S Y BSc. Botany (Sem IV)

            SY BSC Paper III  (Sem-IV)                   

 Current Trends in Plant Science I     USBO403                  

Unit I: Horticulture and Gardening

Horticulture is a segment of the agriculture industry. The term horticulture literally means the culture of a garden. However, the term has taken on a broader context. Horticulture includes the production and use of plants for food, comfort, and beautification. A direct relationship exists between horticulture and science. The area of science most closely associated with horticulture is botany. Botany is the study of plants and plant processes. The field of science that deals with the cultivation of horticultural plants is known as horticulture science.

Science is applied across the horticulture industry. The application of science to horticulture is called horticulture technology. Successfully raising horticultural plants takes more than just daily watering. Time, patience, and an understanding of diverse scientific processes are needed to produce optimal plant growth.

The horticulture industry is the combination of scientific, technological, and production activities that ensure the satisfaction of the consumer. The horticulture industry can be divided into three areas: pomology, olericulture, and ornamental horticulture. Each area is unique and includes many career opportunities.

 

 Branches of Horticulture:

Horticulture is a wide field and includes a great variety and diversity of crops. The science of horticulture can be divided into several branches depending upon the crops it deals with. The following are the branches.

01.  Pomology (Fruit crops) Pomology is refers to cultivation, production and marketing of fruit crops. The workers in the field is called pomologist.


Pomology is the planting, harvesting, storing, processing, and marketing of fruit and nut crops. Fruit crops include both large and small fruits. Examples of large fruits are peaches, apples, and pears. Small fruits include strawberries, raspberries, and blueberries. Almonds, pecans, and walnuts are popular nut crops.

 

India is the second largest producer of fruit after Brazil. A large variety of fruit crops are grown in India. Of these, mango, banana, citrus, papaya, guava, pineapple, jackfruit, litchi, grapes, apple, pear, peach, plum, walnut etc. are important ones. India accounts for 10 per cent of the total world production of fruits. It leads the world in production of mango, banana and acid lime besides recording highest productivity in grape. The leading fruit growing states are Maharashtra, Karnataka, Andhra Pradesh, Bihar and Uttar Pradesh.

 

 

02.  Olericulture (Vegetables crops): Olericulture is refers to cultivation, production and marketing of vegetables. The workers in the field are called olericulturists.


More than 40 vegetables belonging to Solanaceaeous, cucurbitaceous, leguminous, cruciferous, root crops and leafy vegetables are grown in Indian tropical , sub-tropical and temperate region.

Important vegetables grown in India are onion, tomato, potato, brinjal, peas, beans, okra, chilli, cabbage, cauliflower, bottle gourd, cucumber, watermelon, carrot, radish etc.

India is second in vegetable production next to China in area and production contributing 13.38 percent to the total world production. India occupies first position in cauliflower, second in Onion, third in cabbage in the world.

West Bengal, Orissa, U.P, Bihar, Maharashtra, Karnataka are the important states for Horticultural crop production.

 

3. Floriculture (Flower crops): Floriculture is refers to cultivation, production and marketing of flower crops. The workers in the field are called floriculturists 

Flower cultivation is being practiced in India. Since ages it is an important/integral part of socio-cultural and religious life of Indian people. It has taken a shape of industry in recent years.

India is known for growing traditional flowers such as jasmine, marigold, chrysanthemum, tuberose, crossandra, aster, etc. Commercial cultivation of cut flowers like, rose, orchids, gladiolus, carnation, anthurium, gerbera etc.

The important flower growing states are Tamil Nadu, Karnataka, AP, Maharashtra, West Bengal, Sikkim, J&K, Meghalaya etc.

 

4. Nursery Culture (Ornamentals plant): It refers as a cultivation, production and marketing of perennials ornamentals plants. A person who produces and distributes ornamentals plants is called as a nursery grower. Were a person doe’s research, teaching and extensions activities in this area are called ornamental horticulturist. Nursery is a place where plants are propagated and grown to a desired size. Mostly the plants concerned are for gardening, forestry or conservation biology, rather than agriculture 

05.  Landscape Gardening:  Landscape gardening is an aesthetic branch of Horticulture which deals with planting of ornamental plants in such a way that it creates a picturesque effect. It is a very fascinating and interesting subject.


Landscape gardening can be defined as the beautification of a tract of land having a house or other object of interest on it. It is done with a view to create a natural scene by the planting of lawn, trees and shrubs. Landscape gardening is both an art and science of the establishment of a ground in such a way that it gives an effect of a natural landscape. It can be also defined as the imitation of nature in the garden. The expression of landscape may be bold, retired, quiet, etc. This expression will conform to the place and the purpose.

 

Goals of landscape gardening:

Privacy

Safety

Ease of maintenance

Convenience and

Flexibility.

 

 

 

Locations in the garden:

 

01. Edges

These are perennial herbs often used as a short border for lawn or ground cover or dividing beds from roads, walks or paths. These herbs often stand frequent trimming. They consist of live material like the dwarf plants or inert material like bricks, stone slabs or corrugated iron sheets.  The common evergreen edges used for edging are Geranium farreri and Polyanthus. 


02. Hedges (Wall  boundary):

 

A hedge is an artificially created boundary made up of growing plants – a line of thick, woody bushes which do not die down in winter. Countryside hedges around fields usually consist of many different types of plants, but in parks and gardens they may be of one species only.

With the help of plants, live hedges can be formed and used as a fence or a green wall. It serves to screen a particular site or building or hiding of unwanted places. They help to partition the garden into several parts. It provides a natural background to a garden, like a frame to a picture. The characteristics of a good hedge are that it should be thick and dense; it should have foliage from the bottom to top; it should be trim and neat; if it is a flowering hedge its bloom should not clash with the general colour scheme.

Some of the most common foliage hedges are Acalypha with its copper-red leaves, it is suitable for a medium hedge; Clerodendron inerme an evergreen drought-resistant plant used frequently by the roadside, is not eaten by cattle or goats.

03. Lawn:

 

A lawn is an area where grass is grown as a green carpet for a landscape and is the basic feature of any garden. It serves to enhance the beauty of the garden, be it larger or smaller. Proper lawn maintenance plays a crucial part in any landscape design. It enhances the beauty of a garden whether it is small or large. It finds the most important component of a garden giving a view of a green carpet.


A spacious lawn though beautiful will often be monotonous. So, to break the monotony, some beautiful tree or shrub is recommended as single specimen in the lawn.

Plants suitable for planting in lawns as single specimen

Trees: Pinus longifolia, Thuja orientalis

 

 Grass used:


Cynodon dactylon (Family: Gramineae) also known as Bermuda grass is a grass that can be found all over the world. It is used in temperate regions as lawn grass, pasture grass for grazing, and, popularly, as a sports field lawn. this fast-growing plant is considered invasive in many parts of the world.


