Title of Invention

"A PROCESS OF PREPARATION OF TISSUE CULTURE MEDIUM FOR ENHANCING IN-VITRO PLANTLET REGENERATION IN AIR YAM PLANT USING BACTERIAL CULTURE SUPERNATANT."

Abstract This invention relates to a process of preparation of tissue culture medium for enhancing in-vitro plantlet regeneration in air yam plant using bacterial culture supernatant, in place of chemical growth promoter, comprising the steps of; growing Azotobacter chroococcum strain (A chroccoccum) in Jensen medium and/or Gluconacetobacter diazotrophicus strain (G. diazotorphicus) grown in LGI medium given by Covalcante and Dobereiner; adding to both media 75-125 mg/1 D,L tryptophan preferably 100 mg/1 as precursor of indoleacetic Acid (IAA); characterized in that centrifuging the bacterial culture after 4-8 days preferably 4 days for G. diazotrophicus medium and 8 days for A. chroococcum medium at 10,000 rpm for 18-25 minutes preferably 20 minutes to obtain Culture Supernatant; adding Culture Supernatant, obtained from step (ii), to Murashige and Skoog basal medium for nodal explants' growth; wherein Culture Supernatant supplemented medium regenerates healthy roots and shoots simultaneously in 25-30 days as compared to normal 40-45 days.
Full Text Field of Invention;
This invention relates to a modification of the process of preparation of standard tissue culture medium by using culture supernatant.
Background of the Invention;
MS basal media supplemented with various growth regulators are found to be most suitable for regeneration from various explants of particular plants. But the plantlets regenerated on these media are not always healthy, and they take a very long time to grow. The cost per plantlet formation is also more.
Objects of the Invention;
An object of this invention is to modify a process for the preparation of standard tissue culture medium.
Other object of this invention is to modify the process to provide a simple and cost effective medium.
Another objective is to propose a modification wherein the culture supernatant can replace some of phytohermones.
Further objective is to reduce the time period to get complete plant.
Detailed Description of the Invention;
According to this invention there is provided a modification of the process of preparation of tissue culture medium.
Two different microbial strains were grown in their respective growth media. Azotobacter chroococcum strain 103 was grown in Jensen's medium (Jensen 1951) Annexure I whereas Gluconacetobacter diazotrophicus strain 767-50 was grown in LG1 (Cavalcante and Dobereiner, 1988) medium Annexure II. To both the media 100 mg/1 D, L-tryptophan was added as precursor of 1AA. The cultures were monitored for IAA production over several days.
Preparation of culture supernatant and estimation of IAA;
The bacterial cultures were centrifuged at 10, 000 rpm for 20 min. and the supernatant was transferred to a new tube. Amount of IAA in culture supernatant (CS) was estimated by Salkowski's method (Tang and Bonner, 1974; Glickmann and Dessaux, 1995) To 1 ml of CS, 2 ml of Salkowski Reagent (0.05 M ferric chloride and 35% perchloric acid/100ml) was added. The contents were mixed by shaking and allowed to stand in dark at room temperature for 30 min. for development of pink colour. Optical density of the solution (at 500 nm) was recorded using spectrophotometer. Un-inoculated growth medium was used as control. Maximum IAA production by G. diazotrophicus strain 767-50 was recorded on 4th day (140 ug/ml) and it was on 8th day in case of A. chroococcum strain 103 (150 ug/ml). Table I
Testing of CS in tissue culture media;
To study the effectiveness of CS as source of phytohormones, the tissue culture of an important medicinal plant, air yam (Dioscorea bulbifera L.) was used as the test system. Nodal explants were cultured on MS basal medium (Murashige and Skoog, 1962) supplemented with different concentrations of CS from both the bacterial strains Table III. The following media were used as control:
Bo (MS alone), Control I
Ve (MS + Kinetin (2.0 mg/1) + Adenine sulphate (15mg/l), Control II. V8 (MS + NAA (0.1 mg/1) + Adenine sulphate (15 mg/1), Control III ¥12 (MS + IAA (0.1 mg/1) + Adenine sulphate (15 mg/1), Control IV) , rooting medium: (MS+0.5mg/lIAA)
The response of the explants was monitored upto 30 days of culturing. Data were recorded for percent shoot regeneration as follows:
No. of explants responding for shoot formation X 100 Total no. of explants
Comparative cost of different culture media is given in Table II. The results in tabulated form are presented in Table IV.
The cost/plantlet formation was calculated as given in Table V. The chemicals used were obtained from Hi-Media, India, Ltd.
Different concentrations of the culture supernatant of phyto hormone producing bacteria were added to MS basal medium and explants from five different plant species were cultured on these media. MS basal media supplemented with various growth regulators found most suitable for the particular plant were used as one of the controls, the other being only MS basal medium. The response of the explants on the culture supernatant supplemented media was found to be far more than on the control media. Even the plantlets regenerated on these media were healthier than on the control media.
On the control media only shoots or shoots with very weak roots (plate 1) were regenerated, whereas on B7 and C7 both shoots as well as healthy roots (plate 2) were regenerated simultaneously. The shoots regenerated on the control media were first put on rooting medium (VI9) to get complete plantlets. Therefore the time to get complete plant was reduced from approximately 45 days to approximately 30 days. This leads to the more efficiency of CS.
(Figure Removed)
TABLE 1
Estimation of indole-3-acetic acid (IAA) in culture supernatant

(Table Removed)8
Cost of medium with culture supernatant of G. diazotrophicus (MS+1.5ml/l) =Rs
31.27/1
Cost of medium with culture supernatant of A. chroococcum (MS+1.5ml/1) =Rs
31.25/1
(Table Removed)
The following media were used as control:
Bo (MS alone), Control-I
V6 (MS + Kinetin + Adenine Sulphate (15mg/I), Control-II. Vs(MS +NAA + Adenine sulphate (15mg/I), Control-Ill Vi2 (MS +IAA +Adenine sulphate (15mg/I), Control-IV) , rooting medium: (MS +IAA)
The response of the explants was monitored upto 30 days of culturing. Data were recorded for percent shoot regeneration as follows:
No. of explants responding for shoot formation X 100 Total no. of explants
The results in tabulated form are presented in Table IV.
The cost/plantlet formation was calculated as given in Table V. The
chemicals used were obtained from Hi-Media, India, Ltd.
(Table Removed)
Transformed value
The cost/plantlet formation was calculated as given in Table V. The chemicals used were obtained from Hi-Media, India, Ltd.
(Table Removed)
On the control media only shoots or shoots with very week roots were regenerated, whereas on B7 and C7 both shoots aw well as healthy roots were regenerated simultaneously. The shoots regenerated on the control media were first put on rooting medium (V19) to get complete plantlets. Therefore, the time to get complete plant was reduced from approx. 45 days to approx. 30 days. This leads to the more efficiency of CS.
Annexure I
Composition of Jensen's Medium (Jensen, 1951)

(Table Removed)

Annexure II
Composition of LGI Medium (Covalcante and Dobereiner, 1988)

(Table Removed)
Agar-agar = 8.0 gm/1
100 mg tryptophan was added per liter to act as precursor of Indoie acetic acid.
Total Cost/I = Rs. 43.6578
On five times dilution, the cost of medium per liter =Rs. 06.23







We Claim
1. A process of preparation of tissue culture medium for enhancing in-vitro plantlet regeneration in air yam plant using bacterial culture supernatant, in place of chemical growth promoter, comprising the steps of;
i. growing Azotobacter chroococcum strain (A. chroccoccum) in Jensen medium and/or Gluconacetobacter diazotrophicus strain (G. diazotorphicus) grown in LGI medium given by Covalcante and Dobereiner
ii. adding to both media 75-125 mg/1 D,L tryptophan preferably 100 mg/1 as precursor of indoleacetic Acid (IAA)
iii. characterized in that centrifuging the bacterial culture after 4-8 days preferably 4 days for G diazotrophicus medium and 8 days for A. chroococcum medium at 10,000 rpm for 18-25 minutes preferably 20 minutes to obtain Culture Supernatant
iv. adding Culture Supernatant, obtained from step (ii), to Murashige and Skoog basal medium for nodal explants' growth.
v. wherein Culture Supernatant supplemented medium regenerates healthy roots and shoots simultaneously in 25-30 days as compared to normal 40-45 days.

2. A process of preparation of tissue culture medium for enhancing in-vitro
plantlet regeneration in air yam plant as claimed in Claim 1 wherein
maximum IAA production between 120-160 µg/ml is obtained on 4th day
for G.diazotrophicus strain medium and between 140-160 Mg/ml on 8th
day for A. Chroococcum strain medium.
3. A process of preparation of tissue culture medium for enhancing in-vitro plantlet regeneration in air yam plant as claimed in Claim 1, wherein the Culture Supernatant replaces growth regulator/phytohormones in plant tissue culture media.

Documents:

127-DEL-2006-Abstract-(13-05-2011).pdf

127-DEL-2006-Abstract-(13-10-2010).pdf

127-DEL-2006-Abstract-(20-05-2011).pdf

127-del-2006-abstract.pdf

127-DEL-2006-Claims-(13-05-2011).pdf

127-DEL-2006-Claims-(13-10-2010).pdf

127-DEL-2006-Claims-(16-06-2010).pdf

127-DEL-2006-Claims-(20-05-2011).pdf

127-del-2006-claims.pdf

127-DEL-2006-Correspondence Others-(20-05-2011).pdf

127-DEL-2006-Correspondence-Others-(13-05-2011).pdf

127-DEL-2006-Correspondence-Others-(13-10-2010).pdf

127-DEL-2006-Correspondence-Others-(16-06-2010).pdf

127-del-2006-correspondence-others-1.pdf

127-del-2006-correspondence-others.pdf

127-DEL-2006-Description (Complete)-(16-06-2010).pdf

127-del-2006-description (complete).pdf

127-del-2006-description (provisional).pdf

127-DEL-2006-Form-1-(13-05-2011).pdf

127-DEL-2006-Form-1-(20-05-2011).pdf

127-del-2006-form-1.pdf

127-del-2006-form-18.pdf

127-DEL-2006-Form-2-(13-05-2011).pdf

127-DEL-2006-Form-2-(13-10-2010).pdf

127-DEL-2006-Form-2-(16-06-2010).pdf

127-DEL-2006-Form-2-(20-05-2011).pdf

127-del-2006-form-2.pdf

127-DEL-2006-Form-3-(16-06-2010).pdf

127-del-2006-form-5.pdf

127-DEL-2006-GPA-(16-06-2010).pdf

127-del-2006-gpa.pdf


Patent Number 248511
Indian Patent Application Number 127/DEL/2006
PG Journal Number 29/2011
Publication Date 22-Jul-2011
Grant Date 21-Jul-2011
Date of Filing 18-Jan-2006
Name of Patentee CCS HARYANA AGRICULTURAL UNIVERSITY
Applicant Address HISAR-125004
Inventors:
# Inventor's Name Inventor's Address
1 NEERU NARULA, SANTOSH DHILLON AND VIJAY KUMAR CHOWDHURY DEPARTMENT OF BIOTECHNOLOGY, CCS HARYANA AGRICULTURAL UNIVERSITY, HISSAR-125004, INDIA.
2 PUSHPA KHARB, KANTA DAHIYA, VICTORIA YADAV, PRAVEEN BATRA DEPARTMENT OF BIOTECHNOLOGY, CCS HARYANA AGRICULTURAL UNIVERSITY, HISSAR-125004, INDIA.
PCT International Classification Number C07D 271/08
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA