Introduction:
Potato(Solanum tuberosum L.) is fourth major food crop after wheat, rice and maize. Annually more than a billion people consume potato throughout the world. It is rich in carbohydrate and approximately 90 kcal of energy can be obtained from 100 grams of potato.
The potato was first domesticated in the region of modern day Peru and northwestern Bolivia by pre Columbian farmers around Lake Titicaca. It has spread around the world and become a staple crop in many countries.
In 2018, world production of potatoes was 368 million tons, led by China with 27% of the total. Other major producers were India, Russia, Ukraine and the united states. It remains an essential crop in Europe where per capita production is still the highest in the world, but the most rapid expansion over the past few decades has occurred in southern and eastern Asia.
Seed is considered as the most expensive output in potato cultivation. About half of the total expenses are consumed for seed. However despite spending a huge money, most of the seeds obtained are non-certified, impure and contaminated by various pathogens. Plant biotechnology is offering a great deal in this regard by producing disease free and healthy potato seeds via clonal propagation for large scale or commercial production because it has an advantage of immediate age transfer that saves time, reduce cost and the only way to conserve true type of plants.
Tissue culture technique have become very popular and alternative means of vegetative propagation of plants in recent year.

Objective:

To standardise the method for in vitro micropropagation of potato.
To produce 100 potato plants without contamination.

Need of the Project:

In potato production, major input is of seed and approximately 50% is cost of seed. The planting material is also bulky as tubers are used for planting. Potato production is also characterized by low rate of multiplication.
However, despite spending a huge money most of the seeds obtained are non certified, impure and contaminated by various pathogens.
Potatoes are continuously propagated by tubers. this is one of the most disadvantage as all the tissue borne viruses, fungi and bacteria that have infected the crop during previous year lead to significant loss in yield and tuber quality. one way to keep source tubers disease free is to produce nucleus stocks in disease free areas. However, such environments are not available and not always disease free.
Potato affected by a large no of viral diseases, which reduce the yield. These diseases progressively accumulate in each field multiplication and result in steady depression in yield . Thus seed is required to be replaced after 3-4 years. With the development of micropropagation technique it is used as a tool for production of nucleus potato seed stocks. It has all the advantages of increasing pathogen free plants(chandra and upadhya,1998)
Using micropropagation technique, it is possible not only to reduce field exposure but also to increase rate of multiplication several times. Propagation takes place in laboratory under aseptic, controlled condition and in vitro plantlets are produced all the year in a limited space and in completely disease free environment. Also the material can be stored over long period in a limited space.
The micropropagation for commercial seed production has moved the potato from test tube to field. The rapid in vitro propagation of clonal material is an important area where micropropagation technique is used worldwide for quick multiplication of potato germplasm and for raising commercial food crop.

Advantage of Plant Tissue Culture:

Higher rate of multiplication.
Plants available all year around.
Regeneration of whole plant from single tissue.
Large no of clones can be produced.
New imroved genetically engineered plant can be produced.

Micropropagation:

Micropropagation is the process of vegetative growth and multiplication from plant and tissue by using plant tissue culture method. It carried out in aseptic and favorable conditions on growth medium, using plant tissue culture technique. Tissue culture is based on a concept of totipotency, the ability of plant cell and tissue to develop into whole plant.
Stages of micropropagation:
1. Selection of suitable mother plant
2. Initiation stage
3. Multiplication stage
4. Rooting stage
5. Hardening

Mcropropagation Of Potato:

For the micropropagation of Solanum tuberosum Kufri Pukhraj variety is selected. For this research we are going to conduct 6 different trial as follows in table.

WeekDateExplantSterilizationMediaNo. Of PlantsResultConclusion
20/2-27/2/2122/2/21apical segmentBavistin(0.1%)+HgCl2(0.1)+Ethanol(70%)+DWMS with Stock Solution10Contamination only 2 plant surviveContamination is occur due to handling
9/3-14/3/21 9/3/21apical segmentTween20+Bavistin(0.1%)+HgCl2(0.1)for 10 min+Ethanol(70%)for 30sec+DWMS media10No GrowthDue to explant was fully matured
15/3-21/3/21 15/3/21nodal explantTween20+Bavistin(0.1%)+HgCl2(0.1%)for 10min+NaOCl(10%)for 2min+Ethanol(70%)+DWMS media10No Growth Due to explant was fully matured
22/3-28/3/21 22/3/21nodal explantTween20+Bavistin(0.1%)+Calcium hypochlorite(9%)+Ethanol(70%)+DWMS media10No Growth Due to explant was fully matured
29/3-4/4/2129/3/21sproutsTween20+Bavistin(0.1%)+Sodium hypochlorite(10%)+Ethanol(70%)+DWMS media5GrowthMicropropagation is possible through sprouts
5 /4-11/4/21 8/4/21sproutsTween20+Bavistin(0.1%)+Sodium hypochlorite(10%)for 5min+Ethanol(70%)+DWMS media10Waiting for result

MS media composition:

Sr. No.CompositionConcentration(mg/L)
1NH4NO31650
2KNO31900
3MgSO4.7H2O370
4KH2PO4170
5CaCl2.2H2O440
6MnSO4.4H2O22.3
7ZnSO4.4H2O8.6
8H3BO36.2
9KI0.83
10 Nicotinic acid5
11Thimin HCl1
12Glycine20
13Sucrose30
14Agar8

Composition of stock solution for preparation of MS media:

ChemicalConcentration(mg)Amount in gm for 1 L stockVolume of stock(ml) used for 1 L
A) Macronutrients
NH4NO3165033
KNO3190038
Cacl2.6H2O4408.850
MgSO4.7H2O3707.4
KH2PO41703.4
B) Micronutrients
KI0.830.166
H3BO36.201.240
MnSO4.4H2O22.34.460
ZnSO4.4H2O8.601.7205
NaMoO4.2H2O0.250.050
CoCl2.6H2O0.0250.005
CuSO4.5H2O0.0250.005
C)Iron
FeSO4.7H2O27.285.5605
NaEDTA32.387.460
D) Vitamins
Thimin HCl0.10.020
Nicotinic Acid0.50.1005
Glycine20.400
Meso Inositol100205

Trial 1-
Date 22/2/2021
Procedure:

Explant Sterilization
The apical segment of Potato were excised from the plant and used as explant. Explant were washed thoroughly under running tap water for 30 min and then treated with 0.1 % bavistin for 20 min followed by rinsing 4 time with distilled water. Then 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% ethanol for 30 sec. In last the explant were washed with distilled water.

Preparation of media
MS basal media prepared by mixing separate stock solution of macronutrient, micronutrient, iron source and organic supplement. The final media made for 1L with 8gm/L Agar and 30 gm of sucrose. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at 15 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 30 ml of MS media. Sterilized explant were inoculated in bottles by using sterilized forcep. 4 plants were inoculated in each bottle. Result:
After 2 weeks most of the plants were contaminated.

Trial 1

Trial 2-
Date 9/3/2021
Procedure:

Explant Sterilization
The sprout were excised from the potato used as explant. Explant were washed thoroughly under running tap water for 30 min and washed with tween20. Then explant treated with 0.1 % bavistin for 20 min followed by rinsing 4 time with distilled water. Then 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% ethanol for 30 sec. In last the explant were washed with distilled water.

Preparation of media
MS basal media prepared by mixing separate stock solution of macronutrient, micronutrient, iron source and organic supplement. The final media made for 1L with 8gm/L Agar and 30 gm of sucrose. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at 15 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 30 ml of MS media. Sterilized explant were inoculated in bottles by using sterilized forcep. 4 plants were inoculated in each bottle.

Result of Trial 2

Trial 3-
Date 15/3/21
Procedure:

Explant Sterilization
The sprout were excised from the potato used as explant. Explant were washed thoroughly under running tap water for 30 min and washed with tween20. Then explant treated with 0.1 % bavistin for 20 min followed by rinsing 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 NaOCl(10%) for 2 min and 70% ethanol for 30 sec. In last the explant were washed with distilled water.

Preparation of media
MS basal media prepared by mixing separate stock solution of macronutrient, micronutrient, iron source and organic supplement. The final media made for 1L with 8gm/L Agar and 30 gm of sucrose. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at 15 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 30 ml of MS media. Sterilized explant were inoculated in bottles by using sterilized forcep. 4 plants were inoculated in each bottle.

Result of Trial 3

Trial 4-
Date 22/3/21
Procedure:

Explant Sterilization
The sprout were excised from the potato used as explant. Explant were washed thoroughly under running tap water for 30 min and washed with tween20. Then explant treated with 0.1 % bavistin for 20 min followed by rinsing 4 time with distilled water. Then calcium hupochloride(9%) treatment given for 10 min. Again explant washed with distilled water for 3 time. The final disinfection process was done 70% ethanol for 30 sec. In last the explant were washed with distilled water.

Preparation of media
MS basal media prepared by mixing separate stock solution of macronutrient, micronutrient, iron source and organic supplement. The final media made for 1L with 8gm/L Agar and 30 gm of sucrose. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at 15 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 30 ml of MS media. Sterilized explant were inoculated in bottles by using sterilized forcep.

Result of Trial 4

Trial 5-
Date 29/3/21
Procedure:

Explant Sterilization
The sprout were excised from the potato used as explant. Explant were washed thoroughly under running tap water for 30 min and washed with tween20. Then explant treated with 0.1 % bavistin for 20 min followed by rinsing 4 time with distilled water. Again explant washed with distilled water for 3 time. The final disinfection process was done by NaOCl(10%) for 2 min and 70% ethanol for 30 sec. In last the explant were washed with distilled water.

Preparation of media
MS basal media prepared by mixing separate stock solution of macronutrient, micronutrient, iron source and organic supplement. The final media made for 1L with 8gm/L Agar and 30 gm of sucrose. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at 15 psi for 15 min.

Inoculation of explant
After media sterilization media transfer in sterilized laminar air flow cabinet. Media were poured in 5 sterilized bottle each contain 30 ml of MS media. Sterilized explant were inoculated in bottles by using sterilized forcep.

Trial -6
Date 8/4/21
Procedure:

Explant Sterilization
The sprout were excised from the potato used as explant. Explant were washed thoroughly under running tap water for 30 min and washed with tween20. Then explant treated with 0.1 % bavistin for 20 min followed by rinsing 4 time with distilled water. Then sodium hypochloride(9%) treatment given for 10 min. Again explant washed with distilled water for 3 time. The final disinfection process was done 70% ethanol for 30 sec. In last the explant were washed with distilled water.

Preparation of media
MS basal media prepared by mixing separate stock solution of macronutrient, micronutrient, iron source and organic supplement. The final media made for 1L with 8gm/L Agar and 30 gm of sucrose. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at 15 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 30 ml of MS media. Sterilized explant were inoculated in bottles by using sterilized forcep. 10 explants were inoculated.

Trial 6

Conclusion:
On the basis of 5 trials we have concluded that the trial no. 5 is Standard Operating Procedure for Plant tissue culture of Potato, because in trial no 5 I have inoculated 5 explants and from that there is no contamination and the explants also grown well.

SOP for Plant Tissue Culture of Potato:
Explant Sterilization
The sprout were excised from the potato used as explant. Explant were washed thoroughly under running tap water for 30 min and washed with tween20. Then explant treated with 0.1 % bavistin for 20 min followed by rinsing 4 time with distilled water. Again explant washed with distilled water for 3 time. The final disinfection process was done by NaOCl(10%) for 2 min and 70% ethanol for 30 sec. In last the explant were washed with distilled water.

Preparation of media
MS basal media prepared by mixing separate stock solution of macronutrient, micronutrient, iron source and organic supplement. The final media made for 1L with 8gm/L Agar and 30 gm of sucrose. The pH maintained at 5.6 by using NaOH or HCl. Then media were autoclaved at 121c at 15 psi for 15 min.

Inoculation of explant
After media sterilization media transfer in sterilized laminar air flow cabinet. Media were poured in 5 sterilized bottle each contain 30 ml of MS media. Sterilized explant were inoculated in bottles by using sterilized forcep.

Tissue Culture Work