Paspalum dilatatum   (Family: Gramineae) is a species of grass known by the common name Dallas grass. It is commonly used in making of lawns. It prefer to used in the area were high rain fall is present. It having ability to grown in any kinds of soil.  It has a clump-type growth habit and grows well in association with other grasses and legumes and persists under heavy grazing.

 

 

 

04. Flower beds:

 

A flowerbed is an area of ground in a garden or park which has been specially prepared so that flowers can be grown in it. This can help to display flowers in best possible way.  Choose plants so that your garden offers color and interest throughout the growing season and even in winter. Spring-flowering bulbs are good for early color.


Pentas lanceolata, commonly known as Egyptian starcluster, is a species of flowering plant in the madder family, Rubiaceae A fast-growing, small to medium-sized herbaceous shrub with light green foliage. Each branch tip bears a bunch of small flowers, often for a long period throughout the warmer months. Pentas comes in a variety of colours, including pink, red, mauve and white. It cannot withstand severe frost but will tolerate a fair amount of drought once established.

A species of Petunia with a lax sprawing habit, spreading outward rather than upwards. Flowers are only 2-inches wide, fragrant, violet-colored and cover the plants all summer long. It's an excellent choice for the front of the border or spilling over the edge of a container. P. violacea is a parent to many of the hybrids on the market today. It is a tough plant and has great heat tolerance for a petunia.

 

 

05. Avenue (road side plants in row):

An avenue is the row of trees grown on both sides of roads. Shade and beauty are the sole criteria to be considered while selecting avenue trees. The trees should also be selected according to the length and breadth of the road. Tree planted to line an avenue, traditionally a straight path or road with trees running along each side, is a customary way to emphasise the coming to or arrival at a landscape or architectural feature. Avenues of trees are some of the most strikingly important structural plantings to be found in designed landscapes


Some common road side plants are:

Polyalthia longifolia: 

Commonly known as Ashoka, Belongingto the family Annonaceae.  Leaves are lance-shaped with wavy margins and, depending on their age, either bronze, lime green or dark green. They remain on the tree in all seasons and in a dense arrangement that almost completely cloaks the trunk in lush greenery, from near the tree bottom to the top.  It grows natively in warm humid climates of India, Myanmar, and Malaysia. Ashoka is ever-green, slender but slow-growing flowering tree. It is easy-to-grow, an easy-to-prune tree that flowers abundantly and produces a compact shaped canopy.

 

 Acacia auriculiformis

Belongs to the family Leguminosae. The tree is originally from tropical Australia. It has been widely planted in India. Espeically in southern states. The tree is quick growing of around 15 to 20 meters thronless and evergreen. The leaves are sickle shaped up to 18 cm long and 6 cm broad. Flowers are mildly scented, sessile in dense spike near the end of branches. Flowers are followed by twisted pod shaped fruit. Tree is used for firewood, box making and paper making.  Plants should be planted in staggered rows. Watering in the first dry season is required. Protection from animals is not required. It is used in large scale for tree planting. Grown as a road side tree.

 

 

06. Water garden:

It may have a water course, a water pond and a water foun­tain or any one or more of these features, harbouring water loving and marsh plants. Water gardens, no matter, how tiny, are extremely effective in beautifying the landscape.

 

Water lilies (Nymphaea) are the most popular water plants. Another aspect to be considered is the depth of water.  The large tank is around 3-5 m deep at the deepest point.  A reflecting garden pool is preferably shallow with a depth of 25-30 cm. If hydrophytic plants are to be grown, varying depths have to be provided in the same pond, which may range from 15 to 90 cm, the deepest portion allotted to lotus and water lilies.  In shallow pockets and galleries, swamp plants such as Typha and Cyperus are accommodated. 

 

Salvinia, Pista and other floating plants may be conveniently added to this grouping. Provision is necessary to prevent rain water and through it silt entering into the pool. A slightly raised rim to a height of 10 cm will be helpful in this regard. Provision to drain the pool by providing an outlet at its floor level will help to clean it, as often as is necessary 

A focal point is a highlighted, outstanding feature that draws the eye and grabs attention. In the landscape, this can be nearly any type of feature, from plants to structures to hardscaping to ornamentation. Popular landscaping focal points include…

 

 

 

Types of Garden:

A garden is a planned space which is set aside for the display, cultivation and enjoyment of plants as well as other forms of nature. It incorporate both natural and man-made materials. Depending on the layout, the gardens are categorized into two types viz., formal garden and informal garden.

 

Formal gardens

A formal garden is a neatly ordered garden having geometric and symmetric patterns which are laid out carefully in planned matter. The simplest formal garden would be a box-trimmed hedge enclosing a flowerbed of simple geometric shape. The most elaborate formal gardens contain radiating avenues, path of gravel, lawns, plant-beds, statuary, water pools of geometric shapes with fountains etc.

Trees, shrubs and other foliage in a formal garden should be carefully arranged, shaped and continually trimmed.

 

The basic principles which should be observed while constructing the formal garden are as follows:
 Symmetry: Symmetrical layout the main principle behind formal garden. Symmetry enables the garden to impose order, balance and harmony onto the changing canvas of nature.

 

Flat Ground Plane: formal garden requires a flat plane to create the most visual impact.

 

Well-Planned Pathways: Ideally, paths are wide enough to accommodate two people side by side. Gravel, stone, and brick are good choices for paving. Grass paths also work well for formal gardens.

 

Strong Axis: Paths provide visual sight lines or axes that lead to dramatic end points, which are typically punctuated with statue, gate that arrest and captivates the eye.

 

Defined Borders: One of the most intriguing aspects of any formal garden is its enclosure. Many formal gardens are conceived as a series of rooms defined by tall, clipped hedges or brick walls.


A typical formal garden shows presence of following garden features:

Terrace:

In gardening, a terrace is an element where a raised flat paved or graveled section overlooks a prospect. A raised terrace keeps a house or main building dry and provides a transition between the hard materials of the architecture and softer ones of the garden.

 Pathways: Pathway facilitates the moving in a garden. Garden paths are the routes, which connect different parts of the garden and give access to other garden features and resources that reside in it. While serving the practical purpose, paths also add aesthetic appeal to the gardens. Many gardens are highlighted by their paths that accentuate the planning and other features.


Topiary: Topiary is the art of creating sculptures in the medium of clipped trees, and shrubs. The trees and shrubs used in topiary are evergreen have small leaves or needles with dense foliage and show compact or columnar growth habits. Common plants used in topiary include Pinus spp, Thuja spp., Taxus spp., Polyalthia spp., Eugenia spp Etc. Shaped wire cages are sometimes employed in modern topiary to guide untutored shears, but traditional topiary depends on patience and a steady hand; small-leaved ivy can be used to cover a cage and give the look of topiary in a few months.

 

Hedge: A hedge is a line of closely spaced shrubs and tree planted and trained in such a way, that they form a barrier or mark the boundary of the area. It is a simple form of topiary. Hedges used to separate a road from the adjoining fields or one field from another.

