WO2010113178A2 - Development of viable protocol for in vitro propagation of pomegranate - Google Patents

Abstract

The present invention relates to the efficient propagation of Punica granatum wherein the in vitro protocol for mass multiplication comprises nutrient salts, growth regulators and optimal culture conditions. The multiplication rate observed was higher with the protocol derived.

Description

DEVELOPMENT OF VIABLE PROTOCOL FOR IN VITRO PROPAGATION OF POMEGRANATE.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to Indian Provisional Patent Application No. 858/MUM/2009 filed 31 March, 2009, the entire content of which. is incorporated by reference.

TECHNICAL FIELD OF THE INVENTION:

The present invention relates to methods for in vitro propagation of plants. Specifically, the invention relates to a process for in vitro propagation of Pomegranate (Punica granatum). The invention in particular relates to a commercially viable process for in vitro mass culture of Punica granatum.

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 publications over 1990-2007 to have potent free-radical scavenging ability. 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.

Plants and flowers currently grown in greenhouses using soil as a growth medium result in being infested with disease and pests; Freedom from the disadvantages of soil-medium is achieved by utilizing different substrate materials and growth media.A rapid and efficient procedure is outlined for in vitro clonal propagation of an elite cultivar 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 2.0 mg/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). JI ANZHU SHAO, CHUNLI CHEN, XIUXIN DENG. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, CHINA). Somatic Embryogenesis and Regeneration of in Plantlets in Pomegranate has been tried by some groups but failed to work on a commercial scale.

However, the above cited protocols have got less multiplication rate and the efficiency of the protocol has not been validated upto the hardening stage.

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

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. The inventors of the present invention have developed a procedure which proves the efficiency of the micropropagation protocol of Punica granatum upto field deployment stage. In accordance with the present invention there is provided is a commercially viable process for in vitro mass culture of Puriica granatum for large-scale multiplication of the true to type clones of elite variety with disease free plants of uniform quality which can survive in the field at the rate of as much as about 100% and has thus overcome the specific problems encountered in improving the efficiency of the micropropagation protocol upto hardening stage.

The present invention provides a simple and faster process for production of the true to type clones of elite variety.

The present invention provides a process suitable for commercial production of disease free high yielding clones of uniform quality.

The present invention provides methods for producing quality planting material with high survival rate at low cost

The present invention provides methods to identify explants, media and culture conditions for producing maximum regenerates of Punica granatum plant by mass culture in vitro.

It is an aspect of the present invention to provide a standardized method for surface sterilization of explants without damaging the isolated tissues.

In another aspect of the present invention suitable nutrient media supplemented with optimum growth regulators and other components required for different modes and phases of regeneration, is provided.

It is an 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.

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 meristematici 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 1 -naphthaleneacetic acid (NAA); and culturing the plantlets to produce a true- to-type clone of the Punica granatum. mother plant.

In one embodiment the present invention identifies the morphogenetic potential of Punica Granatum caused by application of growth regulators and light condition.

In one embodiment the present invention has provided the effect of various concentrations of cytokinin used for induction and multiplication of shoots.

In still other preferred embodiments, the meristerriatic explant is from a shoot tip or a nodal bud. Preferably, the shoot tip has bud tissue; more 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.

Figure 1 shows the response of explants in MS (Murashige and Skoog) hormone free media.

Figure 2 shows response of explants in MS basal medium supplemented with 1 mg/L 6- benzyl amino purine (BAP). Jar marked CONTROL contains MS hormone free media. Jar marked A contains explants in MS basal medium supplemented with BAP (lmg/L).

Figure 3 shows response of explants in MS basal medium supplemented with 0.5 mg/L BAP. Jar marked CONTROL contains MS hormone free media. Jar marked B contains explants-in MS basal medium supplemented -with BAP (0.5 mg/L).

Figure 4 shows response of explants in MS basal medium supplemented with 0.2 mg/L BAP. Jar marked CONTROL contains MS hormone free media. Jar marked C contains explants in MS basal medium supplemented with BAP (0.2 mg/L).

Figure 5 shows response of explants in MS basal medium supplemented with 0.4 mg/L BAP. Jar marked CONTROL contains MS hormone free media. Jar marked B contains explants in MS basal medium supplemented with BAP (0.4 mg/L). Figure 6 shows response of explants in IvIS. basal medium supplemented with 0.1 mg/L BAP. Jar marked CONTROL contains MS hormone free media. Jar marked B contains explants in MS basal medium supplemented with BAP (0.1 mg/L).

Figure 7 shows multiplication 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/L, alone or in combination).

Figure 8 shows Rooting. Figure 8A: Rooting in the shoots inhibited in medium devoid of growth regulator. Figure 8B: Shoots rooted on MS medium supplemented with Charcoal (0.25%) show good rooting with thick long roots after 3 weeks of subculture. Figure 8C: Shoots rooted on MS medium supplemented with IAA( lmg/L) show long thick roots with healthy leaves after 3 weeks of subculture.

