Title of Invention	PROCESS FOR DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE INFLORESCENCE SEGMENTS OF SUGARCANE
Abstract	A process of inducing direct somatic embryogenesis, without callus formation, for plant generation in sugarcane using a specifically formulated nutrient media, said process comprising steps of, providing nutrient medium comprising standard nutrients for tissue culture, gelling agent and plant growth regulators; and transferring cut segments of cleaned and sterilized immature inflorescences of sugarcane plant to said nutrient medium and incubating the obtained culture at temperature between 22-28˚ C, exposed to white fluorescent light of Lux between 750-1500 and relative humidity of 60-80% for 3-6 weeks.

Title of Invention

PROCESS FOR DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE INFLORESCENCE SEGMENTS OF SUGARCANE

Abstract

A process of inducing direct somatic embryogenesis, without callus formation, for plant generation in sugarcane using a specifically formulated nutrient media, said process comprising steps of, providing nutrient medium comprising standard nutrients for tissue culture, gelling agent and plant growth regulators; and transferring cut segments of cleaned and sterilized immature inflorescences of sugarcane plant to said nutrient medium and incubating the obtained culture at temperature between 22-28˚ C, exposed to white fluorescent light of Lux between 750-1500 and relative humidity of 60-80% for 3-6 weeks.

Full Text	FORM2 THE PATENTS ACT, 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10; rule 13) 1. Title of the invention. - PROCESS FOR DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE INFLORESCENCE SEGMENTS OF SUGARCANE 2. Applicants) (a) NAME : SECRETARY, DEPARTMENT OF ATOMIC ENERGY (b) NATIONALITY : A department of the Govt. of India (c) ADDRESS : Anushakthi Bhavan, Chatrapathi Shivaji Maharaj Marg, Mumbai 400 001, State of Maharashtra, India 3. PREAMBLE TO THE DESCRIPTION The following specification particularly describes the invention and the manner in which it is to be performed: 11 SEP 2006 FIELD OF INVENTION The present invention relates to a process of inducing plant generation of sugarcane by direct somatic embryogenesis, without an intervening callus phase, in defined culture media. The present invention particularly relates to use of defined nutrient media in process of inducing direct somatic embryogenesis of immature inflorescence segments of sugarcane without callus formation. . BACKGROUND OF THE INVENTION Sugarcane (Saccharum spp. hybrids) is a highly polyploid plant grown in different parts of the world from the tropics to subtropics, and accounts for around 60% of the world's sugar. It is also one of the important cash crops in many developing/developed countries, with a high trade value. The importance of sugarcane has increased in recent years because cane is an important industrial raw material for sugar industries and allied industries producing alcohol, acetic acid, butanol, paper, plywood, industrial enzymes and animal feed. Considering its importance in the agricultural industry, concerted efforts are being made for its improvement using bio-technological approaches. A plant reproduces naturally through the development of zygotic embryos. Formation of the embryo begins with the division of the fertilized eggs or zygote within the embryo sac of the ovule. Through an orderly progression of divisions, the embryo eventually differentiates, matures, and develops* into the new plantlet. Alternatively, the plant can be derived from a single somatic cell or a group of somatic cells. This 2 regeneration process, which differs from the natural pathway, is called somatic embryogenesis. Somatic embryogenesis is an efficient and high volume propagation system for the production of large number of plants within a short period. Plant regeneration through somatic embryogenesis has been reported in sugarcane using young leaf rolls and immature inflorescences. In these and other studies, embryogenic callus was induced in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram and regeneration was obtained by reducing the concentration of the auxin or deleting it from the medium, or by media supplementation with thidiazuron. Successful genetic transformation attempts have mostly employed embryogenic callus/cell cultures as the target tissue in several crop plants, including sugarcane. However, a major limitation of this callus system is the repeated subculture to select embryogenic callus portions among highly proliferating non-embryogenic tissue. This process is not highly reproducible and furthermore increases the chances of somaclonal variation. As these limitations have become unavoidable, strategies to improve plant regeneration must necessarily include manipulation of the explant material to embark upon new morphogenetic pathways. Direct somatic embryogenesis offers several advantages in crop improvement, as cost-effective and large-scale clonal propagation is possible using bioreactors, ultimately leading to automation of somatic seed production and development of artificial seeds. Besides, such a system could also provide a new source for use in genetic transformation. Somatic embryos are referred to as somatic because they are derived from somatic (vegetative) tissue, rather than through 3 sexual methods. Vegetative propagation via somatic embryogenesis has the potential to retain genetic gain of elite clones. The plants derived from direct somatic embryogenesis usually are unicellular in origin and hence genetically uniform. Thus plant generation through somatic embryogenesis provides an efficient means of producing a large number of elite or transgenic plants. In sugarcane, direct somatic embryogenesis has been reported using young leaf rolls and immature inflorescences as the culture system. For this, growing healthy plants are cut down to isolate required explant material that secrets phenolic compounds in culture and therefore inhibits the tissue growth. Whereas the inventors of the present invention have used specifically formulated nutrient media for the immature inflorescences (hereafter called, `explants`) of graminaceous plants, which are an excellent source of young meristematic tissue for the induction of direct somatic embryogenesis in which callus is not formed. Here fresh immature inflorescence material is collected from 9-10 month-old field grown plants. At this stage i.e., when the