In this article we will discuss about the Plant Tumours and Virus Eradication Procedure.
Plant Tumours:
Tumour means uncontrolled growth or proliferation of dis-organised tissues. After its initiation the tumour can grow even in absence of the inciting agent, such as, virus, bacteria, chemical substances etc. Braun (’42) studied plant tumours.
Later various scientists induced tumours on susceptable plants by introducing Agrobacterium tumefaciens through wounds. The tumour inducing substance of this bacterium is a small T-DNA or Ti plasmid. It occurs only in the virulent strains of A. tumefacians. Three types of tumours are usually studied in vitro.
These are:
(a) Crown gall tumour of sunflower produced by Agrobacterium tumefaciens,
(b) Genetic tumours occurring in some tobacco hybrids and
(c) Wound tumours produced by Aurogertus.
All these tumours show some common features. These features are:
(i) Host tissue must be damaged before tumour formation,
(ii) Tumour tissue if grafted on healthy plant it Continues to grow,
(iii) Tumour free from inciting agent when placed on a simple hormone free medium it grows rapidly.
This indicates that tumour tissue produces more hormone than normal tissue and thus causes abnormal growth and proliferations. Virus, bacteria and genetic instability may induce tumour formation.
Crown gall tumour (Fig. 27) is produced by Agrobacterium tumefaciens. Wounding and presence of inciting agent produce tumour. Working with A. tumefaciens Braun concluded that tumour formation takes place in two stages;
(a) Inception phase:
At this stage normal cells are transfoimed into tumour cells,
(b) Developmental phase:
Tumour cells continue to grow forming abnormal proliferation.
Studies on crown gall tissue suggest that the tumour forming substance is a small ring T-DNA or plasmid. The T-DNA is incorporated in the host genome and produces bacterial protein. This protein may be responsible for increased production of auxin and cytokinin which may lead to tumour-like growth.
T-DNA can act as a vector for the introduction of new genes into the host protoplasts. A desirable gene and marker genes are introduced into T-DNA or Ti plasmid (Fig. 28). The reconstructed Ti plasmid is inserted into an intact Agrobacterium. Now the selected plant is infected with this transformed bacterium, thereby introducing a new gene to the host plant.
Indefinite growth of disorganized tumours has been noted on culture. Tumours can grow on simple culture medium and do not require a supply of hormones. In tumours both in vivo and in vitro the hormone concentration is high and this causes disorganised growth of tumours.
Gautheret (’46) noted that callus tissue of Scorzonera hispanica which initially grows in auxin containing medium, can grow on auxin free medium. This is also, noted in tobacco, carrot, sunflower etc. This phenomenon is known as habituation.
Such tissues contain high concentration of hormones and low differentiating potentiality and are often friable. They can grow indefinitely on a simple auxin free medium. Thus habituation may be due to either increased auxin production or decreased auxin breakdown. When habituated tobacco tissue is grafted on healthy plant it rapidly produces tumour.
Callus cultures of hybrid tobacco (Nicotiana glauca × N. langsdorfii) readily form tumours. It has been noted that tumour tissue can grow on a simple medium. From culture of tumour tissue a comparison between biochemical and physiological properties of tumour cells and normal cells can be done.
Eradication of Virus:
Yield of various economic plants is seriously affected by virus infection. In vegetatively growing plants with systematic infection the virus spreads rapidly from generation to generation. Virus infection may also spread through infected seeds. So production of virus free plant is needed.
Heat treatment is used to get virus free plants. But in many plants this method is not satisfactory. By meristem tip culture virus free plants and virus resistant plants can be produced most successfully.
In systematically virus infected plant viruses are not uniformly distributed. The root tip and shoot tip are usually free from virus. By culturing smallest shoot apex (0.5—1 mm. long) with few leaf primordia virus free plants can be obtained.
By meristem tip culture Morel and Martin (’52, ’55) obtained virus free shoots in potato and Dalhia. These shoots failed to form roots and were grafted on healthy seedlings. But later, from meristem tip culture of various plants virus free plants with roots were obtained (Fig. 29).
In meristem tip culture small segment of stem or axillary bud tip is taken and is placed on a suitable culture medium. The size of the shoot tip to be cultured is important. In stem tip culture the meristem dome with first pair of leaves is usually taken.
Depending on the plant the meristem tip taken for culture ranges from 0.5 mm to 1 mm. Very small shoot tips do not produce roots, whereas larger shoot tips may contain virus particles.
The shoot tips may be cultured at 30-40°C to inactivate the virus. Presence of virus in culture should be tested rigorously. Infected materials are immediately destroyed. Only one healthy explant from a diseased plant is cultured.
Stem tip to be cultured may be surface sterilised. According to Hollings (’65) it is not always required. Liquid medium on culture tube with filter paper bridge is most satisfactory for meristem tip culture. The two ends of the filter paper bridge are immersed into the liquid medium. Agar medium is unsatisfactory for meristem tip culture.
Hollings and Stone (’68) noted that a medium containing major and minor elements of Knops and Barthelots solution (without beryllium and titanium) and 40g/l glucose, 10-3 g/l thiamine, 10-3 g/l mesoinositol having a pH 5.5 is the suitable medium for several plants.
For root initiation 10-3 g/1 NAA may be added. Cultures are then transferred to NAA free medium. Holling and Stone (’68) suggested to incubate the cultures below 20°C and illuminate for 22 hours/day. Formation of plantlets from meristem tip requires few weeks to several months.
Better results have been obtained when meristem tip for culture is taken from heat treated plants. In Chrysenthemum 5% of surviving meristem tips are free from leaf mottling virus, whereas 98% virus free tips are obtained when explants are taken after heat treatment.
On culture medium virus inhibiting substances, such as, malachite green, 2-4-D and thiouracil may be added to eliminate viruses. Once a virus free plant is produced it is well tested and then by clonal multiplication many healthy plants can be obtained from it.
Significance of tissue culture in plant pathogen studies:
By tissue culture virus free plants can be obtained.
Host parasite interaction can also be studied from tissue culture.
By tissue culture plants systematically infected by beneficial micro-organism, such as, nitrogen fixing bacteria can be obtained.
Tissue culture may be used to maintain stocks of contamination free special parasites, which may be required for various investigations.