Method for mass propogation of podophyllum hexandrum royle using embryo culture technique
OwnerAmit Chandra Kharkwal, Om Prakash +2inventors
InventorsAmit Chandra Kharkwal, Om Prakash, Amita Bhattacharya, Paramvir Singh Ahuja
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Abstract
The present invention describes an improved method of Podophyllum hexandrum Royle through embryo culture technique by using B5 medium supplemented with 3% sucrose and 0.8% agar under initial dark conditions for embryo germination and initial embling growth followed by light/dark photoperiod regime for further embling development.
Claims14
An improved method for mass propagation of Podophyllum hexandrum Royle using embryo culture technique, said method comprising the steps of:
- (a) placing an embryogenic tissue in a culture initiation medium comprising B-5 vitamins, supplemented with sucrose and agar and having pH of 5.8, at a temperature between 20 to 40° C. and subjected to a light/dark regime for about one week, to obtain embryos with radicals and expanded cotyledons,
- (b) transferring the embryos to a basal MS medium supplemented with vitamins in the presence of about 14 hours of white fluorescent light of intensity of 52 μmol m−2 s−1, 10 hr dark period for 4 weeks to obtain emblings with well developed root, hypocotyl and green cotyledonary leaves,
- (c) growing the emblings to acclamatize the plantlets for one week in bottles containing sand-soil and farm yard manure mix, B5 vitamins, without sucrose at a temperature between 20 to 40° C., light-dark regime and photo-period with light of intensity of 52 μmol m−2 s−1, and
- (d) transferring the acclimatized plantlets to field to obtain healthy young plants with true leaves in 12 weeks.
A method as claimed in 1 wherein BS medium was selected as the most suitable medium out of a range of media like B5, MS and WPM with their full and half strengths.
A method as claimed in 1 wherein culturing of the excised zygotic embryos on B5 medium under dark conditions at 25±2° C. for one week and transfer of the emblings with radicle, hypocotyl and expanded cotyledons after 2 weeks to light/dark photoperiod regime (14h light/10h dark).
A method as claimed in 1 wherein the composition of potting mix has been specified to be 3:1 of sand :soil with 0.6N B5 medium without sucrose and a mix of sand:
- soil:farm yard manure in the ratio of 3:1:1 for Pododphylluim plant growth.
A method as claimed in 1 wherein the green house conditions for successful plant establishment and true leaf emergence was selected out of the use of poly tunnels covered with and without green agro net and planting in Hikkotrays, pots and directly into potting mixes in dug trenches.
A method as claimed in 1 wherein the ex situ raised plants can be transferred to their natural habitat
A method as claimed in 1, wherein the heterogeneous population of Pododphyllum can be raised to conserve genetic diversity, which may be below level of species.
An improved method for mass propagation of Podophyllum hexandrum Royle using embryo culture technique, said method comprising:
- (a) collecting mature pods of P. hexandrum Royle from their natural habitats in the Western Himalayas, India at an altitude ranging from 2700 m-3500 m above mean sea level during the last week of August and first week of September,
- (b) separating the seeds from the pulp immediately after collection, prior to disinfection with 0.1% Bavistin and streptomycin sulphate (0.1%) for 15 minutes followed by a thorough washing of seeds after about 1 minute dip in 70% alcohol and surface sterilization in 0.1% mercuric chloride for 7 minutes, all traces of mercuric chloride were finally washed off by 4-5 rinses in sterile distilled water,
- (c) dissecting the surface sterilized seeds by a sharp excision with a scalpel blade at the base of the microplyar lobe and embryos were carefully squeezed out of their micropylar groove,
- (d) culturing the embryos at 25±2° C. in dark on basal B-5 (Gamborg et al., 1968) medium supplemented with 3% sucrose of pH 5.8 adjusted with 1N KOH and HCl prior to the addition of agar (0.8%) and autoclaved at 121° C.,
- (e) transferring the emblings with fully developed radicals and expanded cotyledons after 2 weeks to about 14 hour photoperiod with light of intensity of 52 ptmolm2sI alternating with 10-h dark period for further growth,
- (f) transferring of the 4 week old emblings (8.0 cms long) with well developed root, hypocotyl and green cotyledonary leaves to jam bottles (covered with lids) containing 120 g of sand:
- soil in the ratio 3:1 enriched with 0.6 N (liquid) of B5 medium without sucrose,
- (g) hardening of the emblings for about 1 week under lab culture conditions (25±2° C.) and 14 h photoperiods with light intensity of 52 μmolm−2s−1 alternating with 10-h dark period),
- (h) transferring the jam bottles containing the emblings to green house for another one week with partially opened lids,
- (i) transferring of at least 8.0 cm long emblings to 4′ pots containing potting mix of sand:
- soil:
- farm yard manure in the ratio:
- 3:1:1 covered with magenta jars for 6 weeks,
- (j) removing the magenta jars from over the plants after about 6 weeks as true leaves start emerging,
- (k) growing the plants again for about 6 weeks for further emergence of true leaves, and
- (l) transferring the healthy plants to natural conditions after 6 months.
Dependencies are inferred from the claim text.