Standardisation of in vitro techniques for the rapid clonal propagation of mango (Mangifera indica L.)

Abstract

Standardization of techniques for the in vitro propagation of mango (Mangifera indica L) varieties was attempted. The studies were carried out at the Plant Tissue Culture Laboratory, Department of Horticulture, College of Agriculture, Vellayani, during 1992-96.

Attempts for the in vitro propagation via somatic embryogenesis, somatic organogenesis and enhanced release of auxillary buds were made. Six monoembryonic and six polyembryonic mango varieties were subjected to the initial response studies. Neelum (monoembryonic) and Vellari Manga (polyembryonic) varieties were selected for further detailed studies. Explants like nucellus, embryo mass, segments of leaf and inflorescence were used.

The effects of culture medium (basal medium, major and minor nutrients, plant growth substances, casein hydrolysate, sucrose, glutamine, coconut water, activated charcoal, polyvinyl pyrrolidone, sodium butyrate, thidiazuron, polyethylene glycol, sodium chloride, silver nitrate, cobalt chloride and agar), culture conditions (light and temperature) and frequency of subculture on the various stages of somatic embryogenesis were studied.

Among the various explants tried, somatic embryogenesis could be induced only from nucellus and embryo mass. In Neelum, somatic embryogenesis could be induced in 66.67 percent cultures of nucellus and 75.00 percent cultures of embryo mass. In vellari manga 83.33 percent cultures of nucellus and 66.67 percent cultures of embryo mass responded.

Somatic embryogenesis from nucellus of Neelum could be best induced on half strength MS basal medium supplemented with GA3 5.0mg/l, 2,4-D 2.0mg/l, GA3 5.0mg/l, sucrose 60.0g/l, glutamine 400.0mg/l, coconut water 200.0ml/l, activated charcoal 2.5g/l and agar 5.0g/l. The ideal treatment for inducing somatic embryogenesis from nucellus of Vellari Manga was half strength MS basal medium supplemented with 2, 4-D 2.0 mg/l, GA3 5.0mg/l, sucrose 60.0g/l, glutamine 600.0 mg/l, coconut water 200.0ml/l, activated charcoal 2.5g/l and agar 6.0g/l. Subculturing in medium of the same composition at an interval of five days increased the percentage induction in Neelum (30.0 percent) and five to ten days in Vellari Manga (40.0 percent).

The best treatment identified for the initiation of somatic embryoids from nucellus of Neelum was half strength MS basal medium supplemented with 2, 4-D 2.0mg/l, GA3 5.0mg/l, BA 1.0mg/l, sucrose 60.0g/l, glutamine 400.0mg/l, casein hydrolysate 500.0mg/l, coconut water 200.0ml/l, activated charcoal 2.5g/l and agar 5.0g/l. The ideal treatment for the initiation of somaticembryoids from nucellus of Vellari Manga was half strength MS basal medium supplemented with 2, 4-D 0.5mg/l, GA3 5.0mg/l, BA 1.0mg/l, sucrose 60.0g/l, glutamine 400.0 mg/l, case in hydrolysate 600.0 mg/l, coconut water 200.0ml/l, activated charcoal 2.5g/l and agar 5.5g/l. Subculturing at an interval of ten days in Neelum and five to ten days in Vellari Manga was beneficial for the initiation of somatic embryoids. The corresponding percentage of initiation of somatic embryoids was 66.67 in Neelum and 55.56 percent in Vellari Manga.

A medium containing B5 major salts and MS minor salts supplemented with abscisic acid 5.0mg/l, sucrose 40.0g/l, casein hydrolysate 100.0mg/l, coconut water 200.0ml/l, polyvinyl pyrrolidone 10.0g/l and agar 4.5g/l was the best for supporting the maturation of the somatic embryoids of Neelum. The best medium for the maturation of the somatic embryoids of Vellari Manga contained B5 major salts, MS minor salts, abscisic acid 4.22mg/l, sucrose 40.0g/l, casein hydrolysate 100.0mg/l coconut water 200.0ml/l, polyvinyl pyrrolidone 10.0g/l and agar 5.0g/l. The size of embryoids was the highest (1.0-1.5cm long) when subcultured at an interval of ten days for Neelum and fifteen days (0.5-1.5cm long) for Vellari Manga.

Incubating the cultures in darkness at 26 ± 20C favoured the induction, initiation and maturation of somatic embryoids of both the varieties.

Near-normal germination of the somatic embryoids of Neelum was observed when cultured on a medium containing B5 major salts and MS minor salts, BA 0.1 mg/l, sucrose 40.0g/l, sodium chloride 0.5g/l, cobalt chloride 10.0 mg/l, polyvinyl pyrrolidone 10.0g/l and agar 5.5g/l. Near-normal germination of the somatic embryoids of Vellari Manga was observed on a medium containing B5 major salts and MS minor salts, BA 1.0 mg/l, sucrose 50.0g/l, sodium chloride 0.5g/l, cobalt chloride 10.0 mg/l, polyvinyl pyrrolidone 10.0g/l and agar 5.5g/l. A few germinated embryoids were planted out. However, they did not survive.

Histological and morphological studies ascertained the status of the somatic embryoids formed. Scanning electron microscope studies depicted the morphological features of the developmental stages of the somatic embryoids.

Attempts to standardize in vitro propagation via somatic organogenesis and enhanced release of auxiliary buds were not successful. However, de-diffrentiation could be induced from leaf segment explants of Neelum and Mulgoa.