concentrations on shoot bud induction and frequency of shoot regeneration on different
plant species were studied (Grzegorczyk and Wysokinska, 2008; Akbaş et al., 2011). Gopi et al. (2006)
reported that using di fferent concentrations and combinations of 6 -benzyl amino purine (BAP), Kinetin
(Kin), indole-3-acetic acid (IAA) and indole-3- butyri c acid (IBA) for direct regeneration, maximum
number of shoots (14.3±1.5) was observed on medium containing 0.5 mg/L BAP and 0.25 mg/L IAA
after four weeks of culture. Akbas et al. (2011) cl aimed that the highest shoot formation using lea f
explants, was obtained on Murashige and Skoog (MS) medium containing 1mg/L BAP and 1 mg/L KIN.
Considering above points in view the current study was undert aken for rapid regeneration of Polianthes
tuberosa under In Vitro condition.
Materials and Methods
An experiment on in vitro regeneration of tuberos e was conducted at Tissue Culture Laboratory,
Department of Biotechnology, Sher-e-Bangla Agri cultural University, Dhaka, Bangladesh from July
2011 to March 2012. Diseas e free bulbs of tuberose were collect ed from Bangladesh Agri cultural
Research Institute, Gazipur for explants in this experiment. Murashige and Skoog (MS) (1962) medium
were used with di fferent hormonal supplements as culture medium for shoot initiation, root initiation and
regeneration of tuberose. The culture tube cont aining medium was sterilized in an autoclave at
temperature of 121°C for 20 minutes at l5 psi pressure. The medium was then cooled at room
temperature before use. The lead of cabinet then was closed well and UV was switched on while turning
off the air flow. The UV light of cabinet was left on for 30 minutes and the surface area is wiped with
70% ethanol. The temperature of culture room was maintained within 25±1 °C with the help of air
conditioner with 16 hours photoperiod and 3000 lux light intensity by using white flores cent lights. Buds
were first sterilized with 70% (v/v) ethanol for few seconds after that surface sterilized by immersing in
0.1% HgCl2 solution for 4-5 minutes and then was washed several times with sterilized distilled water.
Then the bud was cut aseptically into small pieces with the help of sharp as eptic kni fe and the small
segments of bud were used as explants. Three to five segments of explants were directly inoculated into
each test tube containing 10 ml of MS medium suppl emented with di fferent callus induction hormonal
combinations of IAA and KIN. Experiment consisted ten different hormonal combination viz. T0 : Simple
MS, T1 : MS+0.5 mg/L IAA+0.5 mg/L KIN, T2 : MS+0.5 mg/L IAA +1.0 mg/L KIN, T3 : MS+0.5 mg/L
IAA+1.5 mg/L KIN, T4 : MS+1.0 mg/L IAA+0.5 mg/L KIN, T5 : MS+1.0 mg/L IAA+1.0 mg/L KIN, T6 :

*Corresponding Authors Email: jamal4@yahoo.com

1
Department of Horticulture, Sher-e-Bangla Agricultural University , Dhaka-1207, Bangladesh
2
Department of Biotechnology , Sher-e-Bangla Agricultural University , Dhaka-1207, Bangladesh
Efficiency of Artificial Insemination ( AI) 36

