Title of Invention	"A TISSUE CULTURE MEDIUM FORMULATION FOR INDUCTION AND PROLIFERATION OF MULTIPLE SHOOTS IN EXCISED EMBRYO AXES OF COTTON"
Abstract	An improved culture medium formulation useful for induction and proliferation of shoots in excised embryo axes of cotton essentially consisting ingredients mentioned below: Ingredients (mg/L) MgSO4,7H2O 370.00 KH2PO4 170.00 KNO3 1900.00 NH4NO3 1650.00 CaCl2.2H2O 440.00 H3BO3 ' 6.2 ZnSO4.7H2O 8.6 NaMoO4.2H2O 0.25 CuSO4.5H2O 0.025 CoCI2.6H2O 0.0025 Kl 0.83 MnSO4.4H2O . 22.3 FeSO4.7H2O 27.8 Na2 EDTA 37.3 Nicotinic acid 1.00 Pyridoxine HCL 1.00 ThiamineHCL 10.00 MyO-inositol 100.00 BAP (6-Benzylaminopurine) 0.10-0.50 Sucrose 20000.00 Agar 6500.00 and optionally containing NAA in the range of 0.02 mg/1, wherein the pH of the medium ranges from 5.6 to 5.8.

Title of Invention

"A TISSUE CULTURE MEDIUM FORMULATION FOR INDUCTION AND PROLIFERATION OF MULTIPLE SHOOTS IN EXCISED EMBRYO AXES OF COTTON"

Abstract

An improved culture medium formulation useful for induction and proliferation of shoots in excised embryo axes of cotton essentially consisting ingredients mentioned below: Ingredients (mg/L) MgSO4,7H2O 370.00 KH2PO4 170.00 KNO3 1900.00 NH4NO3 1650.00 CaCl2.2H2O 440.00 H3BO3 ' 6.2 ZnSO4.7H2O 8.6 NaMoO4.2H2O 0.25 CuSO4.5H2O 0.025 CoCI2.6H2O 0.0025 Kl 0.83 MnSO4.4H2O . 22.3 FeSO4.7H2O 27.8 Na2 EDTA 37.3 Nicotinic acid 1.00 Pyridoxine HCL 1.00 ThiamineHCL 10.00 MyO-inositol 100.00 BAP (6-Benzylaminopurine) 0.10-0.50 Sucrose 20000.00 Agar 6500.00 and optionally containing NAA in the range of 0.02 mg/1, wherein the pH of the medium ranges from 5.6 to 5.8.