 

Arches: It is one of the important garden features. It is an arc which is supported by pillars and covered totally by clipping and training the climbing plants like Ipomoea spp., Rose spp., Clitoria spp. Etc.


Pergola: A pergola is a garden feature forming a shaded walk or passageway of pillars that support cross beams and a sturdy open lattice, upon which woody vines are trained. Pergolas may link pavilions or may extend from a building’s door to an open garden feature such as an isolated terrace or pool. Sometimes, it may be entirely free-standing structures providing shelter and shade to a length of walkway.

 

Statuary: Sometimes the statuary also becomes a part of the formal garden and beautifies it. A statue is a full-length sculpture of a person, an animal or an event, which is close to life-size or larger. Statues serve dual purpose. They serve as memorials of great people who have contributed to the welfare of the society or as decorative status not only to beautify the garden but also the edification of the visitor.

 

Landscaping: It refers to any activity that modifies the visible features of an area of land. Landscaping is the use of ornamental plants & other elements to fulfill aesthetic & functional purposes. It is both science and art which requires good observation and designing skills. A good landscaper first understands the elements of nature as well as construction and then blends them accordingly.

 

Pavilion: It refers to a free-standing structure sited at a short distance from the main residence, whose architecture makes it an object of pleasure. They often resemble to small classical temples and are built for pleasure and relaxation.

  

Parterre: A parterre is a formal garden construction on a level surface consisting of planting beds, edged in stone or rightly clipped hedging and gravel paths arranged to form a pleasing symmetrical pattern. Many times, it becomes a part of open theatre and ‘orchestra seats’ or ‘stalls’.


Sylvan theater: A sylvan theater which is also knows as Greenery Theater is a type of outdoor theater, situated in a wooden setting. Often adorned with classical columns and statues, a sylvan theater may substitute the lawn and can be used for seating. Sylvan theater includes elaborate arrangements of shrubs, flowers and other greenery.


Informal gardens

Informal garden is an exotic attempt to mimic the nature. It is a landscape casually designed with few straight lines having a nice mixture of foliage, colors, textures, heights and varieties. Informal gardens act as a temporary sanctuary that offers a relaxed ambience from day-to-day stressful life.

Types of Informal Garden

Within the informal garden styles, variations have been evolved. Cottage gardens, woodland gardens and meadow gardens are a few of these variations.

Cottage Gardens/Kitchen Gardens

These are the real working gardens that yield edible crops including fruits, vegetables, herbs as well as flowers. Flowers are the essential part of any working garden as they attract bees and other insects to the vegetable garden to ensure good crop pollination. Cottage garden flowers also attract birds that help control harmful insect populations. Cottage Gardens traditionally have pathways weaving throughout the garden beds to facilitate tending and harvesting crops. Informal fencing and operational gates prevent domestic and wild animals from traipsing through the gardens and destroying the crops.

 

 Wild & woodland Gardens

Wild and woodland garden is a naturalistic style designed to provide a beautiful and relaxing type of garden and provide a habitat suitable not only for plants but also for local animals. These gardens usually include a water source and safe shelter to attract a range of wildlife. Small trees and shrubs provide an abundance of nesting place for birds and hibernating insects. Suitable plants include those that retain their seeds through the winter as a food source for wildlife.

Meadow & Wildflower Gardens

Meadow and wildflower gardening is a modern type of informal gardening. It uses localized plants in a garden setting. These types of gardens are often the best way to plant an area that doesn’t lend itself to more conventional cultivation. Wildflower gardening is helpful to conserve native species threatened by erosion of natural habitats. It’s not possible to create habitats exactly, but growing even small area of wildflowers contributes to the conservation and attracts varieties of insects and other beneficial wildlife into the garden.


National Park:

National parks are areas that aim to protect the natural environment. They are also involved in public recreation and enjoyment activities. In a national park, the landscapes and its flora and fauna are present in their natural state.

 

Sanjay Gandhi National Park.

 

        Sanjay Gandhi National Park was called 'Krishnagiri Park' before 1947 and 'Borivali National Park' from 1947 to 1990. The park was spread over a relatively smaller area of 20.26 square km till 1969, post which it was increased to 104 square km by acquiring various forest reserves adjoining the park. The name was changed again in 1990 and it was rechristened 'Sanjay Gandhi National Park' in the memory of Sanjay Gandhi .

The presence of 2400-year-old Kanheri caves, carved out from rock cliffs, in its premises is another added attraction of this place. The fascinating greenery of this park allows great moments of meditation and self-reflection. The gorgeous sight of the lakes, river, valleys and hills provide an in-depth therapeutic effect on the body and mind.


 Flora & Fauna

Every visitor who comes here enjoys the great panorama of the lush greenery with a remarkable collection of birds, butterflies and various kinds of fauna including the wild leopard. This reputed park is a natural habitat for 36 varieties of mammals, 62 species of reptiles, 5,000 kinds of insects and 800 diverse flowering plants. One would find a varied collection of wild animals including Bonnet and Rhesus monkeys, Indian Hare, Sambar deer, Gray Lungur, Chital, Macaque and many more. Another major attraction of this place is the presence of Atlas Moth, world's largest moth. Karvi, an exotic variety of flora is also found here which blossoms only once in eight years.

 

Interesting facts about Sanjay Gandhi National Park

 

 

Botanical Garden:

 

The botanical garden or botanic garden is a garden dedicated to the collection, cultivation, preservation and display of an especially wide range of plants, which are typically labelled with their botanical names. Botanical Gardens have collections of living flora species for reference. Plant species in these gardens are grown for identification purposes and each plant is labelled indicating its botanical name and its family. The famous botanical gardens are at Kew (England), Indian Botanical Garden, Howrah (India) and at National Botanical Research Institute, Lucknow(India).

 

History of Botanical Gardens

The gardens are as old as human development. The man started to cultivate plants in gardens, to feed himself conveniently with food, to provide drugs, or to cultivate beautiful flowers. Indeed veritably primitive tribes engage in vegetable gardening and frequently, unexpectedly, flower gardening.

In classical civilization, gardens were important features on the grounds of temples or palaces, as well as of the homes of the nobility. The number of plants cultivated by the ancient Egyptians was a source of fascination to neighbouring peoples.

The “Hanging Gardens” of Babylon are added up among the admiration of the ancient world. With Renewable and the widening of men’s horizons, the art of gardening prospered as a result of new enthusiasm. Curious and valuable plants from the recently discovered lands brought a new zest for plant introduction. The sixteenth-century herbalists, as we have seen,  familiarize the world with hundreds of plants, numerous of them growing in gardens. Mounting interest in the growing flowers for the beautification of grounds around homes led to the preaface of species from parts of the world. 

Luca Ghini was the first person to establish a botanical garden on scientific criteria in 1543 at Pisa in Italy.

In India, the botanic gardens have existence at a very early date probably as early as 546 B.C. The famous Indian physician Jivaka Komarabhacca who thrive during the region of King Bimbisara of Magadh (modern Bihar) from 546 to 494 B.C. made an intensive survey of the medicinal plants of India. These gardens have been in existence all over India for thousands of years and have been repeatedly mentioned in ancient Sanskrit literature. They are roled as the botanical gardens of the Old World.

