|
Techniques in the Collection of Root Exudates |
|
|
Techniques in the Collection of Root Exudates
From time to time methods of raising plants aseptically have been described in the literature and some of them are illustrated in. Many of them can be used successfully to collect root exudates under aseptic conditions. Initially, seeds are surface sterilized with anyone of the following chemicals: mercuric chloride, calcium or sodium hypochlorite, freshly prepared chlorine water, hydrogen peroxide, sulphuric acid and ethyl alcohol. An ideal choice for surface sterilization of seeds with soft seed coat is chlorine water, prepared afresh each time until the water receiving chlorine gas turns yellow.
Concentrated sulphuric acid has been used with success for seeds having tough seed coat such as cotton and clovers. It is essential to use dry test tubes or beakers while surface sterilizing seeds with sulphuric acid and it is also advisable that subsequent washings with sterile water must be made as quickly as possible so as to prevent damage to seed by excessive heat generated by the addition of water to acid. In all cases, the surface sterilized seeds have to be necessarily washed in several changes of sterile water before planting.
|
It is emphasised that all parts of the equipment involved in the techniques are sterilized prior to use. Similarly, all the transfers and handling are done in a sterile room under aseptic conditions to prevent microbial contamination.
In the technique referred to in A, a seedling is raised initially in a test tube and then transferred to a one litre capacity Erlenmeyer's flask filled with the nutrient solution and stoppered with a cotton plug in such a way that the sterilized cotton plug separates the root system from the shoot system.
A three-neck distilling flask (capacity 300 ml) serves as a root chamber in the assembly referred to in B. The centre neck of the flask is used for growing plants in a planting tube inserted into the neck while the side tubes are used as inlet and outlet for aeration. The reservoir of nutrient solution is connected to one of the side opening of the root chamber by means of rubber and glass tubing.
An ordinary glass jar with a metal screw type lid serves the purpose for the technique referred to in C. A hole (3.5 cm diameter) is punched in the centre of the metal lid and an aluminium tube fitted on the upper side of the hole. On the inside of the screw type lid of the jar, a disc of aluminium sheet with a central hole (similar to the one on the screw type lid) is placed after sandwiching a piece of cotton gauze in between the lid and the disc. A gauze wrapped cotton plug is inserted into the aluminium tube fitted on top of the lid.
The entire apparatus is assembled as shown in the figure after pouring nutrient solution into the jar. The seedling is raised in the aluminium tube and when it grows to a sufficient height, the cotton plug is removed and a small amount of sterilized sand or vermiculite is placed around the seedling to separate the root system from the shoot system.
|
One end of a glass vessel open at both ends is plugged with cotton wool over which a small amount of sand is placed. This serves as a seedling chamber in the technique referred to in D. The entire vessel is placed in a two-litre conical flask in such a way that the seedling chamber is half-way immersed in the nutrient solution and the growing plant would then be automatically fed with nutrient by the capillarity action of the cotton gauze and the sand. A mechanical aerating device.
In the technique referred to in E, a glazed pot or jar is used to grow plants with arrangement for drainage of excess nutrient by means of a flask attached to the lower end of the pot. A large glass cylinder, stoppered at the top with a large cotton plug and sealed with paraffin is placed over the glazed pot. An aluminium tube is made to pass through the cotton plug, then through the glass cylinder and end up as a loop onthe soil jar. The loop portion of the aluminium tube is drilled with holes. The upper end of the tubing is connected to two aspirator bottles which serve as reservoirs of nutrient. Seeds are sown on sand in the glazed pot and plants are raised by releasing nutrient by means of stoppers in the aspirator bottle assembly.
|
|
| Methods for aseptic culturing of plants |
|
|
a) sterile culture tube
b)1% agar
c) Erlenmeyer's flask
d) sterile cotton
e) outer tube
f) germination tube
g) nutrient solution |
a) root chamber
b) planting tube
c) nutrient solution reservoir
d) to air supply
e) air outlet
f) air filters
g) hood
h) cotton plug
|
|
|
a) glass container
b) lid
c) aluminium cylinder
d) aluminium disc
e) cotton gauze
f) vermiculite
g) gauze-wrapped cotton plug
|
a) glass vessel open top and bottom containing gravel
b) two litre wide mouth Erlenmeyer pyrex flask
c) air pump
d) Koby air filter
e) glass wool fitted outlet tube
f) air-bubble lift pump
|
|
|
a) 4" glazed soil jar
b) drainage collection flask
c) 100mm glass cylinder
d) cotton plug
e) paraffin seal
f) aluminium watering tube
g) nutrient solution
h) distilled water
i) 20-litre aspirator bottle
|
a) pyrex container
b) pyrex tube open at both ends
c)a layer of soil
d) glass wool
e) an annular ring of cotton
f) a layer of acid washed sand |
|
|
|
|
|
|
K and L methods for growing multiple ans single plants respectively in two stages of operation-h cup to hole the chromel wire mesh
i) vessel to contain nutrient solution
j) plants
k1) inlet for nutrient
k2) inlet for aeration
k3) exit for nutrient
1) lid with an upper limb to hold the shoot
m) orifice
n) lid
o) glass beads and
a) cotton gauze
|
|
|
|
|
Seedling can be grown in natural soil and root exudates collected if the technique referred to in F is used. Soil is filled in a glass tubing open at one end and closed at the other end with glass wool. The glass wool end is placed in a flask containing water and the open end is fitted into the neck of a flask by means of an annular ring of cotton. Seeds are sown on the open side of the tube and when the seedling emerges, sterilized sand is placed around the stem to prevent aerial contamination.
In the technique referred to at G, a boiling tube is filled with nutrient solution and a microscope slide and a cover slip dipped in it are used to raise small seedlings. Approximately 0.2 ml of cooled agar is pipetted over one-half of the slide and a previously germinated seed is placed in position on the agar surface followed by the placement of the cover slip on the radicle. The root system grows in between the glass slide and the cover slip.
|
An ordinary glass jar is again used as a container for growing plants aseptically in the technique referred to in H. The jar is filled with sand and a bent glass tube, also filled with sand, is inserted into the sand. Seeds are sown on sand in the jar and when the seedlings are sufficiently tall, the surface of the sand is covered with a thin layer of paraffin covered glass sand. The bent glass tube serves as a means to provide nutrient solution to plants as and when required.A conical flask with a sigmoid side tube is intended to grow plants wherein surface sterilization of seeds is also accomplished. A seed is placed in the sigmoid tube and a small quantity of surface sterilizing agent poured into it by means of a pipette. By tilting the flask to one side, the sterilant could be poured out and seed rinsed with sterile water before pushing the seed into the flask for subsequent growth.
Cross-tube flasks are shown in different views in J. The two openings of the horizontal portion of the tube system are plugged with cotton. The upper opening of the longitudinal stem is connected to a sigmoid tube by means of a short rubber tube, while the lower opening is continuous with the flask. The open end of the sigmoid tube is covered by cotton plug. The sigmoid tube is intended for surface sterilization and subsequent transfer of seed into the basal portion of the longitudinal stem. A seed is introduced into the sigmoid tube, the cotton plug removed and the surface-sterilizing agent poured over the seed. The plug is reintroduced and after half an hour the sigmoid tube is tilted in a manner so as to draw out the sterilizing agent through the cotton plug. This is achieved by pressing dry cotton wool against the wet cotton plug. Subsequently, the sterile seed is conducted by tilting the set-up suitably until the seed is lodged in the basal portion of the longitudinal stem. On germination, the root will grow into the flask and the stem of the seedling passes through the longitudinal stem of the cross-tube system. At this stage of plant growth, the cotton plugs in the horizontal tube are pushed inwards so that they hold the stem in position. The sigmoid tube and the rubber tubing are then removed.
Many seedlings can be grown in the technique referred to in K. The main vessel has a wide opening at the top with narrow side openings for aeration and drainage. The neck of the wide opening has a chrome wire mesh held in position by a glass cup and this in turn is closed by a glass lid. Seeds are sown on the chrome wire mesh and nutrient solution poured into the main stem of the vessel. When the seedlings grow sufficiently tall, the lid is removed and glass beads are poured on the wire mesh to prevent aerial contamination.
A similar technique L for growing single plants takes advantage of a flask, whose neck is fitted with a special lid having a small hole in the middle as shown in' the figure. As usual, the apparatus has outlets for aeration and passage of nutrients. A previously germinated seed is held gently by a pair of forceps and introduced into the hole and kept in position by means of two glass beads. When the seedling establishes, more glass beads are put in position to prevent aerial contamination.
Two superimposed acid washed filter papers and placed in flasks M and moistened with nutrient solution. Seeds are sown on the filter paper and when seedlings have established the filter papers are removed, ground with acid washed sand and extracted for recovery of root exudates. This appears to be the simplest technique so far described.
All the techniques have been designed to grow plants free of microorganisms and therefore periodical checking of the nutrient solution by plating it on agar media is a necessary step in maintaining the efficiency of the technique employed. The techniques could be divided into two lots: (1) where root and shoot systems grow in the same environment, and (2) where both the systems are separated so as to grow the root system aseptically and the shoot system free in the air. Cotton wool, special wax and glass beads have been used to achieve the separation between the root and shoot systems.
|
|
Submit Your College, Institute, Company, Products for FREE