Full Text	This invention relates to a tissue culture medium formulation useful for induction and proliferation of multiple shoots in the excised embryo axes of cotton. Cotton (Gossypium spp.), a high economic value crop is grown in about 75 countries for its textile fiber and various other by products. Over 180 million people are associated with the cotton industry that produces raw cotton every year worth of 20-30 billion US dollars. Thus cotton biotechnology can have a significant impact on world economics (John, M.E. Critical Reviews in Biotechnology 17 :185-208, 1997). Although conventional breeding method has produced steady improvements in agronomic traits, the approach has limitations due to interspecific incompatibility barriers in cotton (Pundir NS, Bot. Gaz. 133:7-26,1972). Modern techniques of genetic engineering have enabled the isolation, amplification and in vitro manipulation of desirable genes (Christou P, Trends in Plant Science 1 (12): 423-431, 1996). Though more than 30 crop species have been genetically modified, however, the technology of gene transfer for most species is not efficient and limited by cultivar restriction for in vitro regeneration (Zapata et al, Theor. Appl. Genet. 98: 252-256, 1999). Most cotton varieties of commercial interest have been found difficult or impossible to regenerate into plants (Trolinder NL and Goodin JR, Plant Cell Tissue and Organ Culture 12: 31-42, 1988). Plant regeneration through somatic embryogenesis via callus phase has been reported to generate undesirable somaclonal variations in cotton (Stelly et al., Genome 32:762-770, 1989). Alternative regeneration methods / explants have been attempted to achieve plant regeneration in cotton (Agrawal et al., Plant Cell Reports 16, 647-52, 1997; Hemphill et al., Plant Cell Reports 17: 273-278, 1998). However, due to its larger size, explants used in these reports are not amicable to transformation events. Therefore, other explants, like embryo axis has been attempted to achieve plant regeneration. The explant has several advantages like: 1) Small size of embryo axis is amicable to both the transformation techniques (Agrobacterium as well as Particle bombardment). 2) Embryo axis takes the least time to develop into single shoot (10-15 days) compared to several months in case of plant regeneration via callus phase. 3) Induction and further proliferation of multiple shoots in the explants as in present invention will enhance the efficiency of transformation by manifold. 4) We can avoid callus phase which gives rise somaclonal variations. 5) The procedure is simple and does not require costly equipments. Embryo axis in cotton normally gives only one shoot per explant in culture conditions, while it is desirable to induce multiple shoots in embryo axis to further enhance the rate of multiplication and the efficiency of transformation. In the present investigation, we have formulated a medium composition to achieve this task. Therefore, the main object of the present invention is to provide a culture medium composition useful for induction and proliferation of multiple shoots in the excised embryo axes of cotton. Another object of this invention is to provide a composition of culture medium containing appropriate concentrations of macronutrients, micronutrients, vitamins and hormones to obtain high frequency shoot induction and proliferation from the excised embryo axes of four cultivars of cotton. Yet another object is to provide an improved culture medium composition which is not a mere admixture but a synergistic mixture capable of inducing multiple shoots and their further proliferation in embryo axis explants of cotton cultivars under a given set of incubation conditions. Delinted seeds of four Indian cultivars of cotton (Gosspypium hirsutum L.) commercially designated as NHH-44; DCH-32; DHY-286; H-8, were surface sterilized and disinfected by the method described earlier (Agrawal et al. Plant Cell Reports 16,647-52, 1997). The disinfected seeds were soaked in the sterile water for 1 hour prior to inoculation on sterile filter paper moistened with 2 ml of sterile distilled water in petridishes. The seeds were incubated in dark for 48 -72 hour for germination. Under aseptic conditions, embryo-axes were excised from germinated seeds, their radicles dissected out and discarded and remaining 2 mm long embryo-axes were used as explants. These explants were cultured using the improved composition of the present invention. Accordingly, the present invention provides an improved culture medium formulation useful for induction and proliferation of shoots in excised embryo axes of cotton essentially consisting ingredients mentioned below: Ingredients (mg/L) MgS04,7H2O 370.00 KH2P04 170.00 KNO3 1900.00 NH4NO3 1650.00 CaCI2.2H2O 440.00 H3B03 6.2 ZnSO4.7H20 8.6 NaMoO4.2H2O 0.25 CuSO4.5H2O 0.025 CoCI2.6H20 0.0025 Kl 0.83 MnS04.4H2O 22.3 FeSO4.7H2O 27.8 Na2EDTA 37.3 Nicotinic acid 1.00 Pyridoxine HCL 1.00 ThiamineHCL 10.00 MyO-inositol 100.00 BAP 0.10-0.50 (6-Benzylaminopurine) Sucrose 20000.00 Agar 6500.00 and optionally containing NAA in the range of 0.02 mg/l, wherein the pH of the medium ranges from 5.6 to 5.8 In an embodiment the medium formulation containing NAA is denoted as medium A and without NAA is denoted as medium B. In yet another embodiment, the pH of the medium may be in the range of 5.6 to 5.8. The process followed in the present invention is as follows: Ten explants excised as above were inoculated in each petridish which were incubated under the conditions mentioned in Table 5. Each treatment had 6 replicates and the experiment was repeated twice.. Table 5 (Table Removed) After 3 weeks of incubation on Medium A in petridishes, the explants were transferred to 100 ml capacity Erlenmeyer flasks containing 30 ml of Medium A and were further incubated for 9 weeks under conditions listed in Table 5. After that number of shoots in each explant were recorded. And then these shoots were transferred to 250 ml capacity Erlenmeyer flasks containing 100 of Medium B and were incubated for further 8 weeks under conditions mentioned in Table 5. After that a final observation of number of shoots per explant was recorded. The formulation and the method described in the present invention was tested on explants of four cotton cultivars. All the cultivars responded to the formulated media sequence. The following examples relates to induction and proliferation of multiple shoots: Example 1 The formulation and the method described in the present invention was tested on explants of cotton cultivar NHH-44. Embryo axis explants (70.00 %) responded on Medium A and resulted on an average of 5. 95 + 1.16 number of shoots per explant. On transfer of this shoot mass onto the Medium B produced on an average 13.08 + 3.34 number of shoots per explant. Example 2 The formulation and the method described in the present invention was tested on explants of cotton cultivar DCH-32. Embryo axis explants (75.00 %) responded on Medium A and resulted on an average of 6.04 + 1.38 number of shoots per explant. On transfer of this shoot mass onto the Medium B produced on an average 15.04 + 3.07 number of shoots per explant. Example 3 The formulation and the method described in the present invention was tested on explants of cotton cultivar DHY-286. Embryo axis explants (60.00 %) responded on Medium A and resulted on an average of 4.55 + 1.25 number of shoots per explant. On transfer of this shoot mass onto the Medium B produced on an average 11.12 + 2.80 number of shoots per explant. Example 4 The formulation and the method described in the present invention was tested on explants of cotton cultivar H-8. Embryo axis explants (43.33 %) responded on Medium A and resulted on an average of 3.88 + 1.11 number of shoots per explant. On transfer of this shoot mass onto the Medium B produced on an average 9.72 + 2.54 number of shoots per explant. Example 5 The formulation (without BAP and NAA) and the method as described in the present invention was tested on the explants of four cotton cultivars namely NHH-44, DCH-32, DHY-286 and H-8. No multiple shoot induction was observed in any of these cultivars. The comparative results on multiple shoot induction, proliferation on formulation with and without BAP and NAA have been given in Table 6. Table 6 Comparative results on the use of formulation with or without 6-benzylamino purine (BAP) and Napthalene acetic acid (NAA) on induction, proliferation of multiple shoots in excised embryo axes of four cultivars of cotton: (Table Removed) The main advantages of the improved formulation of the present invention are: (1) It can be used for rapid propagation of cotton cultivars. 2) It provides higher frequency of shoot development and multiplication rate comparatively in shorter time. (3) Will enhance transformation efficiency. (4) The composition can be used gainfully in Agrobacterium as well as Particle Bombardment Mediated transformation experiments with embryo-axes. We Claim: 1. An improved culture medium formulation useful for induction and proliferation of shoots in excised embryo axes of cotton essentially consisting ingredients mentioned below: Ingredients (mg/L) MgS04,7H2O 370.00 KH2PO4 170.00 KNO3 1900.00 NH4N03 1650.00 CaCI2.2H20 440.00 H3B03 6.2 ZnSO4.7H2O 8.6 NaMoO4.2H2O 0.25 CuS04.5H2O 0.025 CoCI2.6H20 0.0025 Kl 0.83 MnSO4.4H20 22.3 FeS04.7H2O 27.8 Na2EDTA 37.3 Nicotinic acid 1.00 Pyridoxine HCL 1.00 Thiamine HCL 10.00 MyO-inositol 100.00 BAP 0.10-0.50 (6-Benzylaminopurine) Sucrose 20000.00 Agar 6500.00 and optionally containing NAA in the range of 0.02 mg/l, wherein the pH of the medium ranges from 5.6 to 5.8 2. An improved culture medium formulation useful for induction and proliferation of shoots in excised embryo axes of cotton as herein described with reference to the examples.

Full Text

This invention relates to a tissue culture medium formulation useful for induction and proliferation of multiple shoots in the excised embryo axes of cotton.
Cotton (Gossypium spp.), a high economic value crop is grown in about 75 countries for its textile fiber and various other by products. Over 180 million people are associated with the cotton industry that produces raw cotton every year worth of 20-30 billion US dollars. Thus cotton biotechnology can have a significant impact on world economics (John, M.E. Critical Reviews in Biotechnology 17 :185-208, 1997). Although conventional breeding method has produced steady improvements in agronomic traits, the approach has limitations due to interspecific incompatibility barriers in cotton (Pundir NS, Bot. Gaz. 133:7-26,1972). Modern techniques of genetic engineering have enabled the isolation, amplification and in vitro manipulation of desirable genes (Christou P, Trends in Plant Science 1 (12): 423-431, 1996). Though more than 30 crop species have been genetically modified, however, the technology of gene transfer for most species is not efficient and limited by cultivar restriction for in vitro regeneration (Zapata et al, Theor. Appl. Genet. 98: 252-256, 1999). Most cotton varieties of commercial interest have been found difficult or impossible to regenerate into plants (Trolinder NL and Goodin JR, Plant Cell Tissue and Organ Culture 12: 31-42, 1988). Plant regeneration through somatic embryogenesis via callus phase has been reported to generate undesirable somaclonal variations in cotton (Stelly et al., Genome 32:762-770, 1989). Alternative regeneration methods / explants have been attempted to achieve plant regeneration in cotton (Agrawal et al., Plant Cell Reports 16, 647-52, 1997; Hemphill et al., Plant Cell Reports 17: 273-278, 1998). However, due to its larger size, explants used in these reports are not amicable to transformation events. Therefore, other explants, like

embryo axis has been attempted to achieve plant regeneration. The explant has several advantages like:
1) Small size of embryo axis is amicable to both the transformation techniques
(Agrobacterium as well as Particle bombardment).
2) Embryo axis takes the least time to develop into single shoot (10-15 days)
compared to several months in case of plant regeneration via callus phase.
3) Induction and further proliferation of multiple shoots in the explants as in present
invention will enhance the efficiency of transformation by manifold.
4) We can avoid callus phase which gives rise somaclonal variations.
5) The procedure is simple and does not require costly equipments.
Embryo axis in cotton normally gives only one shoot per explant in culture conditions, while it is desirable to induce multiple shoots in embryo axis to further enhance the rate of multiplication and the efficiency of transformation. In the present investigation, we have formulated a medium composition to achieve this task.
Therefore, the main object of the present invention is to provide a culture medium composition useful for induction and proliferation of multiple shoots in the excised embryo axes of cotton.
Another object of this invention is to provide a composition of culture medium containing appropriate concentrations of macronutrients, micronutrients, vitamins and hormones to obtain high frequency shoot induction and proliferation from the excised embryo axes of four cultivars of cotton.
Yet another object is to provide an improved culture medium composition which is not a mere admixture but a synergistic mixture capable of inducing multiple

shoots and their further proliferation in embryo axis explants of cotton cultivars under a given set of incubation conditions.
Delinted seeds of four Indian cultivars of cotton (Gosspypium hirsutum L.) commercially designated as NHH-44; DCH-32; DHY-286; H-8, were surface sterilized and disinfected by the method described earlier (Agrawal et al. Plant Cell Reports 16,647-52, 1997). The disinfected seeds were soaked in the sterile water for 1 hour prior to inoculation on sterile filter paper moistened with 2 ml of sterile distilled water in petridishes. The seeds were incubated in dark for 48 -72 hour for germination. Under aseptic conditions, embryo-axes were excised from germinated seeds, their radicles dissected out and discarded and remaining 2 mm long embryo-axes were used as explants. These explants were cultured using the improved composition of the present invention.
Accordingly, the present invention provides an improved culture medium
formulation useful for induction and proliferation of shoots in excised embryo
axes of cotton essentially consisting ingredients mentioned below:
Ingredients (mg/L)
MgS04,7H2O 370.00
KH2P04 170.00
KNO3 1900.00
NH4NO3 1650.00
CaCI2.2H2O 440.00
H3B03 6.2
ZnSO4.7H20 8.6

NaMoO4.2H2O 0.25
CuSO4.5H2O 0.025
CoCI2.6H20 0.0025
Kl 0.83
MnS04.4H2O 22.3
FeSO4.7H2O 27.8
Na2EDTA 37.3
Nicotinic acid 1.00
Pyridoxine HCL 1.00
ThiamineHCL 10.00
MyO-inositol 100.00
BAP 0.10-0.50
(6-Benzylaminopurine)
Sucrose 20000.00
Agar 6500.00
and optionally containing NAA in the range of 0.02 mg/l, wherein the pH of the
medium ranges from 5.6 to 5.8
In an embodiment the medium formulation containing NAA is denoted as medium A and without NAA is denoted as medium B.
In yet another embodiment, the pH of the medium may be in the range of 5.6 to 5.8.

The process followed in the present invention is as follows: Ten explants excised as above were inoculated in each petridish which were incubated under the conditions mentioned in Table 5. Each treatment had 6 replicates and the experiment was repeated twice..
Table 5
(Table Removed)
After 3 weeks of incubation on Medium A in petridishes, the explants were transferred to 100 ml capacity Erlenmeyer flasks containing 30 ml of Medium A and were further incubated for 9 weeks under conditions listed in Table 5. After that number of shoots in each explant were recorded. And then these shoots were transferred to 250 ml capacity Erlenmeyer flasks containing 100 of Medium B and were incubated for further 8 weeks under conditions mentioned in Table 5. After that a final observation of number of shoots per explant was recorded.
The formulation and the method described in the present invention was tested on explants of four cotton cultivars. All the cultivars responded to the formulated media sequence.

