Introduction :
Rose (Rosa hybrida Linn.) has ever been the world most favourite and unchallenged queen of flowers. There is probably no flower more popular and better known than the rose. It is the adaptability of the plant which makes its appeal so wide. Roses grow and flourish in the wildest eange of conditions of soil, climate and treatments.
Rose belongs to the family Rosaceae. They are mostly low or medium sized ornamental shrub, usually with prickly stems. Most of the cultivated roses are found in the temperate zones of the nor
thern hemisphere and yet Asia seems to be the gene centre where the majority of species are found(Broertjes and Van harten,1988) The chromosome number of rose varies from 2n=2x=14 to2n=8x=56, but most species are diploid or tetraploid. The commercially grown rose cultivars are generally triploid or tetraploid.
Pant tissue culture :
Plant tissue culture is vegetative method of propagation in which plant cell, tissue and organ are used as explants under aseptic or controlled or in vitro conditions, which results in to whole plant in very short time.
Advantages of plant tissue culture :
- Higher rate of multiplication.
- Plants available all year round .
- Large number of clones can be produced.
- Production of secondary metabolites.
- Regeneration of whole plantlets from a single cell or tissue.
- Conservation of threatened plant species.
Application of tissue culture :
- The recovery if disease free clones.
- The genetic improvement of crops.
- Preservation of valuable germplasm by cryopreservation.
- Production of identical sterile hybrid species.
Micro propagation :
Micro propagation is the process of vegetative growth and multiplication from plants tissues or seed, using modern plant tissue culture methods.
Micro propagation method can be divided into five stages :
- Selection of suitable mother plant.
- Initiation stage.
- Multiplication stage.
- Rooting stage.
- Hardening.
Aim and Objectives :
1 .To develop a simple and efficient micro propagation protocol for the rose
2. To produce 50 plant without contamination.
Plan for 2 months Micropropagation of rose :
| Date | Week | Fungicide | Surface sterilization | Media |
| 22/2/2021 | 1 | – | Mercury chloride (HgCl2) (0.1) + Ethanol (70%) + DW | MS media |
| 9/3/2021-15/3/2021 | 2 | Bavistin | Tween20 + Mercury chloride (HgCl2) (0.1) + Ethanol (70%) + DW | MS media |
| 15/3/2021-22/3/2021 | 3 | Bavistin | Tween20+Dw+HgCl2(0.1) 10 min+ NaOCl (10%) 2 min + Ethanol 70%+Dw | MS media with stock |
| 22/3/2021-29/3/2021 | 4 | Bavistin | Tween20+Calcium hypochlorite{Ca(ClO)2 (10%) +Ethanol70%+Dw | MS media with stock |
| 29/3/2021-5/4/2021 | 5 | Bavistin | Tween20+Dw+NaOCl (10%) 10 min +Ethanol 70%+Dw | MS media with stock |
| 5/4/2021-12/4/2021 | 6 | Bavistin | Tween20+Dw+NaOCl(10%) 5 min +Ethanol 70%+Dw | MS media with stock |
For the micropropagation of { Top Secret } Dutch rose plant variety is selected.
| Sr. No | Date | Explant | Sterilization | Media | No. of plants | Result | Conclusion |
| [T1] | 22/2/2021 | Nodes | Mercury chloride (HgCl2) (0.1) + Ethanol (70%) + DW | MS with Stock solution | 10 | _ | Contamination is occur due to handling. |
| [T2] | 9/3/2021 | Nodes | Bavistin(0.1%)+Tween20 + Mercury chloride (HgCl2) (0.1) + Ethanol (70%) + DW | MS media | 10 | 5 Explant grown | 5 Explant contaminate |
| [T3] | 15/3/2021 | Nodes | Bavistin(0.1%) + Tween20+Dw+HgCl2(0.1) 10 min+ NaOCl (10%) 2 min + Ethanol 70%+Dw | MS with Stock solution | 10 | 9 Explant grown | 1 Explant contaminated |
| [T4] | 22/3/2021 | Nodes | Bavistin+Tween20+Calcium hypochlorite{Ca(ClO)2(5%)+Ethanol70%+Dw | MS with Stock solution | 10 | 7 Explant grown | 3 Explant contaminated |
| [T5] | 29/3/2021 | Nodes | Bavistin+Tween20+Dw+NaOCl (10%) 10 min +Ethanol 70%+Dw | MS with Stock solution | 10 | On going process | _ |
| [T6] | 5/4/2021 | Nodes | Bavistin(0.1%)+Tween20+Dw+NaOCl(10%) 5 min +Ethanol 70%+Dw | MS with Stock solution | 10 | On going process | _ |
Procedure :
- Selection of Explant :
Healthy and disease free rose apical segment is used as explant for micropropagation of rose. - Sterilization :
Aseptic condition is required for tissue culture process. Explant is surface sterilise with different chemical to remove the all micro-organism. - Inoculation :
Inoculation is done in laminar air flow to avoid contamination. for this purpose laminar hood is treated with uv light and surface sterilize by using ethanol after sterilization of laminar hood and explant are inoculated in basal ms media for the initiation stage.
MS media composition
| Sr.no | Composition | concentration |
| 1 | NH4NO3 | 1650 |
| 2 | KNO3 | 1900 |
| 3 | MgSO4.7H2O | 370 |
| 4 | KH2PO4 | 170 |
| 5 | CaCl2.2H2O | 440 |
| 6 | MnSO4.4H2O | 22.3 |
| 7 | ZnSO4.4H2O | 8.6 |
| 8 | H3BO3 | 6.2 |
| 9 | KI | 0.83 |
| 10 | Nicotinic acid | 5 |
| 11 | Thiamin HCl | 1 |
| 12 | Glycine | 20 |
| 13 | Sucrose | 30 |
| 14 | Agar | 8 |
COMPOSITION OF STOCK SOLUTION FOR PREPARATION OF MS MEDIA:
| Chemical | Concentration(mg) | Amount in gm for 1 L stock | Volume of stock(ml) used for 1 L |
| (A) Macronutrients | |||
| NH4NO3 | 1650 | 33 | |
| KNO3 | 1900 | 38 | |
| Cacl2.6H2O | 440 | 8.8 | 50 |
| MgSO4.7H2O | 370 | 7.4 | |
| KH2PO4 | 170 | 3.4 | |
| (B) Micronutrients | |||
| KI | 0.83 | 0.166 | |
| H3BO3 | 6.20 | 1.240 | |
| MnSO4.4H2O | 22.3 | 4.460 | |
| ZnSO4.4H2O | 8.60 | 1.720 | 5 |
| NaMoO4.2H2O | 0.25 | 0.050 | |
| CoCl2.6H2O | 0.025 | 0.005 | |
| CuSO4.5H2O | 0.025 | 0.005 | |
| (C) Iron | |||
| FeSO4.7H2O | 27.28 | 5.560 | 5 |
| NaEDTA | 32.38 | 7.460 | |
| (D) Vitamins | |||
| Thimin HCl | 0.1 | 0.020 | |
| Nicotinic Acid | 0.5 | 0.100 | 5 |
| Glycine | 2 | 0.400 | |
| Meso Inositol | 100 | 20 | 5 |
Procedure :
[Trial-1] 22/2/2021
- Explant sterilization : The apical segment of rose were excised from the plant and used as explant. Explant were washed thoroughly under running tap water 30 min and then treated with Hgcl2 (0.1) treatment given for 5 min. Again explant washed with distilled water for 3 time. The final disinfection process was done by 70 % Ehanol for 30 sec. in last the explant were washed with distilled water.
- Preparation of media : MS basal media prepared by mixing of macronutrient, iron source and organic supplement. the final media made for 1L with 8gm/L agar and 30 gm of source. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at15 psi for 15 min.
- Inoculation of explant : After media sterilization media transfer in sterilized laminar air flow cabinet. media were poured in 10 sterilized bottle each contain 50 ml of MS media. sterilized explant were inoculated in bottles by using sterilized forcep. 4 plant were inoculated in each bottle
- Result : After 2 week most of the plants were contaminated.
[Trial-2] 9/3/2021
1.Explant sterilization : The apical segment of rose were excised from the plant and used as explant. Explant were washed thoroughly under running tap water 30 min and then treated with 0.1 Bavistin for 20 min followed by rining 4 time with distilled water. Then Hgcl2 (0.1) treatment given for 10 min. Again explant washed with distilled water for 3 time. The final disinfection process was done by 70 % Ehanol for 30 sec. in last the explant were washed with distilled water.
2.Preparation of media : MS basal media prepared by mixing of macronutrient, iron source and organic supplement. the final media made for 1L with 8gm/L agar and 30 gm of source. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at15 psi for 15 min.
3. Inoculation of explant : After media sterilization media transfer in sterilized laminar air flow cabinet. media were poured in 10 sterilized bottle each contain 50 ml of MS media. sterilized explant were inoculated in bottles by using sterilized forcep. plant were inoculated in each bottle
Result : After 2 week most of the 5 plants were contaminated and 5 plant survive.
[Trial-3] 15/3/2021
1.Explant sterilization : The apical segment of rose were excised from the plant and used as explant. Explant were washed thoroughly under running tap water 15 min and then treated with 0.1 Bavistin for 20 min followed by rining 4 time with distilled water. Then Hgcl2 (0.1) treatment given for 10 min and NaOCl (10%) treatment given for 2 min . Again explant washed with distilled water for 3 time. The final disinfection process was done by 70 % Ehanol for 30 sec. in last the explant were washed with distilled water.
2.Preparation of media : MS basal media prepared by mixing separate stock solution of macronutrient, iron source and organic supplement. the final media made for 1L with 8gm/L agar and 30 gm of source. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at15 psi for 15 min.
3. Inoculation of explant : After media sterilization media transfer in sterilized laminar air flow cabinet. media were poured in 10 sterilized bottle each contain 50 ml of MS media. sterilized explant were inoculated in bottles by using sterilized forcep. plant were inoculated in each bottle
Result : After 2 week most of the 1 plants were contaminated and 9 plant survive.
[Trial-4] 22/3/2021
.Explant sterilization : The apical segment of rose were excised from the plant and used as explant. Explant were washed thoroughly under running tap water 15 min and then treated with 0.1 Bavistin for 20 min followed by rining 4 time with distilled water. Then Calcium hypochlorite{Ca(ClO)2 treatment given for 10 min . Again explant washed with distilled water for 3 time. The final disinfection process was done by 70 % Ehanol for 30 sec. in last the explant were washed with distilled water.
2.Preparation of media : MS basal media prepared by mixing separate stock solution of macronutrient, iron source and organic supplement. the final media made for 1L with 8gm/L agar and 30 gm of source. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at15 psi for 15 min.
3. Inoculation of explant : After media sterilization media transfer in sterilized laminar air flow cabinet. media were poured in 10 sterilized bottle each contain 50 ml of MS media. sterilized explant were inoculated in bottles by using sterilized forcep. plant were inoculated in each bottle
Result : After 2 week most of the 3 plants were contaminated and 7 plant survive.
[Trial-5] 29/3/2021
Explant sterilization : The apical segment of rose were excised from the plant and used as explant. Explant were washed thoroughly under running tap water 15 min and then treated with 0.1 Bavistin for 20 min followed by rining 4 time with distilled water. Then NaOCl (10%) treatment given for 10 min . Again explant washed with distilled water for 3 time. The final disinfection process was done by 70 % Ehanol for 30 sec. in last the explant were washed with distilled water.
2.Preparation of media : MS basal media prepared by mixing separate stock solution of macronutrient, iron source and organic supplement. the final media made for 1L with 8gm/L agar and 30 gm of source. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at15 psi for 15 min.
3. Inoculation of explant : After media sterilization media transfer in sterilized laminar air flow cabinet. media were poured in 10 sterilized bottle each contain 50 ml of MS media. sterilized explant were inoculated in bottles by using sterilized forcep. plant were inoculated in each bottle
Result : On going process
[Trial-6] 5/4/2021
Explant sterilization : The apical segment of rose were excised from the plant and used as explant. Explant were washed thoroughly under running tap water 15 min and then treated with 0.1 Bavistin for 20 min followed by rining 4 time with distilled water. Then NaOCl (10%) treatment given for 10 min . Again explant washed with distilled water for 3 time. The final disinfection process was done by 70 % Ehanol for 30 sec. in last the explant were washed with distilled water.
2.Preparation of media : MS basal media prepared by mixing separate stock solution of macronutrient, iron source and organic supplement. the final media made for 1L with 8gm/L agar and 30 gm of source. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at15 psi for 15 min.
3. Inoculation of explant : After media sterilization media transfer in sterilized laminar air flow cabinet. media were poured in 10 sterilized bottle each contain 50 ml of MS media. sterilized explant were inoculated in bottles by using sterilized forcep. plant were inoculated in each bottle
Result : On going process
Conclusion:
On the basis of 5 trials we have concluded that the trial no. 3 is Standard Operating Procedure for Plant tissue culture of rose, because in trial no 3 I have inoculated 10 explants and from that there is 1 explant contaminated and other explants also grown well. So the SOP of Rose has set
