DESC:
TITLE:
TISSUE CULTURED TEAK PLANTS
FIELD OF INVENTION:
The invention in general relates to the field of applied forestry and/or biotechnology and in particular relates to a new practice for cloning teak (Tectonagrandis. L) for rapid propagation and production of teak plants with tissue culture technique by appropriate procedures necessarily to develop explants with good quality and volume of wood to harvest in short term on a large scale with the new application of exvitro rooting process.The new technique is highly useful to increase productivity of teak plantation by many folds by planting genetically superior cloned planting material.
BACKGROUND OF INVENTION:
Teak {TectonagrandisL.f.) is one of the most valued timbers of India. Although the genus Tectona is native to the tropical regions of Southeast Asia, primarily Indonesia, Myanmar, Bangladesh and Thailand, the cultivation of teak plantation is economically viable in other tropical regions such as Central America andis exclusively planted for the purpose of forestry management, for either commercial or ecological purposes.It is one of the most important hardwoods of the world and used for furniture, cabinet making, various grades of plywood, paneling, all types of construction, poles, heaps, ship building and other purposes.
The practice of raising plantations of teak out of seedlings started since 1840’s and even now this is in vogue (Mohammed Hussain et al., 1976). The need for forest plantation was realizedas early as the mid-nineteenth century.The first attempt at organized plantationin India was a teak plantationestablished in 1842 at Nilambur inKerala, southern India, with the purposeof enriching the forests (Bapat andPhulari, 1995). ChatuMenon, wellknown as the father of indianteak plantations,raised more than a million teakplants between 1842 and 1862(Parameswarappa, 1995). Organized plantations ona large scale were attempted only after1948, and until 1951 plantation was nota regular and extensive activity (Tewari,1992).

Thelow levels of investment in the forestrysector (including afforestation and logging)in the past resulted in low production and shortage of raw materialfor processing industries and also have a major impact on the country’s forest cover. Forest-based industries realizedthat the existing forests will not beable to meet their growing demand forraw materials. In addition, the supply oftimber is limited by restrictions or banson harvesting in natural forests in someIndian states. Recognizing the need,many forest-based industries have initiatedplantations on private lands, withthe collaboration of farmers to whomthey provide financial and technical assistance.

The conventional method of producing planting material of teak for raising plantations is by sowing seeds in the nursery beds and subsequently using stumps (root/shoot cuttings) of the seedlings obtained. Successful attempts on budding and grafting for vegetative propagation started at the beginning of the twentieth century (Fergusen, 1938), for establishing teak seed orchards and it gained importance as it was aimed at the genetic improvement of teak (MohamadHussain and Somasundaram, 1975).

A more quick and direct method for tree improvement of teak is clonal propagation of phenotypically superior trees by rooting shoot cuttings and thereby capturing the immediate genetic gains of the species. The productivity could be increased several folds by planting genetically superior propagates of teak and by raising clonal plantationsbut this method has several limitations and only provides a few propagules from selected individuals.A truly successful method for cloning of mature teak through rooting of shoot cuttings has not been developed so far.

Atissue culture or invitro clonal propagation technique has been developed for producing plants as uniform as possible on a large scale in a short time for the plantation industry. Tissue culture of the speciesbegan in the seventies (Gupta et al., 1977) withmore recent attempts to improve establishment andmultiplication (Devi et al., 1994). However, micropropagationof teak from mature trees has remainedproblematic. Poor explant response and rapid explantbrowning are major hurdles to successful establishment of teak invitro.

BRIEF DESCRIPTION OF RELATED ART
Tissue culture and rapid propagation in recent years in the seedling propagation process is the most widely used in a new practical technology.At present, the world has thousands of plants through tissue culture to be successful.
In this connection already known techniques for propagation of tissue cultured teak are reviewed herewith based on the availability of the data on patentability and search report,

1. Patent application no. CN101558741 (WO-200910065102.0) with title “METHOD FOR TISSUE CULTURE OF DRUPE-TYPE NON-TIMBER PRODUCT FOREST ROOTSTOCK” provides a rapid propagation technique of the economic forest rootstock of the drupaceous type, and in particular to a tissue culture method of the rootstock which is suitable for rapid propagation by adopting a stem section with axillary buds so as to achieve industrial seed culture in 6 to 7 months.
The limitations are subjected to the propagation of fine rootstock varieties in the healthy development of economic forest due to anti-shedding disease and root cancer characteristics. Hence, it is very important to study the propagation technology of fine rootstock and rapid propagation which is of great importance to improve the quality and yield of the economic forest varieties.

2. Patent application no. CN104719155 (WO-201510097702.0) with title “A RAPID PROPAGATION METHOD OF PHOEBE BOURNEI TISSUE CULTURE” provides a method for plant tissue culture in agricultural biotechnology, and in particular to a rapid propagation method for tissue culture of Phoebe bourneiwhich is a precious tree species for garden trees and precious timber species in China by adopting In vitro regeneration system by the process of inducing culture, subculture, rooting, transplanting and so on.
The limitations are subjected to the chances of high mortality rate while transplanting the seedling into the field.

3. Patent application no. CN104381133A (WO-201410648995.2) with title “TISSUE CULTURE BREEDING METHOD OF PHLOX SUBULATA”provides a reliable technical way for the large-scale production of the fine seedling of the conifers through a tissue culture and breeding method. The invention is simple and easy to operate, and has short operation cycle, high survival rate and low cost.
The limitations subject to the use of invitro culture method where the survival rate of seedlings may be problematic when transferring to the normal environmental conditions.

4. Patent application no. CN103461121B (WO-201310396794.3) with title “EX-VITRO ROOTING METHOD FOR TISSUE CULTURE SEEDLINGS OF PINUS MASSONIANA” belongs to the field of plant propagation technology, relates to tissue culture seedling technology of Masson pine as China's major industrial timber species and biomass energy tree species to reduce the production of cost and improve the efficiency methods of exoteric rooting.The features include highest rooting rate of 98%, and high root system quality, thesurvival rate of more than 90% with good economic, social and ecological benefits.
The limitations are subjected to the adaptation of invitro rooting method and woody/multiple root system which in turn develops into multiple shoots which is laborious to handle for plantation.
5. Patent application no. CN103875529A (WO-201410054055.0) with title “BLUEBERRY TISSUE CULTURE PROPAGATION AND EX-VITRO ROOTING METHOD”provides a method for tissue culture propagation and extragenogenesis of blueberry. The invention relates to the technical field of industrialized seedling production and in particular relates to a blueberry tissue culture propagation and ex-vitro rooting method. The method mainly aims to solve the technical problems of low rooting rate, long rooting period, complex rooting program, high rooting cost and in-vitro blueberry rooting.
While the methods adopted by different inventors are varied, the underlying concept is the same that to provide users a functional and practical method of tissue culture technique for the production of large-scale propagation of seedlings with high-quality in short time period.
Acknowledging that the concept is not a novel one, this application seeks to project the advantage of the invention and the methods designed and applied in every step proposed to be patented over former applications from a functional, practical usage, ease of implementation, cost and replacement point of view.
The present invention eliminates the problem of media contaminationto almost zero percentage and use of standardized protocols and unique chemical combinations at each and every step from bud initiation to multiplication by maintaining specific temperatures, humidity and other special measures which playa crucial part in tissue culture/micro-propagation method. The new application of exvitro rooting process helps the survival percentage of the saplings when exposed to the field conditions and also saves time, material, money and manpower.
SUMMARY OF THE INVENTION

The invention pertains to a novel and improved technique for production of teak plants through tissue culture propagation technique which shortens the cultivation period with high survival rate in simple and economical method. In order to comprising of the following steps:
Achieve the above objects, the technical proposal of the present invention comprising the following steps:

1) Selection of Mother Plant: There are nine plus tress or mother plants belonging to the 9 agro climatic conditions labeled by the forest departmentwere identified after analyzing 60 different characteristics and maintained under surveillance from time to time for generations. These identified 9 plants are suitable for rainfall ranging from 400mm to above 12000mm.

2) Screening of the Explant: The teak plants produced are not seed originated and are produced after extracting tissue from a part of (called explant) of the mother plant or plus tree. These 9 clones of explant identified are;
a. MBT400
b. MBT500
c. MBT700 – 2 Clones
d. MBT900 – 2 Clones
e. MBT1100
f. MBT1400
g. MBT2000

3) Surface Sterilization of the Explant: Surface sterilization of the explant was done for 30 times in about 6 hours period of duration. During the process the explant washed in running water, RO (Reversible Osmosis) water, DM (De-mineralized) water and double distilled water.

4) Bud Initiation: Standardizeda unique protocol for bud initiation with various chemical combinations using MS media as the base by maintaining a standard temperature, humidity and artificial light source etc. The media needs to be poured in sterilized bottles and autoclaved to reduce contamination.

5) Bud Elongation: Standardized a protocol for preparation of media without any contamination by using a series of combinations for elongation of the bud with the key ingredient as MS media with phytohormones. The sterilized culture bottles were stored in the growth rooms with optimum temperature of 25-29 ? C and artificial light source for 20 hours and maintained sterile conditions.

6) Multiplication: As the elongated buds reach the desired height, the buds were used for the purpose of inoculation where buds were cut for multiplication and placed them back into the culture bottles filled with sterilized media. The standard protocol for culture media is maintained as in bud elongation.
Then, the buds were shifted to the growth rooms and would be supplied artificial light and temperature under controlled conditions for the growth of the culture.

7) Rooting: As an alternative to routine process of “Invitro Rooting Method” by various tissue culture units, a standardized method of “Exvitro Rooting Process” was established to skip all the conventional rooting methodsand to complete the rooting process within 25 days with nil/negligible mortality.
The plantlets were given antifungal treatment and dipped in rooting hormone and sowed in polybags. Later they shifted to poly-tunnels in the shade house, where the optimum temperature and humidity was maintained between 27 ? C and 90% respectively.
8) Primary Hardening: Once the roots are fully developed, the polytunnels are removed and the plants are kept in the shade house and watered regularly by maintaining the standard temperature and humidity.

9) Secondary Hardening: The plants were shifted out of the shade house and exposed to outside environment or climatic conditions of the nature.

10) After 45 days of secondary hardening, the plants would be ready for plantation, where a proper watering for the plants is essential at this stage.

DETAILED DESCRIPTION OF THE INVENTION:

In general, teak production by the forest departments, nurseries and biotech units in the world propagated by seeds or stumps. Forest departments teak propagation includes the collection and storage of seeds in dark place until the dormancy period breaks, later artificially treats for germination. Then the germinated seeds will be segregated and after the formation of first leaf the shoot portion will be cut and the remaining part of the seedling called “stump” distributed to the farmers for plantation.Biotech units and nurseries also follow the same procedure as forest departments but the stumps will be kept in poly bags and grown to certain heights after which they sold to the farmers or customers. The characteristics and drawbacks of conventional stumps are;
i. Seed origin
ii. Irregular growth and high mortality rate
iii. Harvesting Period about 25-70 years (hence called next generation crop)
iv. Low Yield (maximum 30% of total crop)

The present invention belongs to the technical field of plant tissue culture with proven track record of mother plant and relates to a method of tissue culture propagation and ex-vitro rooting process. The invention simplifies the production procedure of teak plant by improving rapid propagation and production efficiency. This is a simple economical and reliable technical method for the large scale production of teak plants with high survival rate.The detailed description of the invention with the following steps explains the production of teak plants in effective way according to the establishment of standardized protocol from selection to the completion of total process,

Selection of Mother Plant:

The selection of mother plant is a crucial part in tissue culture propagation. The teak plants produced were not seed generated and were extracted tissue (explant) of the mother plant or the plus tree. There were nine (9) mother plants selected and identified based on analyzing 60 different characteristics. The plants were in surveillance from time to time for generations in nine (9) agro-climatic conditions of Telangana and Andhra Pradesh states for micro clonal propagation. The 9 clones identified suitable for rainfall ranging from 400mm to 1200mm are MBT400, MBT500, MBT700 (2 Clones), MBT900 (2 Clones), MBT1100, MBT1400, MBT2000. After successful establishment, the clones were propagated in tissue culture method which involves 6 stages and each stage was involved approximately 45 days’ time,

Surface Sterilization of the Explant:

The invention developed a unique method for selection of culture and storage media with different combination of chemicals. The meristematic tissuescollected from the mother plant were cut several times and isolated in order to get final sterilized part of the tissue. It is very precise and time consuming process.
In general, the surface sterilization of the explant needs to be done twice or thrice. The present invention carried out surface sterilization of the explant with the following steps which include;
1. Cut the tender stem (explant) soak in tap water for 120 minutes, then rinse with running water for 30 minutes, RO (Reverse Osmosis) water, DM (De-mineralized water) and DD (Double Distilled) water for thirty times about 6 hours.

2. The explant is treated with 75% ethyl alcohol for 02 minutes, sodium hypochlorite solution for 20 minutes and in Tween 20 for 05 minutes.

3. Disinfectant rinse clean was done properly by sterile water blot dry

Bud Initiation or Inoculation:

After sterilization, the bud initiation process was done based on the standardized protocol developed with different chemical combinations and hormonal concentration, by cutting the explant into small pieces and incubatingin the induction (nutrient)medium which serves as a nutrient source (micro and macro nutrients) for bud initiationcontaining chemicals such as Kinetin 2mg/liter, IAA 0.5mg/liter.The cultures were multiplied in the incubation room with illumination intensity 2000 lux at 29? for 16 (16+8) hours.

Media is prepared using the following chemical combination during the bud elongation:

1. KNO3 :1900mg/L
2. NH4NO3 : 1650mg/L
3. Cacl2.2H2O : 440mg/L
4. MgSO4.7H2O : 370mg/L
5. KH2PO4 : 170mg/L
6. MnSO4.4H2O :22.3mg/L
7. ZnSO4.7H2O :8.6mg/L
8. H3BO3 : 6.2mg/L
9. KI : 0.83mg/L
10. CuSO4.5H2O : 0.025mg/L
11. Na2MoO4.2H2O : 0.25mg/L
12. CoCl2.6H2O : 0.025mg/L
13. FeSO4.7H2O : 27.8mg/L
14. Na2EDTA : 37.26mg/L
15. Inositol : 100mg/L
16. ThiamineHcl : 0.1mg/L
17. Nicotinic acid : 0.5mg/L
18. Pyridoxine Hcl : 0.5mg/L
19. Glycine : 2.0mg/L
20. Hormone (Kinetin) : 2mg/L
21. IAA : 0.5mg/L
22. Agar : 8g/L
23. Sucrose : 30g/L
24. Cal D : 10mg/L
pH 5.7 throughout the process.

After the preparation and sterilization of the media in the culture bottles, it is cooled in the media storage room. After it solidifies, the explant is inoculated and then the culture bottles are arranged in the growth rooms and will be allowed to grow in controlled conditions.

After 20 days, the bud will initiate and we get around 1cm length of the same, it’ll then be transferred into the bud elongation media for the next stage.

Bud Elongation:

After several sets of changing the bud from one media to the other, finally standardized the media protocol for the elongation of the bud where the key ingredient was MS media with phytohormone such GA3 1.5mg/liter, NAA 0.5mg/liter. The number of explants producing embryogenesis/organogenesis were stored in the sterilized cultured bottles in the growth room and maintained temperature between 25-29?with artificial light source of 2500lux for 20 hours.

Media is prepared using the following chemical combination:

1. KNO3 :1900mg/L
2. NH4NO3 : 1650mg/L
3. Cacl2.2H2O : 440mg/L
4. MgSO4.7H2O : 370mg/L
5. KH2PO4 : 170mg/L
6. MnSO4.4H2O :22.3mg/L
7. ZnSO4.7H2O :8.6mg/L
8. H3BO3 : 6.2mg/L
9. KI : 0.83mg/L
10. CuSO4.5H2O : 0.025mg/L
11. Na2MoO4.2H2O : 0.25mg/L
12. CoCl2.6H2O : 0.025mg/L
13. FeSO4.7H2O : 27.8mg/L
14. Na2EDTA : 37.26mg/L
15. Inositol : 100mg/L
16. ThiamineHcl : 0.1mg/L
17. Nicotinic acid : 0.5mg/L
18. Pyridoxine Hcl : 0.5mg/L
19. Glycine : 2.0mg/L
20. Hormone GA3 : 1.5mg/L
21. NAA : 0.5mg/L
22. Agar : 8g/L
23. Sucrose : 30g/L
24. Cal D : 10g/L

Media is prepared with the above chemical combination and the sterilization and storage process will be the same as in bud initiation. The elongated buds are then placed in the culture bottles and then shifted to the growth rooms for development of the next stage in controlled conditions.

The buds elongate in 40 days after which they’ll be multiplied.

Multiplication:

After 40 days, when the germinated cluster of buds have reached to the desired height, they were cut for multiplication and placed back in the culture bottles filled with sterilized MS media with Phytohormones. Then they will be shifted to the growth rooms and would be supplied with artificial light and temperature and kept under controlled conditions for the growth of the culture where plantlets will be developed.

Media is prepared using following chemical combination:

1. KNO3 :1900mg/L
2. NH4NO3 : 1650mg/L
3. Cacl2.2H2O : 440mg/L
4. MgSO4.7H2O : 370mg/L
5. KH2PO4 : 170mg/L
6. MnSO4.4H2O :22.3mg/L
7. ZnSO4.7H2O :8.6mg/L
8. H3BO3 : 6.2mg/L
9. KI : 0.83mg/L
10. CuSO4.5H2O : 0.025mg/L
11. Na2MoO4.2H2O : 0.25mg/L
12. CoCl2.6H2O : 0.025mg/L
13. FeSO4.7H2O : 27.8mg/L
14. Na2EDTA : 37.26mg/L
15. Inositol : 100mg/L
16. ThiamineHcl : 0.1mg/L
17. Nicotinic acid : 0.5mg/L
18. Pyridoxine Hcl : 0.5mg/L
19. Glycine : 2.0mg/L
20. Hormone GA3 : 2mg/L
21. BAP : 1.5mg/L
22. IAA : 0.5mg/L
23. Agar : 8g/L
24. Sucrose : 30g/L
25. Cal D : 10mg/L

Using the above chemical combinations, media is prepared and stored as per the process mentioned in the above two stages and the cultures are inoculated and placed in the growth rooms under controlled conditions for 60 days after which, they’ll be taken for the root initiation.

Rooting:

In general, rooting methods by various tissue culture units was done by “Invitro Rooting Method”, where the rooting is done using a separate rooting media within the growth rooms. In the present invention, an “Exvitro Rooting Process” was developed, where it skips all the conventional rooting methods with nil/negligible mortality and also saves time, material, money and man power. The process is as follows;

The plantletswere isolated from the growth room after they reach to certain height. When the shoots were fully developed, they were taken to the rooting area where they will be given antifungal treatment using BAVISTINE (2g/L) and the base of the shoot (where the roots need to be initiated) was dipped in standardized rooting hormone (NAA 3000ppm) and transferred to the rooting tubs filled with rooting medium (coco-peat) and sprinkled water liberally. The tubs were covered with polysheets and shifted to the shade house where the temperature of 27? C and humidity of 90% was maintained and are monitored constantly. The formation of roots starts in 25 days and completed in 45 days.

Primary Hardening:
When the roots are fully developed, the plants were gently shifted to red soil filled with polybags and kept in the shade house and watered regularly under controlled conditions of temperature at 27-29 degrees C and 70% humidity.

Secondary hardening:

When primary hardening was completed, the plants were shifted out of the shade house and exposed to the external environmental conditions. The plants were survived at this stage by resisting with the natural conditions. The plants were kept under observation for 30 days.

After 45 days of secondary hardening, the plants were ready for planting, where proper watering was very important at this stage for maintenance of the plants.

ADVANTAGES OF PRESENT INVENTION:

Compared to the prior art, the invention has the following advantages:

1. The stumps are not seed originated and are extracted from the tissue of the mother plant, the plants have 100 percent mother plant characteristics by the application of microclonal propagation method.

2. The mother plants or plus trees selected in the present invention are based on the research observation on 9 agro-climatic conditions where 9 clones of mother plants with proven track record were identified and selected for the research purpose.

3. Surface sterilization: As the buds (clones) of teak (timber yielding plants) are more prone to microorganisms there is a greater chance of contamination when the buds are placed in media as the phenolic oxidation is very high. In present invention, standardized method of surface sterilization cut down the contamination percentage to almost zero.

4. Standardized protocols and unique chemical combinations were developed and used for bud initiation, elongation and multiplication. Specific temperatures, humidity and other standard measures were monitored in each and every stage of the tissue culture process.

5. The developed plants have uniform growth. The trees were straight without bending and the growth was in continuation with the root without multiple shoots.

6. If the cultivation is done as per the guidelines provided of the present invention the harvesting would be possible from 8th year onwards where in general process, it takes about 25-70 years for harvest (so called next generation crop).

7. The resulted harvesting trees average height and girth would be around 70-90 feet and 85 to 110cm respectively.

8. High yield with good quality and volume of the wood would be produced with around 70 percent of heartwood.

BRIEFDESCRIPTION OF DRAWINGS
FIG. 1 shows the process of tissue cultured teak plant production with all the stages from the selection of the mother plant to the secondary hardening.
DETAILED DESCRIPTION OF DRAWINGS
As shown in Fig 1, the invention describes process of tissue culture for the production of teak plant including stages from the selection of the mother plant to the secondary hardening. The stages are described as follows,
1. Mother Plant Selection
2. Collection of suitable explant
3. Sterilization
4. Media Preparation
5. Inoculation
6. Incubation
7. Sub Culture
8. Multiplication
9. Rooting
10. Primary Hardening
11. Secondary Hardening

,CLAIMS:CLAIMS
I Claim,
1. A method of tissue culture of teak plants cloning for rapid propagation and production with tissue culture technique. The method comprises the steps of:
a. Selection of mother plant
b. Screening of explant
c. Surface sterilization of explant
d. Bud initiation
e. Bud elongation
f. Multiplication
g. Rooting
h. Primary hardening
i. Secondary hardening
j. Plantation

2. A method of tissue culture in teak plants according to Claim 1, wherein the selection of mother plant is from nine agro-climatic conditions and having 60 different characteristics.

3. A method of tissue culture in teak plants according to Claim 2, wherein the nine clones identified are suitable for rainfall ranging from 400-1200 mm.

4. A method of tissue culture in teak plants according to Claim 2, wherein nine clones identified are MBT400, MBT500, MBT700 (2 Clones), MBT900 (2 Clones), MBT1100, MBT1400, MBT2000.

5. A method of tissue culture in teak plants according to Claim 2, wherein the clones were propagated in tissue culture method which involves six stages with each stage is approximately 45 days of time.

6. A method of tissue culture in teak plants according to Claim 1, wherein the surface sterilization of explant is carried out and treated with 75% ethyl alcohol for 2 minutes, sodium hypochlorite solution for 20 minutes and in Tween-20 for 5 minutes.

7. A method of tissue culture in teak plants according to Claim 1, wherein bud initiation is done with standard protocol with chemicals such as Kinetin-2mg/Litre, IAA-0.5mag/Litre along with illumination intensity 2000LUX at 29 ? C for 16 hours.

8. A method of tissue culture in teak plants according to Claim 1, wherein bud elongation will be carried out with standard media protocol in MS media with phytoharmones.

9. A method of tissue culture in teak plants according to Claim 8, wherein the phytoharmone is preferably GA3 1.5mg/Litre or NAA 0.5mg/Litre along with growth room temperature maintains at 25-29 ? C with artificial light source of 2500 LUX for duration of 20 hours.

10. A method of tissue culture in teak plants according to Claim 1, wherein the rooting is by exvitro-rooting process involving following steps,
a. Fully developed shoots are treated with BAVISTINE (2g/L)
b. Roots are dipped in standardized rooting hormone of NAA 3000ppm.
c. Plantlets are transferred to the rooting medium (coco-peat) tubs and covered with polysheets.
d. Maintained standard temperature of 27? and humidity of 90%
e. Formation of roots starts in 25 days and process completes in 45 days.