flag leaf initiation is noticed, the inflorescence material is still enclosed within outer leaf base coverings. Thus the process uses specifically formulated media to induce embryogenesis utilizing immature inflorescence material of sugarcane, which is often discarded at the time of cane harvesting which saves the loss of growing plants. Vikrant A, Rashid A (2001) discloses a direct as well as indirect somatic embryogenesis from immature (unemerged) inflorescence of a minor millet, Paspalum scrobiculatum L., (Euphytica 120: 167-173). Sahasrabudhe NA, Nandi M, Bahulikar RA, Rao PS, Suprasanna P (2000) describe a two step approach to scale up green plant regeneration through somatic embryogenesis from in vitro cultured immature inflorescence of 4 a male sterile line and a maintainer line of rice. (J. New Seed. 2(4) : 1-10) . Eudes F, Acharya S, Selinger LB, Cheng KJ (2003) discloses a novel method to induce direct somatic embryogenesis, secondary embryogenesis and regeneration of fertile green cereal plants (Plant. Cell Tiss. Org. Cult. 73: 147-157). This report deals with rapid induction of direct somatic embryogenesis in monocot plants like barley (Hordeum vulgare) , common wheat (Triticum aestivum), durum wheat (T. durum) and derived amphiploids, wild wheat (T. monococcum and T. urartu) , rye (Secale cereale) and oats (Avena sativa) from excised immature scutellum. Ho WJ, Vasil IK (1983) discloses Somatic embryogenesis in Sugarcane (Saccharum officinarum L.) embryogenic cell suspension cultures (Ann. Bot. 51: 719-726). Snyman SJ, Watt MP, Huckett FC. Botha BI (2000) describes direct somatic embryogenesis for rapid, cost effective production of transgenic sugarcane (Saccharum spp. hybrids). [Proceedings of the South African Sugar Technologists Association 74: 186-187 & Snyman SJ, Huckett BI, Watt MP, Botha FC (2001) : A comparison of direct and indirect somatic embryogenesis for the production of transgenic sugarcane, Saccharum spp. hybrids, Acta Hort. 560 105-108]. As outlined above, these have used young leaf rolls as the explant material. The above reports dealt with sugarcane: embryogenic cell suspension culture derived from leaf roll derived-callus None of the prior art teaches an efficient and effective single step process for inducing the rapid multiplication of sugarcane plants by a process without callus formation and the inventors have addressed such drawbacks of the prior art. 5 OBJECT OF INVENTION It is thus an object of the present invention to provide a process of inducing direct somatic embryogenesis in sugarcane -using immature inflorescence of sugarcane plant in specifically formulated nutrient media such that callus is not formed. It is further object of the present invention to provide a chemical process, which utilizes the immature inflorescence material of sugarcane, which is often discarded at the time of cane harvesting. It is further object of the present invention to provide a process, which induces somatic propagation for production of large number of elite sugarcane plants. Yet another object of the present invention is to provide a process, which induces somatic propagation for production of sugarcane plants which are genetically uniform. Yet another further object of the present invention to provide a process, which induces somatic propagation for production of sugarcane plants which can be used for the production of artificial or synthetic seeds. SUMMARY OF INVENTION According to an aspect of the present invention there is provided a process of inducing direct somatic embyogenesis, without callus formation for plant generation in sugarcane, using a specifically formulated nutrient media, said process comprising steps of, 6 (i) providing specifically formulated nutrient medium containing standard nutrients for tissue culture, gelling agent and plant growth regulators; and (ii) transferring cut segments of cleaned and sterilized segments of immature inflorescence of sugarcane plant to said nutrient medium and incubating the obtained culture at temperature between 22-28°C exposing to white fluorescent light of Lux between 750-1500 and relative humidity of 60-80% for 3-6 weeks; DETAILED DESCRIPTION The inventors of the present invention have addressed the need of a process for the induction of propagation of sugarcane plants in nutrient medium by direct somatic embryogenesis that overcomes the disadvantages of the prior art. The present invention is advantageous as it provides a method of inducing one step somatic propagation for rapid process of multiplication of sugarcane plants. The culture medium used does not contain plant growth regulators for root generation unlike other nutrient medium used for somatic propagation of sugarcane plants and achieves induction of somatic propagation without callus formation. It uses explant material from discarded sugarcane inflorescences instead of leaf rolls or apical meristem that require sacrificing of the mother plant. Less genetic variation is observed among plants derived through direct somatic embryogenesis. It avoids use of strong surface sterilization methods (like, mercuric chloride which is toxic) for explant preparation. Also phenolic secretion into the medium is avoided which normally affects growth of explants during in vitro culture. 7 The process of the present invention employs defined nutrient media with use of immature inflorescences for inducing rapid multiplication of plants through direct somatic embryogenesis, without callus formation in culture media. The inflorescence are exposed to the light conditions as mentioned above for 10-12. hrs/day. The somatic embryos formed by the process of present invention are then transferred to separate culture medium for induction of roots and then grown to the rooted plants in green house. Thus large number of genetically uniform sugarcane plants may be generated from the somatic embryos induced by the present process. For the process of the present invention fresh immature inflorescence material collected from 9-10 month-old field grown plants are used. At this stage i.e., when the flag leaf initiation is noticed, the inflorescence material is still enclosed within outer leaf base coverings. The material is washed few times with tap water with few drops of liquid soap and the outer leaf base coverings are removed carefully. The inflorescence explants (hereafter called, 'explants') are surface cleaned with absolute alcohol for 5 min. After removal of outer sheaths, the innermost pink-red, colored inflorescence segments are cut in to 3-6mm long pieces. The process involves providing a medium containing sufficient quantity of nutrients, a level of gelling agent like agar-agar of 0.8-1.0 % and phytagel of 0.15-0.3%, plant growth regulators (either singly or in combinations): Kinetin (0.5-2.5mg/l), Naphthalene acetic acid (0.l-0.5mg/l) , Benzylaminopurine (l-2mg/l), Thiadiazuron (0.5-lmg/l) and 2,4-dichlorophenoxyacetic acid (l-3mg/l), L- glutamine (50-100mg/l), Malt extract (50-100mg/l), Casein hydrolysate (0.5- 8 lg/1), Coconut water (5-15%) and sucrose (2-4%). To such culture medium contained in sterile Petri plates cut segments, 18 per each 9.5 cm diameter are transferred and the cultures are maintained for sufficient period of time (for 3-6 weeks) under suitable environmental conditions (in the culture room at 26±1°C under cool, white fluorescent light (750-1500 lux) for 10-12 hrs/day, with relative humidity of 60-80%) to develop somatic embryos. . Such maintenance in the defined culture medium as above induces somatic embryogenesis. The healthy growing somatic embryo derived plants (emblings) are then cultured on nutrient medium with plant growth regulators for root induction. Once sufficient roots are formed, the adhering gelling agent (agar or phytagel) is washed off under running tap water and rooted plants are grown in small plastic cups with soil in the green house. In the process of present invention the immature inflorescence segments swell in the culture medium and initiation of small embryo-like structures is apparent in the first week of culture. In the next two weeks, the proliferation of embryogenic clumps is seen at the cut end of the explants. Well-developed embryos all over the cultured explants appear within four weeks of culture without any callus formation. The globular embryos develop into club shaped embryos, characteristic of a monocot embryo, with development of scutellum and followed by well-differentiated shoot and root primordia. It has been seen that medium supplemented with kinetin (0.5 -2.5mg/l), naphthalene acetic acid (0.1- 0.5mg/l) and Glutamine (50-100mg/l) with 2-4% sucrose leads to better development of somatic embryo. The well-grown and healthy embryos generally develop into plantlets with good rooting and vigorous growth 9 in medium without plant growth regulators for root induction unlike the prior art methods. The details of the invention, its objects and advantages are explained hereunder in greater detail in relation to non-limiting exemplary illustrations. The examples are merely illustrative and do not limit the teaching of this invention and it would be obvious that various modifications or changes in the procedural steps as well as compositions by those skilled in the art without departing from the scope of the invention and shall be consequently encompassed within the ambit and spirit of this approach and scope thereof. EXAMPLES Example 1 The medium was prepared using stock solutions supplemented with major, minor elements, different plant growth regulators, vitamins and additives either singly or in combinations: kinetin (2.5mg/l), naphthalene aceticacid (0.5mg/l), bezylaminopurine (2mg/l), thidiazuron (lmg/1) and 2,4-Dichlorophenoxyaceticacid (l-3mg/l), L- glutamine (100mg/l), ME (100mg/l), CH (lg/1), CW (5%) and sugars like sucrose (3-4%). The medium was made up with double distilled water. While heat stable compounds were added into the medium before autoclaving, heat labile compounds were filter sterilized and added into medium after autoclaving before pouring into test tubes or 9 cm diameter culture petriplates. The pH of the medium was adjusted to 5.8 using IM NaOH or 1 N HCl prior to autoclaving. The gelling agent (0.8% agar-agar OR 0.2% phytagel) was used for solidification. Sugarcane variety of Saccharum officinarum, Co-671 was used. Fresh plant material of immature inflorescence segments was collected from 10-month 10 old field grown plants. The field-collected material was washed several times with tap water with few drops of liquid soap. The outer old leaf base coverings were removed carefully without damaging the internal young and delicate tissue, followed by the immersion of portion of inflorescence in absolute alcohol for 5 min for surface cleaning. After removing the outer sheaths, innermost inflorescence segments were cut in to 3-6mm long pieces and inoculated on said media. The cultures were maintained in dark initially for 3-6 weeks and thereafter all the cultures were incubated in the culture room at 26±1°C under cool, white fluorescent light (750-1500 lux) for 10-12 hrs/day, with relative humidity of 60-80%. The embryogenic response was observed by the presence of somatic embryos on the cultured explant after four-week culture period and the number of regenerated plants was recorded per explant after 8 week culture period. The immature inflorescence segments of 3-6 mm size showed swelling of the explant and initiation of small embryo-like structures in the first week of culture. This was followed in the next two weeks of culture by the proliferation of embryogenic clumps at the cut end of the explants. Well-developed embryos were observed all over the cultured explants within four weeks of culture. Absence of any callus formation indicated that the process of embryo development was direct with the appearance of globular stage embryos. These embryos exhibited compactly arranged cells with thick cytoplasm. Further the initiation of direct somatic embryos was observed from the lower layer of epidermis with initiation of procambium development. The globular embryos developed into club shaped embryos, characteristic of a monocot embryo, with development of scutellum and followed by well-differentiated shoot and root primordia. 11 The healthy growing somatic embryo derived plants (emblings) were cultured on MS basal media for rooting. Agar was washed off carefully under running tap water and rooted plants were allowed to grow in small cups containing autoclaved soil, covered with polythene sheets and maintained under 25-27°C and 70% relative humidity in the green house. Maximum embryogenic response (54.09 + 2.7 %) with well-developed plants (7.72 ± 0.89) per explant was observed on medium with napthaleneacetic acid, kinetin and glutamine. Somatic embryos that formed on media supplemented with kinetin (2.5mg/l), naphthalene aceticacid (0.5mg/l), Glutamine (100mg/l) with 4% sucrose showed vigorous growth, while those on media with 1 mg/1 thidiazuron and 3% sucrose were stunted in growth. . This thus demonstrates that the process of present invention is capable of inducing somatic growth even without growth regulators for root induction The processes of direct somatic embryo induction to the well-rooted plants were obtained within 6-8 weeks. All the regenerants grew normally under the green house conditions with 95% survival. Example 2 The medium was prepared using stock solutions supplemented with different plant growth regulators, vitamins and additives either singly or in combinations: Kn (11.625mM), NAA (2.685mM), BA (8.88mM), TDZ (5mM) and 2,4-D (4.5-13.5mM), L-glutamine (100g/l), ME (100mg/l), CH (lg/1), CW (5%) and sugars like sucrose (3-4%) . The final volume as made up with double distilled water, while heat stable compounds were added into the medium before autoclaving and heat labile compounds were filter sterilized and added into medium after autoclaving before pouring into test tubes or petriplates. The pH of the medium was adjusted to 5.8 using IN NaOH or 1 N HCl prior to 12 autoclaving and gelled with 0.2% phytagel for solidification. Sugarcane varieties, viz. Coc-86032, Co-8014, Co 7524 (Saccharum officinarum) collected from different part of Maharashtra (Kolhapur, Pune and Parbhani) were used. Fresh immature inflorescences were collected from 10-month old-field grown plants. The inflorescences were cold stored at 8°C for a period of 24 hrs. The field-collected material was washed several times with tap water with few drops of liquid soap. The outer old leaf base coverings were removed carefully without damaging the internal young and delicate tissue, followed by the immersion of portion of inflorescence in absolute alcohol for 5 min for surface sterilization. After removing the outer sheathing leaf bases, innermost inflorescence (rachis) segments were cut in to 3-6mm long pieces and inoculated on said media. All the cultures were maintained at 24 ° C, under light intensity of 750 Lux and relative humidity of 65%. The embryogenic response was seen by the presence of somatic embryos on the cultured explant after four-week culture period and the number of regenerated plants was recorded per explant after 2 months of culture period. The immature inflorescence (rachis) segments showed swelling of the explant and initiation of small embryo-like structures in the first week of culture, while few cultures treated with low temperature treatment for long time period turned brown. This was followed in the next two weeks of culture by the proliferation of embryogenic clumps at the cut end of the explants. Well-developed embryos were observed all over the cultured explants within four weeks of culture. Absence of callus formation indicated that the process of embryo development was direct with the appearance of globular stage embryos. These embryos exhibited compactly arranged cells 13 with thick cytoplasm. Further the initiation of direct somatic embryos was observed from the lower layer of epidermis with initiation of procambium development. The globular embryos developed into club shaped embryos, characteristic of a monocot embryo, with development of scutellum and followed by well-differentiated shoot and root primordia. Good embryogenic response (50%) with well-developed plants (4-5) per explant was observed on medium with NAA, KN and glutamine. Somatic embryos that formed on media supplemented with KN (10\|oM), NAA (0.5uM), L-glutamine (120mg/l) with 4-5% sucrose showed vigorous growth. This thus demonstrates that the process of present invention is capable of inducing somatic growth even without growth regulators for root induction. The processes of direct somatic embryo induction to the well-rooted plants were obtained within 6-8 weeks. All the regenerants grew normally under the green house conditions with 95% survival. 14 WE CLAIM: 1. A process of inducing direct somatic embryogenesis, without callus formation, for plant generation in sugarcane using a specifically formulated nutrient media, said process comprising steps of, (i) providing nutrient medium comprising standard nutrients for tissue culture, gelling agent and plant growth regulators; and (ii) transferring cut segments of cleaned and sterilized immature inflorescences of sugarcane plant to said nutrient medium and incubating the obtained culture at temperature between 22-28° C, exposed to white fluorescent light of Lux between 750-1500 and relative humidity of 60-80% for 3-6 weeks. 2. A process as claimed in claim 1, wherein the gelling agent is selected from agar-agar, phytagel and gelrite. 3. A process as claimed in claim 1 wherein the plant growth regulators are a mixture of an auxin, a cytokinin, an amino acid, organic additives and coconut water. 4. A process as claimed in claim 3, wherein the auxin is selected from naphthalene acetic acid, 2,4-dichlorophenoxyaceticacid, picloram, dicamba and cytokinin is selected from kinetin, benzylaminopurine, thidiazuron, zeatin and 2-imino purine, wherein the ratio of auxin to cytokinin is 1:5. 15 5. A process as claimed in claim 3, wherein the amino acid is selected from L-glutamine, L-arginine and L-tryptophan, in concentration range of 100-200 mg/1 of the nutrient medium. 6. A process as claimed in claim 3, wherein the organic additive is a mixture of malt extract and casein hydrolysate in the ratio of 1:5 to 10. 7. A process as claimed in any of the preceding claims, wherein the inflorescence used is the innermost inflorescence segment after removing the outer leaf coverings that are cleaned with water and sterilized with alcohol. Dated this 8th day of September 2006 Dr. Sanchita Ganguli Of S. Majumdar & Co. (Applicant's Agent) 16 ABSTRACT PROCESS FOR DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE INFLORESCENCE SEGMENTS OF SUGARCANE A process of inducing direct somatic embryogenesis, without callus formation, for plant generation in sugarcane using a specifically formulated nutrient media, said process comprising steps of, providing nutrient medium comprising standard nutrients for tissue culture, gelling agent and plant growth regulators; and transferring cut segments of cleaned and sterilized immature inflorescences of sugarcane plant to said nutrient medium and incubating the obtained culture at temperature between 22-28° C, exposed to white fluorescent light of Lux between 750-1500 and relative humidity of 60-80% for 3-6 weeks. 11 SEP 2006

Full Text

FORM2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention. - PROCESS FOR DIRECT SOMATIC EMBRYOGENESIS FROM
IMMATURE INFLORESCENCE SEGMENTS OF SUGARCANE
2. Applicants)
(a) NAME : SECRETARY, DEPARTMENT OF ATOMIC ENERGY
(b) NATIONALITY : A department of the Govt. of India
(c) ADDRESS : Anushakthi Bhavan, Chatrapathi Shivaji Maharaj Marg,
Mumbai 400 001, State of Maharashtra, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:
11 SEP 2006

FIELD OF INVENTION
The present invention relates to a process of inducing plant generation of sugarcane by direct somatic embryogenesis, without an intervening callus phase, in defined culture media.
The present invention particularly relates to use of defined nutrient media in process of inducing direct somatic embryogenesis of immature inflorescence segments of sugarcane without callus formation. .
BACKGROUND OF THE INVENTION
Sugarcane (Saccharum spp. hybrids) is a highly polyploid plant grown in different parts of the world from the tropics to subtropics, and accounts for around 60% of the world's sugar. It is also one of the important cash crops in many developing/developed countries, with a high trade value. The importance of sugarcane has increased in recent years because cane is an important industrial raw material for sugar industries and allied industries producing alcohol, acetic acid, butanol, paper, plywood, industrial enzymes and animal feed. Considering its importance in the agricultural industry, concerted efforts are being made for its improvement using bio-technological approaches.
A plant reproduces naturally through the development of zygotic embryos. Formation of the embryo begins with the division of the fertilized eggs or zygote within the embryo sac of the ovule. Through an orderly progression of divisions, the embryo eventually differentiates, matures, and develops* into the new plantlet. Alternatively, the plant can be derived from a single somatic cell or a group of somatic cells. This
2

regeneration process, which differs from the natural pathway, is called somatic embryogenesis.
Somatic embryogenesis is an efficient and high volume propagation system for the production of large number of plants within a short period. Plant regeneration through somatic embryogenesis has been reported in sugarcane using young leaf rolls and immature inflorescences. In these and other studies, embryogenic callus was induced in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram and regeneration was obtained by reducing the concentration of the auxin or deleting it from the medium, or by media supplementation with thidiazuron. Successful genetic transformation attempts have mostly employed embryogenic callus/cell cultures as the target tissue in several crop plants, including sugarcane. However, a major limitation of this callus system is the repeated subculture to select embryogenic callus portions among highly proliferating non-embryogenic tissue. This process is not highly reproducible and furthermore increases the chances of somaclonal variation. As these limitations have become unavoidable, strategies to improve plant regeneration must necessarily include manipulation of the explant material to embark upon new morphogenetic pathways.
Direct somatic embryogenesis offers several advantages in crop improvement, as cost-effective and large-scale clonal propagation is possible using bioreactors, ultimately leading to automation of somatic seed production and development of artificial seeds. Besides, such a system could also provide a new source for use in genetic transformation.
Somatic embryos are referred to as somatic because they are derived from somatic (vegetative) tissue, rather than through
3

sexual methods. Vegetative propagation via somatic embryogenesis has the potential to retain genetic gain of elite clones. The plants derived from direct somatic embryogenesis usually are unicellular in origin and hence genetically uniform. Thus plant generation through somatic embryogenesis provides an efficient means of producing a large number of elite or transgenic plants.
In sugarcane, direct somatic embryogenesis has been reported using young leaf rolls and immature inflorescences as the culture system. For this, growing healthy plants are cut down to isolate required explant material that secrets phenolic compounds in culture and therefore inhibits the tissue growth. Whereas the inventors of the present invention have used specifically formulated nutrient media for the immature inflorescences (hereafter called, `explants`) of graminaceous plants, which are an excellent source of young meristematic tissue for the induction of direct somatic embryogenesis in which callus is not formed. Here fresh immature inflorescence material is collected from 9-10 month-old field grown plants. At this stage i.e., when the flag leaf initiation is noticed, the inflorescence material is still enclosed within outer leaf base coverings. Thus the process uses specifically formulated media to induce embryogenesis utilizing immature inflorescence material of sugarcane, which is often discarded at the time of cane harvesting which saves the loss of growing plants.
Vikrant A, Rashid A (2001) discloses a direct as well as indirect somatic embryogenesis from immature (unemerged) inflorescence of a minor millet, Paspalum scrobiculatum L., (Euphytica 120: 167-173). Sahasrabudhe NA, Nandi M, Bahulikar RA, Rao PS, Suprasanna P (2000) describe a two step approach to scale up green plant regeneration through somatic embryogenesis from in vitro cultured immature inflorescence of
4

a male sterile line and a maintainer line of rice. (J. New Seed. 2(4) : 1-10) .
Eudes F, Acharya S, Selinger LB, Cheng KJ (2003) discloses a novel method to induce direct somatic embryogenesis, secondary embryogenesis and regeneration of fertile green cereal plants (Plant. Cell Tiss. Org. Cult. 73: 147-157). This report deals with rapid induction of direct somatic embryogenesis in monocot plants like barley (Hordeum vulgare) , common wheat (Triticum aestivum), durum wheat (T. durum) and derived amphiploids, wild wheat (T. monococcum and T. urartu) , rye (Secale cereale) and oats (Avena sativa) from excised immature scutellum.
Ho WJ, Vasil IK (1983) discloses Somatic embryogenesis in Sugarcane (Saccharum officinarum L.) embryogenic cell suspension cultures (Ann. Bot. 51: 719-726). Snyman SJ, Watt MP, Huckett FC. Botha BI (2000) describes direct somatic embryogenesis for rapid, cost effective production of transgenic sugarcane (Saccharum spp. hybrids). [Proceedings of the South African Sugar Technologists Association 74: 186-187 & Snyman SJ, Huckett BI, Watt MP, Botha FC (2001) : A comparison of direct and indirect somatic embryogenesis for the production of transgenic sugarcane, Saccharum spp. hybrids, Acta Hort. 560 105-108]. As outlined above, these have used young leaf rolls as the explant material. The above reports dealt with sugarcane: embryogenic cell suspension culture derived from leaf roll derived-callus
None of the prior art teaches an efficient and effective single step process for inducing the rapid multiplication of sugarcane plants by a process without callus formation and the inventors have addressed such drawbacks of the prior art.
5

OBJECT OF INVENTION
It is thus an object of the present invention to provide a process of inducing direct somatic embryogenesis in sugarcane -using immature inflorescence of sugarcane plant in specifically formulated nutrient media such that callus is not formed.
It is further object of the present invention to provide a chemical process, which utilizes the immature inflorescence material of sugarcane, which is often discarded at the time of cane harvesting.
It is further object of the present invention to provide a process, which induces somatic propagation for production of large number of elite sugarcane plants.
Yet another object of the present invention is to provide a process, which induces somatic propagation for production of sugarcane plants which are genetically uniform.
Yet another further object of the present invention to provide a process, which induces somatic propagation for production of sugarcane plants which can be used for the production of artificial or synthetic seeds.
SUMMARY OF INVENTION
According to an aspect of the present invention there is provided a process of inducing direct somatic embyogenesis, without callus formation for plant generation in sugarcane, using a specifically formulated nutrient media, said process comprising steps of,
6

(i) providing specifically formulated nutrient medium containing standard nutrients for tissue culture, gelling agent and plant growth regulators; and
(ii) transferring cut segments of cleaned and sterilized segments of immature inflorescence of sugarcane plant to said nutrient medium and incubating the obtained culture at temperature between 22-28°C exposing to white fluorescent light of Lux between 750-1500 and relative humidity of 60-80% for 3-6 weeks;
DETAILED DESCRIPTION
The inventors of the present invention have addressed the need of a process for the induction of propagation of sugarcane plants in nutrient medium by direct somatic embryogenesis that overcomes the disadvantages of the prior art.
The present invention is advantageous as it provides a method of inducing one step somatic propagation for rapid process of multiplication of sugarcane plants. The culture medium used does not contain plant growth regulators for root generation unlike other nutrient medium used for somatic propagation of sugarcane plants and achieves induction of somatic propagation without callus formation. It uses explant material from discarded sugarcane inflorescences instead of leaf rolls or apical meristem that require sacrificing of the mother plant. Less genetic variation is observed among plants derived through direct somatic embryogenesis. It avoids use of strong surface sterilization methods (like, mercuric chloride which is toxic) for explant preparation. Also phenolic secretion into the medium is avoided which normally affects growth of explants during in vitro culture.
7

The process of the present invention employs defined nutrient media with use of immature inflorescences for inducing rapid multiplication of plants through direct somatic embryogenesis, without callus formation in culture media. The inflorescence are exposed to the light conditions as mentioned above for 10-12. hrs/day.
The somatic embryos formed by the process of present invention are then transferred to separate culture medium for induction of roots and then grown to the rooted plants in green house. Thus large number of genetically uniform sugarcane plants may be generated from the somatic embryos induced by the present process.
For the process of the present invention fresh immature inflorescence material collected from 9-10 month-old field grown plants are used. At this stage i.e., when the flag leaf initiation is noticed, the inflorescence material is still enclosed within outer leaf base coverings. The material is washed few times with tap water with few drops of liquid soap and the outer leaf base coverings are removed carefully. The inflorescence explants (hereafter called, 'explants') are surface cleaned with absolute alcohol for 5 min. After removal of outer sheaths, the innermost pink-red, colored inflorescence segments are cut in to 3-6mm long pieces.
The process involves providing a medium containing sufficient quantity of nutrients, a level of gelling agent like agar-agar of 0.8-1.0 % and phytagel of 0.15-0.3%, plant growth regulators (either singly or in combinations): Kinetin (0.5-2.5mg/l), Naphthalene acetic acid (0.l-0.5mg/l) , Benzylaminopurine (l-2mg/l), Thiadiazuron (0.5-lmg/l) and 2,4-dichlorophenoxyacetic acid (l-3mg/l), L- glutamine (50-100mg/l), Malt extract (50-100mg/l), Casein hydrolysate (0.5-
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lg/1), Coconut water (5-15%) and sucrose (2-4%). To such culture medium contained in sterile Petri plates cut segments, 18 per each 9.5 cm diameter are transferred and the cultures are maintained for sufficient period of time (for 3-6 weeks) under suitable environmental conditions (in the culture room at 26±1°C under cool, white fluorescent light (750-1500 lux)
for 10-12 hrs/day, with relative humidity of 60-80%) to develop somatic embryos. . Such maintenance in the defined culture medium as above induces somatic embryogenesis.
The healthy growing somatic embryo derived plants (emblings) are then cultured on nutrient medium with plant growth regulators for root induction. Once sufficient roots are formed, the adhering gelling agent (agar or phytagel) is washed off under running tap water and rooted plants are grown in small plastic cups with soil in the green house.
In the process of present invention the immature inflorescence segments swell in the culture medium and initiation of small embryo-like structures is apparent in the first week of culture. In the next two weeks, the proliferation of embryogenic clumps is seen at the cut end of the explants. Well-developed embryos all over the cultured explants appear within four weeks of culture without any callus formation. The globular embryos develop into club shaped embryos, characteristic of a monocot embryo, with development of scutellum and followed by well-differentiated shoot and root primordia.
It has been seen that medium supplemented with kinetin (0.5 -2.5mg/l), naphthalene acetic acid (0.1- 0.5mg/l) and Glutamine (50-100mg/l) with 2-4% sucrose leads to better development of somatic embryo. The well-grown and healthy embryos generally develop into plantlets with good rooting and vigorous growth
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in medium without plant growth regulators for root induction unlike the prior art methods.
The details of the invention, its objects and advantages are explained hereunder in greater detail in relation to non-limiting exemplary illustrations. The examples are merely illustrative and do not limit the teaching of this invention and it would be obvious that various modifications or changes in the procedural steps as well as compositions by those skilled in the art without departing from the scope of the invention and shall be consequently encompassed within the ambit and spirit of this approach and scope thereof.
EXAMPLES
Example 1
The medium was prepared using stock solutions supplemented with major, minor elements, different plant growth regulators, vitamins and additives either singly or in combinations: kinetin (2.5mg/l), naphthalene aceticacid (0.5mg/l), bezylaminopurine (2mg/l), thidiazuron (lmg/1) and 2,4-Dichlorophenoxyaceticacid (l-3mg/l), L- glutamine (100mg/l), ME (100mg/l), CH (lg/1), CW (5%) and sugars like sucrose (3-4%). The medium was made up with double distilled water. While heat stable compounds were added into the medium before autoclaving, heat labile compounds were filter sterilized and added into medium after autoclaving before pouring into test tubes or 9 cm diameter culture petriplates. The pH of the medium was adjusted to 5.8 using IM NaOH or 1 N HCl prior to autoclaving. The gelling agent (0.8% agar-agar OR 0.2% phytagel) was used for solidification. Sugarcane variety of Saccharum officinarum, Co-671 was used. Fresh plant material of immature inflorescence segments was collected from 10-month
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old field grown plants. The field-collected material was washed several times with tap water with few drops of liquid soap. The outer old leaf base coverings were removed carefully without damaging the internal young and delicate tissue, followed by the immersion of portion of inflorescence in absolute alcohol for 5 min for surface cleaning. After removing the outer sheaths, innermost inflorescence segments were cut in to 3-6mm long pieces and inoculated on said media. The cultures were maintained in dark initially for 3-6 weeks and thereafter all the cultures were incubated in the culture
room at 26±1°C under cool, white fluorescent light (750-1500 lux) for 10-12 hrs/day, with relative humidity of 60-80%. The embryogenic response was observed by the presence of somatic embryos on the cultured explant after four-week culture period and the number of regenerated plants was recorded per explant after 8 week culture period.
The immature inflorescence segments of 3-6 mm size showed swelling of the explant and initiation of small embryo-like structures in the first week of culture. This was followed in the next two weeks of culture by the proliferation of embryogenic clumps at the cut end of the explants. Well-developed embryos were observed all over the cultured explants within four weeks of culture. Absence of any callus formation indicated that the process of embryo development was direct with the appearance of globular stage embryos. These embryos exhibited compactly arranged cells with thick cytoplasm. Further the initiation of direct somatic embryos was observed from the lower layer of epidermis with initiation of procambium development. The globular embryos developed into club shaped embryos, characteristic of a monocot embryo, with development of scutellum and followed by well-differentiated shoot and root primordia.
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The healthy growing somatic embryo derived plants (emblings) were cultured on MS basal media for rooting. Agar was washed off carefully under running tap water and rooted plants were allowed to grow in small cups containing autoclaved soil, covered with polythene sheets and maintained under 25-27°C and 70% relative humidity in the green house.
Maximum embryogenic response (54.09 + 2.7 %) with well-developed plants (7.72 ± 0.89) per explant was observed on medium with napthaleneacetic acid, kinetin and glutamine. Somatic embryos that formed on media supplemented with kinetin (2.5mg/l), naphthalene aceticacid (0.5mg/l), Glutamine (100mg/l) with 4% sucrose showed vigorous growth, while those on media with 1 mg/1 thidiazuron and 3% sucrose were stunted in growth. . This thus demonstrates that the process of present invention is capable of inducing somatic growth even without growth regulators for root induction
The processes of direct somatic embryo induction to the well-rooted plants were obtained within 6-8 weeks. All the regenerants grew normally under the green house conditions with 95% survival.
Example 2
The medium was prepared using stock solutions supplemented with different plant growth regulators, vitamins and additives either singly or in combinations: Kn (11.625mM), NAA
(2.685mM), BA (8.88mM), TDZ (5mM) and 2,4-D (4.5-13.5mM), L-glutamine (100g/l), ME (100mg/l), CH (lg/1), CW (5%) and sugars like sucrose (3-4%) . The final volume as made up with double distilled water, while heat stable compounds were added into the medium before autoclaving and heat labile compounds were filter sterilized and added into medium after autoclaving before pouring into test tubes or petriplates. The pH of the medium was adjusted to 5.8 using IN NaOH or 1 N HCl prior to
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autoclaving and gelled with 0.2% phytagel for solidification. Sugarcane varieties, viz. Coc-86032, Co-8014, Co 7524 (Saccharum officinarum) collected from different part of Maharashtra (Kolhapur, Pune and Parbhani) were used. Fresh immature inflorescences were collected from 10-month old-field grown plants. The inflorescences were cold stored at 8°C for a period of 24 hrs. The field-collected material was washed several times with tap water with few drops of liquid soap. The outer old leaf base coverings were removed carefully without damaging the internal young and delicate tissue, followed by the immersion of portion of inflorescence in absolute alcohol for 5 min for surface sterilization. After removing the outer sheathing leaf bases, innermost inflorescence (rachis) segments were cut in to 3-6mm long pieces and inoculated on said media.
All the cultures were maintained at 24 ° C, under light intensity of 750 Lux and relative humidity of 65%. The embryogenic response was seen by the presence of somatic embryos on the cultured explant after four-week culture period and the number of regenerated plants was recorded per explant after 2 months of culture period.
The immature inflorescence (rachis) segments showed swelling of the explant and initiation of small embryo-like structures in the first week of culture, while few cultures treated with low temperature treatment for long time period turned brown. This was followed in the next two weeks of culture by the proliferation of embryogenic clumps at the cut end of the explants. Well-developed embryos were observed all over the cultured explants within four weeks of culture. Absence of callus formation indicated that the process of embryo development was direct with the appearance of globular stage embryos. These embryos exhibited compactly arranged cells
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with thick cytoplasm. Further the initiation of direct somatic embryos was observed from the lower layer of epidermis with initiation of procambium development. The globular embryos developed into club shaped embryos, characteristic of a monocot embryo, with development of scutellum and followed by well-differentiated shoot and root primordia.
Good embryogenic response (50%) with well-developed plants (4-5) per explant was observed on medium with NAA, KN and glutamine. Somatic embryos that formed on media supplemented
with KN (10|oM), NAA (0.5uM), L-glutamine (120mg/l) with 4-5% sucrose showed vigorous growth. This thus demonstrates that the process of present invention is capable of inducing somatic growth even without growth regulators for root induction. The processes of direct somatic embryo induction to the well-rooted plants were obtained within 6-8 weeks. All the regenerants grew normally under the green house conditions with 95% survival.
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WE CLAIM:
1. A process of inducing direct somatic embryogenesis,
without callus formation, for plant generation in
sugarcane using a specifically formulated nutrient media,
said process comprising steps of,
(i) providing nutrient medium comprising standard nutrients for tissue culture, gelling agent and plant growth regulators; and
(ii) transferring cut segments of cleaned and sterilized immature inflorescences of sugarcane plant to said nutrient medium and incubating the obtained culture at temperature between 22-28° C, exposed to white fluorescent light of Lux between 750-1500 and relative humidity of 60-80% for 3-6 weeks.
2. A process as claimed in claim 1, wherein the gelling agent is selected from agar-agar, phytagel and gelrite.
3. A process as claimed in claim 1 wherein the plant growth regulators are a mixture of an auxin, a cytokinin, an amino acid, organic additives and coconut water.
4. A process as claimed in claim 3, wherein the auxin is selected from naphthalene acetic acid, 2,4-dichlorophenoxyaceticacid, picloram, dicamba and cytokinin is selected from kinetin, benzylaminopurine, thidiazuron, zeatin and 2-imino purine, wherein the ratio of auxin to cytokinin is 1:5.
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5. A process as claimed in claim 3, wherein the amino acid is selected from L-glutamine, L-arginine and L-tryptophan, in concentration range of 100-200 mg/1 of the nutrient medium.
6. A process as claimed in claim 3, wherein the organic additive is a mixture of malt extract and casein hydrolysate in the ratio of 1:5 to 10.
7. A process as claimed in any of the preceding claims, wherein the inflorescence used is the innermost inflorescence segment after removing the outer leaf coverings that are cleaned with water and sterilized with alcohol.
Dated this 8th day of September 2006

Dr. Sanchita Ganguli
Of S. Majumdar & Co.
(Applicant's Agent)

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ABSTRACT
PROCESS FOR DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE INFLORESCENCE SEGMENTS OF SUGARCANE
A process of inducing direct somatic embryogenesis, without callus formation, for plant generation in sugarcane using a specifically formulated nutrient media, said process comprising steps of, providing nutrient medium comprising standard nutrients for tissue culture, gelling agent and plant growth regulators; and transferring cut segments of cleaned and sterilized immature inflorescences of sugarcane plant to said nutrient medium and incubating the obtained culture at temperature between 22-28° C, exposed to white fluorescent light of Lux between 750-1500 and relative humidity of 60-80% for 3-6 weeks.
11 SEP 2006

PROCESS FOR DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE INFLORESCENCE SEGMENTS OF SUGARCANE

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