Title of Invention

A PROCESS FOR THE PRODUCTION OF SOMATIC SEEDLINGS OF PLANTS.

Abstract

A process for the production of somatic seedlings of plants comprising inoculating a plant pro-embryogenic cell/callus mass In a nutrient medium containing H1 hormones such as herein described in a bioreactor, draining out the spent medium, adding fresh nutrient medium into the bioreactor followed by cultivation of the embryos therein, adding H2 hormones such as herein described into the medium and allowing the somatic embryos to germinate to somatic seedlings, draining out 1-12 hormone containing medium and adding fresh nutrient medium containing an inhibitor such as herein described into the bioreactor, allowing the seedlings to develop in the medium to obtain the somatic seedlings.

Full Text

FIELD OF THE INVENTION This invention relates to a process for the production of somatic seedlings. This invention further relates to a process for the production of somatic seedlings of sandalwood plants by using plant cell for tissue culture technology. BACKGROUND OF THE INVENTION It is known in the art to produce somatic seedlings in culture using mainly three different processes. In one process, pro- embryogenic callus mass is placed on solid medium containing hormones (H1) for somatic embryo induction. After about 4-6 weeks somatic embryos are produced asynchronously with embryos at different stages of development/differentiation. The mature embryos are then transferred to a different medium having other hormones (H2) in different culture vessel for germination, thereby raising somatic seedlings. The drawbacks of this process are that a majority of somatic embryos (>90%) are asynchronous and abnormal; often joined with one another and very low number of normal somatic embryos are obtained per gram of pro-embryogenic callus. A second method known in the art for somatic seedling production is direct somatic embryo production from any plant part, on solid medium in presence of one hormone (H1). These embryos are then germinated in a different medium in presence of another hormone (H2) to produce somatic seedlings. This method suffers from the drawbacks of poor response of plants to embryogenesis, ie. very few plants respond to direct embryogenesis, and that a number of embryos produced is very low whereas time taken is very long. Yet another process known in the art is that pro-embryogenic callus mass is cultivated in liquid medium in bioreactor in presence of hormone (H1) to produce somatic embryos. The produced embryos" are then germinated in solid medium in presence of another hormone (H2). Even through the total number of embryos produced by this process are higher than those produced by the first two processes greater than 30% are abnormal. In all of the above three processes, the total time taken from embryogenic callus mass to somatic seedling production is greater than six weeks. The residual effect of hormones H1 and H2 on the subsequent stages are the main reasons for major developmental abnormalities. The somatic seedlings which are apparently normal do not survive in the field due to above mentioned abnormalities. No process has so far been described to take care of the precise exposure time of H1 and H2 and to prevent carry forward effect of H1 and H2 to the subsequent stages. OBJECTS OF THE INVENTION It is therefore an object of the present invention to propose a novel process for the production of somatic seedlings rapidly by proper programming of incubation time of the embryos. It is a further object of this invention to propose a novel process for the production of somatic seedlings which gives greater yield of the seedings from pro-embryogenic cell and is therefore cost-effective. Another object of this invention is to propose a novel process for the production of somatic seedlings which is completed aseptically in one bioreactor without any sub-culture. DESCRIPTION OF THE INVENTION Thus according to this invention is provided a process for the production of somatic seedlings of plants comprising innoculating a plant pro-embryogenic cell/callus mass in a nutrient medium containing H1 hormones in a bioreactor, draining out the spent medium, adding fresh nutrient medium into the bioreactor followed by cultivation of the embryos therein, adding H2 hormones into the medium and allowing the somatic embryos to germinate to somatic seedlings, draining out the H2 hormone containing medium and adding fresh nutrient medium containing an inhibitor into the bioreactor, allowing the seedlings to develop in the medium to obtain the somatic seedlings. In accordance with this invention, an agitated bioreactor is innoculated using plant pro-embryogenic cell/callus mass in a first liquid medium containing H1 hormone(s) favouring somatic embryo induction and maturation upto cotyledonary stage. The agitation in bioreactor is provided in such a manner so that the developing embryos move characteristically with their shoot and root apices oriented acropetally and basipetally respectively, for most of the time and thereby favour normalcy in development. The nutrient medium is charged by aseptically pumping out the spent medium and pouring in fresh nutrient medium without any hormone. The somatic embryos are cultivated in this hormone free (H zero) condition for 2-5 days depending on the plant species. This step prevent residual effect of H1, which interfere with next step hormone for germination. The yield of normal embryos increase significantly because of this step. The hormone(s) required for germination (H2) of somatic embryos are added to the bioreactor, after filter sterilization. The somatic embryos germinate to somatic seedlings. Thereafter, the H2 containing medium is pumped out from bioreactor and fresh medium with an inhibitor (I) for H2 is added to the bioreactor. The germinated somatic seedlings are kept in this inhibitor containing medium which eliminate the residual effect of H2 and favour proper development of somatic seedlings. Such seedlings exhibit much higher survival during green house hardening. The nutrient medium is a liquid medium used for embryogenesis such as the MS plant tissue culture medium proposed by Murashige and Skoog (1962), according to Gamborg et al (1968), McCown plant tissue culture medium proposed by Lloyd & McCown (1981) or CCC, ie. chlorocholine chloride. The hormones used for the culture are H1 and H2 where H1 is used for somatic embryo induction and maturation and is selected from auxius cytokenins etc.. The hormone H1 is added to the medium in a proportion of (3.2-2.5 ppm. The hormone H2 induces germination of the somatic embryos and is selected from gibberellins or gibberellin - cytokinin mixture. H2 is used in a proportion of 0.2 to 1.5 ppm. The The inhibitor is used to minimise the residual effect of H2 and is selected from abscisic acid, ancymidol or chlorocholine chloride singly or in combination. The inhibitor is used in a proportion of 0.2 to 1.0 ppm or 1.0 mg per litre of medium. DESCRIPTION OF THE ACCOMPANYING DRAWIN6S The invention will be apparent from the description with read in conjunction with the accompanying drawing where Fig.l depicts a schematic flow diagram of the process for somatic seedling production according to the invention. Step 1 shows innoculation of the bioreactor with the pro-embryogenic friable cell/callus mass as innoculum. In step 2, the first medium is replaced with a second medium without any hormone, followed by cultivation in the hormone free nation is added to the nutrient medium and in step 4, the hormone containing nutrient medium is replaced with fresh medium containing an inhibitor. The seedlings are kept in this condition to obtain the somatic seedlings. The invention will now be explained in greater detail with the help of the following non-limiting example. EXAMPLE Nutrient Medium : MS (according to Murashige & Skoog, 1962) Hormones : H1 - indole acetic acid 2.5 mg/litre H2 - gibberellic acid 1.5 mg/litre Innoculum biomass : 100 gms fresh weight per litre medium Bioreactor capacity : 2 litres, with various probes, pneuma- tically agitated Incubation step : Step 1: 14 days; Step 2s 2 days; Step 3s 10 days; Step 4: 2 days Temperature : 25oC Light s 1000 lux The unique features of the foregoing process are that induction, maturation and germination of somatic embryos take place in liquid medium with programming of hormone exposure in the sequence H1-Hzero-H2-I . Agitation is provided in a specific manner facilitating acropetal and basipetal orientation of the somatic embryo shoot and root initial apices respectively, for most of the time. All the 4 steps (STEP1 to STEP 4) are completed aseptically in one bioreactor without any sub—culture and there is a significant reduction in incubation time from nearly eight weeks to about five weeks. WE CLAIM: 1. A process for the production of somatic seedlings of plant comprising innoculating aplaat pro-embryogenic cell/callus mass in a nutrient medium containing H1 hormones such as herein described in a bioreactor,draining out the spent medium, adding fresh nutrient medium into the bioreactor followed by cultivation of the embryos therein, adding H2 hormones such as herein described into the medium and allowing the somatic embryos to germinate to somatic seedlings, draining out H2 hormone containing medium and adding fresh nutrient medium containing an inhibitor such as herein described into the bioreactor, allowing the seedlings to develop in the medium to obtain the somatic seedlings. 2. The process as claimed in claim 1 wherein said bioreactor is an agitated bioreactor. 3. The process as claimed in claim 1 wherein the bioreactor is agitated in a manner which facilitates acropetal and basipetal orientation of the somatic embryo shoot and root initial apices for most of the time. 4. The process as claimed in claim 1 wherein said nutrient medium is a liquid nutrient medium such as MS, B5, McCown or CCC. 5. The process as claimed in claim 1 wherein said hormone H1 is a hormone for somatic embryo induction and maturation, such as auxins,cytokinins singly or in combination. 6. The process as claimed is claim 1 wherein said hormone H2 is a hormone for Germination of somatic embryos such as gibberellins, gibbereilin-cytokinin. 7. The process as claimed in claim 1 wherein raid inhibitor used is abscicic acid, ancymidol, CCC. 8. The process as claimed in claim 1 wherein said hormone H1 is added in a proportion of 0.2 to 2,5 ppm. 9. The process as claimed in claim 1 wherein said hormone H2 is added in a proportion of 0.2 to 1.5 ppm. 10. The process as claimed in claim 1 wherein the inhibitor is used in the range of 0.2 to 1.0 ppm 11. Tne process as claimed in claim 1 wherein the cell/callus mass is cultured in the medium containing H1 hormone for a period in the range of 10 to 14 days. A process for the production of somatic seedlings of plants comprising Innoculating a plant pro-embryogenlc cell/callus mass in a nutrient medium containing H1 hormones such as herein described in a bioreactor, draining out the spent medium, adding fresh nutrient medium into the bioreactor followed by cultivation of the embryos therein, adding H2 hormones such as herein described into the medium and allowing the somatic embryos to germinate to somatic seedlings, draining out H2 hormone containing medium and adding fresh nutrient medium containing an inhibitor such as herein described into the bioreactor, allowing the seedlings to develop in the medium to obtain the somatic seedlings.

Documents:

00177-cal-2001-abstract.pdf

00177-cal-2001-claims.pdf

00177-cal-2001-correspondence.pdf

00177-cal-2001-description (complete).pdf

00177-cal-2001-drawings.pdf

00177-cal-2001-form 1.pdf

00177-cal-2001-form 18.pdf

00177-cal-2001-form 2.pdf

00177-cal-2001-form 3.pdf

00177-cal-2001-gpa.pdf

00177-cal-2001-letter patent.pdf

00177-cal-2001-pa.pdf

00177-cal-2001-reply f.e.r.pdf