Title of Invention

A TISSUE CULTURE MEDIEM COMPOSITION FOR PEANUT

Abstract A tissue culture medium composition for peanut which comprises a mixture of the following ingredients in the specified as given below. MgS04,7H20 370 mg/l KH2PO4 170 mg/l KNO3 1900 mg/l NH4NO3 1650 mg/l CaGI2,2H20 440 mg/l H3BO3 6.2 mg/l MnS04 15.6 mg/l ZnS04,H20 8.6 mg/l NaMo04,2H20 0.25 mg/l CuS04,5H20 0.025 mg/l CoGI2,6H20 0.025 mg/l Kl 0.83 mg/l FeS04,7H20 27.8 mg/l Na2EDTA 37.3 mg/l Sucrose 20 g/1 Thiamine HCI 0.5 mg/l Pyridoxine.HCI 0.5 mg/l Nicotinic acid 0.G5 mg/l Myo-inositol 100:00 mg/l Glycine 2.0 mg/l Thidiazuron 1 to 7 mg/l Agar 450 mg/l to 800 mg/l
Full Text This invention relates to a tissue culture medium composition for peanut.
Among the methods of plant development in cultured tissues, somatic embryogenesis is preferred over organogenesis because they develop as bipolar structures bearing both root and shoot meristems necessary for development of complete plant. Thus research directed towards in vitro morphogenesis has resulted in a number of protocols for somatic embryogenesis in various plant species. To be of practical value, somatic embryogenesis should culminate in the formation of plantlets. However, the efficient conversion of somatic embryos into plantlets remains a serious constrain.
In peanut there are several reports on regeneration of plants via somatic embryogenesis but the conversion rate reported is very low. The specific rates reported are tabulated in Table-1
TABLE-1
(Table Removed)
It is apparent from the above table that due to the limitations at the conversion stage the rate -t>f production of plants using the protocols developed by various researchers via somatic embyogenesis are very low, hence plantlets in large number can not be produced by any of these methods.
Therefore these protocols are not suitable -for various biotechnological applications e.g. isolation of variants, synthetic seed technology, genetic trans-formation etc. Thus there is a need to formulate medium compositions in which conversion of peanut somatic embyos can be achieved at-^high frequency.

It 1B therefor© an objective of the present invention ho develops a method and formulate a composition in which the conversion of somatic embryos to plantlets is very high compared to the earlier reported values. Somatic embryogenesls is induced in the immature leaflets following the procedure deacribed earlier (Chengalrayan et al . 1994). In the earlier report we mentioned that the 100"/. of the* embryos germinated to give rise to roots and no shoots. Only 24.7% of the rooted embryos developed stunted shoots on transfer to Murashige and Skoog's (MS) medium, supplemented with 0.5 mg/1 each of benzylamino purine (BAP) and kinetin The present invention therefore provides a composition for the production of plantlets of peanut using the method of somatic embryogenesis.
The concentration of the specific ingredients in the composition is very important to promote somatic embryo conversion of peanut. The perticular combination with specific concentration imparts synergistic effect on the resultant composition in somatic embryo conversion of peanuts.
The composition of the present invention consists of macronutrients such as MgSO4, KH2PO4 „ KMO3, NM4 NO3., CaCl2 micro nutrients such as H3 BO3, MnSO4 NaMoO4, CuSO4, C0CI25 KI„ FeSO4,
Na2EDTA: Vitamins such as Thiminw, Pyridoxine, Nicotinic acid, Myo-inositol,
amino acid such as Qlycinep carbon source such as Sucrose and cytokinin such as
Thidiazuron.
Accordingly the present invention provides a tissue culture medium
composition for peanut which comprises a mixture of the following ingredients in the
specified as given below.
MgS04,7H20 370 mg/l
KH2PO4 170 mg/l
KNO3 1900 mg/l
NH4NO3 1650 mg/l
CaCI2,2H20 440 mg/l
H3BO3 6.2 mg/l
MnS04 15.6 mg/l
ZnS04,H20 8.6 mg/l
NaMo04,2H20 0.25 mg/l
CuS04)5H20 0.025 mg/l
CoCI2,6H20 0.025 mg/l
Kl 0.83 mg/l
FeS04,7H20 27.8 mg/l
Na2EDTA 37.3 mg/l
Sucrose 20 g/1
Thiamine HCI 0.5 mg/l