The following examples relates to induction and proliferation of multiple shoots:
Example 1
The formulation and the method described in the present invention was tested on explants of cotton cultivar NHH-44. Embryo axis explants (70.00 %) responded on Medium A and resulted on an average of 5. 95 + 1.16 number of shoots per explant. On transfer of this shoot mass onto the Medium B produced on an average 13.08 + 3.34 number of shoots per explant.
Example 2
The formulation and the method described in the present invention was tested on explants of cotton cultivar DCH-32. Embryo axis explants (75.00 %) responded on Medium A and resulted on an average of 6.04 + 1.38 number of shoots per explant. On transfer of this shoot mass onto the Medium B produced on an average 15.04 + 3.07 number of shoots per explant.
Example 3
The formulation and the method described in the present invention was tested on explants of cotton cultivar DHY-286. Embryo axis explants (60.00 %) responded on Medium A and resulted on an average of 4.55 + 1.25 number of shoots per explant. On transfer of this shoot mass onto the Medium B produced on an average 11.12 + 2.80 number of shoots per explant.
Example 4
The formulation and the method described in the present invention was tested on explants of cotton cultivar H-8. Embryo axis explants (43.33 %) responded on Medium A and resulted on an average of 3.88 + 1.11 number of shoots per explant.

On transfer of this shoot mass onto the Medium B produced on an average 9.72 + 2.54 number of shoots per explant.
Example 5
The formulation (without BAP and NAA) and the method as described in the present invention was tested on the explants of four cotton cultivars namely NHH-44, DCH-32, DHY-286 and H-8. No multiple shoot induction was observed in any of these cultivars. The comparative results on multiple shoot induction, proliferation on formulation with and without BAP and NAA have been given in Table 6.
Table 6
Comparative results on the use of formulation with or without 6-benzylamino purine (BAP) and Napthalene acetic acid (NAA) on induction, proliferation of multiple shoots in excised embryo axes of four cultivars of cotton:
(Table Removed)
The main advantages of the improved formulation of the present invention are:
(1) It can be used for rapid propagation of cotton cultivars. 2) It provides higher frequency of shoot development and multiplication rate comparatively in shorter time.
(3) Will enhance transformation efficiency.
(4) The composition can be used gainfully in Agrobacterium as well as Particle
Bombardment Mediated transformation experiments with embryo-axes.

We Claim:
1. An improved culture medium formulation useful for induction and
proliferation of shoots in excised embryo axes of cotton essentially consisting
ingredients mentioned below:
Ingredients (mg/L)
MgS04,7H2O 370.00
KH2PO4 170.00
KNO3 1900.00
NH4N03 1650.00
CaCI2.2H20 440.00
H3B03 6.2
ZnSO4.7H2O 8.6
NaMoO4.2H2O 0.25
CuS04.5H2O 0.025
CoCI2.6H20 0.0025
Kl 0.83
MnSO4.4H20 22.3
FeS04.7H2O 27.8
Na2EDTA 37.3
Nicotinic acid 1.00
Pyridoxine HCL 1.00
Thiamine HCL 10.00

MyO-inositol 100.00
BAP 0.10-0.50
(6-Benzylaminopurine)
Sucrose 20000.00
Agar 6500.00
and optionally containing NAA in the range of 0.02 mg/l, wherein the pH of the
medium ranges from 5.6 to 5.8
2. An improved culture medium formulation useful for induction and proliferation of shoots in excised embryo axes of cotton as herein described with reference to the examples.

Documents:

324-del-2000-abstract.pdf

324-del-2000-claims.pdf

324-del-2000-correspondence-others.pdf

324-del-2000-correspondence-po.pdf

324-del-2000-description (complete).pdf

324-del-2000-form-1.pdf

324-del-2000-form-2.pdf

324-del-2000-form-3.pdf

324-del-2000-form-4.pdf

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Patent Number

232855

Indian Patent Application Number

324/DEL/2000

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

21-Mar-2009

Date of Filing

28-Mar-2000

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG NEW DELHI-110 001,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	DINESH CHANDRA AGRAWAL	CHEMICAL LABORATORY,PUNE-411 008,MAHARASHTRA,INDIA
2	ANJAN KUMAR BANERJEE	CHEMICAL LABORATORY,PUNE-411 008,MAHARASHTRA,INDIA
3	SATISH MANOHAR NALAWADE	CHEMICAL LABORATORY,PUNE-411 008,MAHARASHTRA,INDIA
4	SULEKHA HAZRA	CHEMICAL LABORATORY,PUNE-411 008,MAHARASHTRA,INDIA
5	KAZA VENKATA KRISHNAMURTHY,	CHEMICAL LABORATORY,PUNE-411 008,MAHARASHTRA,INDIA
6	ANJAN KUMAR BANERGEE	CHEMICAL LABORATORY,PUNE-411 008,MAHARASHTRA,INDIA

PCT International Classification Number

C12N 5/04

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA