In vitro regeneration of Saccharum officinarum var. Co 92005 using shoot tip explant

Abstract

Micropropagation is a widely implemented technique for production of a large number of plantlets in a very short period of time and in a trifling space of a culture room. Shoot tips and meristem culture techniques offer an opportunity for virus free plants production. In the present study, shoot tip culture technique was used for the production of plantlets of Saccharum officinarum (var. Co 92005). Shoot tips of size 0.5 cm dissected out from actively growing twigs of 6-8 month old plants, were cultured on MS basal medium supplemented with different concentrations of BAP and coconut milk. MS medium with 0.5 mg/l BAP and 20 per cent coconut milk was found to be best for culture initiation and establishment with 100 per cent response and maximum elongation of shoots. The same medium was found better for multiple shoot formation with 100 per cent response and maximum shoot multiplication. Maximum rooting response was observed on MS medium with 2.5 mg/l IBA with 100 per cent response with an average number of roots 2.93 roots/shoot and average root length 2.5 cm within two weeks. The rooted plantlets were transferred to pots filled with coco peat and were hardened under a polytunnel with 90 per cent survival after 15 days.

Full text

I.J.S.N., VOL.8 (1) 2017: 154-157 ISSN 2229 –6441
154
IN VITRO REGENERATION OF SACCHARUM OFFICINARUM VAR.CO
92005 USING SHOOT TIP EXPLANT
1Komal Ramchandra Pawar, 1Swapnil Gorakh Waghmare, 1Ramling Tabe, 1Ashok Patil,
2Ajinkya Rajendra Ambavane
1VSBT College of Agricultural Biotechnology, Mahatma Phule Krushi Vidyapeeth, India.
2College of Horticulture, Kerala Agricultural University, Thrissur, India
*Corresponding authors email: swapnil.waghmare1856@gmail.com
ABSTRACT
Micropropagation is a widely implemented technique for production of a large number of plantlets in a very short period of
time and in a trifling space of a culture room. Shoot tips and meristem culture techniques offer an opportunity for virus fre e
plants production. In the present study, shoot tip culture technique was used for the production of plantlets of Saccharum
officinarum (var. Co 92005). Shoot tips of size 0.5 cm dissected out from actively growing twigs of 6-8 month old plants,
were cultured on MS basal medium supplemented with different concentrations of BAP and coconut milk. MS medium
with 0.5 mg/l BAP and 20 per cent coconut milk was found to be best for culture initiation and establishment with 100 per
cent response and maximum elongation of shoots. The same medium was found better for multiple shoot formation with
100 per cent response and maximum shoot multiplication. Maximum rooting response was observed on MS medium with
2.5 mg/l IBA with 100 per cent response with an average number of roots 2.93 roots/shoot and average root length 2.5 cm
within two weeks. The rooted plantlets were transferred to pots filled with coco peat and were hardened under a polytunnel
with 90 per cent survival after 15 days.
KEYWORDS: Micropropagation, Saccharum officinarum (var. Co 92005), MS Medium, Coconut Milk.
INTRODUCTION
Sugarcane (Saccharum officinarum) belonging to family
Gramineae (Poaceae) is one of the major cash crop grown
in India. Main product of sugarcane is sugar and its juice
is used for the preparation of various products like
jaggery, molasses, bagasse and ethanol. Conventionally
sugarcane propagation is carried out by stem cuttings
using three budded setts. Disease-ridden planting materials
can pass diseases like grassy shoot, red rot, ratoon
stunting, leaf scald, etc. to succeeding crops. This leads to
financial loss due to a reduction in cane yield and sucrose
recovery. Plant tissue culture offers a way to overcome
these issues and to produce disease-free planting material.
Technique of plant tissue culture, ‘micropropagation’ is
the most successful and tranquil way to produce ample
amount of pathogen free and vigorous planting material.
Micropropagation defined as, ‘tremendous increase in the
number of shoots available for rooting results from the
proliferation of the lateral buds or adventitious shoots or
the differentiation of the shoots’. This method has several
advantages over the conventional method of propagation.
Meristematic tissues are mainly used in micropropagation
to produce disease free planting material. According to
Hendre et al. (1983), about 0.2 million plants can be
produced from single sugarcane shoot tip within six
months. According to Dhumale et al. (1994), single
sugarcane shoot tips of size 2-3 mm can give significant
shoot regeneration. Millions of clonally uniform plants can
be produced within a year from single explant by using
micropropagation technique. MS Basal medium
supplemented with benzylamine purine (BAP) and kinetin
(Kn) effectively used for rapid shoot multiplication (Ali
and Afghan, 2001; Singh et al., 2006; Mekonnen et al.,
2014). According to Schenk and Hildebrandt (1972),
sugarcane requires a high concentration of auxin for
rooting. Roots can be regenerated from in vitro
regenerated shoots on half MS medium supplemented with
NAA, IBA and IAA (Baksha et al., 2002; Khan et al.,
2008). For each new variety and clone, efficient in vitro
regeneration protocol is essential to guarantee the rapid
shoot initiation, shoot multiplication, elongation and root
induction (Cheema and Hussai, 2004).
The sugarcane variety Co 92005 recorded more cane yield,
higher sugar yield, and more jaggery recovery compared
to other varieties grown in Maharashtra. Its jaggery quality
is better and fetches a higher market price. This variety is
commercially recommended for jaggery production in
Maharashtra. The present study was undertaken to study in
vitro regeneration in S. officinarum var. Co 92005 using
shoot tip explant, in order to determine an effective
protocol for rapid plantlets multiplication. This paper
presented the first time report of such study in S.
officinarum var. Co 92005.
MATERIALS & METHODS
Collection of plant material
Healthy, fresh 6-8 months old sugarcane plant twigs were
collected from fields of Vidya Pratishthan’s VSBT
College of Agricultural Biotechnology, Baramati,
Maharashtra. The twigs were excised in the morning and
brought to the laboratory in glass bottles containing
distilled water in order to prevent browning.

