EFFECT OF CRYOPRESERVATION, LYOPHILIZATION ON DNA EXTRACTION PROTOCOL OF ACACIA ARABICA, ACACIA SINUATA, PROSOPIS SPICIGERA, ADENANTHERA PAVONINA AND ACACIA AURICULIFORMIS

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About Authors:
Hardik R. Patel
Industrial Biotechnology from Sardar Patel University,
Gujarat, India.
hardikigbt@gmail.com

ABSTRACT
Cryopreservation and lyophilization of plant germplasm has obvious advantages over in vitro storage in term of space saving and improved phytosanitation. We compared cryopreserved and lyophilized leaf as sources for genomic DNA isolation by CTAB protocol and PVP protocol.Our results showed that cryopreservation of leaf tissue yielded high molecular weight genomic DNA. The DNA was suitable for restriction-enzyme digestion and as a template for polymerase chain reaction (PCR) amplification. While these results rule out cryopreserved  tissue as a source for DNA isolation, the ability to freeze-dry, powder, and efficiently store voluminous tissue samples for later use in DNA and protein isolation could be of great benefit to laboratories involved in  molecular genetics and molecular biology.


Reference Id: PHARMATUTOR-ART-1563

MATERIALS AND METHODS

Plant Material
Total 4plant species were collectedforDNA isolation from Indroda Botanical Garden, Gandhinagar.

Table 1: Plant species collected from Indrora Botanical Gardenand its GPS location.

Plants name

Family

Latitude

Longitude

Acacia Arabica

Mimosaceae

N 23° 11.558

E 72° 38.939

Acacia sinuata

Mimosaceae

N 23° 11.566

E 72° 38.944

Adenanthera pavonina

Mimosaceae

N 23° 11.571

E 72° 38.937

Prosopis spicigera

Mimosaceae

N 23° 11.680

E 72° 38.836

LYOPHILIZATION
Surface sterilization

Leaves were sterilized by surface washing under tap water than distilled water to remove dust and other contaminants.

Sample preparation for Lyophilization
The leaves were dried with blotting paper and middle ribs were removed. They were crushed into liquid nitrogen to make fine powder and approximately 15 g were weighed. After lyophilization, the powder was collected in petridish which were sealed with cling film.


Lyophilization process
Plant samples were lyophilized at vacuum of 0.16 mbar and -50 °C for 72 hrs. Lyophilized powder stored was stored in tightly sealed petridishes at room temperature.

The SuperModulyo freeze dryer used for lyophilization at GSBTM does not facilitate the temperature and pressure regulation. The protocol for operation of this instrument is as follows:
1. The powdered samples were frozen for some time at -20°C freezer.
2. The frozen leaves were evenly spread on the product trays without the delay for the leaves to cool down again.
3. The racks with the samples were placed in the dome and covered with the acrylic lid.
4. It was made sure that all the detachable parts associated with vacuum are well greased so as to minimize the chances of any leakage.
5. The “Fridge” switch was turned on. Within 1 hr, the chamber temperature should be around -40°C to -50°C.
6. The “pump” switch was turned on. The pressure reached up to 10-1 mbar.
7. The samples were lyophilized for 1 whole day.
8. After the run was complete, the “Pump” switch was turned off. The knobs were gradually opened to release the pressure.
9. The samples were placed in a dry, air tight environment as soon as possible and stored out of direct sunlight.

CRYOPRESERVATION

Surface sterilization
Leaves were sterilized by surface washing under tap water than distilled water to remove dust and other contaminants.

Weighing of sample
The leaves were dried with blotting paper and approximately 20 g leaves were weighed without middle ribs.

Sample preparation for cryopreservation
Leaves were crushed into liquid nitrogen to make a fine powder and 3 cryovials were filled up for each plant and were cryopreserved at -196 °C cryopreservation was carried out by placing the cryovials in canisters and immersing them in the Cryocane containing liquid nitrogen. DNA extraction was carried out from cryopreserved sample at intervals of 1, 7 and 15 days.

DNA EXTRACTION
DNA extraction was carried out from cryopreserved, lyophilized and oven dried plant samples using two different protocols which are mentioned below as follows:

A. CTAB protocol [Doyle J and Doyle J, 1987]
1. Water bath was set to 65 ºC and CTAB buffer was put into the water bath.
2. 100 mg of plant material (cryopreserved, lyophilized, oven dried samples) was taken in a motor pestle.
3. Liquid nitrogen was added and the sample was crushed to powder form. (In case of already crushed samples, proceed to next step)
4. 3 ml of 3% CTAB buffer, containing 6% PVP (pH 8) and 6.1 µl of β-mercaptoethanol was added.
5. The solution was incubated at 65ºC for 1 hour.
6. The tube was centrifuged at 12,000 rpm for 5’ at 4 0C by using centrifuge (Eppendorf).
7. The supernatant was collected in fresh tube and 5µl/ml of Proteinase K was added and incubated at 65 °C for 1 hr.
8. Equal volume of P: C: I was added, mixed gently, centrifuged 12000 rpm for 15 mins.
9. Supernatant was collected, equal volume of C: I was added, mixed and centrifuged at 12000 rpm for 15 mins.
10. Step 9 was repeated.
11. 5 µl of 20 mg/ml RNase A or 10µl of 10 mg/ml RNase A was added incubated at 65 °C for 1 hour or 37 °C overnight.
12. 1/10th volume of 3M sodium acetate and equal volume of absolute ethanol was added, incubated for 20 mins at -20°C.
13. The samples were centrifuged at 12000 rpm for 10 mins, supernatant was discarded and the pellet was recovered.
14. The pellet was washed twice with 70% ethanol and centrifuged at 10000 rpm for 10 mins.
15. The pellet was allowed to air dry and dissolved in 200 µl of 1X TE buffer.

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