FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
A TISSUE CULTURE MEDIUM AND A METHOD FOR PRODUCING BANANA PLANTS IN VITRO
Gujarat State Fertilizers & Chemicals Ltd.
Of P.O. Fertilizernagar -391 750, Dist. Vadodara, Gujarat, India
Inventor(s): Vadawale A.V, Panara T.G, and Sutaria P.M
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE DISCLOSURE:
The present disclosure relates to a culture media for Banana plants. The present disclosure also relates to a process for producing banana plants.
BACKGROUND:
'Banana' is the common name for herbaceous plants of the genus 'Musa' and for the fruit they produce. Bananas come in a variety of sizes and colors when ripe, including yellow, purple, and red.
Bananas are native to tropical South and Southeast Asia. Edible Bananas originated in the Indo-Malaysian region reaching to northern Australia. Today, Bananas are cultivated throughout the tropics, particularly in every humid tropical region, primarily for their fruit, and to a lesser extent to make fiber, banana wine and as ornamental plants. Bananas constitute the 4th largest fruit crop of the world. World production of Bananas is estimated to be 121.85 million tons from around 10.1 million hectare area. India is the largest producer of Banana in the world with a production of around 26.99 million tons from 0.75 million hectare area. In India, Gujarat ranks first in the productivity of banana with 61.50 tons/hectare.
Banana plants are normally tall and fairly sturdy with succulent, juicy upright stem which is actually a pseudostem that grows 6 to 7.6 meters (20 to 24.9 ft) tall, growing from a rhizome/corm. Each pseudostem can produce a single bunch of bananas. After fruiting, the pseudostem dies, but offshoots may develop from the base of the plant. Suckers spring up around the main plant forming a clump or 'stool' where the eldest sucker replaces the main plant when it fruits and dies and this process of succession continues indefinitely.

Around 5 to 15 tender, smooth, oblong or elliptic, fleshy-stalked leaves are arranged spirally and may grow 2.7 meters (8.9 ft) long and 60 cm (2.0 ft) wide. Each pseudostem normally produces a single inflorescence, also known as the banana heart.
The banana fruits develop from the banana heart, in a large hanging cluster, made up of tiers which are called as hands, with up to 20 fruit to a tier. The hanging cluster is known as a bunch, comprising 3-20 tiers and can weigh from 30-50 kilograms (66-110 lb). Individual banana fruits (commonly known as a banana or 'finger') normally weigh about 125 grams (0.28 lb) comprising approximately of 75% water and 25% dry matter.
Bananas are of interest globally because of their influence on the socio-economics of millions of small and marginal farmers and being source of nutrition for large sector of population. However, banana crop encounters a number of seed born diseases caused by various microorganisms including bacterial species, fungal species, viral species and nematodes due to which the yield of banana crop is constantly decreasing.
Plant improvement by hybridizations or genetic manipulations may cause changes in crop species. An alternative to these methods is plant regeneration by tissue culture techniques which is well established. Although the totipotency of a plant cell is a well known phenomenon, yet each plant or plant part requires specialized studies to invent the conditions that allow such regeneration at high efficiency and frequency. There seems to be a consensus that success in inducing differentiation depends, amongst others, upon the type of explant, physiological condition of the explant and culture medium composition. Thus, tissue culture techniques can be directed to optimize the physiological condition of source plant, the type of

explant, the culture conditions and the culture medium composition to initiate the tissue response for plant regeneration which substantiates the fact that the development of a new process for proliferation of plants by tissue culture is not obvious.
Currently, there have been a few attempts for regeneration of banana species by tissue culture techniques that have brought only modest success which may be due to lack of proper culture conditions and composition of culture medium that is required for plant regeneration.
Some representative patent documents that disclose the use of tissue culture for plant regeneration are discussed herein below:
US Patent Application number 20060143740 discloses a process for selecting banana clones characterized by increased bunch weight and increased total fruit yield, the process involves steps of initiating tissue cultures from apical meristem explants, and propagating and rooting them; hardening, potting, and growing propogated plants in vivo; and growing the mats formed from a cluster of plants under conditions of abiotic stress.
Accordingly, it is desirable to develop a tissue culture medium and a process for production of disease free banana plants with increased yield.
OBJECTS:
Some of the objects of the present disclosure are discussed herein below.

It is an object of the present disclosure to provide a culture medium for the high frequency in vitro shoot induction and multiplication from shoot tip (sucker) explants of banana.
It is another object of the present disclosure to provide a culture medium for root induction and development of root system in banana plantlets.
It is yet another object of the present disclosure to provide a simple and reproducible tissue culture method for regeneration of a large number of disease free Banana plants from their explants.
It is another object of the present disclosure to provide a simple and reproducible tissue culture method for regeneration of Banana plants with increased yield.
SUMMARY OF THE DISCLOSURE:
These and other objects of the disclosure are to a great extent dealt with the disclosure disclosed herein after:
In accordance with one aspect of the present disclosure, there is provided a first culture medium for in-vitro shoot induction and multiplication in Banana plants, the said first culture medium comprising:
• salts;
• amino acids;
• vitamins;
• a carbon source;
• at least one plant growth regulator, and;
• a gelling agent.

