MSc

Nutmeg (Myristica fragrans Houtt.), is dioecious and dimorphic in branching habit with erect growing orthotropic and horizontally growing plagiotropic shoots. The long gestation period and dioecious nature of the crop causes difficulty in the production of quality planting materials of known sex. Vegetative propagation, budding and grafting with orthotropic scion material produces erect growing tree with upright tree architecture. In vegetative propagation, scarcity of orthotropic scion material is a limiting factor in large scale production of planting materials. So the programme “In vitro shoot regeneration and micrografting in nutmeg (Myristica fragrans Houtt.)” was taken up.

The objectives of the study were: (1) To identify the culture conditions for multiple shoot production from orthotrops of gynoecious plants of nutmeg through enhanced release of axillary buds and shoot tip culture and (2) To standardize micrografting technique with in vitro and in vivo shoots as scion. The work was done at CPBMB, College of Horticulture, Vellanikkara.

SH medium (Schenk and Hildebrandt, 1972) was found to be the best basal medium for in vitro culture establishment of nodal segments of nutmeg compared to ½ MS (Murashige and Skoog, 1962) and WPM (Lloyd and Mc Cown, 1980). Surface sterilization of nodal segments by soaking in (0.1%) carbendazim for 10 minutes followed by (0.1%) HgCl2 treatment for six minutes and sterile water wash, recorded 33% survival of cultures. The best explant for culture initiation was nodal segments. The best season for culture establishment was summer months (April-May) compared to rainy season (June- July). Loss of cultures was due to fungal contamination and necrosis of tissues. The media combination SH + Thidiazuron (TDZ) 0.03mg l-1 + Activated Charcoal (A.C.) 0.5% recorded bud expansion in 50% of the cultures within a period of nine days. Nodal segments are superior to shoot tips in culture establishment. Culture condition for culture establishment was 26 ± 20C at a light intensity of 1000 lux. The carbon source; 2% Sucrose + 1% glucose or 5% sucrose supported bud elongation and leaf expansion
Refinement of culture establishment media was attempted with organic supplements; Coconut water (5, 10, 15 and 20% v/v), Casein hydrolysate (10, 25 and 50mg l-1) and Brassinolide (0.05, 0.1 and 0.2mg l-1) with nodal segments from juvenile seedlings, regenerants from coppiced trees and mature trees. In explants from juvenile seedlings and mature trees, 5 to 15%coconut water supported culture establishment. In juvenile explants, shoot elongation was also observed at 10% coconut water. Casein hydrolysate supported bud expansion in juvenile and mature tree explants at 10 to 50 mg l-1. Bud elongation and leaf expansion was observed at 25 mg l-1 concentration. Brassinolide (0.2 mg l-1) supported bud expansion in juvenile explants.

The suggested media for explants from juvenile as well as coppiced trees for culture establishment of nodal segments of nutmeg is SH + 0.03 mg l-1 TDZ + 25 mg l-1 Casein hydrolysate + 2% sucrose + 1% glucose + 0.5% A.C. Casein hydrolysate concentration for explants from mature trees could be 50 mg l-1.

In vitro seed germination was observed in mature seeds in presterilized bottles with water soaked cotton/ little water. Somatic embryos were formed at the cut portion of six month old seeds in the medium of ½ MS + 2% Sucrose + A.C. 0.5%. Proliferation of callus and somatic embryos was observed with the medium B5 + 0.1 mg l-1 Kin + 0.01 mg l-1 NAA + 0.01 mg l-1 GA3 + 10.0 mg l-1 Casein hydrolysate + . 0.5% A.C. The response was obtained two and a half months after inoculation. Three days old in vivo germinated seedlings did not established under in vitro condition even though surface sterilization treatment with 0.1% Emissan for thirty minutes followed by 0.1% HgCl2 for six minutes was given.

Feasibility of grafting in juvenile plants was studied with epicotyl grafting and got 80% success. Grafting was done on twenty days old seedling with scion material from different seedling.

In vitro epicotyl micrografting was done with in vitro raised scion and root stocks. It was also done on in vivo germinated seedlings after surface sterilization. Scion shoot of 2.5 cm length was grafted on twenty days old root stock. Graft was cultured in liquid nutrient medium and survived for two weeks. Later fungal contamination destroyed the cultures. Grafting with in vitro shoots on in vivo raised root stocks did not succeed.