Pyridoxine.HCI 0.5 mg/l
Nicotinic acid 0.05 mg/l
Myo-inositol 100.00 mg/l
Glycine 2.0 mg/l
Thidiazuron 1 to 7 mg/l
Agar 450 mg/l to 800 mg/l
The composition of the present invention makes use of conventional MS medium along with thidiazuron in the concentration of 1 to 7 mg/l.
In one embodiment of the present invention the survival rate of the plant produced by using the composition described in the present invention is more than 80%
The invention is further described by the following examples which are of illustrative nature and should not be construed to limit the scope of the invention.
The composition prepared in the present invention is synergistic mixture and not mere admixture and is not a product of chemical reaction.
The experiments were carried out preparing media which are conventionally known and also with the addition of thidiazuron. The conversion rates obtained are given at the end of each of the examples. Conventional (MS) medium is used in example 1&2. Eaxmple 3-7 illustrate use of MS medium supplemented with BAP and KN as cytokinins. Example 8-10 illustrates the use of the composition of the present invention.
Example 1
Ingredientn Amounts (mg/l)
Macronutrients
MgS04,7H2O 370
KHoP04 "*" 170
KNO-s 1900
NH4i\IO^ 1650
CaCln"-,2HnO 440
Micronutrients
H3B0^ 6.2
MnS0'4,H20 15.6
ZnS04,7H?0 8.6
NaMDa4,2H2a 0.2S
CuB04,5H26 0.025
CDC12,6HJO 0.025
KI *~ 0.83
FeS04,7H20 27.8
Na-jEDTA *" 37.3
Sucrose 20
Vitamins
Thimine.HCl 0.5
Pyrido>:ine.HCl • 0.5
Nicotinic acid 0.05
Myo-inositol 100
Glycine 2
Cytokinin(s)
Benzylamino purine 1
pH 5.8
Agar 0.45"/.
Light ' 20 - 32uE/m2/s
Photoperiod 16h light
Temperature 23 - 27°C
Culture vessel 150 X 25rnm
(test tube)
Percentage o-f conversion obtained 60.0"/.
Example 2
j
Ingredients Amounts Macronutrients
MqB04,7H20 370
KH7P04 *■ 170
KNO^ 1900
NH4NQ3 1650
CaCl2?2H20 440
Micronutrient H"^B0^» tb. 2
MnSO^fH^a 15.6
ZnS04,7H20 8.6
NaMo04,2Ho0 0.25
CuSD4,5H20 0.025
Coc 1 -7, 6H20 0.025
KI "" 0.83
FeS04,7Ho0 27.8
Na2EDTA "" 37.3
Sucrose 20
Vitamin©
Thimine.HCl 0.5
Pyridoxin©.MCI • 0.5
Nicotinic acid 0.05
Myo-inositol 100
Glycine 2
Cytokinin(s)
Bensylamino purine 5
pH 5.8
Agar 0.457.
Light 32uE/m2/s
Photoperiod 16h light
Temperature 23 ~ 27°C
Culture vessel 150 X 25mm
Percentage o-f conversion obtained 707.
Example 3
Ingredients Amounts Macronutrients
H(3SQ4,7H20 370
KH2P04 * 170
KNO-, 1900
NH4N0^ 1650
CaCl2^2H20 440
Micranutrients
H-^BO-; 6.2
MnS0j,H20 15.6
2nS04!,7H20 8.6
NaMo04„2iH20 0.25
CuB04,5H20 0.025
Cocl2,.6HoO 0.025
KI *" 0.83
FeS04,,7H?0 27.8
Na~,EDTA 37.3
Sucrose 20
Vitamins
Thimine.HCl 0.5
Pyridoxin©.HC1 0.5
Nicotinic acid 0.05
Myo~inositol 100
Glycine 2
Cytokinin Benzylamino purine 1
Kinetin 2
pH 5.8
Agar 0.457.
Light 32uE/m2/s
Photoperiod 16h light
Temperature 23 - 27°C
Culture vessel 150 X 25mm
(test tube)
Percentage of conversion obtained 72.0V.
Example 4
Ingredients Amounts img/1)
MacTonutrients
MgS04,7H20 370
KH2P04 * 170
KNOT; 1900
NH4N0^ 1650
CaCloT2HfO 440
Micronutrients
H-^BO, 6.2
MnS04„H^0 15.6
ZnS04,7H?D 8.6
NaMo04,2H20 0.25
CuS04,5H?6 0.025
CDC12,6HJO 0.025
KI ' 0.83
FeS04,7H20 27.8
Na2ED"TA "*" 37.3
Sucrose 20
Vitamins
"Thimine.HCl 0.5
