Tesfa et al.

, Adv Crop Sci Tech 2016, 4:2

Advances in Crop Science and Technology DOI: 10.4172/2329-8863.1000215

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Ex Vitro Rooting of Sugarcane (Saccharum officinarum L.) Plantlets

Derived from Tissue Culture
Melaku Tesfa1*, Belayneh Admassu2 and Kassahun Bantte3
1
Ethiopian Sugar Corporation Research and Training Division, Biotechnology Research Team, Wonji Research Center, Wonji, Ethiopia
2
National Agricultural Biotechnology Research Program, Holetta Agricultural Research Center, Ethiopia
3
Jimma University College of Agriculture and Veterinary Medicine, Jimma, Ethiopia

Abstract
A study was conducted at Holetta National Agricultural Biotechnology Laboratory with the objective to determine
the effect of different concentrations of NAA on ex vitro root development of sugarcane microshoots. Five levels of
NAA (0, 10, 20, 30 and 40 mg/l) and two levels of genotypes were combined in factorial arrangement. The basal end
of the shoots was dipped in NAA solution overnight before the shoots were transferred into a plastic tray containing
a mixed growing medium in green house. The results showed that interaction effect of genotype and NAA was highly
significant (p<0.0001) on rooting percentage, number of roots per shoot, root length. In genotype N52, best root
formation was found on the shoots treated with 20 mg/l NAA by which rooting percentage was 76 ± 5.48 with 5.88 ±
0.04 cm root length and 8.06 ± 0.13 number of roots per plantlets. While in genotype N53 maximum, root formation
was recorded on the shoots dipped in 30 mg/l NAA by which rooting percentage was 70 ± 7.07 with 5.42 ± 0.11
cm root length and 4.52 ± 0.19 number of roots per plantlets. Shoots rooted through this method exhibited 100 %
survival in both genotypes.

Keywords: Microshoot; Ex vitro; NAA; In vitro; Medium to support the plantlets to absorb water and nutrients from the potting
medium. Ex vitro rooting is more advantageous than in vitro rooting
Introduction in reducing cost of labour, chemicals and equipments, and the time
of establishment from laboratory to soil revealed that ex vitro rooting
Sugarcane (Saccharum officinarum L.) is a monocotyledonous crop
reduced more than 50% cost of sugarcane plantlet raised by conventional
that is grown in the tropical and subtropical regions of the world, and
micropropagation [11-13]. Besides, the plantlets produced after ex vitro
almost cultivated on over 23.8 million ha for its sucrose rich stalk [1].
rooting have better developed root system than the ones produced
In Ethiopia, it is cultivated commercially on around 96,000 ha with
after in vitro rooting [14,15]. Furthermore, rooting and acclimatization
annual sugar production of 370,000 ton and is a solely raw material
phase can be carried out simultaneously; hence, it is more time efficient.
for sugar production in the country. Recently, in Ethiopia, sugarcane
production has gained attention, allied with its important potential for Ex vitro rooting has been applied in micropropagation of various
an environment-friendly bio-fuel (ethanol) production, in creating job plants species [11,12,16], but there are very limited reports yet on ex
opportunity to the nation and generating huge electric power [2]. vitro rooting of sugarcane plantlets. Most reports of ex vitro rooting
of plant species have involved treatment with exogenous auxin such
Sugarcane, since commercially propagated vegetatively by stem as indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and
cutting, has a low seed multiplication rate (1:10) which resulted in slow 1-naphthalene-acetic acid (NAA) [12,13,16]. Auxin is applied singly
seed production of newly released improved varieties. Furthermore, or in a combination at different concentrations to improve rooting
the seed builds up diseases and pests during several cycles of field frequency of plantlets in the acclimatization period. Therefore, this
production, which leads to further yield and quality declines over years study was aimed to determine the effect of different concentrations of
[3]. Thus, unavailability of disease-free, true to type planting material is auxin, NAA on ex vitro rooting of two elite sugarcane genotypes.
a major limitation in improving sugarcane productivity.
Materials and methods
Recently, tissue culture technology plays a leading role in rapid
multiplication of disease-free and quality planting material of sugarcane The study was undertaken at the National Agricultural Biotechnology
[4]. Accordingly, Ethiopian Sugar Corporation has established its own Laboratory of the Ethiopian Institute of Agricultural Research, in
tissue culture laboratory at Metahara, Kuraz, Tendaho and Fincha sugar Holetta. The study was undertaken with two elite sugarcane genotypes
factories/projects to produce about 55 million disease free plantlets per
year [2]. To do so, in vitro propagation protocols have been developed
using shoot tip and callus explant for several sugarcane cultivars of
Ethiopian Sugar Estates [5-8]. *Corresponding author: Melaku Tesfa, Ethiopian Sugar Corporation Research and
Training Division, Biotechnology Research Team, Wonji Research Center, Wonji,
In vitro propagation involves four crucial steps namely, initiation, Ethiopia, Tel: 251-913241485; E-mail: [email protected]

multiplication and rooting of microshoots and acclimatization Received February 04, 2016; Accepted March 17, 2016; Published March 23,
of plantlets. However, in vitro rooting process is an expensive, 2016
labour consuming process and can even double the final price of Citation: Tesfa M, Admassu B, Bantte K (2016) Ex Vitro Rooting of Sugarcane
micropropagated plants. Earlier report shown that in vitro rooting (Saccharum officinarum L.) Plantlets Derived from Tissue Culture. Adv Crop Sci
Tech 4: 215. doi:10.4172/2329-8863.1000215
may account for 40% total cost of the intensive manipulation needed
during in vitro propagation [9]. In addition, roots of plantlets raised in Copyright: © 2016 Tesfa M, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
vitro are generally very fragile and not have root hairs [10]. Therefore, use, distribution, and reproduction in any medium, provided the original author and
during early acclimatization period, the roots do not function properly source are credited.

Adv Crop Sci Tech, an open access journal

ISSN: 2329-8863 Volume 4 • Issue 2 • 1000215
Citation: Tesfa M, Admassu B, Bantte K (2016) Ex Vitro Rooting of Sugarcane (Saccharum officinarum L.) Plantlets Derived from Tissue Culture. Adv
Crop Sci Tech 4: 215. doi:10.4172/2329-8863.1000215

Page 2 of 4

viz. N-52 and N-53 obtained from Ethiopian Sugar Corporation, After 45 days, healthy micro-shoots having 4 cm heights were
Research and Training Division. The genotypes were selected based on employed for ex vitro rooting study. The clumps of in vitro shoots were
their higher yield performance and sugar quality. In order to carry out separated to obtain single micro shoot. The basal portion of these rootless
explant sterilization and preparation, actively growing shoot tops were microshoots was dipped in distilled aqueous solution containing auxin,
excised from 5-months-old screen house grown healthy mother plants NAA at different concentrations i.e. 0, 10, 20, 30, & 40 mg/l overnight to
of genotype N52 and N53. The trimmed shoot tops (segment) were induce rooting under ex vitro condition. The experiment was arranged
washed carefully under running tap water for 30 minutes, and reduced in completely randomized design (CRD) with five replications and each
to 10 cm length by cutting off at the two ends. Then washed thoroughly treatment had 50 microshoots. After treated with auxins, the shoots
for 30 minutes with tap water containing a drop of liquid detergent were transferred to polystyrene trays containing autoclaved mixture
solution and two drops of tween-20 and rinsed three times with double of river sand and forest soil in 2:1 ratio. Subsequently, maintained in
distilled water. Subsequently, the explant was taken to a laminar air flow greenhouse, which uses Fan-Pad evaporative cooling system providing
cabinet and immersed in 0.1% (w/v) Bavistin® DF 50 % (Carbendizem) 25–30°C temperature. During experimenting, high humidity level (80
fungicide solution, ascorbic acid (0.2% w/v) and citric acid (0.4% w/v) %-85 %) was maintained by covering the tray with moisten polyethylene
for 30 minutes followed by three times rinsing each for five minutes sheet and red shade cloth and then sprinkled with water three times a
with sterile double distilled water. The shoot tips were washed again day as necessary and sprayed with quarter strength MS basal medium
with 70% ethanol for one minute and rinsed with sterile double distilled at weekly interval.
water three times each for five minute to remove residual ethanol
After 4 weeks, the plantlets were carefully removed from the soil
from the shoot tip surface. Finally, surface sterilized with 50 % (v/v)
mix and data on number of rooted shoots, total number of primary
aqueous solution of Sodium hypochlorite (5.25% w/v active chlorine)
roots and root length were recorded. All microshoots that remain green
containing a few drops of tween-20 for 25 minutes. After pouring out
were considered living and used in calculating rooting percentage.
sodium hypochlorite solution, the explants were rinsed with sterile
Successfully rooted plantlets were subsequently transferred in medium
double distilled water three times each for five minutes to remove all
polyethene bags (15 cm × 20 cm) containing mixture of sand, farm
the trace of the sterilant.
yard manure and soil in 1:1:1 ratio for further hardening and data on
Culture were initiated on [17] medium fortified with BAP, Kinetin survival rate of the plantlets was recorded 4 weeks after transplanting.
and NAA (0.5 mg/l each) [18] and 2% sucrose (w/v) and solidified The collected data were subjected to analysis of variance (F test) using
using agar (agar agar, type I) (0.45 %; w/v) for induction of shoots. SAS program (Version 9.2). The differences among treatment means
The pH of the medium was adjusted to 5.8 followed by autoclaving at were determined by REGQ multiple range test at P<0.05.
121°C at 105 Kpa pressure for 20 minutes. After 30 days of inoculation
the regenerated shoots were transferred to multiplication medium Results and Discussion
supplemented with 2 mg/l BAP + 0.5 mg/l Kinetin (N52) and 1.5 mg/l Statistical analysis of variance showed that the main effect of
BAP and 0.5 mg/l Kinetin (N53) [19]. After 30 days of incubation, genotype and NAA and the interaction effect of genotype and NAA
shoots were maintained on plant growth regulators (PGRs) free MS highly significant (p<0.0001) on rooting percentage, number of roots
medium with 2 g/l activated charcoal for two weeks before transferring per shoot, root length of the two sugarcane genotype (Table 1). The
rooting stage in order to avoid the carry over effect of hormones from present result also showed that rooting was induced ex vitro over the
the multiplication media on ex vitro rooting of the plantlets. entire range of NAA concentration tested including the control shoots
in both sugarcane genotypes (Table 2).

Rooting Root length Number of roots In the control treatment reduced rooting frequency of 36% and
Source of variation DF 28% were obtained in genotypes N52 and N53, respectively (Figure 1A
Percentage (cm) per shoot
MS MS MS and Figure 1B) However, in NAA treated microshoots than 50% of the
Genotype 1 968*** 7.76*** 59.19*** shoot developed roots regardless of the NAA concentration (Table 2).
NAA 4 2307*** 4.73*** 25.56*** Shekafandeh [12] observed increased rooting frequency and number
Genotype*NAA 4 163*** 2.08*** 4.46 *** of roots from zero percent in untreated shoots to 91.7% and 3.3 roots
CV % 10.38 5.60 2.72 per shoot, respectively, when the basal end of the shoots were dipped
in a solution of 1.5 mg/l IAA and 0.3 mg/l IBA for 24 h before culturing
*** = Very highly significant at P≤0.0001; DF = Degree of freedom; NAA =
α-naphthalene acetic acid; MS = Mean square; CV= Coefficient of variation
in soil mixture in Myrtle (Myrtus communis L.) plant. Similar results
Table 1: Effect of different concentration of NAA on ex vitro rooting.
were also reported by Sumaryono and Riyadi [20] in oil palm (Elaeis

Genotypes
Treatment N52 N53
NAA Root length Number of root per Root length Number of root per
Rooting Percentage Rooting Percentage
mg/l (cm) shoot (cm) shoot
0 36d ± 5.48 4.44ed ± 0.30 2.14h ± 0.13 28d ± 0.47 2.58f ± 0.54 1.74i ± 0.05
10 68 ± 4.47
ab
4.64 ± 0.29
cd
5.42 ± 0.08
d
50 ± 7.07
c
4.32 ± 0.24
ed
2.56g ± 0.13
20 76 ± 5.48
a
5.88 ± 0.04
a
8.06 ± 0.13
a
60 bc
± 7.07 4.34 ± 0.21
ed
4.08f ± 0.08
30 70ab ± 7.07 5.04bc ± 0.05 6.36b ± 0.11 70ab ± 7.07 5.42b ± 0.11 4.52e ± 0.19
40 56c ± 5.48 4.68cd ± 0.24 5.68c ± 0.13 54c ± 5.48 4.08e ± 0.19 3.88f ± 0.13
CV% 10.38 5.60 2.72 10.38 5.60 2.72
*NAA =α-naphthalene acetic acid. Values in the same column and variables with different letters are significantly different from each other according to REGWQ at P<0.05.
Table 2: The effect of NAA on rooting percentage, root length and number of roots per shoot.

Adv Crop Sci Tech, an open access journal

ISSN: 2329-8863 Volume 4 • Issue 2 • 1000215
Citation: Tesfa M, Admassu B, Bantte K (2016) Ex Vitro Rooting of Sugarcane (Saccharum officinarum L.) Plantlets Derived from Tissue Culture. Adv
Crop Sci Tech 4: 215. doi:10.4172/2329-8863.1000215

Page 3 of 4

However, when NAA concentration was further elevated to higher

concentration (40 mg/l), rooting frequency, average root length and
average number of roots per shoot, reduced significantly to 56 %, 4.68
± 0.24 cm and 5.68 ± 0.13, respectively. The same trend was observed in
genotype N53, in that rooting frequency increased from 28% in control
shoots to 70 % in treated shoots with a solution of NAA at 30 mg/l. The
average root length and average roots number also increased from 2.58
A B cm ± 0.54 cm and 1.74 to 5.42 cm ± 0.08cm and 4.52 ± 0.19 respectively,
B as the concentration of NAA increased from 0.0 mg/l to 30 mg/l but
Figure 1: Acclimatized plantlets. (A) Genotype N52 (B) Genotype N53. as the concentration of NAA was increased to 40 mg/l the rooting
frequency, root length and root number reduced markedly to 54 %, 4.08
cm ±0.19 cm and 3.88 ± 0.13 respectively (Figures 3-5). This reduction
in rooting response could be due to the fact that higher concentrations
of NAA promote the biosynthesis and buildup of ethylene at the basal
end of the shoot, which have inhibitory effect on the overall rooting
response of sugarcane microshoots [21].
The result of the present study on genotype N52 were in agreement
with earlier results by Pandey et al. [13], who obtained the highest

80
A
Rooting frequency (%)

60

40
RF (%) N53
20 RF (%) N52

0
0 10 20 30 40
NAA concentration (mg/l)
B
Figure 3: Effect of NAA on rooting frequency of both genotypes.
Figure 2: Ex vitro rooting of sugarcane micro-shoots. (A) Genotype N52 at 20
mg/l NAA (B) Genotype N53 at 30 mg/l NAA
7.00
guineensis Jacq.). These results indicated the significance of treating of 6.00
Root length (cm)

microshoots with plant growth regulators during ex vitro rooting before 5.00
culturing in soil medium. 4.00
There was a significant response variation in rooting between 3.00 RL (cm) N53
the two genotypes. Genotype N52 had the highest (76 %) rooting 2.00
RL (cm) N52
frequency with a maximum (5.88 cm ± 0.04 cm) average root length 1.00
and 8.06 ± 0.13 average number of roots per shoot on microshoots 0.00
dipped in 20 mg/l concentration of NAA (Table 2 and Figure 2A). At 0 10 20 30 40
the same concentration of NAA, N53 had only 60 % rooting frequency NAA concentration (mg/l)
with 4.34 cm ± 0.21 cm average root length and 4.08 ± 0.08 average
Figure 4: Effect of NAA concentration on root length of both genotypes.
roots number per shoot. On the other hand, genotype N53 showed a
maximum rooting frequency (70 %) with 5.42 cm ± 0.08 cm and 4.52
± 0.19 numbers of roots per shoot on microshoots treated with 30 mg/l 9
concentration of NAA (Table 2 and Figure 2B). At this concentration of
Number of roots per shoot

8
NAA, genotype N52 gave almost equal rooting frequency (70 %) with 7
comparable root length (5.04 cm ± 0.05cm) and higher (6.36 ± 0.11) 6
root number per shoot than N53.The result of this experiment revealed 5
4 NRPS N53
that genotype N52 was more responsive than N53 for different NAA 3
concentrations. 2 NRPS N52
1
The rate of rooting frequency increased from 36 % in control shoots 0
to 76 % when the basal ends of shoots were dipped in a solution of 0 10 20 30 40
20 mg/l NAA overnight (Figure 3). Similarly, average root length and
NAA concentration (mg/l)
average number of roots increased from 4.44 cm ± 0.30cm and 2.14 ±
0.13 to 5.88 ± 0.04cm and 8.06 ± 0.13, respectively, in genotype N52. Figure 5: Effect of NAA on number of root per shoot in both genotypes.

Adv Crop Sci Tech, an open access journal

ISSN: 2329-8863 Volume 4 • Issue 2 • 1000215
Citation: Tesfa M, Admassu B, Bantte K (2016) Ex Vitro Rooting of Sugarcane (Saccharum officinarum L.) Plantlets Derived from Tissue Culture. Adv
Crop Sci Tech 4: 215. doi:10.4172/2329-8863.1000215

Page 4 of 4

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officinarum L.) genotypes. Adv Crop Sci Tech 2: 143.
CoS96268. Similarly, Martin [16] obtained an average of 5.6 roots per
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of elite sugarcane (Saccharum officinarum L.) varieties of Ethiopian sugar
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on shoot treated with 2 mM NAA with 80% rooting frequency in oil engineering research 2.
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per shoot. Ex vitro rooting reduces the time of acclimatization and grosvenorii, a traditional medicinal plant. Acta physiologiae plantarum 32: 115-
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(HNABL) for provision of Tissue Culture Laboratory with all required consumable 17. Murashige T, and Skoog F (1962) A revised medium for rapid growth and bio
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Adv Crop Sci Tech, an open access journal

ISSN: 2329-8863 Volume 4 • Issue 2 • 1000215