MATERIALS AND METHODS

Plant materials and explants: The present study was carried out at University of Lucknow in the year 2000. The fresh explants of Tobacco were collected from the university campus to see their response under in vitro conditions. The explants were washed in liquid detergent Tween-20 (Himedia) and kept under running tap water until all traces of detergent were completely removed. Surface sterilization of explants were carried out in 0.1% aqueous solution of HgCl₂ for 2 min followed by rinse with autoclaved double distilled water (2-4 times). The surface sterilized explants were inoculated onto a sterile medium containing various hormonal combinations. MS (Murashige and Skoog, 1962) and other medium were used as basal medium throughout the experiment with different combinations and concentration of phytohormones, viz., auxins and cytokines, Sucrose (3%) mayoinositol (0.1%) were also added properly. MS0 and B50 were treated as control. The pH of the solution was checked and adjusted to 5.8 by adding 0.1 N NaOH and 0.1 N HCl (drop by drop) depending upon the solution, followed by the addition of 8% Agar Agar Type-1 (Himedia). The medium was autoclaved at 15l b pressure for 15 to 20 min at 121°C. After dispensing in tubes/vials culture were kept at a temperature of 25±3°C under cool white florescent tubes (200-3000 Lux) at 16-18 h photoperiod and relative humidity (50-69%).

Preparation of Boerhaavia diffusa root extract: The BD-SRIP was obtained from the freshly collected roots of B. diffusa growing in the university campus (Fig. 1). Fine powder was prepared after drying and grinding roots at 37°C in hot air oven. One hundred grams fine dry powder of root was soaked overnight at 4°C in (0.02 M) sodium acetate buffer (500 mL) with pH 5.2 (1: 5 w/v). 0.1% Mercaptoethanol was also added and squeezed through two folds of muslin cloth and the extract was centrifuged at 8000 rpm for 20 min. Supernatant was mixed with 60% Ammonium sulphate and kept overnight at 4°C. The precipitate was collected by low speed centrifugation and dissolved in minimum amount of sodium acetate buffer. This sample was passed through Sephadex G-25 column. Void volume elute from this column was used as virus inhibitor.

Desalting on sephadex G-25 column for obtaining BD-SRIP: Sephadex G-25 matrix (Pharmacia, India) was packed in a column (100x2.6 cm) to a height of 80 cm (void volume = 165 mL) and equilibrated with 20 mM sodium acetate buffer (pH 5.2), containing 0.02% sodium azide and 0.01% (v/v) 2-mercaptoethanol. Low molecular weight nonprotein component was also collected. Both the fractions were tested for their ability to induce resistance on the test host. The extract containing 30 kDa protein (Fig. 2) and possessing viral inhibitory activity was added to the mediumin different concentration or sprayed on test plants and subsequently explants were taken from these treated plants.

Virus inhibition assay method: Inhibition percentage was determined by local lesions assay using host reacting hypersensitivity (N. tabacum cv. Samsum NN) against TMV. The virus inoculum was prepared by crushing infected leaves of tobacco plant (N. tabacum cv. NP-31) with distilled water in a mortar. The pulp was squeezed through two folds of musclin cloth and the solution centrifuged at 6000 rpm for 15 min.

Fig. 1:	Boerhaavia diffusa growing in field

Fig. 2:	SDS polyacrylamide gel electrophoresis of Boerhaavia diffusa root extract, Lane A-BD root extract, Lane B-Mol. et. Marker

The clear supernatant was used as virus inoculum after diluting it suitably with distilled water, so as to produce countable number of local lesions.

Resistance induction in field grown/micropropagated plant: The BD-SRIP solution was applied/sprayed on two lower leaves of test plants. Control sets of plants were sprayed with distilled water. After 2 h carborundum powder (600 mesh) was dusted on the upper surface of all inoculable leaves of a few treated and controlled plants and virus inoculated (TMV = 1: 200 w/v). The leaves were than rinsed with tap water. All the experiments were done in an insect free green house under natural light conditions. The local leisions appearing after 3-4 days were counted separately on treated and controlled set of plants. The remaining plants after 24 h of BD spray were used as explants and inoculated in MS medium containing no BD-SRIP and sterilized BD-SRIP (4%). Controlled plants (non sprayed) were also inoculated on MS medium containing no BD-SRIP.

Degree of inhibition calculated using the formula (C-T/C)x100
Where C = average No. of local lesions on control leaves.
T = average No. of local leaves on treated leaves

Raising of test plants: Test plants (N. tabacum cv. Samsun) were raised in an insect free green house in earthen pots containing sterilized compost and soil in the ratio of (1:1). Initially seeds were sown in bigger pots, when seedlings emerged out; they were separately transplanted in to smaller earthen pots.

Single node stem segment (a node with its axillary bud and small portion of internode on either side) measuring about 2 cm length were taken from test plant (N. tabacum cv. Samsun NN) and used as explants. The experimental material collected from field was surface sterilized in order to establish in vitro.

RESULTS AND DISCUSSION

The explant type and size shows great variation in the proliferation ratio of Tobacco plant. The best profile was seen in the buds as explant type. The size of explants which varies from 1-2 cm, large explant size increase the contamination ratio. The effect of sterilization agent was also prominent as increase in sterilizing % increased. The death rate increases the contamination.

Differentiation of axillary buds was influenced by the size of explant used. With very small explants, the survival rate was less, while using larger explant size the contamination was high.

It was noticed that by using smaller explant size, much lesser differentiation was obtained as compared to 1-2 cm explant size. Therefore, 1-2 cm explant size was used for bud proliferation.

The explants were subjected to 5 min treatment with 0.01 to 0.1% mercuric chloride solution. Sterilized plants were then inoculated on the surface of MS medium. In the optimum treatment by using 0.01% HgCl₂ all segments were obtained infection free, 20% sterilized explants turned brown showing toxicity. Eighty percent explants showed proliferation out of which 70% explants were viable.

Effect of explant position from natural field grown plants on bud proliferation: Nodal segments were taken from different positions of field grown plants and after sterilization were incubated on the MS-medium. The explants taken from upper portion having apical buds showed maximum bud proliferation in comparison to middle and lower portion, which showed poor proliferation. These were 100% viability in these explants and the growth response was fairly good.

Effect of growth hormones on axillary bud proliferation:

The sterilized single node stem segments were transferred to MS medium with auxin, NAA+IBA (0.25+0.25 mg L^-1) with or without hormone. The explants in the medium with no growth hormone showed only 20% proliferation of axillary buds and poor growth. The explants grown in the medium containing NAA+IBA were showed 80% proliferation of buds as well as good growth. The cultures were subcultured after every 4 weeks. On subculturing the explants with out growth hormone in the medium showed better growth and rooting after 15 days of incubation. The number of roots increases from 4-12 cm and length increased 0.5-4.5 cm in the medium containing no growth hormone. Length of shoot increased from 2-7 cm and number of leaves also increased (Table 1).

Testing for induced resistance: The field induced (sprayed with BD-SRIP) Tobacco plants well tested for induced resistance. Control and treated plants challenged with TMV, lesions appeared on the inoculated leaves after 3 days of inoculation. These lesions were counted and degree of inhibition counted. Nighty two percent decrease in lesions number was observed in tobacco plants sprayed with BD-SRIP (Table 2).

Table 1:	Effect of growth hormone on proliferation of different parameter of explants
+ Poor growth; ++ Good growth; +++ Better growth

Table 2:	Test for induced resistance in field induced tobacco plants
Degree of inhibition = C-T/Cx100 = 201-16/201x100 = 92%

Table 3:	Transfer of induced resistance to regenerants

Transfer of induced resistance to regenerants: The BD-SRIP plants showing high degree of resistance were used as elite plants for obtaining explants. Explants were also taken from non sprayed control plants. These explants were inoculated on MS medium. The explants from treated plants were transferred to MS medium to which was added 4 and 100 mL of BD-SRIP after filter sterilization. The proliferated shoots were transferred for rooting. The rooted plants were subjected to hardening and when 4-5 inoculable leaves developed they were inoculated with TMV.

It was found that plants developing from treated explants grown on MS medium containing BD-SRIP were less susceptible to virus infection as compared to explants developing from control plants raised on MS medium. The result have shown that induced resistance induced resistance can be transferred to regenerants and explants grown on MS medium containing BD-SRIP showed high degree of resistance against virus infection. Thus, the results have shown that resistance plants can be developed utilizing BD-SRIP (Table 3).

CONCLUSIONS

The present study showed a procedure for transfer of induced resistance to regenerants. The resistance thus induced was not associated with any phytotoxic symptoms. This procedure would be suitable as an alternative for development of virus resistant plants. Since resistant varieties are not available in most of the crops. The only method perhaps for avoiding viral diseases would be to grow varieties with a high degree of viral tolerance or to increase the tolerance of these varieties by the use of systemic resistance inducing proteins such as present in B. diffusa root extract, so that yield of crop is not much affected by infection.

ACKNOWLEDGMENTS

The authors wish to express gratitude to the former Head of the Department University of Lucknow, Lucknow for providing research facilities.