MS+1.0 mg/L IAA+1.5 mg/L KIN, T7 : MS+1.5 mg/L IAA+0.5 mg/L KIN, T8 : MS+1.5 mg/L IAA+1.0
mg/L KIN and T 9 : MS+1.5 mg/L IAA+1.5 mg/L KIN following Completely Randomized Design with
five replications. Data were taken on days to shoot initiation, days to leaf initiation, days to root
initiation, number of leaves/plantlet, length of leaf, number of roots/plantlet and length of root. Collected
data on di fferent paramet ers were analyzed using a MSTAT-C package computer program. The analysis
of variance was perform ed and means were compared by Least Significant Di fference (LSD) test at 5%
level of signi ficance (Gomez and Gomez, 1984).
Results and Discussion
Days to shoot, leaf and root initiation: Tuberose differed signi ficantly in the number of days required for
shoot initiation. Early shoot was initiated from T 2 (10.5 days) whereas late from T 4 (40.5 days) (Table 1).
Early leaf was initiation was found from T 2 (14.6 days) while late from T4 (50.6 days) (Table 1).
Minimum days required for root initiation was found from T 2 (16.0 days) which was statistically
identical with T 4 (16.9 days) while maximum from T 7 (36.4 days) (Table 1).
Table 1. Effect of different combination of KIN and IAA on days to shoot, leaf and root initiation
Days to initiation of
Hormone (mg/L)
shoot leaf root
T0 No shoot No leaf No root
T1 12.5 h 25.0 g 27.4 e
T2 10.5 i 14.6 h 16.0 h
T3 13.1 g 29.9 e 19.7 g
T4 40.5 a 50.6 a 16.9 h
T5 15.9 f 28.0 f 30.0 d
T6 18.9 e 30.0 e 26.2 f
T7 23.0 d 32.9 d 36.4 a
T8 28.0 c 34.0 c 35.0 b
T9 32.1 b 45.0 b 34.1 c
LSD(0.05) 0.4 0.5 0.9
CV (%) 1.6 1.2 2.9
In column, figure with same letter(s) do not differ significantly at 5% level of significance according to DMRT
Number of leaves/plantlet and length of leaves: Number of leaves/plantlet was varied signifi cantly
among di fferent combinations of KIN and IAA at di fferent days after inoculation. However, maximum
number of leaves was found from T 2 (2.99/plantlet) followed by T9 (2.73/plantlets) while minimum from
T4 (0.54/plantlets) at 60 DAI (Table 2). Length of leaves was vari ed significantly among different
combinations of KIN and IAA at di fferent days aft er inoculation. Longest leaf was found from T 2 (5.48
cm) followed by T 5 (4.19 cm) whereas minimum from T 3 (1.12 cm) which was statistically similar with
T8 (1.25 cm) at 60 DAI (Table 2). Maximum number with longest of leaves roots at 60 DAI on MS
medium supplemented with 0.5 mg/l IAA+1.0 mg/l KIN are shown in Plate 1a.
Number of roots/plantlet and length of roots: Number of roots/plantlet was varied signi ficantly among
the different combinations of KIN and IAA at di fferent days aft er inoculation. Maximum number of
roots/plant was found from T 2 (5.69/plantlets) followed by T 5 (3.47/plantlet) and T1 (3.52/plantlet) while
minimum from T7 (0.64/plantlet) at 60 DAI (Table 3). Longest root was found from T 2 (3.49 cm)
followed by T5 (2.65 cm) whereas shortest from T7 (0.51/plantlet) at 60 DAI (Table 3). Maximum
number with longest roots at 60 DAI on MS medium supplemented with 0.5 mg/l IAA+1.0 mg/l KIN are
shown in Plate 1b.
Table 2. Effect of di fferent combination of KIN and IAA on number of l eaves/plantlet and length of
leaves at different days after inoculation
Number of leaves/plantlet at different
Hormone Length of leaves at different DAI
DAI
(mg/L)
15 30 45 60 15 30 45 60
T0 - - - - - - - -
T1 0.58 c 1.25 c 2.01 c 2.56 c 0.27 e 0.75 e 1.48 f 2.47 e
T2 2.10 a 2.78 a 2.78 a 2.99 a 1.15 a 2.23 a 4.10 a 5.48 a
T3 0.25 e 0.67 f 1.14 f 1.75 f 0.00 f 0.23 g 0.50 h 1.12 f
T4 0.00 f 0.00 h 0.24 i 0.54 h 0.00 f 0.00 h 0.26 i 0.47 g
T5 0.57 c 1.01 d 1.70 d 2.27 d 0.54 c 1.50 c 3.26 b 4.19 b
T6 0.00 f 0.26 g 0.73 g 1.02 g 0.64 b 1.74 b 2.64 c 3.35 c
T7 0.34 d 0.76 e 1.39 e 1.97 e 0.33 de 0.77 e 1.76 d 2.43 e
T8 0.00 f 0.25 g 0.46 h 1.02 g 0.00 f 0.37 f 0.77 g 1.25 f
T9 0.66 b 1.74 b 2.16 b 2.73 b 0.31 de 0.86 d 1.69 e 2.67 d
LSD(0.05) 0.07 0.07 0.11 0.10 0.04 0.04 0.05 0.14
CV (%) 15.85 6.48 6.92 4.99 7.69 4.23 2.92 4.63
In column, figure with same letter(s) do not differ significantly at 5% level of significance according to DMRT

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Roy et al. 37

Table 3. Effect of di fferent combination of KIN and IAA on number of roots/plantlet and length of root
at different days after inoculation
Number of roots/plantlet at different
Hormone Length of root at different DAI
DAI
(mg/L)
15 30 45 60 15 30 45 60
T0 - - - - - - - -
T1 0.99 b 2.01 b 2.89 b 3.52 b 0.56 a 1.45 b 1.91 c 2.26 c
T2 1.99 a 3.90 a 4.47 a 5.69 a 0.50 b 1.10 c 2.50 a 3.49 a
T3 0.33 h 0.74 f 1.11 f 2.17 cd 0.13 de 0.32 f 0.80 f 1.54 f
T4 0.36 g 0.97 e 0.88 g 1.63 e 0.20 e 0.48 f 1.12 e 1.67 e
T5 0.89 c 1.41 c 2.46 c 3.47 b 0.25 c 1.64 a 2.22 b 2.65 b
T6 0.67 e 1.24 d 1.52 de 2.37 c 0.27 c 0.85 d 1.46 d 2.14 d
T7 0.04 i 0.23 g 0.31 h 0.64 f 0.16 d 0.34 e 0.30 i 0.51 i
T8 0.43 f 1.06 e 1.36 e 1.99 d 0.00 f 0.15 g 0.44 h 0.75 h
T9 0.75 d 1.33 c 1.66 d 2.30 c 0.00 f 0.19 g 0.64 g 1.02 g
LSD(0.05) 0.07 0.08 0.21 0.28 0.04 0.08 0.09 0.12
CV (%) 8.80 4.73 10.04 9.36 12.15 9.61 6.11 5.84
In column, figure with same letter(s) do not differ significantly at 5% level of significance according to DMRT

Acclimati zation and establishment of plantlets in soil

Potting mixture containing garden soil, sand and cow dung in 1:2:1 ratio was mixed properly and
autoclaved one hour in 1210 C for 20 minutes at 1.16 kg/cm2 . After cooling soil mixture was taken into 10
cm pots for growing of plantlets in natural condition. When plantlets became 5-8 cm in height with
suffi cient shoot and root system, they were taken out from vials without damaging roots. Medium
attached with root gently washed out in running tap water to prevent further microbial infection. Plantlets
were then transplanted to pot containing sterilized potting mixture. Immediately aft er transplantation
plants along with pots were covered with moist polythene bags to prevent desiccation. To reduce sudden
shock pots were kept in a growth room for 7-15 days under controlled environments (Plate 1c). Interior
of polythene bags was sprayed with distilled water at every 24 hours to m aintain high humidity around
plantlets. After 2-3 days, polythene bags were gradually perforated to expos e plants to natural
environment. Polythene bags were completely removed after 10-15 days when pl antlets, appeared to be
self-sustainable. At this stage, plantlets were placed in natural environment for 3-10 hours daily (Plate
1d).
Finally, after 15-20 days, plantlets were trans ferred to net house for hardening and after that transplant ed
to soil (Plate 1e and Plate 1f). As soon as new leaves started to initiate, plants were watered with ordinary
tap water. Gradually plantlets were adapted to soil.
Survival rates: Survival rates in small hole of pl astic tray at growth chamber was 55.55% while in open
atmosphere was 72.00% (Table 4).
Table 4: Survival rate of in vitro regenerated plantlets of tuberose
Number of transplanted Number of plants Survival rate
Acclimatization
plants survives (%)
1. Initially small hole of plastic 45.00 25.00 55.55
tray at growth chamber
2. Subsequently when moved to 25.00 18.00 72.00
soil in open atmosphere
Discussion
MS medium without growth regulators did not promote root and shoot induction which was resemblance
of the finding of the Rout (2004). Cytokinin is required in optimal quantity for shoot proliferation in
many genotypes but inclusion of a low concentration of auxin along with cytokinin increases the rate o f
shoot multiplication (Tsay et al., 1989; Sharma et al., 1993; Sharma and Singh, 1997; Shasany et al.,
1998; Rout et al., 2000). In present study it was found that low concentration of KIN and IAA provided
the best performance for the all studied characteristics of tuberos e In Vitro.
Conclusion
The results of the present investigation indicated that tuberose could be successfully micro propagat ed
with 0.5 mg/L IAA+1.0 mg/L KIN for rapid shoot and leaf initiation and proliferation.

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Efficiency of Artificial Insemination ( AI) 38

(a) (b) (c)

(d) (e) (f)

Plate 1. (a) Maximum number of leaves along with the longest leaf initiated at 60 DAI on MS medium
supplemented with 0.5mg/l IAA + 1.0 mg/l KIN, (b) Maximum number of roots with the longest root
length at 60 DAI on MS medium supplemented with 0.5 mg/l IAA + 1.0 mg/l KIN, (c) In vivo
acclimatization of regenerated plantlets in growth chamber, (d) In vivo establishment of regenerated
plantlets in open atmosphere, (e) Hardening of plantlets in net hous e with potting and (f) Transplantation
of healthy plantlets to the soil
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