Typically, the salts are selected from preferred Murashige and Skoog (MS) medium with component concentration (mg/L) of NH4NO3 1650, KN03 1900, CaCl2.2H20 440, MgS04.7H20 370, MnS04.4H20 22.3, ZnS04.7H20 8.6, H3B03 6.2, CuS04.5H20 0.025, Na2Mo04.2H20 0.25, CoCl2.6H20 0.025, KI 0.83, Na2EDTA.2H20 37.25 and FeS04.7H20 27.8.
The first culture medium comprises salts in concentrations on the basis of weight by volume.
Typically, the amino acids are selected from the group consisting of Glycine and L-Tyrosine.
Typically the first culture medium comprises Glycine in an amount ranging from 50 to 100 mg/L.
Typically the first culture medium comprises L-Tyrosine in an amount ranging from 70 to 120 mg/L.
Typically, vitamins are selected from the group consisting of Thiamine HC1, Nicotinic acid, Pyridoxine HC1, Myoinositol and Ascorbic acid.
Typically the first culture medium comprises Thiamine HC1 in an amount ranging from 0.001 to 0.3 mg/L.
Typically the first culture medium comprises Nicotinic acid in an amount ranging from 0.01 to 1.0 mg/L.
Typically the first culture medium comprises Pyridoxine HC1 in an amount ranging from 0.01 to 1.0 mg/L.

Typically the first culture medium comprises Myoinositol in an amount ranging from 70 to 120 mg/L.
Typically the first culture medium comprises Ascorbic acid in an amount ranging from 0.5 to 3.0 mg/L.
Typically the first culture medium comprises commercial sugar cubes as a carbon source in an amount ranging from 3 to 5 % w/v.
Typically, plant growth regulator at least one selected from the group consisting of Kinetin, 6-Benzylaminopurine and Adenine sulphate.
Typically, the first culture medium comprises Kinetin in an amount ranging from 1.0 to 5.0 mg/L.
Typically, the first culture medium comprises 6-Benzylaminopurine in an amount ranging from 3.0 to 7.0 mg/L.
Typically, the first culture medium comprises Adenine sulphate in an amount ranging from 5.0 to 10.0 mg/L.
Typically, the gelling agent is selected from the group consisting of agar, in an amount ranging from 0.6 to 0.8 % w/v.
Typically, the pH of the first culture medium ranges between 5.7 and 6.0.
In accordance with another aspect of the present disclosure, there is provided a second culture medium for root induction in Banana Plants, the said second culture medium comprising:
• salts;
• amino acids;
• vitamins;

• a carbon source;
• at least one plant growth regulator, and;
• a gelling agent.
Typically, the salts are selected from preferred Murashige and Skoog (MS) medium with component concentration (mg/L) of NH4NO3 1650, KN03 1900, CaCl2.2H20 440, MgS04.7H20 370, MnS04.4H20 22.3, ZnS04.7H20 8.6, H3B03 6.2, CuS04.5H20 0.025, Na2Mo04.2H20 0.25, CoCl2.6H20 0.025, KI 0.83, Na2EDTA.2H20 37.25 and FeS04.7H20 27.8.
The second culture medium comprises salts in concentrations on the basis of weight by volume.
Typically, the amino acids are selected from the group consisting of Glycine and L-Tyrosine.
Typically the second culture medium comprises Glycine in an amount ranging from 50 to 100 mg/L.
Typically the second culture medium comprises L-Tyrosine in an amount ranging from 70 to 120 mg/L.
Typically, vitamins are selected from the group consisting of Thiamine HC1, Nicotinic acid, Pyridoxine HC1, Myoinositol and Ascorbic acid.
Typically the second culture medium comprises Thiamine HC1 in an amount ranging from 0.001 to 0.3 mg/L.
Typically the second culture medium comprises Nicotinic acid in an amount ranging from 0.01 to 1.0 mg/L.

