DNA Extraction from Dry Rhizomes for DNA Barcoding. Development of a Cost-Effective Method

TABLE OF CONTENT

ABSTRACT

ACKNOWLEDGMENT

TABLE OF CONTENT

LIST OF TABLES

LIST OF PLATES

ABBREVIATIONS

CHAPTER 01

INTRODUCTION

CHAPTER 02

LITERATURE REVIEW
2.1 Zingiberaceae Family
2.1.1 Alpinia galanga – Maha aratta
2.1.2 Alpinia calcarata - Heen aratta
2.1.3 Alpinia purpurata – Red ginger
2.1.4 Alpinia malaccensis – Ran kinihiriya
2.1.5 Alpinia abundiflora
2.1.6 Hedychium coronarium – Ela mal
2.1.7 Hedychium coccineum – Kaula ala
2.2 Plant Sample Collection and Storage for DNA Extraction
2.2.1 Analysis of Fresh Sample
2.2.2 Analysis of Dry Sample
2.3 DNA Extraction Methods for Plants with High Content of Secondary Metabolites
2.3.1 Issues with Secondary Metabolites in DNA extraction
2.3.2 Plant Mini Kit Protocol
2.3.3 CTAB Extraction Method
2.3.4 Modifications of CTAB Method
2.4 DNA Quantification
2.4.1 Gel Electrophoresis
2.5 Purity Testing of Extracted DNA
2.5.1 Restriction Digestion
2.5.2 UV Spectrophotometer

CHAPTER 03

MATERIALS AND METHOD
3.1 Research location
3.2 Plant Materials
3.4 Equipment
3.5 Method
3.5.1 Dry Rhizome Samples Collection and Storage
3.5.2 DNA Extraction
3.5.2 DNA Quantification Using UV Spectrophotometer
3.5.3. Quantification Using Known DNA Sample
3.5.4 Restriction Digestion Reaction
3.5.5 PCR Amplification of Extracted Genomic DNA

CHAPTER 04

RESULTS AND DISCUSSIONS
4.1 DNA Extraction
4.2 DNA Quantification Using UV Spectrophotometer
4.3 Restriction Digestion Reaction
4.4 PCR Amplification
4.5 Estimated Cost Calculation
4.6 Time Consumption for DNA Extraction

CHAPTER 05

CONCLUSION

REFERENCES

APPENDICES

LIST OF TABLES

CHAPTER 03

Table 3. 1: Chemicals for DNA extraction, Gel Electrophoresis, and Restriction Digestion and PCR

Table 3. 2: Preparation of different concentration of λ DNA

Table 3. 3: Preparation of master mix for PCR amplification

Table 3. 4: PCR program (Alpinia test 9)

CHAPTER 04

Table 4. 1: DNA purity information for genomic DNA samples

Table 4. 2: Estimated cost for the SDS extraction buffer (for 20 ml)

Table 4. 3: Estimated cost for the 4x CTAB extraction buffer (for 20 ml)

Table 4. 4: Estimated cost for the CTAB series extraction buffers

Table 4. 5: Estimated time consumption for SDS extraction method (for 5 samples)

Table 4. 6: Estimated time consumption for SDS extraction method (for 5 samples)

Table 4. 7: Estimated time consumption for CTAB series extraction method (for 5 samples)

LIST OF PLATES

CHAPTER 02

Plate 2. 1: Alpinia galanga plant...6 Plate 2. 2: Alpinia galanga inflorescence

Plate 2. 3: Alpinia calcarata plant.7 Plate 2. 4: Alpinia calcarata inflorescence

Plate 2. 5: Alpinia purpurata plant8 Plate 2. 6: Alpinia purpurata inflorescence

Plate 2. 7: Alpinia malaccensis plant.9 Plate 2. 8: Alpinia malaccensis inflorescence

Plate 2. 9: Alpinia abundiflora plant...10 Plate 2. 10: Alpinia abundiflora inflorescence

Plate 2. 11: Hedychium coronarium plant

Plate 2. 12: Hedychium coccineum inflorescence

CHAPTER

Plate 3. 1: Extraction area24 Plate 3. 2: Gel documentation area

Plate 3. 3: Plants that were grown at the University farm

Plate 3. 4: Commercially available Heen Arattan dry rhizomes.27 Plate 3. 5: -80oC Freezer

Plate 3. 6: Incubating samples in a dry bath32 Plate 3. 7: After the first centrifugation