The Indian history, which runs through thousands of years, we find that these gardens flourished with the rise of different dynasties and dwindle down with their fall. During the progress of the Mughals, East India Company, and British, botanical gardens prospered and with their fall, the garden decayed. Now with India’s independence, they are again flourishing up. A network of botanical gardens has come up and is functioning throughout the country with intensive botanical activity.

 

Importance of Botanical Gardens

Botanical gardens and Herbaria are important places for methodical study and exploration on the flora of the region. These are places of great academic and profitable significance. A detailed explanation of botanical gardens is follows. Botanical gardens are the institutions that maintain the living plant collections of different kinds of plants, including ornamental and cultivated ones, wild, medicinal, of economic importance, of various geographical regions, of specific interest, etc. They are of great worth not only to the botanists, horticulturists and foresters but also to the millions of visitors.

A vast botanical garden possesses plant species from various corners of the world. It also includes glasshouses, a library, a herbarium, research laboratories, and several variety of resources including photographs, paintings, illustrations, reprints, note-books and specimens of several types, it is, thus, not simply a garden but a botanical institution.

 

Role of Botanical Gardens

1.      Taxonomic Studies-Botanical gardens give precious information on various plants. Local flora, bonsai, rare plants etc. They act as “outdoor laboratories” for scholars and researchers. It helps us to know about our biodiversity, its features and various other information.

2.      Botanical Research- For botanical research  botanical gardens supply a wide range of plant species, seeds, flowers, and fruits. Research on plants enhance our intellectual life and adds to our knowledge about other life supporting processes.

3.      Conservation-Botanical gardens conserve and generate rare species and genetic diversity. Conservating species may help diversity from fading which could help the upcoming generation with their knowledge about plant species.

4.      Education-They supply facilities for courses in local flora, horticulture, hybridization, plant propagation, etc. These educational programmes include workshops, and training sessions for teachers, students, naturalists, etc.

5.      Public Services-They helps the public in identifying the local and exotic plant species; provide instructions for home gardening, and propagation of plants; supply plant resource; through sale or exchange.

6.      Aesthetics and Resources-They attract people who have made gardening their hobby. These gardens also play a essential role in fulfilling human needs and providing well-being.

7.      Employment-They create job opportunities for a large number of young botanists. It makes people employed by providing work at a different levels of jobs.

 

 

Functions

·         Growing important plants of local flora.

·         Providing living plant material for systematic work.

·         Keeping a record of local flora.

·         Supplying seeds and materials for different side of botanical research.

·         Growing and maintaining rare and endangered plants.

 

Examples

1.      Main Botanical Garden, Moscow. The largest garden is spread over an area of 900 acres.

2.      Royal Botanical Garden, Kew (England). It has an area of 300 acres but grows a very huge number of flora species.

3.      Indian Botanical Garden Howrah, India. It is the largest botanical garden in Asia spread over an area of 273 acres.

4.      Lalbag Gardens, Bangaluru (India). Area of 240 acres, this garden was founded by Hyder Ali.

5.      National Botanical Garden, Lucknow (India). The garden has an area of 70 acres.

6.      Veer Mata JijabaiUdyan (Victoria Garden) The garden has an area of 50 acres.

 

 

Veer Mata JijabaiUdyan (Victoria Garden)

The land where Victoria Gardens/Rani Bagh now stands was previously owned by an affluent Jewish businessman, David Sassoon. He instructed the famous Lundons architect to design the Victoria and Albert Museum. A clock tower was also built along with this, which still exists in the garden, but doesn't tell the time anymore. The interiors of this museum are quite similar to the Magen David synagogue of Byculla. Later, David Sassoon donated this land to the Municipal Corporation of Mumbai.

Features

 

Dr Bhau Daji Lad museum

There is a museum with the name Dr Bhau Daji Lad museum in the garden which is a staff building built in Roman style.  It is made of black marble. It is installed near Mumbai University which is known by the name Kalaghoda and David Sassoon Clock Tower.

Features of Byculla Zoo at Veer Mata JijabaiUdyan

 


Unit II: Biotechnology

Biotechnology, the term, is a combination of two words ‘bio’ and ‘technology’, — ‘bio’ means biological systems or processes, and ‘technology’ refers to methods, systems, and devices used to make useful products from these biological systems. Thus, biotechnology refers to the different technologies that make use of living cells and/ or biological molecules to generate useful products for the benefit of mankind.

Biotechnology also defined as the ‘application of scientific and engineering principles to the processing of material by biological agents to provide goods and services’. The Spinks Report (1980) defined biotechnology as ‘the application of biological organisms, systems or processes to the manufacturing and service industries’.

United States Congress’s Office of Technology Assessment defined biotechnology as ‘any technique that used living organisms to make or modify a product, to improve plants or animals or to develop microorganisms for specific uses’.

 

Introduction to plant tissue culture

 

Plant tissue culture (PTC) was a new addition to the methods of plant breeding that developed around the 1950s. It has a great significance in plant biotechnology especially in the crop improvement programmes. It is gaining attraction as an effective propagation method that embraces cell technology to propagate new plants in artificial environments using tissue fragments from plant media. It is becoming as an alternative means to vegetative propagation of plants. In vitro growing plants are usually free from bacterial and fungal diseases.

PTC is the in vitro aseptic culture of cells, tissues, organs or whole plant under controlled nutritional and environmental conditions often to produce the clones of plants. The resultant clones are true-to-type of the selected genotype. Plant Tissue Culture (PTC) is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. It is widely used to produce clones of a plant in a method known as micropropagation.

 

PTC is widely used in – 

Obtaining disease free plants

Rapid propagation of plants those are difficult to propagate

Somatic hybridization

Genetics improvement of commercial plants

Obtaining androgenic and gynogenic haploid plants for breedingprogrammes

 

Basic Requirements of PTC

The main requirements of plant tissue culture are:

 Laboratory Organisation

 Culture Media

Aseptic Conditions

 

 

Laboratory organization and techniques in PTC:

Laboratory organization:

Tissue culture laboratory, whether for research or for commercial purpose, must have provision for certain basic facilities.

The laboratory should consist of:

1) Store/storage facilities for keeping glassware & chemicals

2) Washing area

3) Area for media preparation

4) Autoclave for sterilization

5) Media Store

6) Laminar air flow cabinets for aseptic manipulations

7) Growth rooms for maintaining cultures under controlled conditions of light and temperature

8) Area for observations of cultures

9) Transfer area

10) Acclimatization

 

1.      Store/storage facilities for keeping glassware & chemicals:

PTC requires large numbers of chemicals; variety of glassware and plasticware and inventory of the same needs to be maintained. Some of the chemicals are not available locally and are thus imported calling for maintaining adequate stock. Like any other store, shelves are integral part. Also, some of the chemicals are to be stored at low temperature and thus provision for double door refrigerator/ deep freezer is made.