Regeneration of Saccharum officinarum VAR.CO 92005
155
Media preparation
For the establishment of explant, MS medium
supplemented with 3 per cent sucrose, 10 per cent (v/v)
coconut water, 0.8 percent agar and different
concentrations (0.1, 0.3, 0.5, 1.0, 2.0 mg/l) of BAP were
prepared. pH of medium was adjusted to 5.75. In each
tubes (25×150 ml) 20 ml media was poured and sterilised
by autoclaving at 121 ℃ temperature and 15 psi pressure
for 20 min.
Preparation of explants
Explants were washed thoroughly under running tap water
and treated with fungicide (Bavistin) (1 per cent v/v) for
10 min. to reduce the fungal contaminants. The explants
were then treated with a solution of the Savlon (1 per cent
v/v) for 10 min. and 70 per cent ethanol for 30 sec. under
aseptic conditions to reduce bacterial contamination. Then
surface sterilized with HgCl2(0.1 per cent w/v) for 10
min. under aseptic conditions to reduce remaining
contamination. The explants were washed three times with
sterile distilled water and then transferred in antioxidant
solution of ascorbic acid (1 per cent v/v) to reduce
browning of explants. After surface sterilization explant
were dissected to get shoot tip of size about 0.5 cm under
aseptic conditions.
Establishment, subculturing and multiplication of
shoots
Excised shoot tips were placed aseptically on medium
supplemented with different concentrations of BAP. All
explants were incubated in culture room at 25 ± 2℃ and
under 2000 lux intensity of white cool fluorescent lamp
with 16:8 photoperiods. Cultures were observed in regular
day interval. Regenerated shoots then subcultured on fresh
medium for its better growth and multiple shoots
production. MS basal media supplemented with different
concentrations of BAP (0.1, 0.3, 0.5, 1.0, 2.0 mg/l) and 20
per cent (v/v) coconut water, 3 per cent sucrose and 0.8
per cent agar were used for multiplication of regenerated
shoots.
Rooting and primary hardening
Multiplied shoots were separated as single shoots for root
initiation. Single shoots were transferred aseptically on
medium containing full strength MS basal media and half
strength MS basal media with different concentrations
(0.5, 1.0, 1.5, 2.0 and 2.5 mg/l) of IBA for root initiation.
Culture bottles were incubated at 25±2 ℃ and 2000 lux
intensity of white cool fluorescent lamp with 16:8
photoperiod. After every three days observations were
taken for root initiation and root elongation. Primary
hardening was carried out in soil, soilrite, cocopeat and
vermicompost separately with ten replicates in a
polyhouse under controlled temperature and humidity.
RESULTS & DISCUSSION
Surface sterilization of explant with 1% Bavistin for 10
min., 1% savlon for 10 min., 70 % ethanol for 30 sec. and
0.1 % HgCl2for 10 min. had shown improved culture
establishment without any contamination. The shoots
became green and callusing at the cut end started within 3-
4 days after inoculation. After 8-9 days of inoculation
shoots were developed in all tubes. Regenerated shoots
were subcultured after 8 days of inoculation on fresh
medium to diminish browning of explant at the base. Per
cent response of shoots regeneration was 100 per cent on
all the concentrations of BAP tested. However, the best
elongation of shoot (2.05 ± 0.07 cm) was observed on the
medium supplemented with 0.5 mg/l BAP (Table 1).
TABLE 1: Effect of different concentrations of BAP on establishment of explant and on its length
Concentration of
BAP (mg/l)
Number of shoots
inoculated/bottle
Number of shoots
established/bottle
% Response
Average length
of shoots (cm)
0.1
6
6
100
1.40 ± 0.08
0.3
6
6
100
1.40 ± 0.10
0.5
6
6
100
2.05 ± 0.07
1.0
6
6
100
1.06 ± 0.13
2.0
6
6
100
1.60 ± 0.13
For the multiplication of regenerated shoots, MS basal
medium supplemented with different concentrations of
BAP were used. Among the media compositions tested,
MS medium supplemented with 0.5 mg/l had shown
maximum rate of multiplication (30 ± 0.31 shoots/culture
bottle) with an average length (3.4 ± 0.05 cm) of shoots
(Table 2).
TABLE 2: Effect different concentrations of BAP on shoot multiplication
Concentration of
BAP (mg/l)
Number of
explants/bottle
Average number of
multiple shoots/bottle
Average number
of shoots
Average length of
shoots (cm)
0.1
5
16 ± 0.24
3.2 ± 0.24
1.0 ± 0.06
0.3
5
14 ± 0.28
2.8 ± 0.28
1.5 ± 0.10
0.5
5
30 ± 0.31
6.0 ± 0.31
3.4 ± 0.05
1
5
12 ± 0.14
2.4 ± 0.14
2.1 ± 0.09
2
5
10 ± 0.54
2.0 ± 0.54
1.8 ± 0.12
For root induction, the shoots were inoculated on half and
full strength MS basal medium supplemented with various
concentrations of IBA. It was found that the full strength
medium was superior to half strength MS basal medium
for root induction. The root initiation was observed after
10 days of inoculations on rooting medium. The maximum
rooting was observed on full strength MS basal medium
supplemented with 2.5 mg/l IBA with 100 per cent
response of the explants with an average 2.93 ± 0.11
numbers of roots (Table 3).

