EFFECT OF LYOPHILIZATION AND CRYOPRESERVATION ON PLANT LEAVES OF TERMINALIA ARJUNA TERMINALIA CATAPPA, TERMINALIA CHEBULA, JATROPHA GOSSYPIFOLIA, JATROPHA CURCAS

Pharma courses

Pharma Admission

pharma courses

pharma admission



PVP protocol [KIM Cet al., 1997]

  1. Approximately 100 mg of sample was ground in a pestle in 5 µl (one drop) of 1% (v/v) 2-mercaptoethanol.
  2. After grinding, 300 µl of extraction was added to the homogenate, which was transferred to a 1.5 ml tube and the tube was flicked at the bottom occasionally to keep the extract mixed.
  3. The homogenate was incubated at room temperature for 1 hr.
  4. Freshly prepared PVP (6% of final volume) and one half volume of 7.5 M ammonium acetate were added separately.
  5. The mixture was incubated on ice for 30 min and centrifuged for 10 min in a micro centrifuge (10 000 at 4 °C).
  6. The supernatant was transferred to a fresh tube to which was added 1 volume of isopropanol, and left at -20 °C for 30 min to precipitate the DNA.
  7. After centrifugation at 10,000 for 10 min, the supernatant was discarded and the DNA pellet was air-dried.
  8. The DNA pellet was resuspended in 500 µl TE buffer (10 mM Tris–HCl pH 8.0, 0.1 mM EDTA pH 8.0) or distilled water.
  9. Two µl of RNase (1 mg/ml) was added to the solution and incubated at 37°C for 15min.
  10. One volume chloroform: isoamyl alcohol (24:1) was added and emulsified by inverted shaking to remove both RNase and plant pigments.
  11. The above step was repeated once again.
  12. After centrifugation (10,000 at 4 °C) for 5 min, the supernatant was transferred to a fresh tube to which 1 volume of isopropanol was added and left at –20 °C for 10 min.
  13. After centrifugation at 10,000 for 10 min, the pellet was washed with 1 ml 80% ethanol and air-dried.
  14. The DNA pellet was re-dissolved in 30 µl TE or distilled water.

AGAROSE GEL ELECTROPHORESIS
Agarose gel electrophoresis was carried out to check for the presence of genomic DNA isolated from plant samples.

Method
1.    The required amount of agarose was weighed, 0.8% agarose was prepared in 0.5X TBE buffer. After heating the solution and letting it to cool, required amount of ethidium bromide was added.
2.    The tank was filled up with 0.5X TBE buffer and the gel was immersed in it.
3.    5 µl of sample with 2 µl of bromophenol blue was mixed and the samples were loaded in the respective lanes.
4.    The gel was run at 80-100 V for 1 hour.
5.    The agarose gel was observed under the Gel-Documentation system (Bio-Rad).
6.    The image was captured, required lanes were labeled and the image was saved.

RESULTS AND DISCUSSION
High quality of DNA, RNA and protein are required for meaningful molecular biology studies. Several extraction methods have recently been developed for DNA, RNA and protein extraction. However, many of these techniques require either fresh tissue or tissue stored and strictly maintained at ultralow low temperatures at -50 ?C to -70 ?C [Saha S et al., 1997]. At this ultra-low temperature, all cellular divisions and metabolic processes are stopped, allowing conservation for a theoretically unlimited period of time [Engelmann, 2004]. Generally, only a limited number of freshly collected tissue samples can be extracted at any given time, while ultralow freezer space for storage of bulky tissue samples is usually at a premium in most laboratories. A solution to this problem is lyophilization of the plant tissues by grinding them as dry powder for efficient storage in limited freezer space [Saha S et al., 1997].

In this study, we had tried optimizing a protocol for lyophilization, cryopreservation and DNA extraction of five socio-economically and medically important plant species. We had also tried to study whether the lyophilized plant leaves were suitable as a source for DNA isolation. A comparative analysis was also performed to find out which among cryopreservation or lyophilization was a better method for storage of plant leaf tissues, by isolation of DNA from the cryopreserved and lyophilized leaves. Three plant species, Terminalia arjuna, Terminalia catappa andTerminalia chebula belonging to Combretaceae family and two plant species Jatropha curcas and Jatropha gossypifolia, belonging to Euphorbiaceae family were selected for the study. The selected plant species have medicinal value which can be used for the treatment of human disease and diagnosis.

For optimization of DNA extraction method, two protocols were performed one of which was a modified CTAB protocol [Doyle J and Doyle J, 1987] and the other was PVP protocol [Kim C et al., 1997]. The CTAB protocol gave better results than PVP protocol for DNA isolation from cryopreserved leaf samples while PVP protocol gave better results only in case of lyophilized leaves.

In case of lyophilization, the concentration and the purity (A260/A280) of the DNA extracted was in the range of 887 ng/µl - 1200 ng/µl and 1.130 - 1.334 respectively. The purity and concentration of Jatropha curcas was found after storage of the lyophilized plant leaves for up to 15 days [Table 2]. The residual moisture content post lyophilization was as low as 22.8 % in Terminalia catappa as high as 35.06 % in Terminalia chebula [Table 3, Figure 5.6]. The gel images are shown in Figure 5.8.                   

In case of cryopreservation, the concentration and the purity of the DNA extracted was in the range of 40 ng/µl - 1642 ng/µl and 0.917 – 1.778 respectively. Bands of DNA were observed in cryopreserved leaf samples of Terminalia chebula stored for up to 15 days and for the remaining four species for up to 7 days [Table 4, Figure 5.7].

Many factors affect the preservation of DNA, including the type of plant, the chemical and physical environment in which that plant is stored, and the duration of storage and the preservation method. However, the interactions of these factors and their resultant effects on DNA preservation are difficult to predict [Dawson M et al., 1998] and the protocol for DNA extraction needs to be further optimized. The ratio of purity of DNA was found to be less than 1.8 with high concentration in lyophilized samples of Terminalia arjuna, Terminalia catappa, Terminalia chebula; therefore it indicates the presence of other impurities like polyphenols, polysaccharides, etc.

The purity of the DNA extracted from both cryopreserved and lyophilized leaves was in the range of 0.917 – 1.778. A ratio less than 1.8 indicates the probable presence of proteins and/or UV absorbers and a ratio higher than 2.0 indicates that the sample may be contaminated with chloroform or phenol [Reddy J, 2009].

Table 2: Concentration and purity of the genomic DNA extracted by CTAB and PVP protocol after preservation of the sample through lyophilization

Sr. No.

Name of plant species

Residual moisture content (%)

Protocol for DNA extraction

Days of storage

Purity of the DNA extracted (A260
/280)

Conce
ntration in (ng/µl)

1.

Terminalia arjuna

30.2667




Protocol 1(CTAB)

1

0.986

365

7

1.677

110

15

1.073

220

Protocol 2
(PVP)

15

1.187

555

2.

Terminalia catappa


22.8



Protocol

1(CTAB)

1

1.412

85

7

1.64

60

15

1.026

200

Protocol 2
(PVP)

15

1.474

140

3.

Terminalia chebula


35.0667



Protocol

1(CTAB)

1

1.158

293

7

1.5

387

15

1.049

160

Protocol 2
(PVP)

15

1.737

82.5

4.

Jatropha curcas


23.7333



Protocol

1(CTAB)

1

1.934

885

7

1.655

300

15

1.176

100

Protocol 2
(PVP)


 

Pages

FIND MORE ARTICLES