Title of Invention

A PROCESS FOR IN VITRO PLANTLET PRODUCTION OF THE BAMBOO OXYTENANTHERA STOCKSII

Abstract

"A process for regenerating plants from mature explants of 0. Stocks" This invention relates to a process for regenerating plants from mature explants of 0. Stocks, which comprises the steps of placing explants obtained from mature 0.stocksii plant on a first step medium capable of inducing the explants to produce multiple axillary buds, said medium containing a cytokine in an amount in the range of from 0.05-2.0 mg/1 and culturing the said explants until a bunch of multiple axillary buds are produced and, Obtaining continuous outshoot cultures in the second step medium, Transferring the outshoot cultures from step (b) to a third step medium containing an audio in an amount in the range from 2.0-12.5 mg/1, said medium being capable of inducing roots from the outshoot clusters, Elongation of root initials formed at step (c ).

Full Text

FIELD OF INVENTION This invention relates to a process for in vitro plantlet production of the bamboo, Oxytenanthera stocks. EXISTING STATE OF ART Oxytenanthera stocks Munro, is a medium sized bamboo. Culms are slender strong with a small hollow and grown up to 9 m tall. The intern odes are 15-30 cm long and 2,5 to 4 cm in diameter with few branches at the node. It is distributed in Karnataka and Go and commonly cultivated in the Coastal belts. Tat grows in a well-drained deep loamy soil. It is, used for making umbrella handles, baskets, punt poles and construction purposes. The last reported flowering of this bamboo occurred in 18S4 and 1SR9 yet the seed production is not known. Planting is done using rhizomes, which not only limits quantity of propagates but also damages mother plants. Though a vegetative propagation method was reported using calm cuttings, large scale production was not practiced due to limitations imposed by bulkiness of propagates and poor rooting percent. Many bamboo species are multiplied using micro propagation methods because of their monocarp nature. Nadirs et al (1984) demonstrated that it is possible to produce enough number of plantlets of bamboo using auxiliary bud proliferation method. Tissue culture protocols for multiplication from mature tissues of bamboo are limited to very few species only (Chattered et al, 1993; Sabena and Dawn, 1994; Ramayana and Yakandawala, 1997) because of the difficulties in continuous multiple shoot production and rooting Nadir et al (1984) produced multiple shoots from mature nodes of Dendrocalamus strict us, Rumbas vulgarism and B. arundinaceous but the shoots could not be rooted except for D. strictus where 20% rooting occured. Catered et al (1993) failed to multiply 10 year old D. strictus nodes but obtained 30% rooting along with regeneration of auxiliary shoots when nodal segments were cultured upside down on a medium containing phloroglucinol• In Ambush glacises the axillary buds produced multiple shoots during first subculture but further multiplication did not occur (Bain, 1985). Similarly, B. Tulia produced multiple shoots only up to four subcultures (Serena and Shawn, 1994). Nodal segments were induced to produce multiple shoots and no rooting was reported in D. strictus (Macarena et al, 1988). Explants from small branches of Thyrsostachys olive, Dendrocalamus as per and Ambush nana produced multiple shoots but the cultures did not multiply after 2 months and none of the shoots could be rooted (Vogvijitra, 1988). . Similarly Sabena and Bowen (1993) successfully multiplied four-year-old plants of D. longispathus and 70% rooting was obtained but verifications of cultures was noticed. Kumar (1994) initiated axillary shoot cultures from D. strictus, B. arundinaceous and Thyrsostachys sameness but no information on age of the tissue, shoot multiplication and root development is available, problems like high contamination rate and poor bud breaking were noticed. Plantlets of D. gigantean were produced from single node segments of 5-years-old plants and 77.5% rooting were reported and the cultures were supported with the fibrous material obtained from fluffs (Bluffer equiangular Robe•) fruits to avoid nitrifications (Ramayana and Yakandawala, 1997). The known process was attended with several disadvantages. In bamboos, in vitro regeneration techniques like micro propagation and somatic embryo genesis were successful mostly from seedling and embryos (Zamora, 1994; Banik,1995; Shirgurkar, 1996). However, there is no information available on the seed production of 0. stocks. Almost all the reports available on the micro propagation of mature tissues of bamboos discussed various problems such as establishment of contamination free cultures, poor rooting, difficulty in multiplication of established cultures, and nitrification of multiple shoots (Catered ec al, 1993; Serena and Nahant, 1994; Kumar, 1994; Ramayana and Yakandawala, 1997). Plantlet production in bamboo through micro propagation mainly depends on the establishment of contamination free cultures and in getting high percentage of rooting (Ramayana and Yakandawala, 1997). When the explants are collected from field grown clumps and nodal segments carry a wide range of microbes (Chang, 1991), To date there have been no published reports available on micro propagation and plantlet production of the species Oxytenanthera stocks. OBJECT OF THE INVENTION An object of this invention is to propose a process for in vitro probation of 0.stocksii plants. An object of this invention is to propose a process for which are true to type. Still another object of this invention is to propose a process for which require less space for probation. Yet another object of this invention is to propose a process for and where large scale propagation can be practiced easily. DETAILED DESCRIPTIOH OF THE INVENTION According to this invention there is provided a process for regenerating plants from mature expands of O.stockii, which comprises the steps of placing explants obtained from nature 0. stocks plant on a first step medium capable of inducing the explants to produce multiple axillary buds, said medium containing a cytokinln in an amount in the range of from 0.05-2.0 mg/1 and culturing the said explants until a bunch of multiple axillary buds are produced and obtaining continuous outshoot cultures in the second step medium transferring the outshoot cultures from step (b) to a third step medium containing an audio in an amount in the range from 2.0 - 12.5 mg/l, said medium being capable of inducing roots from the outshoot clusters. Elongation of root initials formed at step (c). The present method utilizes a multi-step culturing process for producing Oxytenenthera stockier plantlets from mature plants. In this process, at the first step axillary buds are induced to elongate in a solid medium, which helps to screen contaminants also, kit the second step, the contamination free cultures are transferred to liquid medium for shoot proliferation. The well-developed shoots are transferred to root induction medium for a short period followed by root elongation medium as a third step. Finally, the rooted plantlets were potted out. The explants are obtained from non-prismatic structures of the mature plants. The branch complements of current year culms are the preferred sources of explants. Three days prior to the collection of explants the mother plants are sprayed with the 1.0 percent solution of fungicide, Banister. In the first step axillary bud induction medium is normally a solid nutrient medium containing a balanced concentration of inorganic salts and organic nutrients, vitamins and supplemented with plant growth hormone (preferably cytokinlns). Many such nutrient media are known and commercially available. For example, the step one medium can contain Marshier and Slog (MS) salts, mesoinositol (100 mg/1) Thiamine HCl (O.lmg/1) Nicotinic acid (0.5 mg/1) Pyridoxine Hal (0.5 mg/1) Lysine (2.0 mg/1) Sucrose (20.0 g/1) agar (0.7%) and 6-benzylaminopurine (BA) (0.05-2.0 mg/1). The pH of the medium is adjusted to about Soot 6.0. Preferably, the pH of the solid medium is 5.7. Single node segments from the branch complement of current year culms are used as explants. The diameter of the explants is preferably 2.5-3.0 mm. The outer branch sheaths covering the axillary buds are removed and the nodes are washed under running tap water for 30 minutes. The explants are surface sterilized by shaking in 1.0% antiseptic for 5 minutes and rinsed thrice in sterile dematerialized water. To disinfect the fungus and bacteria the explants are immersed in the solution containing 0.1% Baiting and 0.1% Kanamycin for 30 minutes in shaker (50 rpm) followed by 0.1% Sodium hypo chlorite for 5 minutes and 0.1% Mercuric chloride for 5 minutes, fitter each sterility wash the explants are rinsed three times with dematerialized sterile water All the cultures are maintained at about 25 - 27>C with a 16h photoperiod provided by cool white fluorescent tubes. In the second step medium continuous shoot multiplication is obtained. MS liquid medium is used with BA (0.1-1.0 mg/1). The pH of the medium is adjusted to 5.0-5.5. The cultures are placed in the liquid medium without any support. The elongated shoots are transferred to fresh medium with the interval of 15-18 days. When the cluster of tissues is developed after 2 to 3 passages the mother tissue is removed. Large clusters of shoots developed in a period of 15-18 days are separated at their base into small clusters having 3-5 shoots and sub-cultured. After about 15-25 passages, the small cluster with 3-5 shoots are separated and transferred to root induction medium Rooting involved two step procedure in which the shoots treated with axing for root induction are transferred to axing free medium for root elongation. For root induction, MS basal liquid medium supplemented with IBA (2.0-12.5 mg/l) is used. After a short period. of treatment, the cultures are transferred to half-strength MS liquid medium without any growth regulators Seventy percent of the axing treated cultures produce roots. After 15-20 days in the root elongation medium, the rooted plantlets arc planted in the root train s containing vermiculite and placed under a tightly closed politest for acclimatization. The pollens maintains 90-95 percent humidity. After 15 days the politeness’ are loosened gradually and placed in the shade house. The survival rate of the plantlets is 85 percent. After 15-20 days of hardening in the shade house, plants are transplanted in playas containing soil: sand: compost (1:1:3) After 60-75 days in playas all the plants show 3-5 rhizome formation followed by new clump development from the rhizome, which ensures the successftil establishment of the plantlets Following are the definitions for the terms used in the present description. "Axillary bud proliferation' is a process of inducing multiple (axillary) shoe( by incorporation growth regulators (usually cytokines) into the culture medium^ The result is tote product ere miniature hay branched shook system. "Branch complement" refers to the array of branches that develop at a single calm node, including the primordial one and any that arise by proliferation from buds at its proximal nodes. 'Branch sheath' refers to the sheathing organ borne singly at each node of an aerial elative branch of any order exciting the neck sheath and leaf sheath A "calm" refers to the segmented aerial axis that emerges from a rhizome and forms a part of a bamboo plant. An "explants" is a piece of tissue taken from a donor plant for culturing. "Non-rhizomatised structures" refers to those parts of bamboo, which are not arising directly from the rhizomes or from the basal nodes of the calm, egg, Branch. A "plantlet" is a plant asexually reproduced by tissue culture in the present case. A "propagate" is a plant asexually produced in the present case. "Passage" refers to the process by which the multiple shoots produced are first subdivided and then transferred to fresh culture medium. The term is synonymous with the term "subculture". FIELD OF INVENTION This invention relates to a process for in vitro plantlet production of the bamboo, Oxytenanthera stocks. EXISTING STATE OF ART Oxytenanthera stocks Munro, is a medium sized bamboo. Culms are slender strong with a small hollow and grown up to 9 m tall. The intern odes are 15-30 cm long and 2,5 to 4 cm in diameter with few branches at the node. It is distributed in Karnataka and Go and commonly cultivated in the Coastal belts. Tat grows in a well-drained deep loamy soil. It is, used for making umbrella handles, baskets, punt poles and construction purposes. The last reported flowering of this bamboo occurred in 18S4 and 1SR9 yet the seed production is not known. Planting is done using rhizomes, which not only limits quantity of propagates but also damages mother plants. Though a vegetative propagation method was reported using calm cuttings, large scale production was not practiced due to limitations imposed by bulkiness of propagates and poor rooting percent. Many bamboo species are multiplied using micro propagation methods because of their monocarp nature. Nadirs et al (1984) demonstrated that it is possible to produce enough number of plantlets of bamboo using auxiliary bud proliferation method. Tissue culture protocols for multiplication from mature tissues of bamboo are limited to very few species only (Chattered et al, 1993; Sabena and Dawn, 1994; Ramayana and Yakandawala, 1997) because of the difficulties in continuous multiple shoot production and rooting Nadir et al (1984) produced multiple shoots from mature nodes of Dendrocalamus strict us, Rumbas vulgarism and B. arundinaceous but the shoots could not be rooted except for D. strictus where 20% rooting occured. Catered et al (1993) failed to multiply 10 year old D. strictus nodes but obtained 30% rooting along with regeneration of auxiliary shoots when nodal segments were cultured upside down on a medium containing phloroglucinol• In Ambush glacises the axillary buds produced multiple shoots during first subculture but further multiplication did not occur (Bain, 1985). Similarly, B. Tulia produced multiple shoots only up to four subcultures (Serena and Shawn, 1994). Nodal segments were induced to produce multiple shoots and no rooting was reported in D. strictus (Macarena et al, 1988). Explants from small branches of Thyrsostachys olive, Dendrocalamus as per and Ambush nana produced multiple shoots but the cultures did not multiply after 2 months and none of the shoots could be rooted (Vogvijitra, 1988). . Similarly Sabena and Bowen (1993) successfully multiplied four-year-old plants of D. longispathus and 70% rooting was obtained but verifications of cultures was noticed. Kumar (1994) initiated axillary shoot cultures from D. strictus, B. arundinaceous and Thyrsostachys sameness but no information on age of the tissue, shoot multiplication and root development is available, problems like high contamination rate and poor bud breaking were noticed. Plantlets of D. gigantean were produced from single node segments of 5-years-old plants and 77.5% rooting were reported and the cultures were supported with the fibrous material obtained from fluffs (Bluffer equiangular Robe•) fruits to avoid nitrifications (Ramayana and Yakandawala, 1997). The known process was attended with several disadvantages. In bamboos, in vitro regeneration techniques like micro propagation and somatic embryo genesis were successful mostly from seedling and embryos (Zamora, 1994; Banik,1995; Shirgurkar, 1996). However, there is no information available on the seed production of 0. stocks. Almost all the reports available on the micro propagation of mature tissues of bamboos discussed various problems such as establishment of contamination free cultures, poor rooting, difficulty in multiplication of established cultures, and nitrification of multiple shoots (Catered ec al, 1993; Serena and Nahant, 1994; Kumar, 1994; Ramayana and Yakandawala, 1997). Plantlet production in bamboo through micro propagation mainly depends on the establishment of contamination free cultures and in getting high percentage of rooting (Ramayana and Yakandawala, 1997). When the explants are collected from field grown clumps and nodal segments carry a wide range of microbes (Chang, 1991), To date there have been no published reports available on micro propagation and plantlet production of the species Oxytenanthera stocks. OBJECT OF THE INVENTION An object of this invention is to propose a process for in vitro probation of 0.stocksii plants. An object of this invention is to propose a process for which are true to type. Still another object of this invention is to propose a process for which require less space for probation. Yet another object of this invention is to propose a process for and where large scale propagation can be practiced easily. DETAILED DESCRIPTIOH OF THE INVENTION According to this invention there is provided a process for regenerating plants from mature expands of O.stockii, which comprises the steps of placing explants obtained from nature 0. stocks plant on a first step medium capable of inducing the explants to produce multiple axillary buds, said medium containing a cytokinln in an amount in the range of from 0.05-2.0 mg/1 and culturing the said explants until a bunch of multiple axillary buds are produced and obtaining continuous outshoot cultures in the second step medium transferring the outshoot cultures from step (b) to a third step medium containing an audio in an amount in the range from 2.0 - 12.5 mg/l, said medium being capable of inducing roots from the outshoot clusters. Elongation of root initials formed at step (c). The present method utilizes a multi-step culturing process for producing Oxytenenthera stockier plantlets from mature plants. In this process, at the first step axillary buds are induced to elongate in a solid medium, which helps to screen contaminants also, kit the second step, the contamination free cultures are transferred to liquid medium for shoot proliferation. The well-developed shoots are transferred to root induction medium for a short period followed by root elongation medium as a third step. Finally, the rooted plantlets were potted out. The explants are obtained from non-prismatic structures of the mature plants. The branch complements of current year culms are the preferred sources of explants. Three days prior to the collection of explants the mother plants are sprayed with the 1.0 percent solution of fungicide, Banister. In the first step axillary bud induction medium is normally a solid nutrient medium containing a balanced concentration of inorganic salts and organic nutrients, vitamins and supplemented with plant growth hormone (preferably cytokinlns). Many such nutrient media are known and commercially available. For example, the step one medium can contain Marshier and Slog (MS) salts, mesoinositol (100 mg/1) Thiamine HCl (O.lmg/1) Nicotinic acid (0.5 mg/1) Pyridoxine Hal (0.5 mg/1) Lysine (2.0 mg/1) Sucrose (20.0 g/1) agar (0.7%) and 6-benzylaminopurine (BA) (0.05-2.0 mg/1). The pH of the medium is adjusted to about Soot 6.0. Preferably, the pH of the solid medium is 5.7. Single node segments from the branch complement of current year culms are used as explants. The diameter of the explants is preferably 2.5-3.0 mm. The outer branch sheaths covering the axillary buds are removed and the nodes are washed under running tap water for 30 minutes. The explants are surface sterilized by shaking in 1.0% antiseptic for 5 minutes and rinsed thrice in sterile dematerialized water. To disinfect the fungus and bacteria the explants are immersed in the solution containing 0.1% Baiting and 0.1% Kanamycin for 30 minutes in shaker (50 rpm) followed by 0.1% Sodium hypo chlorite for 5 minutes and 0.1% Mercuric chloride for 5 minutes, fitter each sterility wash the explants are rinsed three times with dematerialized sterile water All the cultures are maintained at about 25 - 27>C with a 16h photoperiod provided by cool white fluorescent tubes. In the second step medium continuous shoot multiplication is obtained. MS liquid medium is used with BA (0.1-1.0 mg/1). The pH of the medium is adjusted to 5.0-5.5. The cultures are placed in the liquid medium without any support. The elongated shoots are transferred to fresh medium with the interval of 15-18 days. When the cluster of tissues is developed after 2 to 3 passages the mother tissue is removed. Large clusters of shoots developed in a period of 15-18 days are separated at their base into small clusters having 3-5 shoots and sub-cultured. After about 15-25 passages, the small cluster with 3-5 shoots are separated and transferred to root induction medium Rooting involved two step procedure in which the shoots treated with axing for root induction are transferred to axing free medium for root elongation. For root induction, MS basal liquid medium supplemented with IBA (2.0-12.5 mg/l) is used. After a short period. of treatment, the cultures are transferred to half-strength MS liquid medium without any growth regulators Seventy percent of the axing treated cultures produce roots. After 15-20 days in the root elongation medium, the rooted plantlets arc planted in the root train s containing vermiculite and placed under a tightly closed politest for acclimatization. The pollens maintains 90-95 percent humidity. After 15 days the politeness’ are loosened gradually and placed in the shade house. The survival rate of the plantlets is 85 percent. After 15-20 days of hardening in the shade house, plants are transplanted in playas containing soil: sand: compost (1:1:3) After 60-75 days in playas all the plants show 3-5 rhizome formation followed by new clump development from the rhizome, which ensures the successftil establishment of the plantlets Following are the definitions for the terms used in the present description. "Axillary bud proliferation' is a process of inducing multiple (axillary) shoe( by incorporation growth regulators (usually cytokines) into the culture medium^ The result is tote product ere miniature hay branched shook system. "Branch complement" refers to the array of branches that develop at a single calm node, including the primordial one and any that arise by proliferation from buds at its proximal nodes. 'Branch sheath' refers to the sheathing organ borne singly at each node of an aerial elative branch of any order exciting the neck sheath and leaf sheath A "calm" refers to the segmented aerial axis that emerges from a rhizome and forms a part of a bamboo plant. An "explants" is a piece of tissue taken from a donor plant for culturing. "Non-rhizomatised structures" refers to those parts of bamboo, which are not arising directly from the rhizomes or from the basal nodes of the calm, egg, Branch. A "plantlet" is a plant asexually reproduced by tissue culture in the present case. A "propagate" is a plant asexually produced in the present case. "Passage" refers to the process by which the multiple shoots produced are first subdivided and then transferred to fresh culture medium. The term is synonymous with the term "subculture". WE CLAIM 1. Aprocess for regenerating plants from mature explants of 0.stocksii, which comprises the steps of (a) Placing explants obtained from mature 0.stocksii plant on a first step medium capable of inducing the explants to produce multiple maxillary buds, said medium containing a cytokine in an amount in the range of from 0,05-2.0 mg/l and culturing the said explants until a bunch of multiple auxiliary buds are produced and b) Obtaining continuous multicolor cultures in the second step medium c) Transferring the outshoot cultures from step (b) to a third step medium containing an audio in an amount in the range from 2.0-12.5 mg/l, said medium being capable of inducing roots from the outshoot clusters d) Elongation of root initials formed at step (c). 2. A process for regenerating plants from mature explants of 0.stocksii, as claimed in claim 1, wherein said excised maxillary buds are obtained from a mature bamboo plant over 100 years old. 3. The process for regenerating plants from mature explants of 0.stocksii, as claimed in claim 1, wherein said first step medium further comprises mineral salts, vitamins, amino acids, a gelling agent, 6-benzylaminopurine in an amount in a range from 0.05-"2mg/l and sucrose each in an amount sufficient to induce auxiliary bud proliferation. 4. The process for regenerating plants from mature explants of O.stocksii, as claimed in claim 1, wherein in the second step medium further comprises mineral salts, vitamins and sucrose each in an amount sufficient to ensure multiple shoot production• 5. The process for regenerating plants from mature explants of O sticks, as claimed in claim 1, wherein in the third step medium for induction of roots from in vitro formed multiple shoots transferred to a medium containing indole-3-butyric acid 2.0-12.5 mg/1. 6. The process for regenerating plants from mature explants of O.stocksii, as claimed in claim 1, wherein the elongation of root initials takes place in a medium devoid of growth regulators. 7. The process for regenerating plants from mature explants of O.stocksii, substantially as herein described.

Documents:

307-mas-2000-abstract.pdf

307-mas-2000-claims filed.pdf

307-mas-2000-claims grand.pdf

307-mas-2000-correspondnece-others.pdf

307-mas-2000-correspondnece-po.pdf

307-mas-2000-description(complete) filed.pdf

307-mas-2000-description(complete) grand.pdf

307-mas-2000-form 1.pdf

307-mas-2000-form 19.pdf

307-mas-2000-form 26.pdf

307-mas-2000-form 3.pdf

307-mas-2000-other documents.pdf