FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
& THE PATENT RULES , 2003
PROVISIONAL SPECIFICATION (See Section 10; ru|e 13)
DEVELOPMENT OF VIABLE PROTOCOL
FOR IN VITRO PROPAGATION
OF POMEGRANATE.
RELIANCE LIFE SCIENCES PVT.LTD an Indian Company having its Registered Office at Dhirubhai Ambani Life Sciences Centre, R-282, TTC Area of MIDC, Thane Belapur Road, Rabale, Navi Mumbai - 400 701 Maharashtra India.
The following specification particularly describes and ascertains the nature of thjs invention and the manner in which it is performed:-

FIELD OF THE INVENTION:
The present invention relates to the process for invitro propogation of Pomogranate (Punicagranatum). The invention in particular relates to a commercially viable process for in vitro mass culture.
BACKGROUND OF THE INVENTION
The.. Pomegranate (Punica granatum) is, a species, of fruit-bearing deciduous, shrub. Although it was previously given its own family Punicaceae, recent genetic evidence shows that it is a member of the family Lythraceae. The pomegranate is believed to have originated, in the area from Iran.east to northern India, but has been cultivated around the Mediterranean for so long (several millennia) that its true native range is not accurately known
Pomegranates are grown throughout Asia and the Mediterranean. The pomegranate is native from Iran to the Himalayas in northern India and was cultivated and naturalized over the whole Mediterranean region since ancient times. It is widely cultivated throughout India and the drier parts of southeast Asia, Malaya, the East Indies and tropical Africa. The tree was introduced into California by Spanish settlers in 1769. In this; country it is grown for its fruits mainly in the drier parts of California and Arizona.
Pomegranate juice is a popular drink in the Middle East, and is also used in Iranian and Indian cuisine; it began to be widely marketed in the US in 2004. Providing 40% of an adult's daily vitamin C requirement per 100 ml serving, pomegranate juice 'is also a good source of folic acid and antioxidant polyphenols. The most abundant polyphenols in pomegranate juice are the hydrolyzable tannins punicalagins shown in 38 peer-reviewed research pubHcatibrtsover 1990-2007 to have potent free-radical scavenging lability.
Research suggests that pomegranate juice may be effective against prostate cancer and osteoarthritis: In-2007, six clinical trials in the United States, Israel and Norway have been approved to examine the effects of pomegranate juice consumption on parameters of

prostate cancer or prostatic hyperplasia, diabetes or lymphoma.The juice may also have antiviral and antibacterial effects against dental plaque.
A rapid and efficient procedure is outlined for in vitro clonal propagation of an eliteciltivar
of pomegranate (Punica granatum L. cv. Ganesh) using nodal stem segments of a
mature tree wherein maximum number of shoots were developed on a medium containing
2.0mg/l zeatin riboside. (In vitro propagation of pomegranate (Punica granatum L. cv.
Ganesh) through axillary shoot proliferation from nodal segments of mature tree: Plant
Tissue and Cell Culture Facility, Post-Graduate Department of Botany, Utkal
University, Bhubaneswar, Orissa, INDE, Scientia Horticulturae, Volume 79, Issues 3-4, 26 February 1999, Pages 175-183 )
Colchicine treated shoots were propagated in a study by JIANZHU SHAO et al.( In vitro
induction of. tetraploid in pomegranate (Punica granatum).JlANZHU SHAG", CHUNLI
CHEN, XIUXrN DENG .National Key Laboratory of Crop Genetic Improvement,
Huazhong Agricultural University, Wuhan 430070, CHINE). Somatic Embryogenesis
and; Regeneration'of in Plantlets in Pomegranate has been tried by some groups which
failed to work on a. commercial scale.
However, the above cited protocols have got less multiplication rate and the efficiency of
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the protocol has not been validated upto the hardeningstage. The inventors of the present invention have developed a protocol, which proves the efficiency of the

micropropagation protocol of Punica granatum upto Held. In accordance with the present invention there is provided is a commercially viable process for in vitro mass culture of Punica granatum for large-scale multiplication of the true to type clones of elite var ety with disease free plants of uniform quality which can survive in th'e field at the rate of as.much as about 100% and has thus overcome the specific problems: encountered in proving the efficiency of the micropropagation protocol upto hardening stage:
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OBJECT OF THE INVENTION
The principal aim of the present invention is to develop a commercially viable process for in vitro culture of Punica granatum and commercial production of plantlets as well
field establishment
It s still an object of this present invention to provide a simple and faster process, for
production of the true to type clones of elite variety.
It is still an object of this invention to develop a process suitable for commercial
production of disease free high yielding clones of uniform quality.

It is still an object of this invention to produce quality planting material with high
survival rate at low cost
It is also an object of the present invention to identify explants, media and culture conditions for producing maximum regenerates of Punica granatum plant by invitro mass culture.
It is an important aspect of the present invention to provide a standardized method for
surface sterilization of explants without damaging the isolated tissues.
In other aspect of the present invention there is provided the best suitable nutrient media
supplemented with optimum growth regulators and other components required for
different modes and phases of regeneration.
it is an important aspect of the present invention to provide the optimum growth conditions with respect to physical parameters like temperature, relative humidity, photoperiod and light intensity for all the stages of culture.

It is also an additional aspect of the present invention to provide a hardening protocol that proves the efficient hardening of the micropropagated plants with as much as about 100% rate of survival in the field.
SUMMARY OF THE INVENTION
The present disclosure provides an efficient in vitro micropropagation technique for Punica granatum. and commercial production of plantlets as well field establishment
In one aspect, the invention provides methods for producing true-to-type clones of Punica
granatum mother plants comprising steps of selecting an Punica granatum mother plant;
isolating shoot tips with meristematic explant, culturing the meristematic explant in
initiation medium to generate shoots, where the initiation medium lacks hormones;
culturing the shoots in proliferation and elongation medium to generate elongated shoots,
where the proliferation and elongation media comprises BAP and Kinetin, culturing the
elongated shoots in rooting medium to generate, plantlets, where the rooting medium
comprises NAA; and culturing the plantlets to produce a true-to-type clone of the Punica
granatum.mother plant.
In one embodiment the present invention has studied the morphogenetic. potential of Punica Granatum by application of growth regulator and light condition.
In one embodiment the present invention has provided the effect of various concentration

of

ytokinin used for induction and multiplication of shoots.

In still other preferred embodiments, the meristematic explant is from a shoot tip or a hocal bud. Preferably, the shoot tip has bud tissue; most preferably, apical bud tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be

better understood by reference to one or more of these drawings in combination with the
detailed description of specific embodiments presented herein. '
Fig.l: Illustrates explain the legend of the figures to be presented.
Figure1 : CONTROL - MS + Hormone free media
A - Response of explants in MS basal medium supplemented with BAP(lmg/l).
Figure2 : CONTROL - MS + Hormone free media
B-Response of explants in MS basal medium supplemented with BAP(0.5mg/l)
DETAILED DESCRIPTION OF THE INVENTION
Defmtions:
The term "microprppagation" as used herein refers to the in vitro regeneration of plants
frorti organs, tissues, cells or protoplasts and the true-to-type propagation of a selected
genotype using in.vitro culture technique.
The term "callus" as used herein refers to an unorganized or undifferentiated mass of
proliferative cells produced either in culture or in nature.
The term "true-to type propagation" as used herein means that all characteristics present
in mother plant will also be present in next generation, i:e., the plantlets will be the true
type of the mother plant.
The term "MS" as used herein refers to Murashige and Skoog's medium.
The term "IBA" as used herein refers to Indole-3-butyric acid.

The

term "NAA" as used herein refers to Naphthalene acetic acid

The term "BAP" as used herein refers to 6-benzyI amino purine.
The present invention provides a process for invitro mass culture of Punica granatum. The method involves the progati'on of meristematic explants in hormone free'media.
The present invention shows that it was possible to explore the morphogenetic potential of punica grahatum by application of growth regulator and light condition.The use of different. concentrations of cytokinin was used for the induction and multiplication of shoots.The

regulatory action of cytokinin helped the inyitro shoot induction and multiplication was well observed.The maximum shoot induction and multiplication was well observed in MS medium supplemented with BAP & kinetin ( lmg/1) within 4 weeks of culture under ±16 h photoperiod. This medium gave good multiplication rate of 1: 6.Whereas no multiplication of shoots was observed in MS medium supplemented with BAP( 0.1 mg/l).At lower concentrations, of BAP the rate of shoot multiplication declined.
The interaction of photoperiod and plant growth regulators have significant effect on shoot morphogenesis.The variation of response reul'ted due to varying concentrations of BAP in the . VIS medium.Whereas rate of shoot proliferation was seen in the MS medium supp emented with BAP(0.2 mg /I) but no multiplication of shoots was observed.
The elongated shoots rooted maximum in MS basal medium supplemented with Charcoal(
0.25%) and MS half-strength medium supplemented with NAA ( 0.2mg/l); The rooting
ability was reduced with, the decreasing concentration of NAA in the | medium. The
percentage of shoots forming roots varied with different concentrations of NAA.Thus an
attempt was made to develop an invitro protocol for mass multiplication by manipulating
the nutrient salts, growth regulators and culture conditions.
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In Greenhouse after 4-5 weeks of transfer of plantlets to cocopits it was observed that Plants survived well in cocopit with 80% survival rate of plants and less mortality.
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Thus it is clear that plant propagated through tissue culture provides many advantages with
respect to plants propagated through greenhouse.Time factor is one area .where tissue
culture has obvious benefits over the other.Thus this proves that plants produced from
tissue culture helps in rapid multiplication of stock plant to produce a large number of
progeny plants. |
The following, examples are included to demonstrate preferred embodiments of the
invention. It should be appreciated by those of skill, in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function

well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present; disc osure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from ,the spirit and scope of the invention.
EXAMPLE 1: Collection of Explant and surface sterilisation
The mother plants were collected from the .elite variety of Bhagwa plants from Akluj, Sholapur. Actively growing shoot tips.with apical meristem were collected from the fully grown plants.
Plants brought from the Greenhouse are replete with micro-organisms and dust particles as surface contaminants.To avoid contamination explants are first washed with Sterile Distilled Water (DW) without intermittent shaking. Different trials of surface sterilization can be done by altering the time and treatment with chemical sterilant like HgC12 to get the

best

results.

Example 2: Initiation
Shoot tips were placed on semisolid basal MS ( Murashige.and Skoog 1962 ) hormone free media. The cultures were then 25 °C under 12 hr photoperiod with.white fluorescent lamps. The cultures were then subcultured on a fresh medium with same constituents after 4 weeks. After innoculating the explant onto a new medium they are then kept for incubation on culture rack at 21 - 25 °C at constant temperature and 55 - 60 % humidity in the growth room..
After Here
The main feature of this stage is detection and elimination control of contamination.Actively growing young shoot tips were surface sterilized with HgC12 for 5 min and then it was asceptically placed on semisolid basal MS hormone free media.
2 - 3 weeks of initiation it was observed that it has produced 97.2% clean culture, the treatment with HgC12 for 5 min was the best treatment.

Example 3: Multiplication
Different media trials for Multiplication were carried out by using different combination
ofBAP & kinetin
After 2 - 3 weeks of initiation, the contamination free plants with well developed shoots were inoculated onto a shoot multiplication medium designed to promote rapid axillary branching.Meristerh proliferation and multiplication was initiated from the apical, explants of pomegranate ( punica granatum) within 10-15 days of inoculation onto MS basal medium supplemented with BAP & kinetin ( 0.1 -0.5 mg/1) alone & combination Different media trials for multiplication was carried out. Of these MS basal medium, supplemented with BAP & kinetin was the most effective for shoot proliferation and multiplication.Maximum shoot multiplication was observed in this media',with, healthy plants showing good growth and good height.Maximum percentage of shoot multiplication was observed in the basal region of the plant and maximum percentage of shoot proliferation was seen in the apical region of the plant.
.At lower concentration of BAP( 0.1 mg/1) the rate of shoot multiplication declined.Thus, the above data signifies that MS medium containing higher concentration lof BAP are
better suited for multiple shoot formation.
....
Example 4: Rooting
Different media trials for rooting was carried out using IAA,IBA,NAAl alone or iii
combination.
Elongated shoots( 1- 2 cm long ) were rooted on MS basal medium supplemented with
Charcoal and various concentrations, of NAA.Root initiation took place within 10 -12
days of transfer to MS basal medium supplemented with charcoal and NAA.However
Good rooting was observed on MS basal medium supplemented with charcoal '
MS half strengh medium supplemented with NAA (0.2mg/l) showed cluster long thick
roots with healthy leaves. The rooting ability was reduced with the decreasing
concentration of lAA.The percentage of shoots forming roots and days to rooting
significantly varied with different concentrations of NAA.(0-1 mg/1) and good response in
rooting was given by MS half strength basal medium supplemented with NAA (
0.2mg/l).

Example 5: Hardening
After 4 weeks of Rooting, plantlets grown are ready for hardening. These plantlets are
washed & trated with in fungicide . The plantlets are then transplanted to'plastic sterile
trays containing cocopit. The plantlets establish well in the soil withing 2- 3 weeks.of
transfer. Rooted plants were planted in cocopeat and growm under greenhouse
conditions.
Rooted plantlets grown invitro' were washed thoroughly to remove the adhering gel,
transplanted to plastic sterile pots containing cocopit.About 96% of the rooted plantlets
establish in the greenhouse withing2-3 weeks of transfer
The primary hardened plantlets were further hardened in shade house for asperiod of six
weeks and then transferred to the field. Plants are growing well under field'.conditions &
field evaluation of tissue culture plants are under progress.
Example 6: Field Results -
Thetissue cultured Pbmogranate plants of the present invention showed promising results
at tie field level in the trial plot designed near Sholapur near Akluj. The plants showed
ear y flowering, vigorous growth and stem thickness and higher number of biomass
which all would contribute to superior physiological parameters.
Number ofTC plants in field: 300Nos.
Number of Gooty kalam planted. 44 Nos.
Planting distance: Row to row-15 feet's
Plant to plant-10 feet's
Total 17 lines planted with 15 lines of TC pomegranate and 2 lines of conventional
planting material {Gooty kalam). In each line 22 plants are planted.
Table 1

Characters
Vegetative growth
Stem /stump

TC Pomegranate
Fast and more vigorous
Thick stump so it can bear maximum number of fruit with weight. So easy to take more yield on tree and less damage of stump breaking.

Conventional Pomegranate
(Control)
Slow and less vigorous
Thin stump, so limitation in taking more yields. Chances, of breaking the stump due to more fruits.

Leaves More number of leaves with maximum branches Less leaves with minimum branches •
Pest infestation (Aphids) Flower formation Less Early . More late

REFERENCES

1] Vidyasagar, K, National Conference on Plant Biotechnology, Lady Doak College, Matiurai, April 2006.
2] Phytotechnology laboratories inc
3] Podds, J. H. and Roberts, L. W. (1995). Experiments in plant tissue culture.Cambridge
University press, Cambridge, ' • •
4] Fowler, M. R, (2000). Plant cell culture, laboratory techniques. In Encyclopedia of cell technology (ed. R.E.Spier), pp.994-1004. Wiley, New York.
5] Gamborg, O. L. (2002). Plant tissue culture. Biotechnology. Milestones. In vitro Cellular and Developmental Biology—Plant, 38, 84-92.
6] Sugiyama, M. (1999). Organogenesis in vitro. Current Opinion in Plant Biology, 2,
61-4
7] Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN .
8J Designing a Plant Tissue Culture lab-Mark P. Bridgen and John W. Bartok, Jr. Departments of Plant Science and Agricultural Engineering,University of Connecticut Storrs, Connecticut 06268

Thus, While we have described fundamental novel features of the invention, it will.be understood that various omissions and substitutions and changes in the form and details may be possible without. departing from the spirit of the invention. For 'example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention
Dated this 31st day of March , 2009
For Reliance Life Sciences Pvt. Ltd

K. V. Subramaniam President