In Vitro Micropropagation Of Potato

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.

Week	Date	Explant	Sterilization	Media	No. Of Plants	Result	Conclusion
20/2-27/2/21	22/2/21	apical segment	Bavistin(0.1%)+HgCl2(0.1)+Ethanol(70%)+DW	MS with Stock Solution	10	Contamination only 2 plant survive	Contamination is occur due to handling
9/3-14/3/21	9/3/21	apical segment	Tween20+Bavistin(0.1%)+HgCl2(0.1)for 10 min+Ethanol(70%)for 30sec+DW	MS media	10	No Growth	Due to explant was fully matured
15/3-21/3/21	15/3/21	nodal explant	Tween20+Bavistin(0.1%)+HgCl2(0.1%)for 10min+NaOCl(10%)for 2min+Ethanol(70%)+DW	MS media	10	No Growth	Due to explant was fully matured
22/3-28/3/21	22/3/21	nodal explant	Tween20+Bavistin(0.1%)+Calcium hypochlorite(9%)+Ethanol(70%)+DW	MS media	10	No Growth	Due to explant was fully matured
29/3-4/4/21	29/3/21	sprouts	Tween20+Bavistin(0.1%)+Sodium hypochlorite(10%)+Ethanol(70%)+DW	MS media	5	Growth	Micropropagation is possible through sprouts
5 /4-11/4/21	8/4/21	sprouts	Tween20+Bavistin(0.1%)+Sodium hypochlorite(10%)for 5min+Ethanol(70%)+DW	MS media	10	Waiting for result

MS media composition:

Sr. No.	Composition	Concentration(mg/L)
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	Thimin 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

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 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.

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.

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.

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.

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.

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.

Tissue Culture Work