DETAILED DESCRIPTION OF THE INVENTION Definitions:

The term "micropropagation" as used herein refers to the in vitro regeneration of plants from 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 "fungicide" as used herein means any chemical substance that destroys and inhibits the growth of fungi.

The term "insecticide" as used herein means any substance, synthetic or organic, which inhibits, kills, or destroys insects.

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-benzyl amino purine. The term "IAA" as used herein refers to jndole-3-acetic acid. The term "BA" as used herein refers to benzyl adenine.

The present invention provides a process for in vitro mass culture of Punica granatum. The method involves the progation of meristematic explants in hormone free media.

The present invention shows that it was possible to explore the morphogenetic potential of Punica granatum 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 invitro shoot induction and multiplication was well observed. The maximum shoot induction and multiplication was well observed in MS medium supplemented with BAP & kinetin (lmg/L) 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 has significant effect on shoot morphogenesis. The variation of response resulted due to varying concentrations of BAP in the MS medium. Whereas rate of shoot proliferation was seen in the MS medium j supplemented with BAP (0.2 mg /1), 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 in vitro protocol for mass multiplication by manipulating the nutrient salts, growth regulators and culture conditions.

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 the plants and less mortality.

Thus, 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 disclosure, 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.

EXAMPLES

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 meristems were collected from fully grown plants. Plants brought from a 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 HgCl₂ to get the best results.

Preparation of the Mother Plant: In certain embodiments, the mother plant from which the explants are harvested is subject to screening -to identify healthy specimens and/or treatment to either maintain a disease-free state or to treat existing disease. Health can be determined by assessing the plants for their size, weight, general growth, appearance, and absence of infection or contamination. Decontamination can be performed by spraying the plants with agents such as fungicides, insecticides, pesticides or the like. Preferred fungicides for the pretreatment of the mother plant include Bavistin®, Captan®, Dithane®, Thiram®, Thiovit®, or combinations thereof at a concentration of about 0.05% to 0.2%. Preferred insecticides for the pretreatment of the mother plant include, but are not limited to, Rogor®, Nuvacron, Fastac®, Ultracid® 40-WP, Thiodane® at a concentration of about 0.005% to 0.02%.

Explants: The present invention provides a method for efficient in vitro mass culture of Punica granatum using explants from meristematic tissue. Since meristematic cells are undifferentiated, the use of such tissue as an explant allows regeneration of true-to-type clones of the mother plants. In preferred embodiments, shoot tip or nodal buds are used as explants. In the most preferred embodiments, the contemplated explant is shoot tip with bud tissue. Apical meristem bud tissue is particularly preferred, as it is an active part of the plant and relatively contamination free. Preferably, the explant used in the present invention is selected from healthy, fresh, disease-free plants. The explants, may be isolated from mother plants growing in various locations, both wild and cultivated.

Preparation of the Explants for Culture: In some embodiments, the explants are cleaned prior to inoculation in the media. Cleaning is performed using methods known to those of skill in the art, for example, by shaking explants in a mild detergent, such as Tween-20. In other embodiments, the explants are sterilized prior to inoculation in the media. Sterilization can be performed using any method known to those of skill in the art, for example, by treatment with fungicide, a surface sterilizing agent, or combinations thereof. The explant may be subjected to a single round of sterilization or multiple rounds of sterilization. For example, the explant may go through a primary sterilization step with the fungicide Bavistin and then go through a secondary sterilization with a surface sterilizing agent like sodium hypochlorite or mercuric chloride.

Example 2: Initiation

Shoot tips were placed on semisolid basal MS (Murashige and Skoog 1962) hormone free media. The cultures were then 25 ⁰C under 12 hr photoperiod with white fluorescent lamps. The cultures were then subcultured on a fresh medium with same constituents after 4 weeks. After inoculating the explant onto a new medium they were then kept for incubation on culture rack at 21 - 25 ⁰C at constant temperature and 55 - 60 % humidity in the growth room. The main feature of this stage is detection and elimination and control of contamination. Actively growing young shoot tips were surface sterilized with HgCl₂ for 5 min and then it was aseptically placed on semisolid basal MS hormone free media.

After 2 - 3 weeks of initiation it was observed that it has produced 97.2% clean culture. Treatment with HgCl₂ for 5 min produced the best result. Example 3: Multiplication

Different media trials for multiplication were carried out by using different combinations of BAP & 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. Meristem 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/L) alone & combination

Different media trials for multiplication were 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/L) the rate of shoot multiplication declined. Thus the above data signifies that MS medium containing higher concentrations of BAP are better suited for multiple shoot formation:

Example 4: Rooting

Different media trials for rooting were ' carried out using IAA, IBA, NAA alone or in combination. Elongated shoots (1- 2 cm long) were rooted on MS basal medium supplemented with Charcoal and various _jConcentrations of NAA. Root initiation took place within 10 -12 days of transfer to MS i 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 IAA. The percentage of shoots forming roots and days to rooting significantly varied with different concentrations of NAA. (O.lmg/1 ) and good response in rooting was given by MS half strength basal medium supplemented with NAA (0.2mg/l). i

Example 5: Hardening

After 4 weeks of Rooting, plantlets grown are ready for hardening. These plantlets are washed and treated with 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 in vitro 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 withing 2 - 3 weeks of transfer. The primary hardened plantlets were further hardened in shade house for a period 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 -

A) The tissue cultured Pomegranate plants of the present invention showed promising results at the field level in the trial plot designed near Sholapur near Akluj. The plants showed early flowering, vigorous growth and stem thickness and higher number of biomass which all would contribute to superior physiological parameters.

Number of TC plants in field: 300 Nos. Number of Gooty kalam planted: 44 Nos. Planting distance: Row to row -15 feet Plant to plant - 10 feet

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

B) Further the plantation of tissue cultured plants were done at Subhash Viswanath Mane farm A/P. Velapur, Malshirus, Solapur and compared with conventional plants

Total No. of plants: 500 plants

Conventional: 44 plants

Pruning was done 4 times at six months intervals.

Table 2

REFERENCES

1) Vidyasagar. K, National Conference on Plant Biotechnology, Lady Doak College,Madurai, April 2006.

2) MURASHIGE & SKOOG (MS) MODIFIED MULTIPLICATION MEDIUM. Product ID: M555. Phytotechnology laboratories, Inc (Shawnee Mission, KS).

3) Dodds, J. H. and Roberts, L. W. (1995). Experiments in plant tissue culture. Cambridge University press, Cambridge.

4) Fowler, M. R. (2000)^ Planf 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

8) 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

Claims

CLAIMSWe claim:

1. A method for producing a trύe-to-type clone of a Punica granatum mother plant comprising culturing a meristematic explant of Jatropha curcas in media with phytohormones at a concentration from about 0.01 mg/L to about 10 mg/L and producing a true-to-type clone of a Punica granatum mother plant from said meristematic explant.

2. A method for producing a true-to-type clone of an Punica granatum mother plant comprising: selecting an Punica granatum mother plant; isolating a meristematic explant from said plant; culturing said meristematic explant in initiation medium to generate shoots, wherein the initiation medium lacks hormones; culturing said shoots in a multiplication medium to generate proliferation of shoots, wherein the multiplication medium comprises an amount of one or more cytokinins suitable to allow shoot proliferation and multiplication; culturing the proliferated shoots in a rooting medium to generate plantlets, wherein the rooting medium comprises an effective amount of an auxin; and culturing said plantlets to produce a true-to-type clone of the Punica granatum mother plant.

3. The method of claims 1 or 2, wherein the Punica granatum mother plant is of an elite variety.

4. The method of claims 1-3. wherein the multiplication medium comprises a cytokinin selected from BAP or Kinetin or both.

5. The method of claim 4, wherein the multiplication medium comprises BAP at a concentration greater than 0.1 mg/L..

6. The method of claim 5;¹ wherein' the -multiplication medium ' comprises BAP at a concentration of 0.2 mg/L, 0.4 mg/L, 0.5 mg/L, or 1.0 mg/L.

7. The method of claim 4, wherein the multiplication medium comprises kinetin at a concentration between 0.1mg/L and 0.5 mg/L.

8. The method of claim 4, wherein the multiplication medium comprises kinetin and BAP at concentrations between O.lmg/L and 0.5 mg/L each.

9. The method of claims 1-8, wherein the rooting medium comprises one or more auxins selected from the group consisting of indole-3 -acetic acid (IAA), indole-3-butyric acid (IBA), naphthalene acetic acid (NAA) or a combination thereof.

10. The method of claim 9, wherein the auxin is at a concentration between 0.1 mg/L and 0.2 mg/L.

1 1. The method of claims 2-10, wherein said meristematic explant is from a shoot tip or a nodal bud.

12. The method of claim 11, wherein said meristematic explant is from a shoot tip.

13. The method of claim 12, wherein said shoot tip comprises apical bud tissue.

14. The method of claims 2-13, wherein the, initiation medium is hormone-free MS medium.

15. The method of claim 14, wherein the medium is semisolid.

16. The method of claim claims^~2-15, ^rwherein the rooting medium is half-strength MS medium supplemented with an auxin.

17. The method of claim claims 2-16, wherein the initiation further comprises exposure to a regulated light condition.

18. The method of claim 17, wherein the meristematic explant is exposed to white fluorescent lamp for up to 12 hours.

19. The method according to any of claims 2-18, further comprising hardening the rooted plantlets by: treating the plantlets with fungicide; and transplanting in sterile soil-free medium.

20. The method of claim 20, wherein the hardened plantlets are transplanted to soil.

21. A Punica granatum plant propagated according to a method as claimed above exemplified herein substantially in the examples and figures.