 

2.      Washing area

The washing area should have adequate supply of good quality running tap water. Depending upon the size of the laboratory, washing area can either have separate tanks for soaking or used glass ware/ plasticware or may manage with plastic buckets / tubs.

The first step in washing involves dipping in mild acid solutions (not always essential) followed by dipping in soap solution; manual / brushing on the motor washing; washing with tap water and final rinse with good quality water.

 

3.      Area for media preparation

Media room can be considered as the kitchen of the tissue culture facility. Usually, it consist of a working table in the centre and benches along the walls. The tops of the working table is usually covered with wood / laminated board. Use of granite / stone is avoided as it leads to brackage of glassware with minimal of mishandling. The benches are required to keep small equipments such as balances (top pan macro balance and analytical micro) for weighing chemicals; pH meter; magnetic stirrer; hot plate; media dispenser; gas stove; etc.

 

4.      Autoclave for sterilization

Autoclave is either kept inside the media preparation area or is kept in a separate room. While the media preparation room can be air-conditioned, autoclave, it is kept in a well ventilated room as it generates lot of heat. At pressure of 15 Ibs; and temperature of 121° C for 15 – 25 min. (depending on the quantity of media), the media gets sterilized. For sterilizing contaminated cultures, 30 – 40 min. duration is chosen. The dry cycle is run for sterilizing instruments, lab coats; plates for dissection, etc.  The duration of dry cycle is usually one hour. To avoid spreading of contaminants, it is advisable to autoclave contaminated jars for an hour and immediately discard agar.

 

5.      Media Store

 In large tissue culture facilities a provision for separate room is made which is fitted with UV lights and adequate shelves. Sterilized media is stored for at least 3 days prior to inoculation. The double door autoclave opens directly in media store and media which it is still in molten stage is shifted to shelves.

 

6.      Inoculation area

While smaller laboratories have transfer hoods fitted with UV lights, laminar air flow cabinets are common. They can be kept either in a quiet corner of the general laboratory or in larger laboratories separate transfer room is created. Transfer room has high level of cleanliness and is comparable to operation theatre in a hospital as all aseptic manupulations are to be carried out here. This area has restricted entry and all those working here follow hygine.

 

7.      Laminar air flow cabinets for aseptic manipulations

All the aseptic manipulations are carried out inside the Laminar Airflow cabinets. There is a unidirectional flow of sterile air through HEPA filters that flows in the cabinet where cultures are prepared and transferred to the media. They are also fitted with UV lights to prevent growth of microbes in the cabinet while it is not in use. The culture media / mother cultures are provided to operators / researchers on a trolley which also serve as a size bench for them.


It is advisable to have fire extinguishers in this area as there is use of heat / fire and rectified spirit. Till few years back, spirit lamps were used to sterilize instruments that were dipped in rectified spirit and flamed. However, these have been replaced with hot bead sterilizer where temperature of 200 °C. is maintained and after every operation, instruments are kept in hot beat steralizers for a couple of minutes to sterilize them. The laminar air flow cabinets are monitored continuously for the air pressure and strict schedule is followed for cleaning for pre-filters. While working, operators surface sterilize their hands and arms with rectified spirit; wear cap and face mask to avoid any infection.

 

8.      Growth Rooms:

Growth rooms are rooms in which culture are maintained under controlled conditions of light and temperature. The growth room typically has one entry and has no windows. To maintain high level of cleanliness this room is maintained by passing air through filters; has plastic paints on the walls and have air ducks opening at individual shelf level to maintain uniform temperature. The temperature is constantly maintained and fluctuation of as low as ± 2 ° C is premisible. Usually light is provided by cool white florecent lamps which is gradually getting replaced by CFL / LED bulbs. Depending on the nature of the experiments, shaking machines (horizontal / rotary) are prevalent. BOD incubators with or without shaking provisions are also used in research laboratories. You will learn about specific requirements for suspension culture / single cell culture/ protoplast culture in the subsequent chapters.

 

9.      Area for observations of cultures:

While in research laboratories no separate provision is made in commercial laboratories, a separate room is made for taking observations and observing cultures under the microcopes. Compound microscope enables detection of bacteria and fungi in culture; a provision for stereo microscope can also be made for dissecting small size meristematic dome for getting clean explants from virus infected plants. However, these operations are carried out in laminar flow cabinet.

 

 

10. Transfer area:

A provision for separate room is made where cultures are washed of agar and transplanted to potting mix. Many a times, potting mix is sterilized & thus provision for boiler is made. The plantlets are usualy transferred in portrays.

 

11. Acclimatization

The plantlets produced under in vitro conditions, although green in colour, does not photosynthesize effectively and lack mechanisms to control water loss. These plantlets thus upon transplantation, must be gradually and carefully acclimatized to the natural conditions.

 

12. Green House:

In tissue-culture propagation, greenhouse is required to raise and maintain mother plants; and to harden the plantlets gradually to the natural environment. In the greenhouse, the humidity near the pad is close to 95 – 100% and at the other end (far side) is ~ 80%. The temperature of the greenhouse is usually ± 10 ° C of the ambient temperature. During certain periods (peak summer and winter), providing conducive temperature is a challenge as most species thrive between 20 – 30 ° C.

 

Media preparation

Tissue culture media can be either prepared or brought as ready mix. As said earlier, while developing a media, one has to test and alter individual nutrients / growth regulators and thus a large number of combinations are to be tested and thus prepared. Also, for a commercial tissue culture lab, where the requirement is in bulk, the ready-made media proves to be far more expensive than the one which is prepared with individual ingredients. However, in countries where skilled manpower is either not available or is far too expensive, standard plant tissue culture media, especially for micropropagation are being used. More commonly, stock solutions are prepared and kept for usage over a period of time.

 

For stock solutions, the usually,

1) major salts at 20 x concentration; 50 ml to be used for each liter of the media to be prepared; 2) minor salts at 200 x concentration; 5 ml per liter to be used;

3) Iron at 200 x concentration; 5 ml. to be used per liter and

4) Organic nutrients except sucrose at 200 x- 5 ml per liter to be used.

Sugar is weighted and added to water. If it is liquid media, the growth regulators dissolve in either dilute HCl or dilute solution of NaOH and stock solutions is prepared at 1 µmole per liter or 10 µmole per liter concentration. The individual salts are to be mixed in water and added in a particular sequence, making final volume of 1 liter for major salt, minor salt, iron and organic nutrients and 100 ml in case of growth regulators. The iron stock is kept in brown colour bottles so as to prevent photooxidiatation. The stock solutions are usually stored in the plastic / glass bottles and stored in refrigerator. If they are to be used over a longer period of time, then they can be stored in the deep freeze.

Before using the stock solutions they are to be thoroughly shaken and checked for any contamination and precipitation. For preparing stocks solutions, for usual tissue culture single distilled water is used but in case of protoplast / genetic transformation / nanotechnology etc., double distilled water is used. In case of micropropagation to reduce cost of media, RO water is also used.

