Fig: Explants Inoculated In MS Medium With Various Concentration
A B C
Plant tissue culture of Sugarcane
K. K. Wagh College of Agril .Biotechnology,
(Affiliated to MPKV, Rahuri)
Saraswatinagar, Panchavati, Nashik-3
“In Vitro Regeneration of Sugarcane
(Saccharum officinarum L.)”
Mr. Patil Pavan Ramnath
Under the guidance of
Prof. R. S. Choudhary
Department: Plant Biotechnology
Sugarcane is an important cash crop of India.
Sugarcane (Saccharum spp.), a C4 perennial grass,
is a member of the Gramineae family.
It constitutes a major source of edible sugars.
Sugarcane is an oldest crop known to man, a major
crop of tropical and sub-tropical regions worldwide.
India is the second largest country in sugarcane
production in the world
(thousand metric tons, TMT)
Brazil 734 000
India 342 382
China 115 124
Thailand 95 950
Pakistan 55 309
Mexico 49 735
Philippines 34 000
Table 1. Top Sugarcane Producers Countries-2013-14
1. To standardize the source of explants and sterilization procedure
2. Initiation and multiplication of Sugarcane (Saccharum officinarum )
3. To standardize the growth regulators for micropropagation
4. Hardening *(Depend on duration)
Sugarcane was selected for micropropagation.
The bud, leaf roll and Meristem part was used.
This variety Co86032 (Naina) mainly collected
from in K. K. Wagh College of Agricultural
Engineering Farm (Puria Park), Nashik.
Petri plates, glass jars, beakers, conical flasks, tissue culture bottles,
glass rods, pipettes.
All the glass wares were sterilized and cooled for use. All the glass
wares were sterilized and cooled for use.
Scalpel, Forceps, Scissors, Steripot, Laminar air flow chamber, Auto
clave, Growth room, Cold room, Magnetic stirrer, Weighing balance, pH
meter, Digital camera.
All the instruments were sterilized and observed to be free from
Murashige and Skoog (1962) basal medium was
used for all the experiments. MS media were prepared
from stocks solution. Modification to the medium was
done by adding growth regulators and other organic
Collection and source of sample
Sugarcane variety Co86032.
This variety mainly collected from K. K. Wagh College
of Agricultural Engineering Farm (Puria Park), Nashik.
Sugarcane bud, leaf roll and Meristem part were used.
Preparation of media for initiation and then it stored
in growth room.
Sample was collected from college farm.
The explants used for micropropagation are bud, leaf
roll and meristem part of sugarcane. 15
Protocol for sugarcane
Wash With tap water , Cut the explant avoiding any injury and
treat it with Bavistin solution 1(w/v)
Wash twice and add explant in three various combination such
as 0.1 %Mercuric Chloride, 1.0 % Sodium Hypochlorite and 0.1
%Mercuric Chloride + 1.0 % Sodium Hypochlorite 70% ethanol For
all Combinations. It carried out each 5 min.
Wash thrice with distilled water 3 min for removing residue of
sterilizing agents. 16
The explants were inoculated into the MS media bottles
Different hormone concentration of BAP and Kinetin was used for
the initiation named SO-1,SO-2 and SO-3
The bottles then transferred in Plant Growth Room (PGR) with the
photoperiod of 16 hours profuse light and 8 hours dark period. 17
After complete initiation the explants were transfer into
Multiplication media contains MS medium with different
conc. of BAP and KN hormone named SOM-1,SOM-2
The used conc. of BAP was 0.5mg/L, 1.0mg/L and 1.5mg/L and KN
was 0.5mg/L, 1.0mg/L and 1.5mg/L 18
The growth of explants were checked every day
Multiplication seen in 70-75 days
Microbial/bacterial contamination was checked and the contaminated
bottles were discarded.
The media used for rooting is MS + NAA different concentration +
3% & 7% Sugar , named as SOR-1 and SOR-2
Growth of roots carried out in 55-60 days
After that hardening is done in greenhouse.
After 14-15 days of culture hardening is done in greenhouse.
Coco peat media is used as hardening media; ratio of 1:4(w/w)
soil : coco peat
Hardening is done successfully but successful percentage is not
SO1 10 1 1 5 0 2 1
SO2 10 2 1 4 1 2 0
SO3 10 3 0 4 1 1 1
Total 30 6 2 13 2 5 2
STANDARDIZATION OF EXPLANT
Various explant i.e. Meristem, Nodal Segment, Leaf Roll .
Type of explant
Total no. of
No growth Contamination
1 Meristem 10 8 1 1
2 Nodal Segment 10 9 0 1
3 Leaf Roll 10 3 5 2
Better growth is observed in meristem And nodal part.(Table.)
STANDARDIZATION OF EXPLANT
Total no. of explants
STANDARDIZATION OF GROWTH HORMONE
Standardize effect of shooting hormone of different
Concentration of BAP & KIN
MS + 1.0 mg/L
BAP + 0.5 mg/L
10 5 2 2 1
MS + 2.0 mg/L
BAP + 1.0 mg/L
10 7 2 0 1
MS + 3.0 mg/L
BAP + 1.5 mg/L
10 9 1 0 0
STANDARDIZATION OF GROWTH HORMONE
SO1 SO2 SO3
Total no of bottles
Growth observed in
Batch Total no of
1 SOR-1 10 8 2 0
2 SOR-2 10 9 1 0
Total no of
Explant used No of bottles Growth
1 Leaf role 6 4 1 1
2 Leaf segment 6 1 2 3
3 Nodal region 6 0 5 1
Results Was Not satisfactory as contamination percentage was high.
Just in four bottle callus growth is observed but after multiplication it turns
brown and dead.
Leaf role Leaf
No of bottles
The project “In vitro regeneration of Sugarcane (Saccharum officinarum L.)” was
successfully carried out and following outcome was obtained.
Present investigation indicates the in vitro regeneration of Sugarcane explant
possible with hormone.
Protocol For sterilization followed with 1.0% sodium hypo chloride, 0.1%
Mercuric chloride and 70% ethanol; Good result obtained in with combination of
1.0% sodium hypo chloride and 0.1% Mercuric chloride for 3 min each.
For inoculation three different explants where obtained such as bud/node,
meristem and leaf roll respectively. Result obtained in bud/node and meristem.
Protocol For initiation followed with various concentration of BAP and Kinetin,
named as SO-1,SO-2 and SO-3. Result obtained in SO-2 and SO-3.
For multiplication various concentration of BAP and KN where used which
named as SOM-1, SOM-2 and SOM-3. Good result obtained in SOM-2 SOM-3.
Microbial contamination observed in this stage.
Rooting observed in two various concentration of NAA and Sugar named as
SOR-2 and SOR-2. Both concentrations have good results and no microbial
After 14 days of culture on MS medium meant for rooting, the sufficiently
rooted plantlets were transplanted to small hykotrays for hardening. Coco peat
and soil used as hardening media in ratio of 1:4(w/w). Not sufficient result
where obtained in hardening stage.
Callus is formed in various concentration of 2, 4-D named as SIC-1, SIC-2, SIC-
3 and SIC-4. Result obtained in SIC-3 and SIC-4 (80 and 72%). 45
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