NOVEL NUTRIENT MEDIA FOR MICROPROPAGATION OF JUGLANS SPP AND METHOD THEREOF

Abstract

The present disclosure relates improved nutrient media and method for micropropgation of Juglans sp Chandler. The said media comprises NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L, Ca3(PO4)2- 182.5 mg/L, ZnO- 17.0 mg/L, K2SO4- 779 mg/L, KCl- 779 mg/L, MgSO4.7H2O- 779 mg/L, MnSO4.H2O- 33.5 mg/L, CuSO4.5H2O- 0.25, KH2PO4- 182.5 mg/L, H3BO3- 5.0 mg/L, FeEDDHA- 120.00 mg/L, Nicotinic acid- 1.00 mg/L, Glycine- 2.00 mg/L, Thiamine-HCl-2.00 mg/L, AgNO3 - 6.00 mg/L, Casein Hydrolysate-100.00 mg/L, Phloroglucinol- 50.00 mg/L, TDZ - 0.50 mg/L, BAP- 0.50 mg/L, IBA- 0.10 mg/L, myo-inositol- 150.00 mg/L, Sucrose- 30.00 g/L and Agar- 6.50 g/L.

Specification

Description:F O R M 2
THE PATENTS ACT, 1970
(39 of 1970)
The patent Rule, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

NOVEL NUTRIENT MEDIA FOR MICROPROPAGATION OF JUGLANS SPP AND METHOD THEREOF

Name in Full Nationality Address of the Applicant
DR. YASHWANT SINGH PARMAR UNIVERSITY OF HORTICULTURE AND FORESTRY Indian Dr. Y S Parmar University of Horticulture and Forestry,
Nauni, Solan 173 230.
Himachal Pradesh INDIA

DR PANKAJ KUMAR Indian Assistant Professor
Department of Biotechnology, College of Horticulture,
Dr. Y S Parmar University of Horticulture and Forestry,
Nauni, Solan 173 230.
Himachal Pradesh INDIA


The following specification describes the invention and the manner in which it is to be disclosed-

TECHNICAL FIELD
[0001] The present invention relates to the field of Plant Biotechnology. More particularly, the present invention relates to novel nutrient media formulation with different concentrations of macro and micronutrients and an novel and improved methodology to establish Juglans spp (walnut) successfully.

BACKGROUND
[0002] Background description includes information that may be useful in understanding the present invention.
[0003] In vitro propagation is a vital technique in modern plant biotechnology, providing significant benefits for the propagation of recalcitrant plant species that are difficult to reproduce using conventional methods, with walnut (Juglans regia L.) being a prime example. This method enables the efficient multiplication of high-quality, true to type plants, overcoming the limitations of traditional propagation techniques.
[0004] Despite ongoing efforts to optimize establishment protocols for walnuts, successful propagation remains particularly challenging due to the unique attributes of this species. High levels of phenolic compounds causing oxidative browning, along with complex nutrient and hormonal requirements, are significant barriers that hinder tissue culture success and complicate medium optimization efforts. Overcoming these challenges is critical to developing a stable, efficient medium that promotes healthy growth and regeneration in vitro. Although the MS (Murashige and Skoog) medium has remained a constant foundation for in vitro culture establishment since its introduction in 1962, it has not been proven universally effective for walnuts.
[0005] For walnut, in vitro propagation DKW medium (Driver and Kuniyuki, 1984), and modified DKW medium (mDKW1 and mDKW2) given by Asharafi et al. (2010) have been commonly used. However, low success rates due to necrosis and slow growth have been reported with both standard and modified DKW formulations.
[0006] The phenol content and slow growth becomes more prominent in the lateral-bearing walnut variety such as Chandler which is of critical importance in Juglans spp. due to its superior yield potential compared to terminal-bearing varieties. In lateral-bearing genotypes, floral buds develop along the sides of the branches in addition to terminal bearing, enabling more frequent fruit production across multiple growing points. This trait significantly increases the number of nuts produced per tree, leading to higher overall yields. Additionally, lateral-bearing varieties are precocious, meaning they start producing fruit earlier in their life cycle, which results in faster returns for growers.
[0007] There is abundant literature disclosing the various media to micropropgate the walnut sp Chandler at different stages. Previous studies on J. regia using DKW medium supplemented with 1.0 mg/L BAP and 0.01 mg/L IBA reported explant survival rates of 50% by Revilla et al. (1989) and 56.25% by Tarinejad et al. (2013). Additionally, Kushnarenko et al. (2022) enhanced survival to 62.7% by incorporating Plants Preservative Mixture (PPM) into the same DKW medium. Further investigations by Revilla et al. (1989) achieved a maximum of 1.81 shoots per explant on MS medium supplemented with 1.0 mg/L BAP and 0.1 mg/L IBA. Dirlik et al. (2022) reported a multiplication rate of 1.05 shoots per explant using 4.0 mg/L BAP and 0.01 mg/L IBA. Ehteshamnia and Gholami (2014) achieved a maximum of 3.5 shoots per explant on DKW medium. In findings by Vahdati et al. (2004); Yegizbayeva et al. (2021); Ribeiro et al. (2022) have achieved root under in vitro conditions followed by hardening to outside environment conditions.

