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

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About Authors:
1Hardik R. Patel*, 2Upanita C. Patel
1Industrial biotechnologist, 2Microbiologist
Anand, Gujarat, India.
*hardikigbt@gmail.com

INTRODUCTION:
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-1587

Objective: To improve and apply advanced techniques for plant genetic resources conservation through cryopreservation and lyophilization

To optimize a protocol for cryopreservation and lyophilization of following plants.
o   Terminalia arjuna
o   Terminalia catappa
o   Terminalia chebula
o   Jatropha curcas
o   Jatropha gossypifolia

To optimize a protocol for DNA extraction from said plants after cryopreservation and lyophilization

Taxonomical classification of plant species and its description

Family:  Combretaceae and Euphorbiaceae
Combretaceae are trees, shrubs, and lianas comprising about 20 genera and 600 species. The leaves are simple, alternate or opposite, entire, stipules small or absent. The flowers are bisexual or sometimes unisexual, usually actinomorphic. The perianth arises from near the summit of a tubular epigynous zone; calyx of usually 4 or 5 distinct to slightly connate sepals; corolla commonly of 4 or 5 distinct petals, occasionally absent. The androecium of 4-10 stamens is adnate to the epigynous zone, commonly in two whorls, often strongly exserted. The gynoecium is a single compound pistil of 2-5 carpels; style and stigma 1; ovary inferior, with 1 locule containing 2-6 apical ovules pendulous on long funiculi. The nectary is usually a disk (often hairy) above the ovary.  The fruit is 1-seeded, often a flattened, ribbed, or winged drupe [Lim T, 2012].


Euphorbiaceae is a spurge family of flowering plants, in the order Malpighiales, containing some 7,500 species in 275 genera. The family consists of annual and perennial herbs and woody shrubs or trees, rarely climbers. Flowers are of one sex, with male and female flowers usually borne on the same plant. Petals are rarely present. Flowers of Euphorbia are in cup-shaped clusters called Cynthia, each of which seems to be a single female flower, consisting of a single pistil surrounded by several male flowers, each of which has a single stamen. These clusters of reduced flowers are enclosed by involucres (whorl) of bracts (modified leaves) that resemble a corolla, or whorl of flower petals.

Male flowers of the other genera have one to many stamens, free or joined. Female flowers have three-chambered ovaries that are superior. There are as many styles as there are ovary cavities. The fruit is a three-chambered capsule. Leaves are usually simple and are alternate (or rarely opposite or whorled) in arrangement along the stems. The stems of many species contain milky latex [Tripathi S and Srivastava M, 2008].

A.  Terminalia arjuna
Terminalia arjuna
is a medicinal plant of the genus Terminalia. The plantis about 20-25 metres tall; usually has a buttressed trunk, and forms a wide canopy at the crown, from which branches drop downwards. It has oblong, conical leaves which are green on the top and brown below; smooth, grey bark; it has pale yellow flowers which appear between March and June; its glabrous, 2.5 to 5 cm fibrous woody fruit, divided into five wings, appears between September and November [Biswas M et al., 2011].


Figure 1:Terminalia arjuna

·         Binomial name Terminalia arjuna
·         Scientific classification [Nema R et al., 2012]
Kingdom: Plantae
Division
:   Magnoliophyta
Class:
        Magnoliopsida
Order:
                   Myrtales
Family:
     Combretaceae
Genus:
      Terminalia
Species:
     T. arjuna

·         Geographical location
Terminalia arjuna
is usually found growing on river banks or near dry river beds in West Bengal and South and Central India.  Terminalia arjunais common throughout India especially in the sub Himalayan tracts and Eastern India. They are widely grown in Bandhavgarh National Park, Pench Tiger Reserve and Kanha National Park in India [Biswas M et al., 2011].

·         Chemical constituents and components
Terminalia arjuna
consists of various components such as triterpenoids, tannins, flavonoids, oligomeric proanthocyanidins and saponin glycosides [Nema R et al., 2012].

·         Medicinal uses
Terminalia arjuna
is used for the treatment of coronary artery disease, heart failure, edema, angina, hypertension and hypercholesterolemia [Biswas M, et al., 2011]. Its bark possesses diuretic, prostaglandin enhancing and coronary risk factor modulating properties. It is also considered as beneficial in the treatment of asthma [Devi R et al., 2007].

B. Terminalia catappa
Terminalia catappa
is a large tropical tree in the Leadwood tree family, Combretaceae. It has corky, light fruit that is dispersed by water. The seed within the fruit is edible when fully ripe, tasting almost like almond.  It grows to 35 metres (115 ft) tall, with an upright, symmetrical crown and horizontal branches. As the tree gets older, its crown becomes more flattened to form a spreading, vase shape. Its branches are distinctively arranged. The leaves are large, 15–25 centimeters (5.9–9.8 in) long and 10–14 centimeters (3.9–5.5 in) broad, ovoid, glossy dark green and leathery. The trees are monoecious, with distinct male and female flowers on the same tree. Both are 1 centimeter (0.39 in) in diameter, white to greenish, inconspicuous with no petals; they are produced on axillaries or terminal spikes.  The fruit is a drupe 5–7 centimeters (2.0–2.8 in) long and 3–5.5 centimeters (1.2–2.2 in) broad, green at first, then yellow and finally red when ripe, containing a single seed [Hnawia E et al., 2011].


Figure 2: Terminalia catappa

·         Binomial name Terminalia catappa
·         Scientific classification [Hnawia E et al., 2011]
Kingdom:
Plantae
Division:
   Magnoliophyta
Class:
        Magnoliopsida
Order:
      Myrtales
Family:
     Combretaceae
Genus:
      Terminalia
Species:
     T. catappa

·         Geographical location
The tree has been spread widely by humans and the native range is uncertain. It has long been naturalised in a broad belt extending from Africa to Northern Australia and New Guinea through Southeast Asia and Micronesia, in subtropical and tropical zones of Indian Subcontinent and Pacific Oceans. It has also been planted extensively throughout the tropics. Subtropical and tropical maritime climates with annual rainfall generally 1000–3500 mm (40–140 in); elevations below 300–400 m (1000–1300 ft)It is distributed in the forest of Western UP and Haryana [Thomson L and Evans B, 2006].

·         Chemical constituents and components
The leaves contain several flavonoids (like kaempferol or quercetin), several tannins (such as punicalin, punicalagin or tercatin), saponines and phytosterols [Hnawia E et al., 2011].

·         Medicinal uses
Astringent in thrush, antidisenteric, mild diuretic, leaf-antiseptic, anti-inflammatory, diaphoretic, antiindigestion, antidysentery, for treating mouth infections, headache, colic [Deharo E et al., 2011].

C.Terminalia chebula
It is belongs to the family combretaceae. It is commonly called as Black myrobalan. It is a deciduous tree, younger stems glabrescent, woody. Leaves are 10 – 20 cm long, sub – opposite, simple; estipulate; petiolate; laminae broadly elliptic to elliptic – oblong, rarely ovate, the bases obtuse, the margins entire, the tips acute, glabrescent. Flowers are 2 mm long , 3-4 mm in diameter; bracts nearly glabrous, 1.5-2.0 mm long; calyx outside glabrous, inside densely villous, calyx-segments triangular; stamens 3-4 mm long; ovary glabrous, ovoid, 1 mm long; style glabrous, 2.5- 3.0 mm long. Seed is rough, ellipsoid, 1.0-2.0 cm by 0.2 -0.7 cm and without ridges [SuryaPrakash D et al., 2012;].


Figure 3: Terminalia chebula

·         Binomial name  Terminalia chebula
·         Scientific classification
Kingdom:
Plantae
Division:
   Magnoliophyta
Class:
        Magnoliopsida
Order:      Myrtales
Family:
     Combretaceae
Genus:
      Terminalia
Species:
     T. chebula

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