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TABLE 3: Effect of different concentrations of IBA on root initiation
Concentration of
IBA (mg/l)
Full strength medium
Half strength medium
% Response
Average number of
roots/explant
% Response
Average number of
roots/explant
0.5
0
0
0
0
1.0
46.00
1.42 ± 0.02
40.00
1.25 ± 0.07
1.5
66.66
1.00 ± 0.07
36.00
1.45 ± 0.05
2.0
80.00
1.87 ± 0.08
86.00
1.57 ± 0.04
2.5
100.00
2.93 ± 0.11
66.66
1.20 ± 0.06
In primary hardening after 10 days of transplanting in
polytunnel, shoots were green and growth was observed.
The survival of plantlets obtained was 90 per cent in
cocopeat as compared to soil (32 %), soilrite (46 %), and
vermicompost
DISCUSSION
For the establishment of meristem culture, MS medium
supplemented with BAP was similar to results of Biradar
et al. (2009) who determined maximum frequency of
establishment (72 %) in sugarcane var. CoC-671was with
2.0 mg/l BAP. Dhumale et al. (1994) reported that the MS
basal medium supplemented with BAP (3 mg/l) and NAA
(1 mg/l) had shown better shoot development. The
remarks propose that the use of cytokinin and its
concentration changes according to the genotype. MS
medium supplemented with 0.1 mg/l to 2.0 mg /l BAP can
be used for the establishment of the sugarcane shoot tips
as it has given 100 per cent establishment. Nevertheless,
for higher elongation of shoots, moderate concentration of
BAP is enough. MS medium supplemented with 0.5 mg/l
BAP had shown maximum rate of multiplication similar to
results obtained by Biradar et al. (2009) who reported that
MS medium containing 1.0 mg/l of BAP had given
maximum multiplication of shoots with desirable quality;
shoots were well-grown, easily separable and healthy
plantlets. Also 0.5-1 mg/l BAP is good for shoot
multiplication of sugarcane (Alam et al., 1995). Sugarcane
(var. CO-86032) was able to form maximum shoots on
MS medium supplemented with 0.3 mg/l BAP (Godheja et
al., 2014). For normal development of the shoot from the
bud-meristem, requires low levels of the growth regulators
(Sreenivasan and Jalaja, 1983).
The maximum rooting was observed on full strength MS
basal medium supplemented with 2.5 mg/l IBA as reported
by Khan et al. (2008) that the IBA is essential for rooting
in sugarcane. Biradar et al. (2009) have observed 80 per
cent rooting of sugarcane cultivar CoC- 671 on medium
supplemented with 2 mg/l NAA, whereas 70 per cent
rooting was obtained on medium supplemented with 3
mg/l NAA. Many researchers also reported that 5 mg/l
NAA was good for rooting (Larkin, 1982; Shukla et al.,
1994; Alam et al., 1995; Islam et al., 1996) and more than
5 mg/l NAA inhibits rooting. Similar findings were
observed in present study.
In present study it was observed that, the only 6-7 months
old plants give better establishment of shoots within a
period of one week whereas, explants of younger mother
plants (3-4 months) had took more than two weeks. Low
concentration (0.5mg/l) of cytokinine is effective for the
shoot growth and multiplication of shoots. Full strength
MS basal had shown better root initiation as compared to
half strength. Cocopeat is the best suitable medium as
compared to soil and soilrite for primary hardening of
sugarcane.
ACKNOWLEDGEMENT
The authors are thankful to the VSBT College of
Agricultural Biotechnology, Baramati for their financial
and technical support.
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