[0008] It is evident from the prior art, that there is only 50-55% survival of the explants and despite using very high concentrations of growth hormones for shoot regenerations, there is normally 1-3 shoot regerating from the explant.

[0009] In the view of foregoing, there exists a need of an efficient method and media for callus production of Juglans spp which ensures maximum survival of explants after initial steps i.e surface sterilization followed maximum shoot regeneraton from explant. It will be advantageous to provide a media which promotes callus induction as well as shoot regeneration which can reduce the hassle of making different media swith different compositions which can in turn help in micropropagation of walnut at large scale.

OBJECTS OF THE INVENTION
Some of the objects of the present disclosure, which at least one embodiment herein satisfy, are listed herein below.
[0010] The principal object of the present subject matter is to provide an novel medium formulation for micropropgation of Juglans spp.
[0011] Another object of the present subject matter is to provide an improved method for maximum explant survival of Juglans spp.
[0012] Another object of the present subject matter is more efficient methodology for the multiplication of Juglans sp.
[0013] Another object of the present subject matter is the methodology for acclimatization of micro shoot without rooting.
These and other objects and advantages will become more apparent when reference is made to the following description and accompanying drawings.

SUMMARY
[0014] This summary is provided to introduce concepts related to novel nutrient media and method for micropropgation of walnut. The said media comprises NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L, Ca3(PO4)2- 182.5 mg/L, ZnO- 17.0 mg/L, K2SO4- 779 mg/L, KCl- 779 mg/L, MgSO4.7H2O- 779 mg/L, MnSO4.H2O- 33.5 mg/L, CuSO4.5H2O- 0.25, KH2PO4- 182.5 mg/L, H3BO3- 5.0 mg/L, FeEDDHA- 120.00 mg/L, Nicotinic acid- 1.00 mg/L, Glycine- 2.00 mg/L, Thiamine-HCl-2.00 mg/L, AgNO3 - 6.00 mg/L, Casein Hydrolysate-100.00 mg/L, Phloroglucinol- 50.00 mg/L, TDZ - 0.50 mg/L, BAP- 0.50 mg/L, IBA- 0.10 mg/L, myo-inositol- 150.00 mg/L, Sucrose- 30.00 g/L and Agar- 6.50 g/L.
[0015] Furthermore, the method to make media of the said invention comprises- Mixing NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L to make Stock 1; mixing KH2PO4- 182.5mg/l and Ca3(PO4)2- 182.5 mg/l to make Stock 2; mixing MgSO4.7H2O- 779 mg/l, MnSO4.H2O- 33.5 mg/l, CuSO4.5H2O- 0.25 mg/l, K2SO4 - 779 mg/l and KCl - 779mg/l to make Stock 3;mixing FeEDDHA- 120 mg/l, H3BO3- 5.0 mg/l and ZnO -17.0 mg/l to make Stock 4;mixing Nicotinic acid- 1.0 mg/l, Thiamine-HCl- 2.0 mg/l and Glycine 2.0 mg/l to make Stock 5; adding AgNO3 (6.0 mg/L), casein hydrolysate (100 mg/L), and phloroglucinol (50 mg/L) follwd by mixing; adding growth hormones TDZ (0.5 mg/L), BAP (0.5 mg/L), and IBA (0.1 mg/L) followed by mixing; adding sucrose (30 g/L), along with myo-inositol (150 mg/L) and agar (6.5 g/L) and adjusting the pH of the medium to 5.5-5.8 followed by sterilization in an autoclave at 121.6°C and 15 pounds per square inch pressure for 15-20 minutes.
[0016] Moreover, the said invention provides a method of microppgation and novel media which assutres highest multiplication rate of 4- 5 shoots/callus within 18-19 days.
[0017] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The illustrated embodiments of the subject matter will be understood by reference to the drawings that are consistent with the subject matter as claimed herein,
Fig. 1- Overview of in vitro establishment, propagation and culture techniques for the walnut, progressing through to the hardening stage
Selection of shoot tip explant from walnut tree of cultivar Chandler.
a) Surface sterilization of shoot tip explants.
b) Inoculation of shoot tip segments on invented PKW medium.
c) Shoot induction and callus formation at shoot tip base on PKW medium
d) For subculturing after 20 days on the same medium combination, vertical excisions were made on the callus.
e) Excise the regenerated shoot after forming 2-3 axillary buds and culture on fresh PKW medium.
f) Formation of multiple shoots and new callus on sub-cultured shoot tip.
g) Newly formed shoots were excised along with callus and culture on fresh PKW medium.
h) Multiplication of shoots arises from a single callus at the shoot tip base.
i) Shoots along with callus were excised and culture on fresh medium containing 3.0 mg/L IBA followed by incubation for 2 days.
j) Transfer of shoot on acclimatization medium containing 1:1:1 sterile mixture of cocopeat, vermiculite and perlite. After 15-20 days root initiation will occur which indicates the successful acclimatization.
k) Transplant the acclimatized plant on soil medium containing 1:1:1 sterile mixture of cocopeat, vermiculite and perlite.

DETAILED DESCRIPTION
[0019] The detailed description of various exemplary embodiments of the disclosure is described herein with reference to the accompanying drawings. It should be noted that the embodiments are described herein in such details as to clearly communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the scope of the present disclosure as defined by the appended claims.
[0020] It is also to be understood that various quantities and ratios may be devised that, although not explicitly described or shown herein, embody the principles of the present disclosure. Moreover, all statements herein reciting principles, aspects and embodiments of the present disclosure, as well as specific examples, are intended to encompass equivalents thereof.
[0021] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0022] It should be noted that the description merely illustrates the principles of the present subject matter. It will thus be appreciated that those skilled in the art will be able to devise various arrangements that, although not explicitly described herein, embody the principles of the present subject matter and are included within its scope.
[0023] Disclosed herein is an improved media and method for micropropagation of walnut.
[0024] In a prefered embodiment of the invention, an improved media for micropropagation of walnut (var. Chandler) comprises of NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L, Ca3(PO4)2- 182.5 mg/L, ZnO- 17.0 mg/L, K2SO4- 779 mg/L, KCl- 779 mg/L, MgSO4.7H2O- 779 mg/L, MnSO4.H2O- 33.5 mg/L, CuSO4.5H2O- 0.25, KH2PO4- 182.5 mg/L, H3BO3- 5.0 mg/L, FeEDDHA- 120.00 mg/L, Nicotinic acid- 1.00 mg/L, Glycine- 2.00 mg/L, Thiamine-HCl-2.00 mg/L, AgNO3 - 6.00 mg/L, Casein Hydrolysate-100.00 mg/L, Phloroglucinol- 50.00 mg/L, TDZ - 0.50 mg/L, BAP- 0.50 mg/L, IBA- 0.10 mg/L, myo-inositol- 150.00 mg/L, Sucrose- 30.00 g/L and Agar- 6.50 g/L.
[0025] In an embodiment of the invention, constituents/ingredients of the media can be mixed as such or stocks solutions of the major and micro constituents can be made and mixed in the appropriate concentrations to make the media.
[0026] In an embodiment of the invention, the stock solutions to prepare the improved media of the instant invention comprises of-
Constituents Quantity (mg/l)
Stock 1
NH4NO3 1400
Ca(NO3)2.4H2O 1034
KNO3 1034
Stock 2
KH2PO4 182.5
Ca3(PO4)2 182.5
Stock 3
MgSO4.7H2O 779
MnSO4.H2O 33.5
CuSO4.5H2O 0.25
K2SO4 779
KCl 779
Stock 4
FeEDDHA 120
H3BO3 5.0
ZnO 17.0
Stock 5
Nicotinic acid 1.0
Thiamine-HCl 2.0
Glycine 2.0

[0027] In an embodiment of the invention, the method of preparation of the invented media for micropropagation of walnut comprises steps as below-
i. Mixing NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L to make Stock 1,
ii. Mixing KH2PO4- 182.5mg/l and Ca3(PO4)2- 182.5 mg/l to make Stock 2.
iii. Mixing MgSO4.7H2O- 779 mg/l, MnSO4.H2O- 33.5 mg/l, CuSO4.5H2O- 0.25 mg/l, K2SO4 - 779 mg/l and KCl - 779mg/l to make Stock 3.
iv. Mixing FeEDDHA- 120 mg/l, H3BO3- 5.0 mg/l and ZnO -17.0 mg/l to make Stock 4.
v. Mixing Nicotinic acid- 1.0 mg/l, Thiamine-HCl- 2.0 mg/l and Glycine 2.0 mg/l to make Stock 5.
vi. Adding AgNO3 (6.0 mg/L), casein hydrolysate (100 mg/L), and phloroglucinol (50 mg/L) follwd by mixing.
vii. Adding growth hormones TDZ (0.5 mg/L), BAP (0.5 mg/L), and IBA (0.1 mg/L) followed by mixing.
viii. Adding sucrose (30 g/L), along with myo-inositol (150 mg/L) and agar (6.5 g/L).
ix. Adjusting the pH of the medium to 5.5-5.8 followed by sterilization in an autoclave at 121.6°C and 15 pounds per square inch pressure for 15-20 minutes.
[0028] In a preferred embodiment of the invention, the improved nutrient media of the instant invention ensures both walnut culture establishment and multiplication.
[0029] In a preferred embodiment of the invention, an improved method for shoot regeneration and multiplication (Fig. 1) of walnut var. Chandler comprising steps as below-
i. Obtaining shoot tip segmenst as explants from parent plant followed by cleaning and washing with detergent and water.
ii. Soaking in a 1.0% (w/v) solution of activated charcoal for 10 minutes.
iii. Washing with 0.5% (w/v) fungicide and 3-4 drops of detergent followed by washing with water.
iv. Sterlising the explants, under aseptic conditions, with 0.1% (w/v) mercuric chloride (HgCl2) for 3.0 minutes, followed by 3-4 rinses with autoclaved distilled water.
v. Transferring explants, under axenic conditions, to media comprising NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L, Ca3(PO4)2- 182.5 mg/L, ZnO- 17.0 mg/L, K2SO4- 779 mg/L, KCl- 779 mg/L, MgSO4.7H2O- 779 mg/L, MnSO4.H2O- 33.5 mg/L, CuSO4.5H2O- 0.25, KH2PO4- 182.5 mg/L, H3BO3- 5.0 mg/L, FeEDDHA- 120.00 mg/L, Nicotinic acid- 1.00 mg/L, Glycine- 2.00 mg/L, Thiamine-HCl-2.00 mg/L, AgNO3 - 6.00 mg/L, Casein Hydrolysate-100.00 mg/L, Phloroglucinol- 50.00 mg/L, TDZ - 0.50 mg/L, BAP- 0.50 mg/L, IBA- 0.10 mg/L, myo-inositol- 150.00 mg/L, Sucrose- 30.00 g/L and Agar- 6.50 g/L.