Pyridoxine.HCl 0.5
Nicotinic acid 0.05
M/o-inositol 100
Glycine 2
Cytokinin(s)
Benzylamino purine 2
Kinetin 2
pH 5.8
Agar 0.457.
Light 32uE/m2/s
Photoperiod 16h light
Temperature 23 - 27°C
Culture vessel 150 X 25mm
(test tube)
Percentage of conversion obtained 80.07.
Example 5
Ingredients Amounts Macronutrients
MgSa4„ 714^0 370
KH2P04 " 170
KNO-.n; 1900
NH4N0-^ 1650
CaCl2/2H20 440
Micronutrien.ts
H-^IHO^ 6.2
MnB04,H20 15.6
ZnS04!l7H20 8.6
WaMa04,2H20 0.25
CuS04.,5H2a 0.025
Cocl^,6HoO 0.025
KI 0.83
FeS04 ,,7H20 27.8
NaoEDTA "" 37.3
Sucrose 20
Vitamins
Thimine.HCl 0.5
Pyrido«ine.HCl 0.5
Nicotinic acid 0.05
Myo-inositol 100
Glycine 2
Cytokinin Benzyl amino purine 3
Kinetin 2
pH 5.8
Agar 0.457.
Light 32uE/m2/s
Photoperiod 16h light
Temperature 23 - 27°C
Culture vessel 150 X 25mm
(test tube)
Percentage of conversion obtained 76.0%
Example 6
Ingredients Amounts
liac ronu t r i en t s
MqSa4»7H2Q 370
KH2P04 "*" 170
KNO^r 1900
CaCl2?2H20 440
Clicronu.tr tents
H-^BO^ 6 - 2
MnS04»H20 15.6
Zn804,7H20 8-&
NaMo04,2H->0 0.25
CuS04,5H26 0.025
Cocl-,.6HoO 0.025
KI 0.83
FeS04,7H-,a 27.9
MaoEDTA *" 37.3
Sue rose 20
Vitamins
Thimine.HCl O.S
Pyridoxine.HCl 0.5
Nicotinic acid 0.05
Myo-inositol 100
Glycine 2
Cytokinin(s)
Benzylamino purine 1
Kinetin 3
pH 5.8
Agar 0.457.
Light 32uE/m2/s
Photoperiod 16h light
Temperature 25 ± 2°C
Culture vessel 150 X 25mm
Percentage o-f conversion obtained 72.07.
Example 7
Ingredients Amounts liacronutrients
MgS04,7H20 370
KH2P04 170
KNO^r 1900
NH4N0-^ 1650
CaCl2»2H20 440
MjLcronutrients
H-^BO^ 6.2
MnS04!,H2O 15.6
7.nSD4,7H?0 8.6
NaMo04.2H70 0.25
CuS0455H26 0.025
Cocl2,6H20 0.025
KI "' *~ 0.83
FeS04,7H20 27.8
Na-7EDTA 37.3
Sucrose 20
Vitamins
Thimine.HCl 0.5
Pyridoxine.HCl 0.5
Nicotinic acid 0.05
Myo-inositol 100
Glycine 2
Cytokinin(s)
Benzylamino purine 3
Kinetin 3
-~-pt4- S.8
Agar 0.45X
Light 32uE/m2/s
photoperiod 16h light
Temperature 23 - 27°C
Culture vessel 150 X 25mm
(test tube)
Percentage o-f conversion obtained 687.
Example 8
Irusredients Amounts (mg/l)
Macronutrients
MgS04,7Ho0 370
KH->F04 *" 170
KN0-, 1900
NH4N0-^ 1650
CaCl2^2H20 440
Micronutrients
H-^B03 6.2
Mn804,H20 15.6
ZnB04„7H20 8.6
NaMD04.2H20 0.25
CuS04,5H26 0.025
Coclo.6H20 0.025
KI "*" J~ 0.83
Fe804,7H20 27.8
Na0EDTA * 37.3
Sucrose 20
Vitamins
Thimine.HCl 0.5
Pyridoxins.HC1 0.5
Nicotinic acid 0.05
Myo-inositol 100
Glycine 2
Cytokinin
Thidiazuron 1
pH 5.8
Agar 0.457.
Light 32uE/m2/s
Photoperiod 16h light
Temperature 23 - 27°C
Culture vessel 150 X 25mm
Percentage o-f conversion obtained 827.
Example 9
Inqredierits Amounts (mg/1)
Macronutrients
M0804,7H20 370
KH0PO4 170
KN6^ 1^00
NH4N0-* l^50
CaCl2;2H20 440
Micronutrients
H-»s80->j 6.2
MnS04,Ho0 15.6
ZnS04,7H-,0 8-6
NaMo04,2Ho0 0.25
CuB04,5H76 0.025
Coc1n,6H^0 0.025
KJ. 0.83
FeS04,7H20 27.8
Na2EDTA ' 37.3
Sucrose 20
Vitamins
Thimine.HCl 0.5
Pyridoxins.HC1 0.5
Nicotinic acid O.05
Myo-inositol 3.00
Glycine 2
Cytokinin Thidiazuron 3
PH 5.8
Agar 0.457.
Light 32uE/m2/s
Photoperiod j^h light
Temperature 23 - 27°C
Culture vessel 150 x 25mm
(test tube)
Percentage o-f conversion obtained 86%
E>i ample 10
Ingredients Amounts (mq/l>
Macronutrients
MgS04„7H20 370
KH2P04 *" 170
KN0-* 1900
NH4N03 1650
CaCl2.,2H2Q 440
Micronutrients
H^BOi.; 6.2
linS04,H20 15.6
ZnS04,7H?0 8.6
NaMoa4,2H^0 0.25
CuS04,5H20 0.025
Cocl^,6HoO 0.025
KI 0.83
FeS04,7H^0 27.8
Wa2EDTA " 37.3
Sucrose 20
Vi taniins
Thimine.HCl 0.5
Pyridoxins.HC1 0.5
Nicotinic acid 0.05
liyo-inasitol 100
Glycine 2
Cytok.inin Thidiaauron 5
pH 5.8
Agar 0.45%
Light - 32uE/m2/s
Photoperiod 16h light
Temperature 23 - 27°C
Culture vessel 150 X 25mm
'.test tube)
Percentage of conversion obtained 94%
Example 11
Inqredients Amounts (mq/1)
Macronutrients
MqS04f,7HoO 370
KH7P04 **" 170
KN0-, 1900
NH4N0-, 1630
CaCl2^2H20 440
Micronutrients
H-3BO-, 6.2
MrtB04,Ho0 15-6
ZnS04f7Ho0 8.6
NaMo04,2H20 0.25
CuS04 5H20 0.025
Coc 1216H 20 0.0 25
KI *" 0.83
FeS04,7Ho0 27.8
NaoEDTA "' 37.3
Sue rose 20
Vitamins
Thimine.HCl 0.5
Pyridoxins.MCI • 0.5
Nicotinic acid 0.05
Myo-inositol 100
Glycine 2
Cytokinin Thidiasuron 7
pH 5. S
Agar 0.457.
Light 32uE/m2/s
Photoperiod 16h light
Temperature 23 - 27DC
Culture vessel 150 X 25mm
(test tube)
Percentage of conversion obtained 94%
It is evident from the above examples that the conversion rate obtained from the use of the composition of the prsent invention is much higher than those obtained -from the media wherein Thidiazuron is not. present. It is also observed that the preferred concentration of the Thidiazuron is in the range of 1-7 mg/lit more preferrably 5.0 mg/liter.
Advantages of the present invention
1. Several agencies around the world are now engaged in
developing improved varieties of crops using somaclonal
variation, protoplast fusion and genetic transformation
approaches. Often the mode of regeneration used for these
purposes is somatic embryagenesis. The final step involved
in this pathway of morphogenesis is plant conversion. The
limitation encountered in conversion of somatic embryos of
peanut can now be overcome using this procedure.
2. In the total cost of peanut production, the cost of seed
alone forms 35.2-46.68%. in rainfall groundnut and 26.23-
37.47'/. in irrigated groundnut.
The plant populations recommended in India for various habit groups are more than twice in plant populations used in other major groundnut growing countries. As the cost of seed is high and a substantial amount of seed gets consumed for cultivation, seedlings raised by somatic embryogenesis
might find .Its application in substituting natural seeds which can then be released -for edible purposes. Effective use of somatic embryos for agricultural purposes will depend on efficient conversion protocol in addition to a reliable regeneration protocol. The protocol developed in this laboratory results in 94% conversion which is comparable to seed germination i.e. 90-95% in bunch types and Q5-90% in spreading types..
3. Once induced somatic embryos of peanut can be proliferated in culture for several passages in batches. In the long run the work might get extended to other important genotypes and with improvement, of technology, the somatic embryogenesis system can serve as a source of planting material round the year. In that case the protocol described here for conversion of somatc embryos to plantlets will be of immense importance-