Plate 3. 8: After the second centrifugation..32 Plate 3. 9: DNA pellet

Plate 3. 10: The results displaying on the monitor

Plate 3. 11: The loading sequence of genomic DNA in the agarose gel

Plate 3. 12: The loading sequence of DNA samples after the restriction digestion

Plate 3. 13: The PCR machine

Plate 3. 14: The PCR program displaying on the monitor

CHAPTER 03

Plate 4. 1: Gel image of genomic DNA samples without dilution

Plate 4. 2: Gel image of genomic DNA samples after the dilution

Plate 4. 3: Gel image of restriction digestion DNA samples

Plate 4. 4: Gel image for the PCR product after the gel electrophoresis

ABBREVIATIONS

Abbildung in dieser Leseprobe nicht enthalten

ABSTRACT

Alpinia calcarata (heen aratta) is widely used as ayurvedic medicine in Sri Lanka. Medicinal uses are treatments for indigestion, impurities of blood, throat inflammation and voice improvement, cough, respiratory ailments, bronchitis, asthma, diabetic and fever. The medicinal valuable part of the Heen aratta plant is the rhizome. The dry rhizomes are supplied to the Ayurveda medicine shops by local collectors and import from India like foreign countries. The collectors identify the Heen aratta plant by the external morphological characters. This method is not enough to identify the Heen aratta plant correctly, because there are many related species to Alpinia calcarata and their morphological characters are almost the same. The related species are Alpinia galanga (Maha aratta), Alpinia malaccensis (Ran kinihiriya) and etc. High possibility to adulterate with these species. Then expected medicinal value from the Heen aratta dry rhizomes reduce. If so, a morphological taxonomic characterization method like DNA barcoding should be developed. High-quality DNA required for the DNA barcoding and the DNA extraction method should be a cost-effective method that suitability with the commercial requirements. This research was done to develop a low-cost method for DNA extraction from the dry rhizomes of Heen aratta. Three modified method methods were followed to extract the DNA from dry rhizomes, SDS, 4x CTAB, and CTAB series methods. After the extraction, the quality and the quantity of the extraction DNA should be checked. The quantity of the DNA mainly checked by using UV spectrophotometer. The purity was also can be checked by using UV spectrophotometer. Based on the absorption ratio that gets from the UV spectrophotometer purity of the extracted DNA can be determined. The purity of the extracted genomic DNA was confirmed by restriction digestion reaction. The suitability of genomic DNA for the downstream application was confirmed by PCR amplification. The estimated cost calculation for DNA extraction was done for each three methods to further select of lowest method among them.

Keywords: Cost-effective method, Heen aratta, DNA extraction, Restriction digestion, PCR amplification

ACKNOWLEDGMENT

I would like to offer my earnest appreciation and gratitude to, Dr. L.M.H.R. Alwis, Senior Lecturer, Faculty of Animal Science and Export Agriculture, Uva Wellassa University for her valuable time, advice and supervising me in the research project and familiarize me to the scientific research disciplines.

I would like to thank, Dr. P.E. Kaliyadasa, Senior Lecturer, Faculty of Animal Science and Export Agriculture, Uva Wellassa University for supervising me to finalize the thesis.

I must thank the officials of Biotechnology Laboratory Ms. Harshani, Ms. Aparna and other staff members of the laboratories of Department of Export Agriculture, Uva Wellassa University for their support in the laboratory work.

Further, I like to thank my family members and friends for help and encouragement towards for success of my research.

CHAPTER 01

INTRODUCTION

Alpinia calcarata and other related species are included in family Zingiberaceae, that is one of the valuable medicinal plants in the traditional medicine system. These plants are perennial aromatic herb and distributed through India, China, Thailand, Malaysia, Indonesia, and Sri Lanka. Many Alpinia species are very valuable because of their medicinal properties such as properties of hypertensive, anti-emetic, anti-oxidant and anti-inflammatory effects (Bhadra & Bandyopadhyay, 2015).

This research was conducted to develop a low-cost DNA extraction protocol from the different plant parts of the Alpinia species. This research was especial focused on the dry rhizomes of Alpinia calcarata. Several easy methods are commercially available to extract DNA from the dry rhizomes. As an example DNA mini kit but they are commercially available kits are highly expensive (Cheng et al., 1997). The advantages of these kits are the purity of the DNA is higher than extracted DNA through CTAB and SDS protocols. However these methods could be modified for obtain better purity.

Most Alpinia species contain a high content of phenolic compounds and other secondary metabolites. These compounds retard the DNA extraction and affect the further downstream applications such as PCR amplification like processes (Bhadra & Bandyopadhyay, 2015). Moreover, these compounds resulting problem with either nucleic acid or protein extractions and could degrade proteins or interfere with enzymes used in subsequent manipulations of these extracts (Dehestani and Tabar, 2007).

It is particularly challenging while extracting DNA from storage organs like rhizomes, bulbs, etc. Even if good quality DNA extracted, certain secondary metabolites tend to co-precipitate with DNA and degrade during long-term storage. Traditional morpho taxonomic identification of members of Zingiberaceae is severely hampered by the apparent phenotypic similarity of their vegetative parts (Bhadra & Bandyopadhyay, 2015).

The widely used genomic DNA extraction procedures depend on lengthy protocols that use hazardous chemicals or expensive commercially available kits. As an example CTAB method and its modifications (Porebski, et al., 1997) regents such as like liquid nitrogen, hydrochloric acid, sodium hydroxide, 2-mercaptoethanol, phenol and chloroform that are either toxic or caustic and therefore require the use of a fume hood. Further procedures are lengthy with a minimum of 5-6 hours per extraction and are also expensive. Such methods are therefore not suitable for large scale DNA extractions in laboratories with minimum resources (Dehestani and Tabar, 2007).

In this research two protocols were followed. The first method was modified from the protocol of Nagaraj et al., 2015. This method was SDS based method. The extraction buffer was prepared according to the protocol without any modification, but at the DNA isolation step some modifications were done. The modifications were mortar and pestles which used to crush the sample taken out from the refrigerator which was ultra-frozen (-76oC). This was very important when crush the sample without minimum degradation of the samples. This method did not use liquid nitrogen. If so, crush the plant samples like powder used ultra-frozen mortar and pestles. This modification was done after doing trials using ultra frozen and without it. The DNA quantity was higher when using ultra frozen mortar and pestles (Khan et al., 2007).

The other important modification was using pre-heated buffer in 65oC for 15 minutes and crushed plant sample to preheated buffer. Proteinase K was added to the mid-step in extraction protocol to remove extra proteins in the samples. After the quantification of the extracted DNA from this method could be observed the DNA concentration is very high. Most of the times it was higher than 400 ng/µl, but purity was very low. When following this method there was no sample between 1.8-2.0 all samples were below at this range after the DNA quantification by using UV spectrophotometer.

Because of the low purity of the extracted DNA sample through the above SDS based method need to be tested another protocol. This modified method based on the protocol of Bhadra, 2016 and plant samples were overnight frozen in -76oC. The extraction buffer was prepared according to protocol with small modification and added β-mercaptoethanol directly to the extraction buffer, the modification was β-mercaptoethanol was added in the first step of the extraction protocol. There were a few other modifications. The RNase solution did not add. The final pellet was dissolved in TE buffer behalf for sterilized water. The extracted DNA samples after the quantification the purity were better the SDS method. Therefore testing above modification will be important for Alpinia calcarata to test the feasibility in extracting DNA successfully in effective way.

1.1 Objectives

1.1.1 Main Objective

- Development of cost-effective method for DNA extraction from dry rhizomes of Alpinia calcarata (Heen aratta) for DNA Barcoding

1.1.2 Specific Objectives

- To confirm the quantity of the extracted DNA
- To confirm the purity of the extracted DNA

CHAPTER 02

LITERATURE REVIEW

2.1 Zingiberaceae Family

Zingiberaceae, includes medicinally and economically important monocotyledons mostly distributed in South and South-East Asia. Most often used gingers belong to the genera Alpinia, Amomum, Curcuma and Zingiber and to a lesser extent Boesenbergia, Kaempferia, Elettaria, Elettariopsis, Etlingera and Hedychium (Rahman and Islam, 2015). Members of this family are perennials that frequently have fleshy rhizome (underground stems). Height of the plants about 6 m in height. Some species are epiphytic, supported by other plants and having aerial roots exposed to the atmosphere. Apparent short aerial stems are forming from the rolled-up sheathing bases of the leaves. The commonly green sepals differ in texture and color from the petals. Bracts (leaflike structures) are spirally arranged, and the flower clusters are spiral and cone (Bhadra & Bandyopadhyay, 2015).

The Zingiberaceae flower resembles an orchid because of its labellum (two or three fused stamens) joined with a pair of petal-like sterile stamens. In the slender flower, tubes contain nectar. The flowers are brightly colored and pollinated by insects. The rhizomes are variously colored ranging from pale yellow, deep yellow, greenish-blue, pink or combinations of these in different species. Scale leaves protect the young rhizomes and axillary buds. True aerial stem present in some genera and absent in others. Leaves are arranged transverse or parallel to the rhizome (Sahoo, 2012).

These family plants contain a higher amount of volatile oils and oleoresins. The plants are characterized according to the presence of these constituents. They have good export value. Normally, the rhizomes and fruits are aromatic, tonic and stimulant. Some are used as food which contains a higher amount of starch and also uses as diaphoretic juice. There are many health benefits from the ginger family. Use to cure stomachache and improve digestion. Also use in motion sickness, nausea, arthritis, and headaches, reducing cold-sweating and vomiting. The recent researches have been discovered extract from the rhizomes of the ginger family has anticancer activity mainly it can be control of prostate cancer (Rahman and Islam, 2015).

2.1.1 Alpinia galanga – Maha aratta

This plant is a perennial herb. The normal average height of the plant is about 5 feet. The shape of the leaves is oblong-lanceolate, slightly aromatic tuberous root have. The length of the rhizome is 3.5-7.5 cm and the thickness is about 2 cm. The leaves are oblong-lanceolate, acute, glabrous, ligules are short and rounded. Flowers are greenish-white in color, bracteate, bracts are ovate-lanceolate. Tubular calyx, corolla lobes oblong, broadly elliptic, shortly 2-lobbed at the apex, with a pair of subulate glands at the base of the claw. The fruits are small and cherry type, color is orange-red (Jia et al., 2017).

Alpinia galanga distributed throughout Indonesia, China, and Arabic gulf areas, Malaysia, Egypt, and Sri Lanka. It favors growing in open, sunny, forests and brushwood. In Sri Lanka, it is normally cultivated in the mid and low country in Sri Lanka (Bhadra & Bandyopadhyay, 2015).

There are so many medicinal uses from this plant such as treating for microbial infections, inflammations, rheumatic pains, tumors, kidney disease, diabetes, and also use in treatment in HIV. The seeds also have a medicinal value they used to clean the mouth. Also, it has improved digestion. The rhizome contains a higher amount of essential oils and is used as a spice. Often flowers and young shoots are used as a spice or a vegetable (Arambewela and Wijesinghe, 2006).

The major chemical compound that contains in this plant is 1’S-1’-acetoxychavicol acetate (ACE). This compound is a type of Phenylpropanoid and acts as an efflux pump inhibitor which provokes resistance in mycobacterium and hence it acts as a new target for the discovery of anti-TB agents. The other types of compounds that contain in the Alpinia galangal are Diterpene and Curcuminoid (natural phenols). They act as enhance antifungal activity and inhibit the proliferation of human melanoma (Jia et al., 2017).

There are many bioactivities from this plant. Antimicrobial activity can be seen in the essential oils obtained from fresh and dried rhizomes. The ethanolic extracts from this plant have antifungal activity. Aqueous acetone extract of the rhizomes has anti-inflammatory activity. The crude extract of this plant has hepatotoxicity. Hot water polysaccharide has immunomodulatory activity. The extract from the rhizome has anti-diabetic activity. Ethanol and water extract from this plant have anti-oxidant activity. An aqueous acetone extract from the fruit of Alpinia galangal shown anticancer activity (Jia et al., 2017).

Due to copyright issues, these pictures have been removed by the editorial staff.

Plate 2. 1: Alpinia galanga plant Plate 2. 2: Alpinia galanga inflorescence

(Source: tropical.thferns.info) (Source: pioneerherbal.com)

2.1.2 Alpinia calcarata - Heen aratta

This plant widely uses in Sri Lanka for medicinal purposes. After the maturing of the rhizome, it forms branches and colors ranging from light to dark brown. The leaf of the plant is simple, alternative, 25-32 cm long, 2.5-5 cm broad. The flowers are irregular, bisexual and pedunculate. Terminal dense flowers are found in panicle 8.5 cm long. This plant distributed throughout the tropical countries containing India. Sri Lanka and Malaysia (Rahman and Islam, 2015).

The important part that uses for traditional medicine is rhizome and treatment for indigestion, impurities of blood, throat inflammation, voice improvement and to maintain youthful vigor. It is also used in treatment for the aphrodisiac. For respiratory-related problems use decoction from a rhizome as medicine. Both the ethanolic and aqueous extracts from the rhizomes of Alpinia calcarata have so many bioactivities such as antibacterial, anthelmintic, antifungal, antioxidant, gastroprotective, aphrodisiac and antidiabetic effects. Recent researches have been shown hot ethanol and hot water extracts have anti-inflammatory and antioxidative properties. And also cytotoxic properties have been observed in alcoholic extract of the rhizomes. The major chemical constituents that contain in the volatile oil of the rhizomes. 1,8‑cineol (42%), camphene (7.6%), α and β‑pinene (11.30%), α‑fenchyl acetate (14.7%), camphor (5%), borneol (2.5%) (Rahman and Islam, 2015).

Due to copyright issues, these pictures have been removed by the editorial staff.

Plate 2. 3: Alpinia calcarata plant Plate 2. 4: Alpinia calcarata inflorescence

(Source: asia-medicinalplants.info) (Source: researchgate.net)

2.1.3 Alpinia purpurata – Red ginger

Red ginger is a tall herbaceous plant and evergreen plant. Normally these plants popular for ornamental and cut flowers. This plant native to New Caledonia, New Hebrides, Yap, British Solomon Islands, Protectorate, Bismark Archipelago, and Bougainville. Optimum growth can be observed in well-fertilized and wet soil. From the rhizomes of the plants, leafy cane-like stems arise. The height of the stem is normally about 3-15 feet and width can be around 2-4 feet. The length of the single inflorescence is about 12 inches. The rhizome is laterally and highly branched. After the inflorescence, the older shoots become dried. The rhizome and stalk contain aromatic chemical constituents (Kobayashi et al., 2007).

The leaves are deep green color leaves arrange in alternative and lacking a petiole, long sheath wraps around the stem. Leaf-blade is oblong, 12-32 inches long and 4-9 inches wide, with a pointed apex. The main factor that affects the flower yield and rate of development is sunlight detected by the plant. The temperature requirement for this plant is 60[0]F. When temperature decline below 50[0]F, red ginger grows very slowly, turns yellowish-green, and produces small, tight cone-like inflorescences. The highest flower yield can be obtained during the summer and plants are flowering annually. Extreme temperature can be led to the yellowing of the foliage. The plants can be grown up to 1600 ft above from the Mean Sea Level (MSL).

Propagation is done by the offshoots and from the rhizomes. Aerial offshoots that are developed from the inflorescence have a higher growth rate. Several cultivars do not produce offshoots. These cultivars should propagate by the rhizomes. The high amount of irrigation to the plants leads to the best flower quality (Kobayashi et al., 2007).

Due to copyright issues, these pictures have been removed by the editorial staff.

Plate 2. 5: Alpinia purpurata plant Plate 2. 6: Alpinia purpurata inflorescence

(Source: tropical.theferns.info) (Source: monaconatureencyclopedia.com)

2.1.4 Alpinia malaccensis – Ran kinihiriya

This plant distributed in Assam, Myanmar, Thailand, Peninsular Malaysia to China-Xiang and Yunnan. It is cultivated in India, Sri Lanka, Java, and Southern Java. Alpinia malaccensis is a perennial herbaceous plant. The average height of the plant is about 3-4 m. The ligule is two clefts, up to 1 cm and slightly tomentose. Leaf length is around 8 cm. The leaf blade is oblong-lanceolate or lanceolate, up to 90x15 cm and abaxially pubescent. The base is acute and the apex is acuminate. Racemes grow up to 47 cm, the rachis is stout and densely yellow pubescent, bracteoles are white. The calyx is campanulate, 1.5 cm, densely sericeous and red-tipped. Corolla is white and sericeous, tube is 1 cm, lobes are oblong-lanceolate and 2.5-3 cm. The ovary is villous. The capsule is yellow, 2 cm diameter and dehiscent irregular (Sahoo et al., 2012)

The main chemical constituents that can find in the methanol and petroleum ether extracts are phenylephrine and 2-pentene. The major chemical constituent present in the essential oil derived from the rhizome is (E) -methyl cinnamate (85.7%). The other minor components are α -phellandrene (1.9 %), β -pinene (1.6 %) and 1,8-cineole (1.5 %); p -cymene (1.6 %), α -pinene (1.1 %), limonene (1 %) (Sahoo et al., 2012).

Extract from the fruits of this plant used to treat sores, wounds, and boils (Burkill, 1966). Vomiting can be reduced by the drinking infuse solution that can be prepared by soaking ripe and unripe fruits in a salt solution. The essential oil used as a treatment in rheumatism and arthritis. Mishing tribe in Assam India use rhizomes in the treatment of sores. A decoction of the fruit or crush seeds utilizes by the Philippines for gastralgia with tympanites. Leaf extract is used for children by the people who live in Java as vomit reducer and rhizome oil utilize as massage oil. A decoction from the rhizomes used as a medicine for intestinal disorders and extract from the rhizomes used as medicine for scabies (Sahoo et al., 2012).

Due to copyright issues, these pictures have been removed by the editorial staff.

Plate 2. 7: Alpinia malaccensis plant Plate 2. 8: Alpinia malaccensis inflorescence

(Source: bambooland.com.au) (Sorce: bambooland.com.au)

2.1.5 Alpinia abundiflora

Tall herbs, leafy stem 3-3.5cm high. The leaves are normally, 60-100 cm long and 10-15 cm, oblong and broadly lanceolate, acuminate at apex, glabrous or sparsely puberulous below, ligules 1.5-1.8 cm long. Inflorescence terminal, peduncle 30-40 cm long, covered with imbricating red bracts. Bracts 10-15 x 1-2 cm, outer bracts are sterile, larger inner fertile and smaller. Flowers 4-7 per bract, 2-3 cm across. Calyx tube 1.1.5 cm long. Corolla almost equal to calyx. Labellum white, striped with pink, 5-lobbed. Ovary 0.3-.4 cm long, 3-lobed. Capsule triangular ovoid, 2-2.5 cm across and smooth. These plants are widely distributed in South India and Sri Lanka. In Sri Lanka, this can be highly observed in Horton Plains National Park. This plant highly use for ornamental purposes (Kobayashi et al., 2007).

Abbildung in dieser Leseprobe nicht enthalten

Plate 2. 9: Alpinia abundiflora plant Plate 2. 10: Alpinia abundiflora inflorescence

(Source: commons.wikimedia.org) (Source: commons.wikimedia.org)

2. 1.6 Hedychium coronarium – Ela mal

This plant is a perennial herbaceous. The average height of the plant is 3-6 m from the rhizomes. Rhizomes are highly branched and fleshy. The diameter of this rhizomes is about 2.5-5 cm. the rhizomes are spread under the soil surface. The shape of the leaves is lance with sharp-pointed, simple, 2-ranked, alternately disposed, 8-24 in (20-61 cm) long and 2-5in (5-12.7 cm) wide. Margins can be observed whole leaf surface. A prominent midrib located on dorsal face, smooth and glabrous on surfaces, intense green and glossy. Clusters of white flowers are emerging from the between of the bracts, single bract has 2-3 flowers. Flowers are zygomorphic, hermaphrodite, calyx glaberous, less than half the length of the corolla tube, tubular calyx 4 cm long. Fruits are oblong and capsule contains numerous seeds (Pachurekar and Dixit, 2017).

This plant contains a large number of secondary metabolites such as terpenoids, steroids, flavonoids, and alkaloids (Sahoo et al., 2012). The main chemicals that contain rhizomes of this plant are hedychicoranarian, peroxycoronarian D, 7β hydroxylcalcaratarin A and E, 7β- hydroxyl-6-oxo-labda-8, 12-diene-15,16-dial. The chemical content of the oil, β- pinene (20.0%), linalool (15.8%), α-pinene (10.1%), 1,8-cineole (10.7%) and α-terpineol (8.6%) in the leaf while the root consists mainly of β- pinene (23.6%), α- humulene (17.1%), β- caryophyliene (13.0%), α- piene (6.9%) and elemol (6.9%) (Chen et al., 1997).

This plant has many bioactivities such as anti-inflammatory, anti-tumor, anti-allergic, analgesic, antihelmintic and significant cytotoxic effects . The flowers of this plant used as traditional medicine for fever, arthritis and eyäe disease. The extract from the flowers is known as “Gulbakawali Ark” in India and it is world popular as an eye tonic and that avoids “motiabind” (Cataract). This plant used as medicine for yellowish skin or white eyes in Bangladesh. The combination of leaf extraction with Coccinia grandis applied for three days (Pachurekar and Dixit, 2017).

Due to copyright issues, this picture has been removed by the editorial staff.

Plate 2. 11: Hedychium coronarium plant

(Source: kiefernursery.com)

2.1.7 Hedychium coccineum – Kaula ala

Also called as orange ginger lily. In the Himalayas which produces beautiful orange bottle-brush like spikes of orange flowers. Flowers are very showy, bright orange-red, the normal length of the inflorescence is about one foot and growing in the upward direction. Each flower has long projecting stamen with bright red filament and two times longer than the linear petals. Leaves are sheathing at the base, with a long lance-shaped blade, 5-8 cm broad and up to 45 cm long. The normal height of the plant is 2 m. Orange ginger lily is widely distributed throughout the Himalayas and in North-Eastern India and can grow up to elevation between 450-2000 m. Flowering occurs in July-August. Mainly this plant used for ornamental purposes (Pachurekar and Dixit, 2017).

Due to copyright issues, this picture has been removed by the editorial staff.

Plate 2. 12: Hedychium coccineum inflorescence

(Source: mybageecha.com)

2.2 Plant Sample Collection and Storage for DNA Extraction

When the plant is sampled in the field or laboratory, the process of plant DNA isolation may be regarded to begin. It is vital to harvest and store properly. Labeling is also a significant problem, particularly when analyzing large numbers of samples. A sampling of small pieces of plant leaves for DNA was done using plastic pipette tips to punch a small leaf disk, nylon membrane can then be squashed with tissue. This approach allows the processing and storage of large numbers of samples. For storage, samples are often frozen. The sample must stay frozen until DNA is extracted as frozen tissue thawing often results in DNA degradation. Alternatively, new material or tissue that has been dried for storage may extract DNA (Khan et al., 2007).

2.2.1 Analysis of Fresh Sample

Before extraction, fresh samples can be immediately obtained or stored for hours or days at suitable low temperatures. Refrigeration (4°C if necessary) generally enables several days of extraction to be postponed. It is desirable to store circumstances that do not promote fungal development in these tissues. Alcohol can assist maintain certain tissues for analysis. Fresh tissue analysis is likely the preferred choice in most instances for DNA extraction (Bhadra and Bandyopadhyay, 2015).

2.2.2 Analysis of Dry Sample

For many types of tissue, long-term storage of dry tissue is possible. The easiest way to sample most leaves is to put the fresh leaf in a paper bag and store it in an air-conditioned setting. A large number of samples can be stored in any office filing cabinet. Forced drying may disrupt and degrade DNA in the tissue. Conditions that generate herbarium specimens of the highest quality also lead to excellent conservation of DNA. In many research, dry seeds can also be used. Dry sample analysis may be useful when collecting samples for a single study over an extended period of time. Upon completion of sampling, drying enables extraction (Cheng et al.,1997).

2.3 DNA Extraction Methods for Plants with High Content of Secondary Metabolites

2.3.1 Issues with Secondary Metabolites in DNA extraction

Pure, intact and high-quality DNA isolation is so essential for any plant genetic research, particularly due to high quantities of compounds in plant tissues that may interfere with subsequent manipulation of DNA. Most plant species are high in polysaccharides, polyphenols, multiple pigments and other secondary metabolites (Sahu et al., 2012). Which makes DNA in molecular biology studies unusable for downstream study. While the new DNA-based techniques are extremely specific, reproducible and sensitive and distinguished by high discriminatory strength, fast processing time and low cost, they are severely restricted by the existence of inhibitors in plant tissues. These secondary metabolites, which are especially abundant in fruit trees, medicinal plants and some desert shrubs in the end, are not entirely removed during the remaining classical extraction protocol as contaminants in the final DNA preparations. Polysaccharides create DNA viscous, glue-like and non-amplifiable in the PCR response by inhibiting Taq enzyme and interfering with precise restriction enzyme activity (Porebski et al., 1997).

These cytoplasmic compounds may come into contact with nuclei and other organelles when the cells are interrupted. Polyphenols covalently attach to DNA in their oxidized forms, giving it a brown color and reducing their maintenance time. In the presence of these compounds, studies are difficult because of long and tedious extraction procedures and often do not lead to good yield and quality standards (Dehestani and Tabar, 2007).

2.3.2 Plant Mini Kit Protocol

A number of commercial DNA extraction kits are now present on the market, differing in isolation technology, sample type, and quantity; time per run, volume of elution, yield of DNA and prospective downstream applications (Dehestani and Tabar, 2007). These kits are most frequently based on the purification of solid-phase nucleic acids . A spin column is used and operated under centrifugal force. This results in a rapid and effective purification of DNA compared to standard techniques like CTAB or SDS Plant samples, however, generally contain elevated levels of secondary metabolites whose content differs from species to species. Therefore, when used with distinct plant species or tissues for further SSR applications, distinct commercial kits or DNA extraction techniques will yield distinct outcomes (Fu et al., 1998).

The mini kit for the extraction of DNA from DNeasy plant is a commonly used kit. This kit uses several procedures that use easy salt buffers such as Tris-EDTA and can produce greater DNA yields when requiring comparatively tiny quantities of genomic DNA for genotyping purposes using PCR. These are frequently used for downstream applications requiring bigger amounts of DNA, such as platforms for hybridization of DNA-DNA. The issue with these techniques is that in order to precipitate clean DNA and get rid of cell debris in the form of polysaccharides, proteins and salts they require several' wash and spin' measures. The DNeasy kit makes it easier for PCR applications to generate clean DNA, as salt contamination can hamper the efficiency of DNA amplification and priming specificity. The primary drawback of using the DNeasy kit is that from a comparatively large original sample have produced very small DNA returns constantly (Aboul-Matty and Oraby, 2019).

2.3.3 CTAB Extraction Method

The use of cationic detergent CTAB (cetyl trimethylammonium bromide) promotes the separation of polysaccharides during purification, while additives such as polyvinylpyrrolidone can help remove polyphenols. Extraction buffers based on CTAB are commonly used in plant tissue purification of DNA. One choice for purifying DNA using CTAB exploits the distinct solubilities of polysaccharides and DNA in CTAB depending on the sodium chloride concentration. Polysaccharides are insoluble at higher salt concentrations, whereas DNA is insoluble at lower concentrations. Consequently, it is possible to precipitate differentially by changing the salt concentration in lysates containing CTAB, polysaccharides, and DNA. Polyphenols are compounds containing more than one phenolic ring (e.g. tannin), a structure that connects to DNA very effectively. They occur naturally in crops, but they also occur when crops are damaged by tissue (browning). Polyphenols are synthesized by freed polyphenol oxidase after crop tissue homogenization. The addition of polyvinyl pyrrolidone by binding the polyphenol stops the interaction of DNA and phenolic rings. Most DNA extraction procedures based on cetyl trimethylammonium bromide (CTAB) were adapted to the inner parts of each plant species (Fu et al., 2012).

2.3.4 Modifications of CTAB Method

Doyle and Doyle method

The commonly used Doyle & Doyle (1987) and Doyle & Doyle (1990) methodologies, based on the CTAB extraction buffer and the maceration of fluid water leaves lately gathered, he maceration of recently collected leaves with liquid nitrogen, with 3% CTAB-based adaptations, using dried young leaves in an oven at 45oC for 72 hours. The use of dried leaves without the use of liquid nitrogen for DNA isolation. This technique is an inexpensive CTAB-based technique with changes for high-quality genomic DNA extraction. Rich in proteins, polysaccharides, and polyphenols, this technique can also be used for plan samples. Advantages of this technique may be extracting Quality DNA, PCR amplification of genomic DNA obtained from separate plant species using the two protocols used using Random Amplified Polymorphic DNA (RAPD), PCR amplification of the neomycin phosphotransferase gene (nptII) was used to assess the effectiveness of the current protocol to generate DNA of good quality appropriate for identification (Borges et al., 2009).

Modified Protocol by Elias et al.,

The advantages of this method Use of dried leaf material compared to the grinding in liquid nitrogen of lately gathered or frozen leaves. One of the problems during plant collection is storing the material before analyzing it, particularly when laboratory installations are remote. For instance, this technique is helpful when collecting samples in situ and on-farm plants. Usually, when using freeze-dried leaves, they must be immersed in liquid nitrogen immediately and transported to a freezer of -20oC or -80 ° C until they are used. Even sun-dried leaves can be used, or young leaves assembled in folded newspaper sheets that are then left to dry at room temperature or in the sun in plant presses. In addition, the young leaves can be filled with silica gel in two sheets of filter paper to be left for dehydration in the laboratory. For further study, the fine powder acquired after grinding can be readily stored at -20oC in 1.5 mL microtubes (Borges et al., 2009).

Modified protocol by Porebski et al.,

A comparatively fast, cheap and coherent DNA extraction protocol comprising big amounts of polyphenols, tannins, and polysaccharides. The technique includes a modified extraction of CTAB, using elevated salt levels to remove polysaccharides, using polyvinyl pyrrolidone (PVP) to remove polyphenols, prolonged treatment with RNase, and extraction of phenol-chloroform. The existence of secondary metabolites like polyphenols, tannins, and polysaccharides inhibits the action of enzymes. Polysaccharides are visually obvious in DNA obtained by their vicious, glue-like texture and render DNA unmanageable in the polymerase chain response (PCR) by inhibiting Taq polymerase activity (Porebski et al., 1997).

The purity of DNA becomes increasingly hard when young, expanding leaf and shoot material is restricted or when the plant does not undergo active shoot elongation at the moment of collection. DNA from previous material was found to be hard to obtain and unstable for long-term storage. Leaves contain enhanced amounts of polyphenols, tannins, and polysaccharides with maturity. When younger growing leaves and shoots are not accessible during the collection moment, it becomes essential to deal with such parts in mature leaves. A DNA extraction protocol from fully developed extended leaf tissue that would produce DNA appropriate for PCR, particularly for RAPDs, and would be compatible for many distinct wild and grown strawberry species. The protocol outlined here is comparatively fast and cheap and offers smooth, continuously amplifiable DNA in the polymerase chain reaction using the RAPD method. Extraction buffer consist of, 100 mM tris, 1.4 M NaC1, 20 mM EDTA, pH 8.0, 2% CTAB, 0.3% ~-mercantoethanol (Porebski et al., 1997).

2.4 DNA Quantification

Nucleic acid quantitation is a critical step in sample preparation that helps to guarantee optimum downstream assay efficiency. A popular misconception is that all techniques of quantitation are identical and equally accurate. But in reality, estimates of concentration may vary depending on the technique used (Biology, 2011).

2.4.1 Gel Electrophoresis

Agarose gel electrophoresis is a nucleic acid or protein separation technique commonly used for visualization and purification in biotechnology, biochemistry, molecular biology, genetics, and clinical chemistry. This can be achieved by forcing nucleic acid (negatively charged) molecules through an agarose (porous) matrix with an electrical field (electrophoresis). In electrophoresis, cation moves towards cathode and anion move towards the anode. This is defined as the migration of charged particles through a solution (e.g., 0.5X TAE buffer) under the influence of an electrical field (Green and Sambrook, 2019).

The gel offers resistance to the migration of DNA. For bigger molecules, the rate of migration will decrease, and the bigger molecule will migrate slowly than lower molecules. Resistance is directly proportional to an agarose gel's porous nature. Smaller pores provide greater resistance. DNA molecules migrate through gel matrices at rates inversely proportional to the strand length log10 or base pair number (Green and Sambrook, 2019).

The Rate of Migration of DNA through Agarose Gels

Several variables influence the rate of DNA motion through gels. The current that flows through the gel. Under the law of Ohm,

V = IR

I is the current (in amperes) where V is the voltage, and R is the resistance (in ohms). Due to the fact that buffers frequently used for electrophoresis are mildly alkaline (pH 7.8–8.0), DNA molecules that travel through the gel carry a negative charge and migrate to the anode at a speed affected by the current applied (Biology, 2011).

The concentration of agarose. Hydrogen bonds and hydrophobic interactions hold spongy hydrocolloids together. Under the impact of an electrical current, linear DNA molecules rotate through a sequence of pores whose efficient diameters are determined by the agarose concentration in the gel. The linear relationship between the DNA electrophoretic mobility logarithm (μ) and the gel concentration (ι) is outlined,

logµ = logµo - Krι

Where μ0 is the free electrophoretic movement of DNA and Kr is the retardation coefficient of the gel and the size and shape of the migratory molecules (Samar et al., 2019). The DNA's conformation. Super helical circular (form I), circular nicked (form II) and linear (form III) DNAs migrate at different rates through agarose gels (Thorne, 1966). The relative mobility of the three forms depends mainly on the concentration and type of agarose used to create the gel, but also on the strength of the applied current, the ionic strength of the buffer, and the density of superhelical bends in the form I DNA (Green and Sambrook, 2019).

In most cases, the best way to distinguish between the different conformational forms of DNA is simply to include a sample of untreated circular DNA in the gel and a sample of the same DNA linearized by digestion with a restriction enzyme that cleaves the DNA in one place only. The presence of dyes in the gel and electrophoresis buffer. Intercalation of the coloring results in a reduction in the adverse charge of the double-stranded DNA and an increase in both its rigidity and length. Consequently, a factor of about 15% delays the rate of migration of the linear DNA–dye complex through gels (Biology, 2011).

The voltage used. The speed of migration of linear DNA fragments at low voltages is proportional to the applied voltage. However, the mobility of high-molecular-weight fragments improves differently as the power of the electric field is increased. As the voltage increases, the efficient range of separation in agarose gels reduces. Agarose gels should operate at no more than 5–8 V / cm to achieve maximum resolution of DNA fragments > 2 kb in size. The type of agarose, two main agarose categories are standard agaroses and low-melting temperature agaroses (Kirkpatrick, 1990). A third and increasing class comprises of agaroses with intermediate melting / gelling temperature, demonstrating the characteristics of each of the two main groups. Different kinds of agaroses are used for specific uses in each class (Green and Sambrook, 2019).

The electrophoresis buffer, DNA's electrophoretic mobility is influenced by the electrophoresis buffer's structure and ionic strength. If there are no ions (For example, if water replaces an electrophoresis buffer in a gel or reservoir), electrical conductivity is low and DNA is slow to migrate, or not at all. In a strong ionic resistance buffer. Electrical conductivity is very effective, generating important quantities of heat even when mild voltages are applied. The gel melts and the denatures of DNA in the worst case (Biology, 2011).

Electroendo-Osmosis

In agarose gels, electroendo-osmosis (EEO) affects the velocity at which nucleic acids migrate to the positive electrode. This method is due to groups of ionized acids (generally sulfate) connected to the agarose gel's polysaccharide matrix. The acidic groups produce strongly loaded counterions in the buffer that migrate to the adverse electrode through the gel, causing a large flow of liquid to migrate in a direction opposite to that of the DNA.The higher the agarose adverse charge density, the higher the EEO flow and the lower the nucleic acid fragment separation. Smaller DNA fragment retardation (< 10 kb) is minor, but bigger DNA molecules can be considerably retarded, particularly in PFGE. Buying agarose from reputable merchants and using kinds of agarose that show low EEO concentrations is best to prevent issues. Agaroses sold as "zero" EEO are undesirable for two purposes: chemically modified by adding strongly loaded groups that neutralize the sulfated polysaccharides in the gel but may prevent later enzyme responses; and adulterated by adding locust bea case (Biology, 2011).

Electrophoresis Buffers

There are several distinct buffers available for indigenous, double-stranded DNA electrophoresis. These include Tris–acetate and EDTA (pH 8.0; TAE) (also known as E buffer), Tris–borate (TBE), or Tris–phosphate (TPE) at a concentration of approximately 50 mM (pH 7.5–7.8). Usually, electrophoresis buffers are focused and stored at room temperature. All of these buffers operate well, and mainly a matter of personal preference is the decision between them. TAE has the lowest buffering capacity of the three and will be exhausted if electrophoresis is carried out for extended periods of time. When this happens, the anodic portion of the gel becomes acidic and the blue bromophenol migrates from bluish-purple to yellow through the gel to the anode color changes. This shift starts at pH 4.6 and ends at pH 3.0. During electrophoresis, regular replacement of the buffer or recirculation of the buffer between the two reservoirs can prevent TAE exhaustion. TBE and TPE are mildly costlier than TAE, but their buffering capability is considerably greater. Double-stranded linear DNA fragments migrate 10% quicker through TAE than through TBE or TPE; TAE's resolution strength is slightly better than TBE or TPE for high-molecular-weight DNA and worse for low-molecular-weight DNA. This distinction likely explains the observation that in TAE electrophoresis in extremely complicated mixtures such as mammalian DNA yields a better resolution of DNA fragments. Because of this, Southern blots used to analyze complex genomes are generally derived from gels prepared in and run as the electrophoresis buffer with TAE. The supercoiled DNA resolution in TAE is better than in TBE (Green and Sambrook, 2019).

Gel-Loading Buffers

Before loading into the gel slots, gel-loading buffers are mixed with the samples. These buffers serve three purposes: to increase the sample density, to ensure that the DNA sinks evenly into the well; to add color to the sample, thereby simplifying the loading process; and to contain colors that move towards the anode at predictable rates in an electrical field. Bromophenol blue migrates quicker than xylene cyanol FF through agarose gels, some 2.2-fold, regardless of the concentration of agarose. Bromophenol blue migrates through agarose gels at about the same pace as linear double-stranded DNA 300 bp, whereas xylene cyanol FF migrates at about the same pace (Green and Sambrook, 2019).

Analysis of DNA fragments

DNAs separated by migration through agarose gels can be identified by coloring with low inherent fluorescence, powerful DNA affinity, and elevated fluorescence quantity after binding to nucleic acids. The higher the quantity output boost, the higher the signal to noise ratio. By illuminating the gel with UV light at one wavelength and recording at another, bands of DNA stained with these colors are visualized. Methods for staining and visualizing DNA in gels with three colors are defined here: ethidium bromide, SYBR Gold, and SYBR green 1. Images of stained gels are captured by a CCD camera with a suitable converter screen and filter under UV illumination (Green and Sambrook, 2018).

Recovering DNA from Gels

DNA purification from agarose gels is sometimes an essential step in DNA fragment subcloning. Over the years, a multitude of methods have been published to recover bands of DNA from slices of agarose and polyacrylamide gels. To some extent, many of these techniques likely operate, but the fact that none of them has been commonly adopted must imply a lack of effectiveness, reproducibility, or robustness. Traditional methods related familiar problems include, difficulties in ligating, digesting, or radiolabeling the recovered DNA, inefficient recovery of large fragments of DNA, inefficient recovery of small amounts of DNA. Given these issues, designing a protocol that does not require isolation of DNA from gels is best wherever feasible. PCR is often a better choice, which is faster and cheaper. If PCR is impractical, however, it is best to use one of the many commercial products now available for DNA regeneration from agarose gels. Most manufacturers of these kits provide data on the efficiency with which DNA fragments of different sizes can be recovered from gels casted with different agarose concentrations and on the purity of the recovered DNA as determined by its ability to be used as a template or substrate (Biology, 2011).

Binding the DNA to a silica surface is the main step used in many of the business kits. A segment of an agarose gel that contains the interesting DNA band is dissolved in a chemotropic buffer that disrupts the hydrogen bond between the agarose polymer's sugar pats. The transferred DNA is then caught on silica beads or a membrane, retrieved by H2O elution or a buffer with low salt or ethanol concentration. Examples of kits of this type include: QIAEXII Gel Extraction Kit (QIAGEN), Wizard SV Kit (Promega), Ultra Silica Bead Kit (Thermo Fisher), NucleoSpin Extract IIa (Clontech), and GenElute (Sigma-Aldrich) (Green and Sambrook, 2018).

Non-Kit-Based Techniques to Recover DNA from Agarose Gels

An unfortunate trade-off between the effectiveness with which DNA is retrieved from the gel and its purity is a prevalent issue with older, non-kit-based methods. If enzymatic reactions are to be used with DNAs isolated from gels, it may be best to choose a method that promises a small output but delivers DNA free from important contamination (Green and Sambrook, 2018).

DNA ladder

In the field of molecular biology, the molecular weight or base-pair length of nucleic acids must be determined. Typically, double-stranded DNA standards are made from DNA from bacteriophages or plasmids. This method is intensive labor, material, and machinery, particularly in preparing big amounts of DNA. For a set of molecular weight markers, restriction enzyme-digested bacteriophage DNA such as lambda DNA is commonly used. The array of fragment lengths obtained in bp depends on the nature of the enzyme, the sequence of DNA, and the conditions used. Each mixture of enzymes, DNA, and circumstances will provide a distinctive range of specified length DNA fragments. In the areas of molecular biology, genetics, biochemistry, genetic engineering, forensics and the like, polymerase chain reaction (PCR) has been commonly used. PCR is an easy, efficient and convenient technique that can be used in a very short time to prepare sufficient quantities of oligonucleotides. A DNA ladder can be prepared using the method of multiplex PCR (Wang et al., 2011).

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