vi. Incubating the culture vessel at 25°C under a photoperiod of 16 hours of light and 8 hours of darkness, in a relative humidity of 70-80% for 15-20 days.
vii. Subculturing of established explant, by vertically excising the extra portion of callus formed at the base of shoot tip explant.
viii. Multiplying micro shoots by vertically excising and separating multiple new micro shoots along with callus.
ix. Subculturing shoots into fresh medium after 15 days by vertically excising callus formed at the bottom of shoot while retaining some portion of callus to obtain shoot multiplication.
[0030] In a preferred embodiment of the invention, the nutrient media for regenerating roots from micropropagated shoots comprises NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L, Ca3(PO4)2- 182.5 mg/L, ZnO- 17.0 mg/L, K2SO4- 779 mg/L, KCl- 779 mg/L, MgSO4.7H2O- 779 mg/L, MnSO4.H2O- 33.5 mg/L, CuSO4.5H2O- 0.25, KH2PO4- 182.5 mg/L, H3BO3- 5.0 mg/L, FeEDDHA- 120.00 mg/L, Nicotinic acid- 1.00 mg/L, Glycine- 2.00 mg/L, Thiamine-HCl-2.00 mg/L, AgNO3 - 6.00 mg/L, Casein Hydrolysate-100.00 mg/L, Phloroglucinol- 50.00 mg/L, TDZ - 0.50 mg/L, BAP- 0.50 mg/L, IBA- 0.10 mg/L, myo-inositol- 150.00 mg/L, Sucrose- 30.00 g/L and Agar- 6.50 g/L.
[0031] In a preferred embodiment of the invention, an improved method for rooting and hardening of plantlets of walnut comprising steps as below-
i. Preparing the nutrient media for root regeneration.
ii. Transferring micropropagated shoots into the media containing 3.0 mg/L IBA under aseptic conditions.
iii. Incubating in dark conditions for 48 hours.
iv. Transferring to an ex vitro root initiation medium comprising of sterile cocopeat, perlite, and vermiculite amixed in 1:1:1, supplemented with half-strength liquid MS medium for fertigation.
v. Root formation after 20 days.
[0032] In another embodiment of the invention, subculturing step is repeated as often as necessary to produce callus and shoots until the desired amount has been obtained.
[0033] In a preferred embodiment of the invention, nutrient media for Callus regeneration, shoot regeneration, shoot multiplication is same.
[0034] Shoot tips of walnut cultivar Chandler have been collected from fruit crop nursery at Dr. Y.S.P. University of Horticulture and Forestry, Nauni, Solan, H.P.
The following examples illustrate the invention without limiting it.
EXAMPLES
Hereinafter, the present disclosure is being described in further detail through examples. However, the following examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples.

Example 1
Method of preparation of media of the instant invention-
[0035] The method of preparation of the improved media for micropropagation of walnut comprises steps as below-
i. Mixing NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L to make Stock 1,
ii. Mixing KH2PO4- 182.5mg/l and Ca3(PO4)2- 182.5 mg/l to make Stock 2.
iii. Mixing MgSO4.7H2O- 779 mg/l, MnSO4.H2O- 33.5 mg/l, CuSO4.5H2O- 0.25 mg/l, K2SO4 - 779 mg/l and KCl - 779mg/l to make Stock 3.
iv. Mixing FeEDDHA- 120 mg/l, H3BO3- 5.0 mg/l and ZnO -17.0 mg/l to make Stock 4.
v. Mixing Nicotinic acid- 1.0 mg/l, Thiamine-HCl- 2.0 mg/l and Glycine 2.0 mg/l to make Stock 5.
vi. Adding AgNO3 (6.0 mg/L), casein hydrolysate (100 mg/L), and phloroglucinol (50 mg/L) follwd by mixing.
vii. Adding growth hormones TDZ (0.5 mg/L), BAP (0.5 mg/L), and IBA (0.1 mg/L) followed by mixing.
viii. Adding sucrose (30 g/L), along with myo-inositol (150 mg/L) and agar (6.5 g/L).
ix. Adjusting the pH of the medium to 5.5-5.8 followed by sterilization in an autoclave at 121.6°C and 15 pounds per square inch pressure for 15-20 minutes.
[0036] Method for shoot regeneration and multiplication of walnut var. Chandler -
Steps comprises as below-
i. Obtaining shoot tip segmenst as explants from parent plant followed by cleaning and washing with detergent and water.
ii. Soaking in a 1.0% (w/v) solution of activated charcoal for 10 minutes.
iii. Washing with 0.5% (w/v) fungicide and 3-4 drops of detergent followed by washing with water.
iv. Sterlising the explants, under aseptic conditions, with 0.1% (w/v) mercuric chloride (HgCl2) for 3.0 minutes, followed by 3-4 rinses with autoclaved distilled water.
v. Transferring explants, under axenic conditions, to media comprising NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L, Ca3(PO4)2- 182.5 mg/L, ZnO- 17.0 mg/L, K2SO4- 779 mg/L, KCl- 779 mg/L, MgSO4.7H2O- 779 mg/L, MnSO4.H2O- 33.5 mg/L, CuSO4.5H2O- 0.25, KH2PO4- 182.5 mg/L, H3BO3- 5.0 mg/L, FeEDDHA- 120.00 mg/L, Nicotinic acid- 1.00 mg/L, Glycine- 2.00 mg/L, Thiamine-HCl-2.00 mg/L, AgNO3 - 6.00 mg/L, Casein Hydrolysate-100.00 mg/L, Phloroglucinol- 50.00 mg/L, TDZ - 0.50 mg/L, BAP- 0.50 mg/L, IBA- 0.10 mg/L, myo-inositol- 150.00 mg/L, Sucrose- 30.00 g/L and Agar- 6.50 g/L.
vi. Incubating the culture vessel at 25°C under a photoperiod of 16 hours of light and 8 hours of darkness, in a relative humidity of 70-80% for 15-20 days.
vii. Subculturing of established explant, by vertically excising the extra portion of callus formed at the base of shoot tip explant.
viii. Multiplying micro shoots by vertically excising and separating multiple new micro shoots along with callus.
ix. Subculturing shoots into fresh medium after 15 days by vertically excising callus formed at the bottom of shoot while retaining some portion of callus to obtain shoot multiplication.
[0037] Method for rooting and hardening of plantlets of walnut
Steps are as below-
i. Preaparing the nutrient media for root regeneration.
ii. Transferring micropropagated shoots into the media containing 3.0 mg/L IBA under aseptic conditions.
iii. Incubating in dark conditions for 48 hours.
iv. Transferring to an ex vitro root initiation medium comprising of sterile cocopeat, perlite, and vermiculite amixed in 1:1:1, supplemented with half-strength liquid MS medium for fertigation.
v. Root formation after 20 days.

