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The present study entitled “In vitro conservation of chethikoduveli (Plumbago rosea L.) using encapsulation and vitrification techniques” was carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to standardise the protocol for short term and long term conservation of axillary buds using encapsulation and vitrification techniques and to assess the genetic fidelity of recovered and regenerated plantlets using molecular markers.

The study was carried out in three phases viz., enhancement of multiplication rate, short term conservation by encapsulation technique and long term conservation using vitrification technique.

Axillary buds from in vitro raised cultures were used as the explants in all the experiments. Different media (Gamborg, White, SH and MS) and different levels of additives (chitosan, adenine sulphate and thidiazuron) were tried for the enhancement of multiplication. Standardized medium, MS supplemented with BA 1.5mg L-1 and IAA 1mg L-1, for shoot proliferation was used as the control treatment in the study.Among the different media tried, MS medium was observed to give the best response with respect to shoot multiplication (3.75 shoots/explant).

Among the different levels of each additive tested, best response was obtained with chitosan 15 mg L-1, adenine sulphate 40 mg L-1 and thidiazuron 4 mg L-1 and 5mg L-1 in MS supplemented with BA 1.5 mg L-1 and IAA 1 mg L-1. Thidiazuron at both levels gave maximum shoot proliferation (11.5 shoots/explant). But shoots obtained were stunted, and vitrified with callusing at the base. Adenine sulphate 40mg L-1, gave significantly better response with 4.25 shoots/explant. Though chitosan 15mg L-1gave maximum shoots/explants (3.67) among different levels of chitosan tried, it was found to be on par with the control. Hence, MS medium supplemented with BA 1.5mg L-1, IAA 1mg L-1 and adenine sulphate 40 mg L-1 was selected as the medium for further conservation studies.

In short term conservation studies, encapsulated axillary buds were used as explants. Effect of different additives (sucrose 10 per cent, sucrose 15 per cent, mannitol 10 per cent, mannitol 15 per cent) in encapsulation matrix (sodium alginate 2.5 per cent and calcium chloride 100 mM), different storage media (liquid MS, sterile distilled water) on shoot regeneration and proliferation were tried at two temperature regimes of 4˚C and 25˚C.

Encapsulated axillary buds could be stored upto 30 days in all combinations, beyond which the regeneration percentage deteriorated. Maximum regeneration of 83.33 and 79.17 per cent was obtained with mannitol 10 per cent in encapsulation matrix and liquid MS as storage medium at 25˚C and 4˚C respectively after 30 days of storage. In this treatment at 25oC, the days to bud initiation was the least (11.60 days); Shoots/explant (6.4), shoot length (1.87cm) and nodes per shoot (2.48) was the highest.

Two cryopreservation techniques of simple vitrification and encapsulation vitrification were attempted for long term conservation studies. In simple vitrification, preconditioned (0.5 M sucrose for 7 days), precultured (0.5 M sucrose for 3days) axillary buds were exposed to vitrification solutions, PVS2 (glycerol 30 per cent, ethylene glycol 15 per cent and DMSO 15 per cent in MS with sucrose 0.4 M, pH 5.7) and PVS3 (glycerol 50 per cent and sucrose 50 per cent in MS, pH 5.7) for 0 to 210 minutes at 30 minutes interval. The best response in terms of survival (62.22 per cent) and regeneration (47.78 per cent) was obtained in PVS2 exposure for 30 min after 2 h of cryopreservation ie., storage in liquid nitrogen. In encapsulation vitrification, preconditioned, encapsulated, precultured axillary buds were given exposure to vitrification solutions as above, maximum survival (78.89 per cent) and regeneration (70.00 per cent) was also obtained in the same treatment. The survival and regeneration percentage was found to be on par with different periods of cryopreservation. Among the cryopreservation treatments, encapsulation vitrification was found to be the best.

The cryoregenerated explants were then inoculated on to the best proliferation medium to study the regeneration and multiplication. Encapsulation vitrification in PVS2 for 30 minutes gave best response with respect to bud initiation (28.89 days), shoots/ explant (2.56) and shoot length (2.88 cm).

The genetic fidelity of the plantlets subjected to short and long term conservation was assessed using 5 RAPD and 4 ISSR markers. Among these, two ISSR primers produced 5-7 bands. The banding patterns of the conservation regenerated plantlets and the control were compared. The profiles generated did not show any polymorphism and was identical to those of control, which indicated the genetic stability.

In the study, maximum multiplication rate (4.25 shoots/explant) was obtained in MS media supplemented with BA 1.5mg L-1, IAA 1mg L-1 and adenine sulphate 40mg L-1. With respect to short term conservation, the encapsulated axillary buds with the additive in encapsulation matrix (mannitol 10 per cent), storage media (liquid MS) and storage temperature (25˚C) gave maximum regeneration (83.33 per cent). Encapsulated axillary buds could be stored upto 30 days in all combinations, beyond which the regeneration percentage deteriorated. In long term conservation the maximum survival (78.89 per cent) and regeneration (70.00 per cent) was obtained in encapsulation vitrification. The genetic stability was maintained in plantlets regenerated from short and long term conservation.