Typically the second culture medium comprises Pyridoxine HC1 in an amount ranging from 0.01 to 1.0 mg/L.
Typically the second culture medium comprises Myoinositol in an amount ranging from 70 to 120 mg/L.
Typically the second culture medium comprises Ascorbic acid in an amount ranging from 0.5 to 3.0 mg/L.
Typically the second culture medium comprises commercial sugar cubes as a carbon source in an amount ranging from 3 to 5 % w/v.
Typically, plant growth regulator is at least one selected from the group consisting of 6-Benzylaminopurine, Adenine sulphate andTndole-3-butyric acid.
Typically, the second culture medium comprises 6-Benzylaminopurine in an amount ranging from 0.5 to 5.0 mg/L.
Typically, the second culture medium comprises Adenine sulphate in an amount ranging from 0.5 to 5.0 mg/L.
Typically, the second culture medium comprises Indole-3-butyric acid in an amount ranging from 0.5 to 5.0 mg/L.
Typically, the gelling agent is selected from the group consisting of agar, in an amount ranging from 0.6 to 0.8 % w/v.
Typically, the pH of the first culture medium ranges between 5.7 and 6.0.
In accordance with yet another aspect of the present disclosure there is provided a method for regenerating large number of viable banana plants starting from a small tissue (explant) of banana plant; said method comprising the following steps:
(a) cutting and chopping the banana suckers to obtain explants;

(b) sterilizing the explants by treating with at least one sterilizing agent to obtain sterilized explants;
(c) culturing the sterilized explants in a pre-sterilized first medium for shoot induction and multiplication to develop several proliferating shoots;
(d) harvesting the developed shoots;
(e) culturing the harvested shoots in pre-sterilized second medium for root induction and development to generate functional roots; obtaining the regenerated plantlets; and
(f) transferring the regenerated plantlets to the soil for growing Banana plant.
Typically, the banana suckers are obtained from plants grown in the plant nursery/field.
Typically, the selected banana sucker is a 2 to 3 months old sword sucker.
Typically, the banana suckers are chopped to obtain explants of a size of 1.5 cm x 1.0 cm with an intact growing meristem.
The explants are treated by conventional methods to remove the contaminants such as fungus, bacteria etc. which are potentially harmful to regeneration.
Typically, the sterilization of the explants is effected by treating the explants with at least one sterilizing agent selected from the group consisting of Bavistin, Carbendazim, Isopropyl alcohol, Mercuric chloride etc.
Typically, the first medium is sterilized by autoclaving before culturing the explants.

Typically, the pH of the first medium is maintained in the range of 5.7 to 6.0.
Typically, the culturing of sterilized explants in the pre-sterilized first medium for shoot induction and multiplication is effected at a temperature in the range of 20 to 30°C in a light/dark period of 16/8 hour and light intensity of at least 3000 to 3500 lux.
Typically, the incubation period of next sub-culturing is around 30 to 40 days.
In accordance with one embodiment of the present disclosure, further minority of developed shoots is re-inoculated in fresh medium and incubated for a period of 5 to 6 weeks at a temperature in the range of 20 to 30°C in a light/dark period of 16/8 hour and light intensity of at least 3000 to 3500 lux to allow further multiplication and shoot development.
Typically, the second medium is sterilized by autoclaving before culturing the explants.
Typically, the pH of the second medium is maintained in the range of 5.7 to 6.0.
Typically, a majority of developed shoots is harvested from the base of the culture and inoculated in a pre-sterilized second medium.
Typically, the culturing of harvested shoots is effected at a temperature in the range of 25 to 30°C in a light/dark period of 16/8 hour and light intensity of at least 3000 to 3500 lux for a period of 30 to 40 days till the functional roots are visible thereby resulting in the development of regenerated plantlets.

The well developed plantlets, so formed, if desired, can be transferred to the soil for growing Banana plant.
Typically, the plantlets regenerated by the process of the present disclosure can be used for the production of disease free banana plants with higher yield than conventional planting material.
In accordance with another embodiment of the present disclosure, after separating the developed shoots from the culture, the base of the culture along with smaller buds is re-inoculated in second medium and incubated for a period of 30 to 40 days for further multiplication and development of rooting system. The well rooted plantlets developed after incubation are further treated for acclimatization and preliminary hardening.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
The disclosure will now be described with reference to accompanying drawings.
Figure 1 illustrates explants obtained from banana plants.
Figure 2 illustrates multiple shoot proliferation of banana plantlets.
Figure 3 illustrates root induction of banana plantlets.

Figure 4 illustrates Banana plantlets under primary hardening stage in green House.
Figure 5 illustrates Banana plantlets under secondary hardening stage in Net House.
Figure 6 illustrates fully developed plantlet of Banana.
Figure 7 illustrates growth of in-vitro regenerated plantlets on transferring to soil.
Figure 8A illustrates field performance of in-vitro regenerated plantlets and Figure 8 B illustrates field performance of conventional plantlet (sucker).
Figure 9 illustrates bunch with the highest weight (62 & 61 kg) of in-vitro regenerated plantlet.
Figure 10 illustrates a graph showing comparison between bunch weight of fruits harvested from Banana plants raised by the process of present disclosure and that of conventional planting material.
DETAIL DESCRIPTION OF THE DISCLOSURE:
Conventional methods for regeneration by tissue culture techniques have various drawbacks associated with them which may include lack of proper culture conditions and composition of culture medium that is required for plant regeneration. There have been a few attempts for regeneration of banana plants. In