[0038] In the view of foregoing, it is evident that the present invention provides an efficient nutrient media and method for micropropagating walnut such that the callus formation, shoot regeneration and multiplication can be done in a single media with 70- 71% regeneration rate for cultured explants, with regeneration occurring at an average interval of 20-21 days.

a. Unique features-
1. To date, various medium formulations have been used to establish walnut cultivars, but they have consistently shown low survival and regeneration rates. To address this challenge, nutrient medium of instant application is disclosed, which incorporates distinct concentrations of macro and micronutrients along with specific additives tailored for walnut cultures. A key innovation of our formulation lies in the precise and unique concentration of salts, which significantly enhances culture success by promoting healthier growth and better regeneration. This advancement provides a more reliable foundation for the in vitro propagation of walnut cultivars.

2. Another unique feature of this invention is the method for culture establishment and multiplication. Specifically, the callus is excised vertically from the sides (as shown in Figure 1) and then transferred to fresh medium, ensuring successful establishment and enhanced multiplication rates. A highest multiplication rate of 4.33 shoots/callus after 19.88 days was achieved.

3. The instant invention discloses a method to subculture the callus to ensure high shoot regeneration.The callus is excised after the first subculturing, and the regenerated shoot must possess 2-3 axillary buds. After each subculture, the shoot forms a new callus at the base, which should be retained to support further multiplication. This approach ensures continued shoot regeneration and enhances the multiplication rate, as the callus plays a crucial role in maintaining the health and growth potential of the microshoots throughout the tissue culture process.

4. For acclimatization, the successfully established shoots are transferred to a root induction medium containing 3.0 mg/L IBA for a period of 2 days only. After this short exposure to IBA, the shoots are then shifted to a sterile soil medium composed of a 1:1:1 ratio of cocopeat, perlite, and vermiculite. This stepwise process ensures proper root development and smooth acclimatization, facilitating the transition from in vitro conditions to soil, thereby improving the survival and establishment of the plants.

[0039] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

[0040] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.



SHWETA SEN
Dated- 28th October, 2024 IN/PA No-3010
PATENT AGENT FOR APPLICANT
, Claims:WE CLAIM
1. A novel media for micropropagation of walnut (var. Chandler) comprises of NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L, Ca3(PO4)2- 182.5 mg/L, ZnO- 17.0 mg/L, K2SO4- 779 mg/L, KCl- 779 mg/L, MgSO4.7H2O- 779 mg/L, MnSO4.H2O- 33.5 mg/L, CuSO4.5H2O- 0.25, KH2PO4- 182.5 mg/L, H3BO3- 5.0 mg/L, FeEDDHA- 120.00 mg/L, Nicotinic acid- 1.00 mg/L, Glycine- 2.00 mg/L, Thiamine-HCl-2.00 mg/L, AgNO3 - 6.00 mg/L, Casein Hydrolysate-100.00 mg/L, Phloroglucinol- 50.00 mg/L, TDZ - 0.50 mg/L, BAP- 0.50 mg/L, IBA- 0.10 mg/L, myo-inositol- 150.00 mg/L, Sucrose- 30.00 g/L and Agar- 6.50 g/L.
2. A method of preparation of the novel media for micropropagation of walnut comprises steps as below-
i. Mixing NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L to make Stock 1,
ii. Mixing KH2PO4- 182.5mg/l and Ca3(PO4)2- 182.5 mg/l to make Stock 2.
iii. Mixing MgSO4.7H2O- 779 mg/l, MnSO4.H2O- 33.5 mg/l, CuSO4.5H2O- 0.25 mg/l, K2SO4 - 779 mg/l and KCl - 779mg/l to make Stock 3.
iv. Mixing FeEDDHA- 120 mg/l, H3BO3- 5.0 mg/l and ZnO -17.0 mg/l to make Stock 4.
v. Mixing Nicotinic acid- 1.0 mg/l, Thiamine-HCl- 2.0 mg/l and Glycine 2.0 mg/l to make Stock 5.
vi. Adding AgNO3 (6.0 mg/L), casein hydrolysate (100 mg/L), and phloroglucinol (50 mg/L) followed by mixing.
