1. The document describes an experiment to standardize the in vitro regeneration of sugarcane (Saccharum officinarum L.) through micropropagation. 2. Various explants, sterilization techniques, growth hormone concentrations, and media formulations were tested to optimize shoot initiation, multiplication, rooting, and hardening of sugarcane plantlets. 3. The most successful protocol involved sterilization with sodium hypochlorite and mercuric chloride, followed by culture of nodal segments and meristems on shoot initiation medium supplemented with 2-3 mg/L BAP and 1-1.5 mg/L kinetin for shooting, and rooting medium with NAA for development of roots

1. 1

2. 2
K. K. Wagh College of Agril .Biotechnology,
Nashik.
(Affiliated to MPKV, Rahuri)
Saraswatinagar, Panchavati, Nashik-3
Department: Plant
Biotechnology

3. “In Vitro Regeneration of Sugarcane
(Saccharum officinarum L.)”
Presented by
Mr. Patil Pavan Ramnath
(BTN-25/2011)
Under the guidance of
Prof. R. S. Choudhary
Department: Plant Biotechnology
(PTC)
3

4. CONTENT
4
 Introduction
 Objective
 Materials
 Method
 Result
 Outcome
 Future prospects
 Reference

5. INTRODUCTION
 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
5

6. 6
Country
Production
(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

7. OBJECTIVES
1. To standardize the source of explants and sterilization procedure
for micropropagation.
2. Initiation and multiplication of Sugarcane (Saccharum officinarum )
3. To standardize the growth regulators for micropropagation
4. Hardening *(Depend on duration)
7

8. MATERIAL
 Plant Materials
 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.
8

9. 9
 Glassware's
 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.
 Instruments
 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
contamination.

10. MEDIA
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
additives.
10

11. MURASHIGE AND SKOOG MEDIA CHEMICAL
(1962)
11
Stock Ingredients MS Medium (Mg / L)
Macronutrients
Ammonium nitrate 1650.00
Potassium nitrate 1900.00
Calcium chloride 440.00
Magnesium sulphate 370.00
Potassium phosphate 170.00
Micro Nutrients
Boric acid 6.20
Potassium iodide 0.83
Manganese sulphate 22.30
Zinc sulphate 8.60
Copper sulphate 0.025
Copper chloride 0.025

12. Stock Ingredients MS Medium (Mg / L)
Iron
Ferrous sulphate 27.25
EDTA 37.25
Vitamins
Nicotinic acid 0.50
Pyridoxine 0.50
Thiamine 0.50
Myoinositol 100.00
Carbon Source Sucrose 30000.00
Amino acid Glycine 2.00
Gelling agent Agar 6000.00 12

13.  Hormones
 BAP (6- Benzyl amino purine)
 Kinetin
 NAA (Naptholic acetic acid )
 Sterilizing agent
 Savlon
 0.1 %Mercuric Chloride
 1.0 % Sodium Hypochlorite
 70 % ethanol
 Distilled Water
13

14. METHOD
 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.
14

15. 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

16. STERILIZATION
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

17. INOCULATION
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

18. MULTIPLICATION
After complete initiation the explants were transfer into
multiplication media.
Multiplication media contains MS medium with different
conc. of BAP and KN hormone named SOM-1,SOM-2
and SOM-3
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

19. 19
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.

20. ROOTING
20
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.

21. HARDENING
21
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
more.

22. RESULT
22

23. EXPLANTS
23
Plate 1. Sugarcane explants: Node, Meristem and Leaf
Roll
A. Node, Meristem and Leaf Roll
B. Explant: Meristem
C. Explant: Leaf Roll
A B C

24. MEDIA BOTTLES WITH VARIOUS
CONCENTRATION
24
Plate 2. Media bottles of different concentration
A. Initiation media
B. Rooting media
A B

25. STERILIZATION TREATMENT
25
Plate 3: Sterilization treatment.
A. Sterilization with Bavistin.
B. Different Sterilization agent.
A B

26. 26
INOCULATION
Plate 4 : Inoculation of Explants.
A. Cutting of explants.
B. Inoculation of explants.
A B

27. INOCULATED BOTTLES
27
Plate 5: Inoculated bottles.
A. Explants inoculated in MS medium with various
concentration of growth hormone.
B. Leaf roll inoculated in SIC medium.
A B

28. 28
SHOOTING
Plate 6: Different hormone concentration bottles. (Obs. After 10th days)
SO-1
SO-2
SO-3

29. 29
Plate 7: Different hormone concentration bottles.(Obs. After 15th day)
A. SO-1
B. SO-2
C. SO-3
A B C

30. SHOOTING & ROOTING
30
Plate8: Shooting and Rooting
A. Secondary shooting & Rooting MS+BAP & NAA (30th day)
B. Secondary shooting MS+3mg/LBAP& 1.5mg/L KN (28th day)
Root
Shoot
A B

31. MULTIPLICATION
31
Plate 9: Multiplication
A. and B. Multiplicated bottles of sugarcane explant.

32. 32
ROOTING
Plate10: Rooting of different concentration
A. SOR-1
B. SOR-2
C. SOR-2 (After 14 day)
A B C

33. HARDENING
33
Plate11:Hardening of Sugarcane
A. and B.: Hardening of Sugarcane in maintained
conditions

34. CALLUS
34
Plate12: Callus culture
A. SIC-3
B. SIC-4
A B

35. 35
STATISTICAL DATA

36. STERILIZATION TREATMENT
Batch
No.
Total
Number
of bottles
0.1% HgCl2
1.0%Sodium
Hypo chloride
+0.1%HgCl2
1.0%Sodium
Hypo chloride
Good
Contam
ination
Good
Contam
ination
Good
Contam
ination
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
36

37. STANDARDIZATION OF EXPLANT
 Various explant i.e. Meristem, Nodal Segment, Leaf Roll .
37
Sr.
no.
Type of explant
Total no. of
explants
Growth
observed
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.)

38. STANDARDIZATION OF EXPLANT
38
0
2
4
6
8
10
12
Meristem Nodal
Segment
Leaf Roll
Total no. of explants
Growth observed
No growth
Contamination

39. STANDARDIZATION OF GROWTH HORMONE
 Standardize effect of shooting hormone of different
Concentration of BAP & KIN
39
Sr.
no
Bat
ch
Concentration of
Growth hormone
for shoot
Total no
of bottles
Growth
observed
in
Growth
/Browning
Contamina
tion
No
growth
1
SO
1
MS + 1.0 mg/L
BAP + 0.5 mg/L
Kinetin
10 5 2 2 1
2
SO
2
MS + 2.0 mg/L
BAP + 1.0 mg/L
Kinetin
10 7 2 0 1
3
SO
3
MS + 3.0 mg/L
BAP + 1.5 mg/L
Kinetin
10 9 1 0 0

40. STANDARDIZATION OF GROWTH HORMONE
0
2
4
6
8
10
12
SO1 SO2 SO3
Total no of bottles
Growth observed in
Growth /Browning
Contamination
No growth
40

41. ROOTING
Sr. no
Batch Total no of
bottles
Growth
observed in
Contaminati
on
No growth
1 SOR-1 10 8 2 0
2 SOR-2 10 9 1 0
41
0
2
4
6
8
10
12
SOR-1 SOR-2
Total no of
bottles
Growth observed
in
Contamination
No growth

42. CALLUS CULTURE
Sr.
no.
Explant used No of bottles Growth
Observed
No
growth
Contaminated
bottles
1 Leaf role 6 4 1 1
2 Leaf segment 6 1 2 3
3 Nodal region 6 0 5 1
42
Callus culture)
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.

43. CALLUS CULTURE
43
0
1
2
3
4
5
6
7
Leaf role Leaf
segment
Nodal
region
No of bottles
Growth Observed
No growth
Contaminated bottles

44. OUTCOME
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.
44

45.  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
contamination.
 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

46. FUTURE PROSPECTS
 Callus culture.
 Multiplication and
46

47. REFERENCE
47
 Anita P., Jain R.K., Sehrawat, A.R and. Punia, A. (2000). Efficient and cost- effective
micropropagation of two early maturing varieties of sugarcane (Saccharum spp.).
Indian Sugar. 50: 611-618.
 Cheema, A.S., Singh, H and Gosal, S. S (1992). Response of different genotypes to
callus induction and plant regeneration in sugarcane. Crop Improvement. 19: 6- 13.
 Chen, W. H., Davey, M. R, Power, J. B. and E. C. Cocking (1998). Sugarcane
protoplasts: factors affecting division and plant regeneration. Plant Cell Rep. 7(5):
344 – 347.
 Chowdhury, M.K.U and Vasil, I. K. (1992). Stably transformed herbicide resistant
callus of sugarcane via microprojectile bombardment of cell suspension cultures and
electroporation of protoplasts. Plant Cell. 11(10): 494-498.

48.  Gupta, J.N., Kaur, R., and Cheema, G.S. (1995). Plants regenerated from
protoplasts of sugarcane. Current Sci. 68 (6): 650-653.
 Kaur, A., Gosal, S. S., Gill, R. and Thind, K. S. (2001). Induction of plant
regeneration and somaclonal variation for some agronomic traits in sugarcane
(Saccharum officinarum L.). Crop Improvement. 28(2): 167- 172, 6 ref.
 Lal, N and Singh, H.N. (1994). Rapid clonal multiplication of sugarcane
through tissue culture. Plant Tissue Cult. 4: 1-7.
 Prajapati, B. S., Patel C. L., Patel, S. R. and Patel, A.A (2000). A Regeneration
of tissue culture plantlets through callus culture in sugarcane cultivar CoC
671. Indian J. Genet. Plant Breed. 60(2): 255-257, 8.
 Singh, B., Yadav, G. C. and Lal, M. (2001). An efficient protocol for
micropropagation of sugarcane using shoot tip explants. Sugar Tech.
3(3):113-116, 10.
48

49.  Wageningen.Sugarcane ethanol, Academic Publishers The
Netherlands, 2008
 Wrigley G. 1982. Tropical agriculture: the development of
production. Fourth ed. Longman Inc. New York. 496 pp.
 http://www.agricoop.nic.in
 www.Jagarnjosh.com
49

50. 50
THANK
YOU…..

51. MEDIA WITH DIFFERENT CONCENTRATION
51
Initiation
Sr.
no.
Batch
Name
Concentration
1 SO-1 MS + 1.0 mg/L BAP + 0.5 mg/L Kinetin
2 SO-2 MS + 2.0 mg/L BAP + 1.0 mg/L Kinetin
3 SO-3 MS + 3.0 mg/L BAP + 1.5 mg/L Kinetin
Multiplication
Sr.
no.
Batch
Name
Concentration
1 SOM-1 MS + 0.5 mg/L BAP + 0.5 mg/L Kinetin
2 SOM-2 MS + 1.0 mg/L BAP + 1.0 mg/L Kinetin
3 SOM-3 MS + 1.5 mg/L BAP + 1.5 mg/L Kinetin

52. 52
Rooting
Sr.
no.
Batch
Name
Concentration
1 SOR-1 MS + 3 mg/L NAA+ 30 gm/l sugar (3%)
2 SOR-2 MS + 5 mg/L NAA +70 gm/l sugar (7%)
Callus Induction & Multiplication
Sr.
no.
Batch
Name
Concentration
1 SOC-1 MS + 1 mg/L 2, 4-D
2 SOC-2 MS + 2 mg/L 2, 4-D
3 SOC-3 MS + 3 mg/L 2, 4-D
4 SOC-4 MS + 4 mg/L 2, 4-D

53. 53
Callus Regeneration
Sr.
no.
Batch
Name
Concentration
1 SOCR-0 MS basal media were taken as control
2 SOCR-1 MS + 0.5 mg/L BAP
3 SOCR-2 MS + 0.5 mg/L BAP + 1.0 mg/L NAA
4 SOCR-3
MS + 0.5 mg/L BAP + 0.125 mg/L Kinetin + 0.5
mg/L 2, 4-D
5 SOCR-4 MS + 0.5 mg/L BAP + 0.5 mg/L Kinetin