A method for an efficient in-vitro system of micropropagation of rose scented Geranium, Pelargonium graveolens L. Herit of Bourbon type of Reunion origin for producing a large number of viable in-vitro plants from explants with a multiplication ratio of 1:15-1:20, the said method comprising the steps of:
- i. treating mother plants growing in open field conditions with systemic fungicides and insecticides in a conventional manner, at one week intervals, for four weeks;
- ii. selecting a mother plant from healthy elite plants of rose scented geranium Pelargonium graveolens L. Herit of Bourbon or Reunion type from the mother plants, having a desired oil characteristic from open field conditions as the mother plant grown in an open field;
- iii. transferring the treated mother plant from the open field conditions to a green house with green house conditions set at 80-90% relative humidity, 22+.sub.−2° C. temperature, with 50% light cut;
- iv. selecting and collecting nodal explants with a small multiplication ratio of 1:15-1:20 from the mother plant growing in the green house conditions for 4 months;
- v. cleaning the nodal explants;
- vi. surface sterilizing the nodal explants;
- vii. cutting the nodal explants into small pieces of approximately 2-12 mm length or diameter;
- viii. inoculating the small pieces of nodal explants on a shoot regeneration and multiplication media for shoot regeneration and multiplication comprising modified MS media supplemented with 200-400 mg of inositol per liter, growth hormones selected from the group consisting of auxins like NAA 0.05-5 mg per liter, cytokinins like kinetin 2.5-7.5 mg per liter and BAP 0.15-3 mg per liter to produce shoot cultures of inoculated nodal explants;
- ix. incubating the inoculated nodal explants for a photoperiod of a day ranging from 0-24 hours and a night or a dark period ranging from 24-0 hours for a period of 2-10 days at a temperature of 18-25 degrees centigrade, scoring the incubated shoots of the inoculated nodal explants to produce cultures free of contamination if any, separating uncontaminated cultures from contaminated cultures;
- x. relocating the uncontaminated cultures of nodal explants and keeping them in a growth room wherein illumination is provided and the illumination has a light intensity of about 50-90 mu.mol m.sup.−2s.sup−1 and provided for a duration of about 10-20 hours of light daily, at a temperature of 18-25° C. for at least 3-6 weeks to produce healthy cultures of nodal explants;
- xi. transferring the healthy cultures of nodal explants on the same media and same culture conditions of shoot regeneration and multiplication, for a period of 2-4 weeks for further growth into multiple healthy shoots, and evaluating them to select the healthy multiple shoots from the nodal explants;
- xii. harvesting the healthy multiple shoots;
- xiii. dissecting the healthy multiple shoots into 0.2-1 cm segments and transferring to media for shoot growth comprising an MS media supplemented with 200-600 mg inositol per liter, growth hormones selected from the group consisting of auxins such as NAA 0.02-2.0 mg per liter, and cytokinins such as BAP 0.2-2.0 mg per liter;
- xiv. incubating cultures from the healthy multiple shoots in the growth room wherein lighting is provided having a light intensity of about 50-90 mu.mol m.sup.−2s.sup−1 and providing illumination for a period of about 10-20 hours of light daily;
- at 18-25 degree centigrade of temperature for a period of 3-6 weeks, multiplying the cultures-from the healthy multiple shoots in the same media and the same culture conditions, and continuing the multiplication up to 10 cycles or until the vigor of plant multiplication is diminished;
- xv. transferring the healthy multiple shoots of 3-6 cm height on to media for rooting comprising MS media of 0.1-1.0 strength, and maintaining for a period of 4-6 weeks or until formation of well developed rooted plants;
- xvi. removing the rooted plants from a container, washing the rooted plants with water to remove agar adhering to the rooted plants, drenching with 0.02-0.2% fungicide like Bavistin and planting on a soil mixture comprising pre-sterilized red soil, cocopeat and decomposed soil from a farm yard into 1:1:1 proportion and keeping them in a green house with 70-80% relative humidity, shaded 40-60% at a temperature of about 18-28 degrees centigrade for primary hardening for 3-6 weeks;
- xvii. transferring the rooted plants outside of the green house under shade with 15-30% light cut for secondary hardening for 2-6 weeks;
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- xviii. transferring the hardened rooted plants to an open field.