vii. Adding growth hormones TDZ (0.5 mg/L), BAP (0.5 mg/L), and IBA (0.1 mg/L) followed by mixing.
viii. Adding sucrose (30 g/L), along with myo-inositol (150 mg/L) and agar (6.5 g/L).
ix. Adjusting the pH of the medium to 5.5-5.8 followed by sterilization in an autoclave at 121.6°C and 15 pounds per square inch pressure for 15-20 minutes.
3. A method for shoot regeneration and multiplication of walnut var. Chandler comprising steps as below-
i. Obtaining shoot tip segmenst as explants from parent plant followed by cleaning and washing with detergent and water.
ii. Soaking in a 1.0% (w/v) solution of activated charcoal for 10 minutes.
iii. Washing with 0.5% (w/v) fungicide and 3-4 drops of detergent followed by washing with water.
iv. Sterlising the explants, under aseptic conditions, with 0.1% (w/v) mercuric chloride (HgCl2) for 3.0 minutes, followed by 3-4 rinses with autoclaved distilled water.
v. Transferring explants, under axenic conditions, to media comprising NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L, Ca3(PO4)2- 182.5 mg/L, ZnO- 17.0 mg/L, K2SO4- 779 mg/L, KCl- 779 mg/L, MgSO4.7H2O- 779 mg/L, MnSO4.H2O- 33.5 mg/L, CuSO4.5H2O- 0.25, KH2PO4- 182.5 mg/L, H3BO3- 5.0 mg/L, FeEDDHA- 120.00 mg/L, Nicotinic acid- 1.00 mg/L, Glycine- 2.00 mg/L, Thiamine-HCl-2.00 mg/L, AgNO3 - 6.00 mg/L, Casein Hydrolysate-100.00 mg/L, Phloroglucinol- 50.00 mg/L, TDZ - 0.50 mg/L, BAP- 0.50 mg/L, IBA- 0.10 mg/L, myo-inositol- 150.00 mg/L, Sucrose- 30.00 g/L and Agar- 6.50 g/L.
vi. Incubating the culture vessel at 25°C under a photoperiod of 16 hours of light and 8 hours of darkness, in a relative humidity of 70-80% for 15-20 days.
vii. Subculturing of established explant, by vertically excising the extra portion of callus formed at the base of shoot tip explant.
viii. Multiplying micro shoots by vertically excising and separating multiple new micro shoots along with callus.
ix. Subculturing shoots into fresh medium after 15 days by vertically excising callus formed at the bottom of shoot while retaining some portion of callus to obtain shoot multiplication.
4. A nutrient media for regenerating roots from micropropagated shoots comprises NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L, Ca3(PO4)2- 182.5 mg/L, ZnO- 17.0 mg/L, K2SO4- 779 mg/L, KCl- 779 mg/L, MgSO4.7H2O- 779 mg/L, MnSO4.H2O- 33.5 mg/L, CuSO4.5H2O- 0.25, KH2PO4- 182.5 mg/L, H3BO3- 5.0 mg/L, FeEDDHA- 120.00 mg/L, Nicotinic acid- 1.00 mg/L, Glycine- 2.00 mg/L, Thiamine-HCl-2.00 mg/L, AgNO3 - 6.00 mg/L, Casein Hydrolysate-100.00 mg/L, Phloroglucinol- 50.00 mg/L, TDZ - 0.50 mg/L, BAP- 0.50 mg/L, IBA- 0.10 mg/L, myo-inositol- 150.00 mg/L, Sucrose- 30.00 g/L and Agar- 6.50 g/L.
5. An improved method for rooting and hardening of plantlets of walnut comprising steps as below-
i. Preaparing the nutrient media for root regeneration.
ii. Transferring micropropagated shoots into the media containing 3.0 mg/L IBA under aseptic conditions.
iii. Incubating in dark conditions for 48 hours.
iv. Transferring to an ex vitro root initiation medium comprising of sterile cocopeat, perlite, and vermiculite amixed in 1:1:1, supplemented with half-strength liquid MS medium for fertigation.
v. Root formation after 20 days.

6. The nutrient media as claimed in claim 1 comprises the stock solutions viz., stock solution 1, 2, 3, 4 and 5 wherein each stock is prepared as below-
i. Mixing NH4NO3- 1400 mg/L, Ca(NO3)2.4H2O- 1034 mg/L, KNO3- 1034 mg/L to make Stock 1,
ii. Mixing KH2PO4- 182.5mg/l and Ca3(PO4)2- 182.5 mg/l to make Stock 2.
iii. Mixing MgSO4.7H2O- 779 mg/l, MnSO4.H2O- 33.5 mg/l, CuSO4.5H2O- 0.25 mg/l, K2SO4 - 779 mg/l and KCl - 779mg/l to make Stock 3.
iv. Mixing FeEDDHA- 120 mg/l, H3BO3- 5.0 mg/l and ZnO -17.0 mg/l to make Stock 4.
v. Mixing Nicotinic acid- 1.0 mg/l, Thiamine-HCl- 2.0 mg/l and Glycine 2.0 mg/l to make Stock 5.




SHWETA SEN
Dated- 28th October, 2024 IN/PA No-3010
PATENT AGENT FOR APPLICANT

Documents