 

Various steps to prepare media are as follows:

1) Weight required quantity of gelling agents, sugar source and add them to distilled water (three fourth of the total volume of the medium), and put them for melting. It can be achieved either on a gas stove; water bath; or in an autoclave. As soon as the temperature of 100° C is reached autoclave is switched off. On the gas stove or water bath, melting is achieved with constant stirring.

2) Add the stock solutions as per the stock concentration, growth regulators are also added at this stage. The pH is adjusted (usually to 5.8) using 0.1 NaOH or 0.1 N HCl and media dispensed into glass vials for sterilization. However, If any, thermolabile substances is to be added then media volume is made minus the amount of the thermolabile substances to be added.

3) The media are dispensed either manually or through a dispenser. Precaution is to be taken to ensure that uniform quantity of media is dispensed in each vial and the media is poured while it is still in the liquid form.

4) The culture vial is closed either with a lid or with cotton plug. The lids / plugs permits the free exchange of gases, but restricts entry of the microbial contaminants.

5) The prepared media, vials are then put in iron autoclavable plastic racks / baskets; covered with aluminium foil and put for sterilization in an autoclave at 120° C (1.06 kg cm-2 ). Depending on the size of the vial and the quantity of the media, autoclaving time can vary from 15 to 25 minutes. In case thermolabile substance is to be added, the vials with lids such as petridishes / jars are sterilized.

6) The thermolabile substance is added through the filter membrane with pour size 0.22 – 0.45 µm through filter assembly, its pH is checked and media is poured into presteralized vials. All these operations are carried out in laminar air flow cabinets.

7) The media is allowed to cool at room temperature. Usually, the culture rack is put in a slanting position so that slants are formed, thus increasing the surface area.

8) Media is stored in media store room for three days so as to check for any contamination.

9) If any contamination occurs, it is to be taken seriously as there could have been a problem associated with the autoclaving cycle. Usually, the entire batch of the media of that particular autoclave cycle is discarded. It is important to maintain history and maintain stock details of who prepared the stock and on which date. Sometimes if stocks are not prepared properly the results are visible across all the cultures and it is important to trace back the history and take timely action. If due to any reason the cultures on a pre-defined media are not growing properly, then cultures are to be transferred on to a fresh media without losing any time.

 

 

Totipotency:  Cell potency is a cell ability to differentiate into other cell types. The more cell types a cell can differentiate into, the greater its potency. Potency is also described as the gene activation potential within a cell.

Definition: Totipotency (Lat. totipotentia, "ability for all [things]") is the ability of a single cell to divide and produce all of the differentiated cells in an organism. Example, Spores and zygotes are a type of totipotent cells.

Types of totipotent:

The term “totipotent” has two basically different interpretations:

(i) capable of developing into a complete organism or

(ii) capable of differentiating into any cell types of an organism.

 

How totipotent cell regenerate:

 A plant after prolonged culture, it has been observed that calluses in some species (e.g. Ntcotiana tabacum, Citrus aurantifolia etc.) maybe- come habituated. This means that they are now able to grow on a standard maintenance medium which is devoid of growth hormones.

The cells of habituated callus also remain totipotent and are capable to regenerate a plant without any major manipulation.

A typical crown gall tumour cell has the capacity for unlimited growth independent of exogenous hormones. It shows totally lack of organ genic differentiation. So such tissue is considered to have permanently lost the totipotentiality of the parent cells.

Importance of Totipotency in Plant Science:

• The ultimate objective in plant protoplast, cell and tissue culture is the reconstruction of plants from the totipotent cell. Although the process of differentiation is still mysterious in general, the expression of totipotent cell in culture has provided a lot of information’s.

• On the other hand, the totipotentiality of somatic cells has been exploited in vegetative propagation of many economical, medicinal as well as agriculturally important plant species. Therefore, from fundamental to applied aspect of plant biology, cellular totipotency is highly important.

• Recent trends of plant tissue culture include genetic modification of plants, production of homozygous diploid plants through haploid cell culture, somatic hybridization, mutation etc. The success of all these studies depends upon the expression of totipotency. In many cases, successful and exciting results have been obtained.

• Plant breeders, horticulturists and commercial plant growers are now more interested in plant tissue culture only for the exploitation of totipotent cells in culture according to their desirable requirement. Totipotent cells within a bit of callus tissue can be stored in liquid nitrogen for a long period. Therefore, for germplasm preservation of endangered plant species, totipotency can be utilized successfully.

 

Organogenesis:

Organogenesis involves the regulation of cell division, cell expansion, differentiation of cell and tissue type, and patterning of the organ as a whole. Although it is highly essential to have insight into how cells are differentiating and developed to give rise to a complete organ, however, it is often directed to technical difficulties as these processes landed deep in tissues and thus are difficult to access or visualize. An adult plant consists of highly organized tissues and organs; tissue like meristem, cortex, phloem and epidermis which possess the cells of uniform shape and specialized function and several of these tissues are organized together to form an organ, such as leaves, roots, flowers and the vascular system. The process of initiation and development of an organ is called organogenesis which is an important way to regenerate plants from the culture. The two distinct phases of Organogenesis in plant tissue culture involves firstly dedifferentiation and secondly redifferentiation. Dedifferentiation deals with the isolation of the explants tissues with an acceleration of cell division and a consequent formation of a mass of undifferentiated cells (called callus).

The developmental processes occur after the first callus formation is called by the name redifferentiation. In this process, the tissue called organ primordia is differentiated from a single or a group of callus cellsis generated into small meristems with cells densely filled with protoplasm and strikingly large nuclei. The Polarity (monopolar) of the longitudinal axis of the organizing growing points of the organs can be seen some time after the formation of meristem tissues and different types of specialized cells again undergo differentiation to give rise to the vascular system which is formed by connecting the new organs to their parent explants or callus mass.


As we have already discussed about the organogenesis process which relies on the production of organs either directly from an explants or callus structure, this can also be explained by the following characteristics.

• The ability of non-meristematic plant tissues to form different organs de novo.

• The formation of adventitious organs

• The production of roots, shoots or leaves

• These organs may arise out of pre-existing meristems or out of differentiated cells

• This may involve a callus intermediate but often occurs without callus.

 

Events during organogenesis

It is a general rule that the organ formation would be through a process of differentiation in the undifferentiated mass of parenchyma. Most of the parenchymatous cells are highly vacuolated and with inconspicuous nuclei and cytoplasm, sometimes with lignification. In this group of cells, regions showing random cell division would occur, leading to radial files of differentiated tissues. These scattered cell division regions would form regions of high mitotic activity resulting in the formation of meristematic centres, otherwise termed as meristemoids. These meristemoids may be either on the surface of the calli or embedded in the tissue. Continued cell division in these meristemoids would produce small protuberances on the surface of the calli, giving nodular appearance to the tissues. From the meristemoids, the primordia of organs by repeated mitotic activity form either shoot or root.