We claim :
1. A tissue culture medium composition for peanut which comprises a mixture of the following ingredients inithe specified as given below.
MgS04,7H20 370 mg/l
KH2P04 170 mg/l
KN03 1900 mg/l
NH4N03 1650 mg/l
CaGI2,2H20 440 mg/l
H3B03 6.2 mg/l
MnS04 15.6 mg/l
ZnS04,H20 8.6 mg/l
NaMo04,2H20 0.25 mg/l
CuSG4,5H20 0.025 mg/l
CoGI2,6H20 0.025 mg/l
Kl 0.83 mg/l
FeS04,7H20 27.8 mg/l
Na2EDTA 37.3 mg/l
Sucrose 20 g/1
Thiamine HGI 0.5 mg/l
Pyridoxine.HGI 0.5 mg/1
Nicotinic acid 0.G5 mg/1
Myo-inositol 100.00 mg/i
Glycine 2.0 mg/l
Thicliazuron 1 to 7 mg/l
Agar 450 mg/l to 800 mg/l
2. A tissue culture medium composition for peanut substantially as herein
described with reference to the examples.


Documents:

1406-del-1996-abstract.pdf

1406-del-1996-claims.pdf

1406-del-1996-complete specification (granted).pdf

1406-del-1996-correspondence-others.pdf

1406-del-1996-correspondence-po.pdf

1406-del-1996-description (complete).pdf

1406-del-1996-form-1.pdf

1406-del-1996-form-2.pdf

1406-del-1996-form-4.pdf

1406-del-1996-form-6.pdf


Patent Number 197001
Indian Patent Application Number 1406/DEL/1996
PG Journal Number 38/2008
Publication Date 19-Sep-2008
Grant Date 30-Mar-2007
Date of Filing 26-Jun-1996
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI- 110001, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 DR. MRS. SULEKHA HAZRA NATIONAL CHEMICAL LABORATORY, PUNE, MAHARASHTRA, INDIA.
2 MR. KUDITHIPUDI CHENGALRAYAN NATIONAL CHEMICAL LABORATORY, PUNE, MAHARASHTRA, INDIA.
3 MISS. VANDANA BHASKAR MHASKE NATIONAL CHEMICAL LABORATORY, PUNE, MAHARASHTRA, INDIA.
PCT International Classification Number A01H 4/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA