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1
2
K. K. Wagh College of Agril .Biotechnology,
Nashik.
(Affiliated to MPKV, Rahuri)
Saraswatinagar, Panchavati, Nashik-3
De...
“In Vitro Regeneration of Sugarcane
(Saccharum officinarum L.)”
Presented by
Mr. Patil Pavan Ramnath
(BTN-25/2011)
Under t...
CONTENT
4
 Introduction
 Objective
 Materials
 Method
 Result
 Outcome
 Future prospects
 Reference
INTRODUCTION
 Sugarcane is an important cash crop of India.
 Sugarcane (Saccharum spp.), a C4 perennial grass,
is a memb...
6
Country
Production
(thousand metric tons, TMT)
Brazil 734 000
India 342 382
China 115 124
Thailand 95 950
Pakistan 55 30...
OBJECTIVES
1. To standardize the source of explants and sterilization procedure
for micropropagation.
2. Initiation and mu...
MATERIAL
 Plant Materials
 Sugarcane was selected for micropropagation.
 The bud, leaf roll and Meristem part was used....
9
 Glassware's
 Petri plates, glass jars, beakers, conical flasks, tissue culture bottles,
glass rods, pipettes.
 All t...
MEDIA
Murashige and Skoog (1962) basal medium was
used for all the experiments. MS media were prepared
from stocks solutio...
MURASHIGE AND SKOOG MEDIA CHEMICAL
(1962)
11
Stock Ingredients MS Medium (Mg / L)
Macronutrients
Ammonium nitrate 1650.00
...
Stock Ingredients MS Medium (Mg / L)
Iron
Ferrous sulphate 27.25
EDTA 37.25
Vitamins
Nicotinic acid 0.50
Pyridoxine 0.50
T...
 Hormones
 BAP (6- Benzyl amino purine)
 Kinetin
 NAA (Naptholic acetic acid )
 Sterilizing agent
 Savlon
 0.1 %Mer...
METHOD
 Collection and source of sample
 Sugarcane variety Co86032.
 This variety mainly collected from K. K. Wagh Coll...
Preparation of media for initiation and then it stored
in growth room.
Sample was collected from college farm.
The explant...
STERILIZATION
Wash With tap water , Cut the explant avoiding any injury and
treat it with Bavistin solution 1(w/v)
Wash tw...
INOCULATION
The explants were inoculated into the MS media bottles
Different hormone concentration of BAP and Kinetin was ...
MULTIPLICATION
After complete initiation the explants were transfer into
multiplication media.
Multiplication media contai...
19
The growth of explants were checked every day
Multiplication seen in 70-75 days
Microbial/bacterial contamination was c...
ROOTING
20
The media used for rooting is MS + NAA different concentration +
3% & 7% Sugar , named as SOR-1 and SOR-2
Growt...
HARDENING
21
After 14-15 days of culture hardening is done in greenhouse.
Coco peat media is used as hardening media; rati...
RESULT
22
EXPLANTS
23
Plate 1. Sugarcane explants: Node, Meristem and Leaf
Roll
A. Node, Meristem and Leaf Roll
B. Explant: Meristem...
MEDIA BOTTLES WITH VARIOUS
CONCENTRATION
24
Plate 2. Media bottles of different concentration
A. Initiation media
B. Rooti...
STERILIZATION TREATMENT
25
Plate 3: Sterilization treatment.
A. Sterilization with Bavistin.
B. Different Sterilization ag...
26
INOCULATION
Plate 4 : Inoculation of Explants.
A. Cutting of explants.
B. Inoculation of explants.
A B
INOCULATED BOTTLES
27
Plate 5: Inoculated bottles.
A. Explants inoculated in MS medium with various
concentration of growt...
28
SHOOTING
Plate 6: Different hormone concentration bottles. (Obs. After 10th days)
SO-1
SO-2
SO-3
29
Plate 7: Different hormone concentration bottles.(Obs. After 15th day)
A. SO-1
B. SO-2
C. SO-3
A B C
SHOOTING & ROOTING
30
Plate8: Shooting and Rooting
A. Secondary shooting & Rooting MS+BAP & NAA (30th day)
B. Secondary sh...
MULTIPLICATION
31
Plate 9: Multiplication
A. and B. Multiplicated bottles of sugarcane explant.
32
ROOTING
Plate10: Rooting of different concentration
A. SOR-1
B. SOR-2
C. SOR-2 (After 14 day)
A B C
HARDENING
33
Plate11:Hardening of Sugarcane
A. and B.: Hardening of Sugarcane in maintained
conditions
CALLUS
34
Plate12: Callus culture
A. SIC-3
B. SIC-4
A B
35
STATISTICAL DATA
STERILIZATION TREATMENT
Batch
No.
Total
Number
of bottles
0.1% HgCl2
1.0%Sodium
Hypo chloride
+0.1%HgCl2
1.0%Sodium
Hypo c...
STANDARDIZATION OF EXPLANT
 Various explant i.e. Meristem, Nodal Segment, Leaf Roll .
37
Sr.
no.
Type of explant
Total no...
STANDARDIZATION OF EXPLANT
38
0
2
4
6
8
10
12
Meristem Nodal
Segment
Leaf Roll
Total no. of explants
Growth observed
No gr...
STANDARDIZATION OF GROWTH HORMONE
 Standardize effect of shooting hormone of different
Concentration of BAP & KIN
39
Sr.
...
STANDARDIZATION OF GROWTH HORMONE
0
2
4
6
8
10
12
SO1 SO2 SO3
Total no of bottles
Growth observed in
Growth /Browning
Cont...
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
...
CALLUS CULTURE
Sr.
no.
Explant used No of bottles Growth
Observed
No
growth
Contaminated
bottles
1 Leaf role 6 4 1 1
2 Lea...
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...
OUTCOME
The project “In vitro regeneration of Sugarcane (Saccharum officinarum L.)” was
successfully carried out and follo...
 For multiplication various concentration of BAP and KN where used which
named as SOM-1, SOM-2 and SOM-3. Good result obt...
FUTURE PROSPECTS
 Callus culture.
 Multiplication and
46
REFERENCE
47
 Anita P., Jain R.K., Sehrawat, A.R and. Punia, A. (2000). Efficient and cost- effective
micropropagation of...
 Gupta, J.N., Kaur, R., and Cheema, G.S. (1995). Plants regenerated from
protoplasts of sugarcane. Current Sci. 68 (6): 6...
 Wageningen.Sugarcane ethanol, Academic Publishers The
Netherlands, 2008
 Wrigley G. 1982. Tropical agriculture: the dev...
50
THANK
YOU…..
MEDIA WITH DIFFERENT CONCENTRATION
51
Initiation
Sr.
no.
Batch
Name
Concentration
1 SO-1 MS + 1.0 mg/L BAP + 0.5 mg/L Kine...
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 s...
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...
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Plant tissue culture of Sugarcane

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Sugarcane micro propagation

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Plant tissue culture of Sugarcane

  1. 1. 1
  2. 2. 2 K. K. Wagh College of Agril .Biotechnology, Nashik. (Affiliated to MPKV, Rahuri) Saraswatinagar, Panchavati, Nashik-3 Department: Plant Biotechnology
  3. 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. 4. CONTENT 4  Introduction  Objective  Materials  Method  Result  Outcome  Future prospects  Reference
  5. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 22. RESULT 22
  23. 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. 24. MEDIA BOTTLES WITH VARIOUS CONCENTRATION 24 Plate 2. Media bottles of different concentration A. Initiation media B. Rooting media A B
  25. 25. STERILIZATION TREATMENT 25 Plate 3: Sterilization treatment. A. Sterilization with Bavistin. B. Different Sterilization agent. A B
  26. 26. 26 INOCULATION Plate 4 : Inoculation of Explants. A. Cutting of explants. B. Inoculation of explants. A B
  27. 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. 28 SHOOTING Plate 6: Different hormone concentration bottles. (Obs. After 10th days) SO-1 SO-2 SO-3
  29. 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. 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. 31. MULTIPLICATION 31 Plate 9: Multiplication A. and B. Multiplicated bottles of sugarcane explant.
  32. 32. 32 ROOTING Plate10: Rooting of different concentration A. SOR-1 B. SOR-2 C. SOR-2 (After 14 day) A B C
  33. 33. HARDENING 33 Plate11:Hardening of Sugarcane A. and B.: Hardening of Sugarcane in maintained conditions
  34. 34. CALLUS 34 Plate12: Callus culture A. SIC-3 B. SIC-4 A B
  35. 35. 35 STATISTICAL DATA
  36. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 46. FUTURE PROSPECTS  Callus culture.  Multiplication and 46
  47. 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. 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. 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. 50 THANK YOU…..
  51. 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. 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. 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

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