This was discovered by Torrey in 1966. The meristemoids consist of a spherical mass of small isodiametric meristematic cells with dense cytoplasm and a high nucleo-cytoplasmic ratio. Normally, callus tissues accumulate starch and other crystals before organogenesis, but the substances disappear during meristemoid formation.

During the initial stages of meristemoid formation, the cytoplasmic protrusions enter the vacuoles thus distributing the vacuoles around the periphery of each cell or dispersed throughout the cytoplasm. The nucleus is in the centre with maximum possible size. Thus cells in the meristemoids resemble the cells of highly active meristem in tissue culture.

 

 

Organ culture:-

01.  Root cultures:

Root cultures can be established in vitro from explants of the root tip of either primary or lateral roots and can be cultured on simple media. The growth of roots in vitro is potentially unlimited, as roots are indeterminate organs. Although the establishment of root cultures was one of the first achievements of modern plant tissue culture, they are not widely used in plant transformation studies. The tips of shoots which contain the shoot apical meristem can be cultured in vitro, producing clumps of shoots from either axillary or adventitious buds. This method can be used for clonal propagation. Shoot meristem cultures are potential alternatives to the more commonly used methods for cereal regeneration as they are less genotypedependent and more efficient.

 

02.  Meristem cultures:

Culture of an actively dividing meristematic tissue of shoot tip, root tip, vegetative bud etc..

• It involves the regeneration of an entire plant from a meristematic tissue.

• They mostly produce virus-free plants.

• More successful in herbaceous plants than in woody plants. 

Application: Production of virus free germplasm. Mass production of desirable genotypes Facilitation of exchange between locations (production of clean material) Cryopreservation (cold storage) or in vitro conservation of germplasm.

 

01.  Anther and pollen culture:

Culture method in the production of Haploids.

• First successfully carried out by Guha and Maheshwari in Datura.

• Anthers bearing uninucleate microspores are isolated, selected and cultured on a suitable solid medium to form androgenic calluses from the pollen mass through dedifferentiation.

• Calluses undergo re-differentiation and give rise to embryos and haploid plants.

• Rice, Wheat, Maize, mustard, Pepper.


Application: Production of haploid plants Production of homozygous diploid lines through chromosome doubling, thus reducing the time required to produce inbred lines Uncovering mutations or recessive phenotypes

 

01.  Embryo culture:

Culture of isolated immature or mature embryos for producing viable plants.

• Mature embryos are excised from ripened ovule/seeds and cultured mainly to avoid inhibition in the seed for germination. Very small globular embryos require a delicate balance of the hormones.

• Embryo is dissected from the ovule/seed and put into culture media.

• This type of culture is relatively easy as the embryos require a simple nutrient medium containing mineral salts, sugar and agar for growth and development.

• Also, multicellular immature embryos are dissected out and cultured aseptically to obtain viable hybrids. Once the embryo is rescued, two genomes are needed to be combined together to produce a fertile plant.

Embryos can also be used as explants to generate callus cultures or somatic embryos. Both immature and mature embryos can be used as explants. Immature, embryo-derived embryogenic callus is the most popular method of monocot plant regeneration.


Application: Overcoming embryo abortion due to incompatibility barriers Overcoming seed dormancy and self-sterility of seeds Embryo rescue in distant (interspecific or intergeneric) hybridization where endosperm development is poor Shortening of breeding cycle

 

 

Recombinant DNA Technology (R-DNA technology):-

Recombinant DNA is a form of artificial DNA that is made through the combination or insertion of one or more DNA strands, therefore combining DNA sequences as per requirement, within different species.

In order to this understand how a fragment of DNA, representing a genetic code is involved in proteins synthesis. • i.e. mRNA transcription from this DNA fragment followed by translation involving rRNA and tRNA that carries the amino acid.

In genetics recombinant DNA technology has many uses

e.g: Agriculture: growing crops of your choice (GM food), pesticide resistant crops, and fruits with attractive colors, all being grown in artificial conditions.

Pharmacology: artificial insulin production, drug delivery to target sites

Medicine: gene therapy, antiviral therapy, vaccination, synthesizing clotting factors

Other uses: fluorescent fishes, glowing plants etc

 

Advantages of Recombinant technology:

• Provide substantial quantity

• No need for natural or organic factors

• Tailor made product that you can control

• Unlimited utilizations

• Cheap

• Resistant to natural inhibitors

 

Disadvantages of Recombinant technology:

• Commercialized and became big source of income for businessmen

• Effects natural immune system of the body

• Can destroy natural ecosystem that relies on organic cycle

• Prone to cause mutation that could have harmful effects

• Major international concern: manufacturing of biological weapons such as botulism & anthrax to target humans with specific genotype

• Concerns of creating super‐human race

 

Steps of Recombinant DNA Technology

1. DNA Isolation 

·         DNA is isolated in its pure form, which means they are devoid of other macromolecules.

·         In rDNA technology, the initial step is to extract the desired DNA in its purest form, that is, free of extraneous macromolecules.

·         Because DNA coexists with other macromolecules such as RNA, polysaccharides, proteins, and lipids within the cell membrane, it must be separated and purified using enzymes such as lysozymes, cellulase, chitinase, ribonuclease, and proteases.

·         Other enzymes or treatments can remove other macromolecules. The DNA eventually precipitates out as fine threads as a result of the presence of ethanol. After that, the pure DNA is spooled out.

 

 

 2. Cutting of DNA/Restriction Enzyme Digestion

·         For this step, the restriction enzymes are quite vital. It helps to identify the location wherein a designated gene is introduced into a vector genome. The said reaction is known as restriction enzyme digestions. 

·         They entail incubating pure DNA with a restriction enzyme of choice at conditions that are appropriate for that enzyme.

·         The 'Agarose Gel Electrophoresis' technology displays the restriction enzyme digestion's progress.

·         This method entails passing the DNA across an agarose gel. When current is applied, negatively charged DNA flows to the positive electrode and is divided into different sizes. This permits the digested DNA fragments to be separated and snipped out.

·         The same method is used to process the vector DNA.


3. Amplifying of DNA

·         Copies of genes are amplified through PCR or polymerase chain reaction. It is essentially a process to increase a single DNA copy into several copies after the desired gene of interest is cut with restriction enzymes. 

·         It allows a single copy or a few copies of DNA to be amplified into thousands or millions of copies.

·         The following components are used in PCR reactions that are conducted on 'thermal cyclers':

1.      Template: DNA that has to be amplified.

2.      Primers: oligonucleotides are tiny, chemically produced oligohnucleotides that are complementary to a DNA region.

3.      Enzyme: DNA polymerase.

4.  Nucleotides: The enzyme is required to lengthen the primers.

·         PCR can be used to amplify the cut DNA fragments, which can subsequently be ligated with the cut vector.

 

4. Joining DNA

·         The vector and a section of DNA are joined in this step. It is achieved with the help of the enzyme DNA ligase. 

·         With the same restriction enzyme, the pure DNA and the vector of interest are cut.