the conventional methods, due to improper choice of explant and inappropriate culture composition, only a few shoots were induced resulting in plants being regenerated at a low frequency and low yield.
Tissue culture is a well established technique but there is still a need for an appropriate culture medium composition and specialized culture conditions that are well suited for a particular plant and allow regeneration of plant at high efficiency and frequency.
In view of the above drawbacks associated with the processes of tissue culture, the inventors of the present disclosure have developed a culture medium, termed as First culture medium capable of efficient shoot induction and multiplication in explants from Banana plant. Further, the inventors have developed a culture medium, termed as Second culture medium capable of root induction and development of rooting system. Still further, the inventors have developed a tissue culture process for regeneration of Banana plants.
The first culture medium capable of efficient in-vitro shoot induction and multiplication contains: salts; amino acids; vitamins; a carbon source; at least one plant growth regulator, and a gelling agent.
The salts used in the first culture medium of the present disclosure are selected from preferred Murashige and Skoog (MS) medium which include but is not limited to (component concentration (mg/L)) NH4NO3, KN03, CaCl2.2H20, MgS04.7H20, MnS04.4H20, ZnS04.7H20, H3BO3, CuS04.5H20, Na2Mo04.2H20, CoCl2.6H20, KI, Na2EDTA.2H20 and FeS04.7H20. The component concentrations (mg/L) of the salts are NH4NO3 1650, KN03 1900, CaCl2.2H20 440, MgS04.7H20 370, MnS04.4H20 22.3, ZnS04.7H20 8.6, H3B03 6.2, CuS04.5H20 0.025, Na2Mo04.2H20 0.25, CoCl2.6H20 0.025, KI 0.83,

Na2EDTA.2H20 37.25 and FeS04.7H20 27.8. The first culture medium comprises
salts in concentrations on the basis of weight by volume. The amino acids in the
first culture medium of the present disclosure include but are not limited to Glycine
and L-Tyrosine. The amount of Glycine in the first culture medium is 50 to 100
mg/L. The amount of L-Tyrosine in the first culture medium is 70 to 120 mg/L.
The vitamins which are also one of the ingredients of the first culture medium
include but not limited to Thiamine HC1, Nicotinic acid, Pyridoxine HC1,
Myoinositol and Ascorbic acid. The amount of Thiamine HC1 used in the first
culture medium of the present disclosure is 0.001 to 0.3 mg/L. The amount of
Nicotinic acid used in the first culture medium of the present disclosure is 0.01 to
1.0 mg/L. The amount of Pyridoxine HC1 used in the first culture medium of the
present disclosure is 0.01 to 1.0 mg/L. The amount of Myoinositol used in the first
culture medium of the present disclosure is 70 to 120 mg/L. The amount of
Ascorbic acid used in the first culture medium of the present disclosure is 0.5 to
3.0 mg/L. The carbon source, which is a vital ingredient of the first culture medium
includes but is not limited to commercial sugar cubes. The amount of carbon
source used in the first culture medium of the present disclosure is present in an
amount ranging from 3 to 5 % w/v. The plant growth regulator in the first culture
medium of the present disclosure includes but is not limited to Kinetin, 6-
Benzylaminopurine and Adenine sulphate. The amount of Kinetin used in the first
culture medium of the present disclosure is 1.0 to 5.0 mg/L. The amount of 6-
Benzylaminopurine used in the first culture medium of the present disclosure is 3.0
to 7.0 mg/L. The amount of Adenine sulphate used in the first culture medium of
the present disclosure is 5.0 to 10.0 mg/L. The gelling agent in the first culture
medium of the present disclosure includes but is not limited to agar. The amount of
gelling agent in the first culture medium of the present disclosure is 0.6 to 0.8 %
w/v.

Typically, the pH of the first culture medium ranges between 5.7 and 6.0.
The present disclosure also discloses a second culture medium capable of efficient root induction and development of rooting system contains: salts; amino acids; vitamins; a carbon source; at least one plant growth regulator, and a gelling agent.
The salts used in the second culture medium of the present disclosure are selected from preferred Murashige and Skoog (MS) medium which include but is not limited to (component concentration (mg/L)) NH4NO3, KN03, CaCl2.2H20, MgS04.7H20, MnS04.4H20, ZnS04.7H20, H3BO3, CuS04.5H20, Na2Mo04.2H20, CoCl2.6H20, KI, Na2EDTA.2H20 and FeS04.7H20. The component concentrations (mg/L) of the salts are NH4NO3 1650, KN03 1900, CaCl2.2H20 440, MgS04.7H20 370, MnS04.4H20 22.3, ZnS04.7H20 8.6, H3B03 6.2, CuS04.5H20 0.025, Na2Mo04.2H20 0.25, CoCl2.6H20 0.025, KI 0.83, Na2EDTA.2H20 37.25 and FeS04.7H20 27.8. The second culture medium comprises salts in concentrations on the basis of weight by volume. The amino acids in the second culture medium of the present disclosure include but are not limited to Glycine and L-Tyrosine. The amount of Glycine in the second culture medium is 50 to 100 mg/L. The amount of L-Tyrosine in the second culture medium is 70 to 120 mg/L. The vitamins which are also one of the ingredients of the second culture medium include but not limited to Thiamine HC1, Nicotinic acid, Pyridoxine HC1, Myoinositol and Ascorbic acid. The amount of Thiamine HC1 used in the second culture medium of the present disclosure is 0.001 to 0.3 mg/L. The amount of Nicotinic acid used in the second culture medium of the present disclosure is 0.01 to 1.0 mg/L. The amount of Pyridoxine HC1 used in the second culture medium of the present disclosure is 0.01 to 1.0 mg/L. The amount of Myoinositol used in the second culture medium of the present disclosure is 70 to 120 mg/L. The amount of Ascorbic acid used in the second culture medium of the

present disclosure is 0.5 to 3.0 mg/L. The carbon source, which is a vital ingredient of the second culture medium includes but is not limited to commercial sugar cubes. The amount of carbon source used in the second culture medium of the present disclosure is present in an amount ranging from 3 to 5 % w/v. The plant growth regulator in the second culture medium of the present disclosure includes but is not limited to 6-Benzylaminopurine, Adenine sulphate and Indole-3-butyric acid. The amount of 6-Benzylaminopurine used in the second culture medium of the present disclosure is 0.5 to 5.0 mg/L. The amount of Adenine sulphate used in the second culture medium of the present disclosure is 0.5 to 5.0 mg/L. The amount of Indole-3-butyric acid used in the second culture medium of the present disclosure is 0.5 to 5.0 mg/L. The gelling agent in the second culture medium of the present disclosure includes but is not limited to agar. The amount of gelling agent in the second culture medium of the present disclosure is 0.6 to 0.8 % w/v.
Typically, the pH of the second culture medium ranges between 5.7 and 6.0.
According to the present disclosure, there is provided a simple and efficient method employing the first culture medium and second culture medium for regenerating large number of viable banana plants starting from a small tissue (explant) of banana plant. The method is described herein below:
To obtain the explants, the plant material may be collected from banana plants grown in the plant nursery/field. The banana suckers are selected from plants which are potentially disease free, morphologically normal, with a plant height of about 2.5 to 3 meters and bunch weight of more than 30 kg. Usually, 2 to 3 months old sword sucker is selected.
In the first step, the selected banana suckers are cut to remove outer portion. The size of the growing tip is kept around 10 to 12 cm. The suckers are then fine

trimmed to a size of 5 to 10 cm while keeping the internal growing region and the lower rhizome portion intact. The fine trimmed suckers are then chopped to a size of 4.5 cm (height) x 2.5 cm (diameter). The chopped suckers are kept in Bavistin solution (0.2 % w/v).
In the next step, the chopped suckers are pretreated and surface sterilized before use to ensure that the explant is free of bacterial and fungal contaminants in the medium by treating with at least one sterilizing agent to obtain sterilized suckers. Many sterilizing techniques are available in the state of art for the purpose of preparing explant culture. Such techniques involve dipping the suckers in the solution containing at least one sterilizing agent. Such sterilizing agents include Bavistin, Carbendazim, Isopropyl alcohol, Mercuric chloride etc. In the present disclosure, the sterilization of the suckers is effected by treating the suckers with Carbendazim (0.2 % w/v) for about 20 to 30 minutes and washing to remove traces of Carbendazim. The suckers are then treated with Isopropyl alcohol (70 % w/v) for 50 to 60 seconds followed by a water wash. The suckers are air dried for 15 to 20 minutes followed by a treatment with Mercuric chloride (0.2 % w/v) for 20 to 30 minutes and draining the suckers. The well drained suckers are then cut to remove the outer leaf primordial to obtain sterilized explants with a size of 1.5 cm (height) x 1.0 cm (diameter).
In the next step, the sterilized explants are placed aseptically for culturing in a first medium for shoot induction and multiplication to develop several proliferating shoots. The first medium is sterilized by autoclaving before culturing the explants. The pH of the first medium is maintained in the range of 5.7 to 6.0. The culturing of sterilized explants in the pre-sterilized first medium is effected at a temperature

in the range of 25 to 30°C in a light/dark period of 16/8 hour and light intensity of at least 3000 to 3500 lux.
In an embodiment of the present disclosure, the incubation period of next sub-culturing is around 30 to 40 days. The culture is constantly checked for any unwanted bacterial/fungal growth (contamination). A large number of shoots are formed after 30 to 40 days.
In the next step, the developed shoots are harvested in sterile environment (laminar air flow) with the help of a sharp scalpel and blade. The culture is cleaned, roots are removed and base is trimmed. The developed shoots (of more than 1.5 cm height) are cut from the base along with little base portion for BM3 production.
In another embodiment of the present disclosure, a minority (about 40%) of developed shoots are treated in S2 fashion. The smaller buds are allowed to grow on their own in BM1 medium. The culture is re-inoculated in fresh medium and incubated for a period of 5 to 6 weeks at a temperature in the range of 20 to 30°C in a light/dark period of 16/8 hour and light intensity of at least 3000 to 3500 lux to allow further multiplication and shoot development.
In the next step, the harvested shoots are cultured in second medium for root induction and development to generate functional roots. A majority (60%) of developed shoots harvested from the base of the culture are graded according to the height and inoculated aseptically in a second medium. The second medium is sterilized by autoclaving before culturing the explants. The pH of the second medium is maintained in the range of 5.7 to 6.0. The culturing of harvested shoots is effected at a temperature in the range of 25 to 30°C in a light/dark period of 16/8

hour and light intensity of at least 3000 to 3500 for a period of 30 to 40 days till the functional roots are visible thereby resulting in the development of regenerated plantlets.
In another embodiment of the present disclosure, after separating the developed shoots from the culture, the base of the culture along with smaller buds is re-inoculated in second medium and incubated for a period of 30 to 40 days for further multiplication and development of rooting system. The well rooted plantlets developed after incubation is further treated for acclimatization and preliminary hardening.
Finally, the well developed plantlets, so formed, if desired, can be transferred to the soil for growing Banana plant. The plantlets regenerated by the process of the present disclosure can be used for the production of disease free banana plants with higher yield than conventional planting material.
The present disclosure is further described in light of the following examples which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure.
Example No. 1: Composition of First Culture medium:
The First Culture medium is described in Table-1
Table-1

INGREDIENTS AMOUNT (mg/L)

Salts
NH4NO3, 1650

KNO3 1900

CaCl2.2H20 440

MgS04.7H20 370

MnS04.4H20 22.3

ZnS04.7H20 S.6

H3BO3 6.2

CuS04.5H20 0.025

Na2Mo04.2H20 0.25

CoCl2.6H20 0.025
KI 0.83
Na2EDTA.2H20 37.25
FeS04.7H20 27.8
Glycine 75
Amino acids L-Tyrosine 100
Thiamine HC1 0.10
Vitamins Nicotinic acid 0.5
Pyridoxine HC1 0.5
Myoinositol 100

Ascorbic acid 2
Carbon source Commercial sugar cubes 50000
Plant growth regulator Kinetin 2-3

6-Benzylaminopurine 4-6
Adenine sulphate 7-9
Gelling agent Agar 7000
Example No. 2: Composition of Second Culture medium: The Second Culture medium is described in TabIe-2
Table-2

INGREDIENTS AMOUNT (mg/L)
Salts NH4NO3, 1650

KNO3 1900

CaCl2.2H20 440

MgS04.7H20 370

MnS04.4H20 22.3

ZnS04.7H20 8.6

H3BO3 6.2

CuS04.5H20 0.025

Na2Mo04.2H20 0.25

CoCl2.6H20 0.025

KI 0.83
Na2EDTA.2H20 37.25
FeS04.7H20 27.8
Glycine 75
Amino acids L-Tyrosine 100
Thiamine HC1 0.10
Vitamins Nicotinic acid 0.5
Pyridoxine HC1 0.5
Myoinositol 100
Ascorbic acid 2
Carbon source Commercial sugar cubes 50000
Plant growth regulator 6-Benzylaminopurine 1-3

Adenine sulphate 1-3

Indole-3 -butyric acid 1-3
Gelling agent Agar 7000
Example No. 3: Effect of First culture medium on shoot multiplication and shoot
development:
Effect of first medium on Shoot Multiplication and shoot development is shown in
Table-3
Table-3

CULTURE MEDIUM NUMBER OF SHOOT SHOOTING
SHOOTS LENGTH
(CMS) (DAYS)
First Culture Medium 9 2.8 24
Conventional Culture 4 2.2 29
Medium
Conclusion: The results show that the number of shoots and shoot length is highest in plants raised by the first medium. Also, the shooting is induced in less number of days as compared to conventional culture medium.

Example No. 4: Effect of Second culture medium on root induction and development (after 5 to 6 weeks from culturing)
Effect of second medium on root induction and development is shown in Table-4
Table-4

CULTURE MEDIUM ROOTING PERCENTA< ROOTING
(DAYS)
Second Culture Medium 100% 15
Conventional Culture Medi 90% 22
Conclusion: The results show that the rooting % is highest in plants raised by the first medium. Also, the rooting is induced in less number of days as compared to conventional culture medium.
Example No. 5: Comparison of Characteristics of in-vitro raised plantlets and Conventional Planting material.
Characteristics of in-vitro raised plantlets and that of Conventional Planting material is shown in Table-5
Table-5

Avera Average Avera Avera Harvesti
S.No Treatment Height stem girth ge Finger ge Bunch ng period
(Mt) (cm) Length Weigh (months)

(cm) t (Kg)
Plantlets raised in-
1 vitro by the Method of Present disclosure 3.4 75 21 38 13
Conventiona
2 1 Planting Material 1 (suckers) 3.2 68 16 26 16
Conventiona 1 Planting
3 Material 2
(TC ratoon
crop) 3.1 71 18 28 14
Conclusion: The results show that the Plantlets raised in-vitro by the Method of Present disclosure show increased Average Height, increased Average stem girth, increased Average Finger Length, increased Average Bunch Weight and decreased harvesting period as compared to the planting materials 1 and 2.
Example No. 6: Comparison in Yield of in-vitro raised plantlets and that of conventional planting material.
Yield in Experiment 1 is shown in Table-6A

Table-6

S.No Treatment Average
bunch
weight (kg) Average yield (Kg/Acre)
Plantlets raised in-vitro by the
1 method of
pesent
disclosure 38 45600
Conventional
2 Planting
Material 1
(sucker) 26 31200
Conventional
3 Planting
Material 2 (TC
ratoon crop) 28 33600
Conclusion: The results show that the Plantlets raised in-vitro by the Method of Present disclosure show increased average per acre yield as compared to the conventional planting materials 1 and 2.

Example No. 7: Comparison of Disease Incidence and survival rate of in-vitro raised plantlets and Conventional Planting material.
Disease Incidence and survival rate in Experiment 1 is shown in Table-7A
Table-7A

Disease
S.No Treatment Survival Incidence
(%) (%)
Plantlets raised in-vitro by the
1 method of
Present
disclosure 96 2
Conventional
2 Planting
Material 1
(sucker) 84 10
Conventional
3 Planting Material 2 (TC 89 4
ratoon crop)

Conclusion: The results show that the Plantlets raised in-vitro by the Method of Present disclosure show increased survival rate and decreased disease incidence as compared to the planting materials land 2.
ECONOMIC SIGNIFICANCE AND TECHNICAL ADVANCEMENT
- The method of the present disclosure is simple and efficient.
- The plantlets raised by the method of the present disclosure have increased Bunch Weight as compared to that of conventional planting material.
- The plantlets raised by the method of the present disclosure give higher yields as compared to that of conventional planting material.
- The plantlets raised by the method of the present disclosure are disease free.

We Claim:
1. A first culture medium for in-vitro shoot induction and multiplication in banana
plants, the said first culture medium comprising:
• salts;
• amino acids;
• vitamins;
• a carbon source;
• at least one plant growth regulator, and;
• a gelling agent.

2. The first culture medium as claimed in claim 1, wherein the salts selected from Murashige and Skoog (MS) medium with component concentration (mg/L) comprise of NH4N03 1650, KN03 1900, CaCl2.2H20 440, MgS04.7H20 370, MnS04.4H20 22.3, ZnS04.7H20 8.6, H3B03 6.2, CuS04.5H20 0.025, Na2Mo04.2H20 0.25, CoCl2.6H20 0.025, KI 0.83, Na2EDTA.2H20 37.25 and FeS04.7H20 27.8.
3. The first culture medium as claimed in claim 1, wherein the amino acids with component concentration (mg/L) comprise of Glycine 75 and L-Tyrosine 100.
4. The first culture medium as claimed in claim 1, wherein the vitamins, with component concentration (mg/L) comprise of Thiamine HC1 0.01, Nicotinic acid 0.5, Pyridoxine HC10.5, Myoinositol lOOand Ascorbic acid 2.
5. The first culture medium as claimed in claim 1, wherein the carbon source is selected from commercial sugar cubes.

6. The first culture medium as claimed in claim 1, wherein the carbon source is in an amount ranging from 3 to 5 % w/v.
7. The first culture medium as claimed in claim 1, wherein the plant growth regulator is at least one selected from the group consisting of Kinetin, 6-Benzylaminopurine and Adenine sulphate, at a concentration range varying from 1 to l0mg/L.
8. The first culture medium as claimed in claim 1, wherein the gelling agent is selected from group consisting of agar at a concentration range varying from 0.6 to 0.8 % w/v.
9. The first culture medium as claimed in claim 1, wherein the pH of the first culture medium ranges between 5.7 and 6.0.
10. A second culture medium for root induction and development in banana
plants, the said second culture medium comprising:
• salts;
• amino acids;
• vitamins;
• a carbon source;
• at least one plant growth regulator, and;
• a gelling agent.
11. The second culture medium as claimed in claim 10, wherein the salts selected
from Murashige and Skoog (MS) medium with component concentration
(mg/L) comprise of NH4N03 1650, KN03 1900, CaCl2.2H20 440,
MgS04.7H20 370, MnS04.4H20 22.3, ZnS04.7H20 8.6, H3B03 6.2,

CuS04.5H20 0.025, Na2Mo04.2H20 0.25, CoCl2.6H20 0.025, KI 0.83, Na2EDTA.2H20 3 7.25 and FeS04.7H20 27.8.
12. The second culture medium as claimed in claim 10, wherein the amino acids with component concentration (mg/L) comprise of Glycine 75 and L-Tyrosine 100.
13. The second culture medium as claimed in claim 10, wherein the vitamins with component concentration (mg/L) comprise of Thiamine HC1 0.01, Nicotinic acid 0.5, Pyridoxine HC1 0.5, Myoinositol lOOand Ascorbic acid 2.
14. The second culture medium as claimed in claim 10, wherein the carbon source
is selected from commercial sugar cubes.
15. The second culture medium as claimed in claim 10, wherein the carbon source
is in an amount ranging from 3 to 5 % w/v.
16. The second culture medium as claimed in claim 10, wherein the plant growth regulator is at least one selected from the group consisting of 6-Benzylaminopurine, Adenine sulphate and Indole-3-butyric acid, at a concentration range varying from 0.5 to 5 mg/L.
17. The second culture medium as claimed in claim 10, wherein the gelling agent is selected from the group consisting of agar at a concentration range varying from 0.6 to 0.8 % w/v.
18. The first culture medium as claimed in claim 10, wherein the pH of the first culture medium ranges between 5.7 and 6.0.

19. A method for regenerating a large number of viable Banana plants by tissue culture technique starting from a small tissue of banana plant, said method comprising the following steps:
(a) cutting and chopping the banana suckers to obtain explants;
(b) sterilizing the explants by treating with at least one sterilizing agent to obtain sterilized explants;
(c) culturing the sterilized explants in a pre-sterilized first medium for shoot induction and multiplication to develop several proliferating shoots;
(d) harvesting the developed shoots;
(e) culturing the harvested shoots in pre-sterilized second medium for root induction and development to generate functional roots; obtaining the regenerated plantlets; and
(f) transferring the regenerated plantlets to the soil for growing Banana plant.

20. The method as claimed in claim 19, wherein said small tissue is an explant.
21. The method as claimed in claim 19, wherein banana sucker is an explant.
22. The method as claimed in claim 19, wherein the banana suckers are chopped to obtain explants of a size of 1.5 cm x 1.0 cm with an intact growing meristem.
23. The method as claimed in claim 19, wherein the explants are treated by conventional methods to remove the contaminants, wherein the sterilization of the explants is effected by treating the explants with at least one sterilizing agent selected from the group consisting of Bavistin, Carbendazim, Isopropyl alcohol, Mercuric chloride etc.

24. The method as claimed in claim 19, wherein the culturing of sterilized explants in the pre-sterilized first medium for shoot induction and multiplication is effected at a temperature in the range of 20 to 30°C in a light/dark period of 16/8 hour and light intensity of at least 3000 to 3500 lux.
25. The method as claimed in claim 19, wherein further minority of developed shoots is re-inoculated in fresh medium and incubated for a period of 5 to 6 weeks at a temperature in the range of 20 to 30°C in a light/dark period of 16/8 hour and light intensity of at least 3000 to 3500 lux to allow further multiplication and shoot development.

26. The method as claimed in claim 19, wherein shoots of about 1.5 to 2 cm height are harvested from the base of the culture and inoculated in a pre-sterilized second medium.
27. The method as claimed in claim 19, wherein the culturing of harvested shoots is effected at a temperature in the range of 25 to 30°C in a light/dark period of 16/8 hour and light intensity of at least 3000 to 3500 for a period of 30 to 40 days till the functional roots are visible resulting in the development of regenerated plantlets.

28. The method as claimed in claim 19, wherein the well developed plantlets are transferred to the soil for growing a Banana plant.
29. The method as claimed in claims 19 to 28, wherein the survival rate of raised plantlets is about 95 to 100%.
30.The method as claimed in claims 19 to 28, wherein the average bunch weight of raised plantlets is about 35 to 40kg.

31.The method as claimed in claims 19 tP 28, wherein the average yield of raised plantlets is about 40 to 50 %.