·         This yields the cut DNA fragment and the cut vector, both of which are now open.

·         Ligation is the process of putting these two parts together with the enzyme 'DNA ligase.'

·         The resulting DNA molecule is a hybrid of the interest molecule and the vector DNA molecules. Recombination is the term used in genetics to describe the merging of different DNA strands.

·         As a result, this new hybrid DNA molecule is known as a recombinant DNA molecule, and the process is known as recombinant DNA technology.

 

5. Insertion of rDNA into a Host

·         Here rDNA is added to the recipient host cell, and the entire process is called transformation. Post insertion, the recombinant DNA multiplies and manifests as manufactured protein under favorable conditions.

·         The recombinant DNA is then transferred into a recipient host cell, most commonly a bacterial cell, in this stage. The term for this procedure is 'Transformation.'

·         Bacterial cells have a hard time accepting foreign DNA. As a result, they are given treatments to make them 'capable' of accepting new DNA. Thermal shock, Ca++ ion therapy, electroporation, and other procedures may be applied.

 

6. Recombinant Cell Isolation

·         A mixed population of converted and non-transformed host cells results from the transformation process.

·         Only the transformed host cells are filtered during the selection procedure.

·         The marker gene of the plasmid vector is used to distinguish recombinant cells from non-recombinant cells.

·         PBR322 plasmid vector, for example, comprises two marker genes (Ampicillin resistant gene and Tetracycline resistant gene). When pst1 RE is utilised, it eliminates the Ampicillin resistance gene from the plasmid, causing the recombinant cell to become Ampicillin sensitive.


Gene cloning:

The term ‘clone’ means, exact copy of the parent. A duplicate or a look alike carrying the same genetic signature or genetic map. Gene cloning is the best application of recombinant DNA technology and could be applied to something as simple as DNA fragment or a larger, sophisticated mammalian specie such as humans.

Enzymes involved in Gene cloning:

Enzymes that modify nucleic acids are used to synthesize, degrade, join, and/or remove portions of nucleic acids. Cloning enzymes are enzymes that are important in nucleic acid cloning procedures. The activities of the cloning enzymes consist of ligases, kinases, phosphatases, and RecA Protein.

1. Ligases are used to join nucleic acid segments, especially when one is cloning a DNA fragment into the vector DNA.

2. Phosphatases remove the 5′-phosphate from nucleic acid strands. This prevents vector downgrading, which would reduce the number of background colonies as well as producing substrate to which a kinase can attach a new radio-labeled phosphate.

3. Kinases add new phosphate groups to nucleic acids. This is usually done in order to label the nucleic acid fragments or the synthetically made oligonucleotide.

4. RecA Protein and AgarACE® Enzyme are used primarily to protect in certain cloning procedures or facilitate the nucleic acid purification. The E. coli RecA Protein is able to facilitate the pairing of homologous DNA sequences. AgarACE® Enzyme is a patented agarose-lysing enzyme produced for the harvest of DNA from agarose gels.

 

Vectors used for Gene cloning:

Genetic vectors are vehicles for delivering foreign DNA into recipient cells. • In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed.

 • Vectors can replicate autonomously and typically include features to facilitate the manipulation of DNA as well as a genetic marker for their selective recognition.

 • The different types of vectors available for cloning are plasmids, bacteriophages, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).

• The cloning vectors are limited to the size of insert that they can carry. Depending on the size and the application of the insert the suitable vector is selected for a particular purpose.

Essential Characteristics of Cloning Vectors:

Regardless of the selection of a vector, all vectors are carrier DNA molecules. These carrier molecules should have few common features in general such as:

• It must be self-replicating inside host cell.

• It must possess a unique restriction site for RE enzymes.

• Introduction of donor DNA fragment must not interfere with replication property of the vector. • It must possess some marker gene such that it can be used for later identification of recombinant cell (usually an antibiotic resistance gene that is absent in the host cell).

• They should be easily isolated from host cell

 

Types of vector:

01.  Plasmids:

Plasmids are extra chromosomal circular double stranded DNA replicating elements present in bacterial cells.

• Plasmids show the size ranging from 5.0 kb to 400 kb.

• Plasmids are inserted into bacterial calls by a process called transformation.

• Plasmids can accommodate an insert size of upto 10 kb DNA fragment.

• Generally, plasmid vectors carry a marker gene which is mostly a gene for antibiotic resistance; thereby making any cell that contains the plasmid will grow in presence of the selectable corresponding antibiotic supplied in the media.

 

02.  Bacteriophage:

The viruses that infect bacteria are called bacteriophage. These are intracellular obligate parasites that multiply inside bacterial cell by making use of some or all of the host enzymes.

• Bacteriophages have a very high significant mechanism for delivering its genome into bacterial cell. Hence it can be used as a cloning vector to deliver larger DNA segments.

 • Most of the bacteriophage genome is non-essential and can be replaced with foreign DNA.

• Using bacteriophage as a vector, a DNA fragment of size up to 20 kb can be transformed.

 

03.  Cosmids: A cosmid, is a type of hybrid plasmid with a bacterial “ori” sequence and a “cos” sequences derived from the lambda phage.

• It is a derived vector.

• The cosmid DNA can be packed in a capsid of lambda phage in vitro to form recombinant phage particles.

• The cosmid gets circularized and behaves like a plasmid.

• Cosmid has an origin of replication, selectable markers, and gene cloning sites of plasmid DNA.

• They lack structural and regulatory genes of lambda DNA.

• Hence there is no lysis and integration of cosmid DNA in the host cell.

commonly used cloning vector suitable for cloning large DNA fragments upto 45 kbp.

 

04.  Bacterial artificial chromosomes (BACs):

Bacterial artificial chromosomes (BACs) are simple plasmid which is designed to clone very large DNA fragments ranging in size from 75 to 300 kb.

• BACs basically have marker like sights such as antibiotic resistance genes and a very stable origin of replication (ori) that promotes the distribution of plasmid after bacterial cell division and maintaining the plasmid copy number to one or two per cell.

• BACs are basically used in sequencing the genome of organisms in genome projects (example: BACs were used in human genome project).

• Several hundred thousand base pair DNA fragments can be cloned using BACs.

 

05.  Yeast artificial chromosomes (YACs):

YACs are yeast expression vectors.

 • A very large DNA fragments whose sizes ranging from 100 kb to 3000 kb can be cloned using YACs.

• Mostly YACs are used for cloning very large DNA fragments and for the physical mapping of complex genomes.

• YACs have an advantage over BACs in expressing eukaryotic proteins that require post translational modifications.

• But, YACs are known to produce chimeric effects which make them less stable compared to BACs.

 

Uses of Vector:

Vectors have been developed and adapted for a wide range of uses. Two primary uses are:

(1) to isolate, identify and archive fragments of a larger genome

(2) to selectively express proteins encoded by specific genes. Vectors were the first DNA tools used in genetic engineering, and continue to be cornerstones of the technology.