FIELD OF INVENTION

The present invention relates to the field of in-vitro regeneration and genetic engineering of plants, more particularly, to a process of regeneration and Agrobacterium-mediated transformation of the plant, Cassava (Manihot Esculenta Crantz).

BACKGROUND OF THE INVENTION

Cassava {Manihot esculenta Crantz) is a woody perennial crop with the ability to grow on land where droughts are frequent and soil is low in nutrients, where cereals and other crops do not grow well. It is cultivated mainly in the tropics for its starchy tuberous roots. Cassava roots are used as a rich source of starch for human nutrition, cattle feed and industrial purpose.

Cassava is vegetatively propagated from nodal cuttings (stakes) in field production systems, while the wild species are seed propagated in nature. Controlled pollination and seed production are mostly used for development of new varieties in breeding programmes. Seed population can also be collected from cassava field collections for storage and preservation as future sources of genetic information. Wild types are more difficult to regenerate in field than cultivated landraces (M esculenta); most of them are perennial and difficult to regenerate through cuttings. Cassava is mainly conserved as live plants in field, but it can also be maintained as seeds, in vitro and by cryopreservation.
Cassava is considered as a highly recalcitrant crop for tissue culture and transformation and is also genotype specific. Although a number of tissue culture and transformation systems have been developed for cassava (Nigel J. Taylor ct al., 2001: Production of embryogenic tissues and regeneration of transgenic plants in cassava: Euphytica 120: 25-34; M.M. Schreuder et al., 2001: Efficient production of transgenic plants by Agrobacterium-mediated transformation of cassava: Euphytica 120: 35-42; Peng zhang et al., 2004: Production of transgenic cassava: I.S. Curtis (ed.), Transgenic Crops of the World-Essential Protocols. 301-319, Kluwer Academic Publishers; B. B. Hankoua et al., 2006: Production of the first transgenic cassava in Africa via direct shoot organogenesis from friable embryogenic calli and germination of maturing somatic embryos: African Journal ot Biotechnology Vol. 5 (19), pp. 1700-1712). They have been applied on a limited number ol' genotypes, especially for African genotypes. So far there is no well established protocol for tissue culture and transformation of various cassava genotypes available. For many economically important plant species, including cassava, regeneration of plants from prolonged periods of tissue culture are prone to cause undesirable somaclonal variation of the original genotype which generally introduces undesirable new traits such as albinism. This limitation of applicability of the system is largely due to variation in morphological responses of different genotypes to regeneration and transformation. Thus, there is a need to provide an efficient regeneration and transformation protocol for production of improved cultivars of cassava plants.

SUMMARY OF THE INVENTION

One aspect of the invention provides a process of somatic embryogenesis of a cassava plant, wherein the process comprises culturing an explant from cassava plants on a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus, transferring the callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IBA and at least 0.3 mg/L CuS04 to obtain somatic embryo, culturing the somatic embryo for 15 to 30 days on a nutrient medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, and at least 0.3 mg/L CuS04 to obtain matured somatic embryo, and culturing the matured somatic embryo for 10 to 30 days on a medium comprising at least 0.1 mg/L BAP and at least 0.3mg/L CuS04 to obtain shoot.

Another aspect of the invention provides a process of regeneration of cassava plant, wherein the process comprises culturing a somatic embryo cotyledon obtained from somatic embryo of cassava plant on a nutrient medium comprising, at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 for 10 to 20 days to obtain shoot clusters; and separating the shoot clusters followed by culturing the shoot clusters onto a nutrient medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04, for 10-30 days to obtain elongated shoots.

Yet another aspect of the present invention provides a process of producing transgenic cassava plant, wherein said process comprises (a) transforming immature leaf lobe explant of the cassava plant with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by (i) wounding the leaf lobe explant and transferring the wounded leaf lobe explant into a suspension ol' Agrobactehum, (ii) transferring the leaf lobe explant from step (i) to a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus (iii) co-cultivating the leaf lobe of step (ii) with Agrobacterium in suspension for a period of 1 to 5 days, (b) selecting the transformed plant material derived from step (a), by transferring the leaf lobe explant to a selection medium comprising at least 4 mg/L 2,4-D, at least 0.3 mg/L CuS04 and appropriate selection agent to kill the Agrobacterium to obtain transformed callus, (c) culturing the transformed callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IBA, at least 0.3 mg/L CuS04 and the appropriate selection agent to obtain transformed somatic embryo, (d) culturing the transformed somatic embryo on a nutrient medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3 mg/L CuS04 and the appropriate selection agent for 15 to 30 days to obtain matured transformed somatic embryo and (e) culturing the matured transformed somatic embryo on a medium comprising at least 0.1 mg/L BAP, at least 0.3mg/L CuS04, and the appropriate selection agents for 10 to 30 days to obtain transformed cassava shoots.
A further aspect of the present invention provides a process of transforming a cassava plant, wherein said process comprises (a) transforming somatic cotyledonary explants obtained from somatic embryo of cassava with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by (i) culturing the somatic cotyledonary explants on a culture medium comprising at least 1 mg/L BAP at least 150 mg/L Myo-inositol and at least 0.3 mg/L CuS04 for 1-3 days, (ii) co-cultivating the somatic cotyledonary explants from step (i) with the Agrobaclerium lor 1 to 5 days, (b) selecting the transformed plant material derived from step (a) by transferring the somatic cotyledonary explants of step (a) to a medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3mg/L CuS04 and appropriate agents to kill the Agrobaclerium and incubating for at least 7 days, (c) transferring the somatic cotyledonary explants from step (b) to selection medium comprising of atleast 0.1 mg/L BAP and atleast 0.3 mg/L CuS04 and the appropriate selection agents to obtain transformed shoot clusters, and (d) transferring the transformed shoot clusters of step (c) to selection medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 and the appropriate selection agents to obtain elongated shoots, to obtain transformed shoot clusters, and transferring the transformed shoot clusters of step (b) to selection medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 and the appropriate selection agents to obtain elongated shoots.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Figure 1 shows the map details of pCAMBIA 1305.1 Figure 2 shows various stages of regeneration of in-vitro cassava plant Figure 3 A shows gel picture of PCR analysis of Putative Transgenic Cassava plants using hpt II primers (Hygromycin gene).

M: Marker (100 bp), P +: Plasmid control, Lanes 1-20: Plant samples, N: Negative control (untransformed plant)

Figure 3B shows Southern blot analysis of Putative Transgenic Cassava plants using fa-32P] dCTP-labelled hpt II gene as a probe.

Lane 1: >DNA digested with Hindlll and EcoRl, Lane 2: Uncut DNA, Lane 3: Control DNA from untransformed cassava plant, Lane 4 to 13: DNA from transformed cassava plants, Lane 14: Eluted probe (50 pg), Lane 15: Eluted probe (100 pg).

DETAILED DESCRIPTION OF THE INVENTION

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Definitions
For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only."

The term "plant" includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same.
The term "Explant" is used to refer to target material for transformation. Therefore, it is used to indicate "somatic tissue" or "any plant parts" as applicable, in the description.
The term "DNA sequence" includes nucleic acids or nucleic acid fragments that consist of a chain of linked units of nucleotides (adenine, guanine, cytosinc, and thymine or uracil). The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring.

The term "Transformation" refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection), can be achieved by any one oi' a number of means including electroporation, microinjection, biolistics , or particle bombardment-mediated delivery, in-plant transformation, and Agrobactehum mediated transformation.

The present invention provides culture media for regeneration and transformation of cassava (Manihot esculenta Crantz) plant, a process for somatic embryogenesis and regeneration of cassava {Manihot esculenta Crantz) and a process for transformation of cassava plants. In addition, a process for producing in-vitro regenerated and transgenic cassava plants are also provided in the present invention.

The present invention particularly provides an efficient tissue culture, regeneration and transformation protocol for production of transgenic cassava plants of three economically important cassava varieties, namely MVD-1, H-226 and Kungumarose. The present inventors found unexpected and surprising results when the highly recalcitrant plant, cassava was regenerated with ease using the culture media and tissue culture techniques as provided in the present invention. The inventors also found that the transformation of the cassava plant could be conducted using the media and protocol as disclosed herein. Transformation of the cassava plant may be carried out through Agrobactehum mediated transformation or by direct gene transfer using the prevalent state of the art. Until now, it has not been feasible to regenerate cassava plants from transformed cassava tissues. However, the present inventors were highly successful when they practiced the process of producing transformed cassava plants as disclosed herein using the nutrient media as disclosed in Table 1. The fine-tuning of the transformation, selection, embryogenesis and regeneration media is often fortuitous and one component in the wrong concentration might inhibit the success of the whole process. The present invention provides a simple and highly efficient process for the regeneration of cassava plants and production of transformed cassava plants, over the conventional methods prevalent in the art. The present invention may be used as a rapid micropropagation system for cassava.

Nodal cuttings of Cassava Varieties: MVD-1, H-226 and Kungumarose were collected from field (Attur, Ammampalayam and Kattukkottai, Salem Dist., Tamil Nadu, India) grown cassava plants (5-months old) and used for raising of in vitro cassava plants.

Somatic embryogenesis and regeneration by explant cultures in Cassava (Manihot esculenta Crantz)

Nodal explants from field grown plants (5-months old) were collected and thoroughly washed with running tap water for 10 minutes. The nodal cuttings were soaked with sterile distilled water containing two drops of Tween-20 for 5 minutes and then washed twice with sterile water. Commercial fungicide (2% Bavistin) was used to wash the cuttings for 10 minutes and again washed two times with distilled water. Finally, the nodal explants were surface sterilized using Tween-20 and 4% sodium hypochlorite solution for 20 minutes. The explants were thoroughly washed four times with sterile distilled water, subsequently placed on half strength N6 basal medium for three weeks in photoperiod cycles (16 hours light/8 hours dark) at 26°C. Buds emerged from nodal regions and developed as in vitro plants.

Young nodal regions are necessary to develop immature leaf lobes. For the same, the nodal cutting from in vitro mother plants were excised and placed on cassava shoot induction medium (CSIM) in petri plates to develop into fresh shoots. The plates were incubated in photoperiod cycles (16 hours light / 8 hours dark) at 26°C for 15 days. Leaf lobes were formed in the in vitro plants.

Immature leaf lobes (1 to 6 mm in length) were excised from in vitro cassava plants and cultured on callus induction medium (CCIM) and incubated in dark at 26°C for 30 days for callus induction. The developed calli thus obtained were transferred into somatic embryo induction medium (SEIM) to obtain somatic embryos. Somatic embryos thus formed were further incubated in photoperiod cycles (16 hours light/8 hours dark) at 26°C for 15 days. Further, the developed somatic embryos which are greenish in colour and globular in shape, were transferred into somatic embryo maturation medium (SEMM) and incubated in photoperiod cycles (16 hours light/8 hours dark) at 26°C for 30 days to obtain matured somatic embryos. Somatic cotyledonary pieces from matured somatic embryos were cut off and kept in shoot induction medium (SIM) for shoot differentiation.

Shoots were differentiated from somatic cotyledonary pieces of matured somatic embryos in 20 days incubation in photoperiod cycles (16 hours light/8 hours dark) at 26°C in the shoot induction medium (SIM). The same medium is used for shoot elongation for another 40 days with same conditions. Well elongated shoots were placed in rooting medium (CRM) and incubated in photoperiod cycles (16 hours light/8 hours dark) at 26°C for 15 days to develop roots. Primary roots were observed. After rooting, well developed plants were acclimatized in sand: soil: coir pith (1:1:1) mixture in the pots in the green house and allowed for establishment. Agrobacterium mediated Transformation in Cassava (Manihot esculenta Crantz)

Nodal cuttings were collected from field grown cassava plants (5-months old) and used for raising in vitro cassava plants. Explants were collected from in vitro cassava plants and used for callus induction. Somatic cotyledonary explants from matured somatic embryos which were developed from calli as described above were used for co-cultivation with Agrobacterium strain LBA4404 harboring a binary vector pCAMBIA 1305.1. The map details of the gene construct is provided in Figure 1.

Somatic cotyledons were developed from 10 days-old somatic embryos which were obtained from calli obtained using the procedure as described above. Somatic cotyledonary pieces were cut off and kept for pre-incubation at 26°C in photoperiod (16 hours light/8 hour dark) cycles for 3 days. Cotyledonary explants were added into a conical flask containing Agrobacterium strain LBA4404 suspension culture harboring a binary vector pCAMBIA 1305.1 and co-cultivated for 3 hours on shaker at 120 rpm at 26°C. The Agrobacterium culture was decanted and the explants were transferred onto sterile filter paper to remove excess of bacterial culture and subsequently cultured onto SHIM supplemented with 100 uM of Acetosyringone and further co-cultivated in dark at 26°C for four days.

Explants co-cultivated with Agrobacterium were washed in a three sequential step of washing with sterile distilled water (3 minutes for each step) and then washing twice with MS liquid (5 minutes) and finally washing with MS liquid with 300 mg/L Cefotaxime (4 minutes). The washed explants were blot dried and transferred to SEMM comprising 200 mg/L of Cefotaxime. The explants were incubated at 26°C for 10 days in photoperiod (16 hours light/8 hours dark) cycles. Healthy explants were further transferred to SIM comprising 20 mg/L Hygromycin and 300 mg/L Cefotaxime as a first selection and incubated at 26°C in photoperiod (16 hours light/8 hours dark) cycles for 15 days. Selected explants were shifted to second selection in the same selection media and conditions mentioned above. Resistant shoot clusters were further transferred to SIM added with 20 mg/L Hygromycin and 300 mg/L Cefotaxime at 26°C for photoperiod (16 hours light/8 hours dark) cycles for 20 days. Resistant shoot clumps were split and kept for rooting in CRM added with 10 mg/L Hygromycin and 200 mg/L Cefotaxime, incubated at 26°C for photoperiod (16 hours light/8 hours dark) cycles. Well rooted plants were kept for pre hardening stage in MS liquid for 7 Days at 26°C for photoperiod (16 hours light/8 hours dark) cycles. Putative plants were then hardened in green house in an appropriate growth condition. Composition of culture media used for regeneration and transformation of cassava is provided in Table 1.

One embodiment of the present invention provides a process of somatic embryogenesis of a cassava plant, wherein said process comprises (a) culturing an explant from cassava plants on a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus (b) transferring the callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IBA and at least 0.3 mg/L CuS04 to obtain somatic embryo (c) culturing the somatic embryo for 15 to 30 days on a nutrient medium comprising at least 1 mg/L BAP, atleast 150 mg/L Myo¬inositol, and at least 0.3 mg/L CuS04 to obtain matured somatic embryo; and (d) culturing the matured somatic embryo for 10 to 30 days on a medium comprising at least 0.1 mg/L BAP and at least 0.3mg/L CuS04 to obtain shoot.

Another embodiment of the present invention provides a process of somatic embryogenesis of a cassava plant, wherein said process comprises (a) culturing an explant from cassava plants on a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus (b) transferring the callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IBA and at least 0.3 mg/L CuS04 to obtain somatic embryo (c) culturing the somatic embryo for 15 to 30 days on a nutrient medium comprising at least 1 mg/L BAP, atleast 150 mg/L Myo¬inositol, and at least 0.3 mg/L CuS04 to obtain matured somatic embryo; and (d) culturing the matured somatic embryo for 10 to 30 days on a medium comprising at least 0.1 mg/L BAP and at least 0.3mg/L CuS04 to obtain shoot, wherein the explant is selected from a group consisting of immature leaf lobe and mature leaf lobe.

An embodiment of the present invention provides a process of regeneration of cassava plant, wherein said process comprises (a) culturing a somatic embryo cotyledon obtained from somatic embryo of cassava plant on a nutrient medium comprising, at least
0.1 mg/L BAP and at least 0.3 mg/L CuS04 for 10 to 20 days to obtain shoot clusters, and
separating the shoot clusters followed by culturing the shoot clusters onto a nutrient medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04, for 10-30 days to obtain elongated shoots.

Another embodiment of the present invention provides a process of somatic embryogenesis of a cassava plant, wherein said process comprises (a) culturing an explant from cassava plants on a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus (b) transferring the callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IBA and at least 0.3 mg/L CuS04 to obtain somatic embryo (c) culturing the somatic embryo for 15 to 30 days on a nutrient medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, and at least 0.3 mg/L CuS04 to obtain matured somatic embryo; and (d) culturing the matured somatic embryo for 10 to 30 days on a medium comprising at least 0.1mg/L BAP and at least 0.3mg/L CuS04 to obtain shoot, wherein the process further comprises culturing the shoot on a rooting medium comprising half strength N6 medium until a root system is developed thereon.

Another embodiment of the present invention provides a process of regeneration oi' cassava plant, wherein said process comprises (a) culturing a somatic embryo cotyledon obtained from somatic embryo of cassava plant on a nutrient medium comprising, at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 for 10 to 20 days to obtain shoot clusters, and separating the shoot clusters followed by culturing the shoot clusters onto a nutrient medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04, for 10-30 days to obtain elongated shoots, wherein the process further comprises culturing the shoot on a rooting medium comprising half strength N6 medium until a root system is developed thereon.

An embodiment of the present invention provides a process of producing transgenic cassava plant, wherein said process comprises (a) transforming immature leaf lobe explant of the cassava plant with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by: (i) wounding the leaf lobe explant and transferring the wounded leaf lobe explant into a suspension of Agrobacterium, (ii) transferring the leaf lobe explant from step (i) to a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus; (iii) co-cultivating the leaf lobe of step (ii) with Agrobacterium in suspension for a period of 1 to 5 days (b) selecting the transformed plant material derived from step (a), by transferring the leaf lobe explant to a selection medium comprising at least 4 mg/L 2,4-D, at least 0.3 mg/L CuS04 and appropriate selection agent to kill the Agrobacterium to obtain transformed callus (c) culturing the transformed callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IBA, at least 0.3 mg/L CuS04 and the appropriate selection agent to obtain transformed somatic embryo (d) culturing the transformed somatic embryo on a nutrient medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3 mg/L CuS04 and the appropriate selection agent for 15 to 30 days to obtain matured transformed somatic embryo, and (e) culturing the matured transformed somatic embryo on a medium comprising at least 0.1 mg/L BAP, at least 0.3mg/L CuS04 and the appropriate selection agents for 10 to 30 days to obtain transformed cassava shoots.
An embodiment of the present invention provides a process of transforming a cassava plant, wherein said process comprises (a) transforming somatic cotyledonary explants obtained from somatic embryo of cassava with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by: (i) culturing the somatic cotyledonary explants on a culture medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol and at least 0.3 mg/L CuS04 for 1 -3 days, (ii) co-cultivating the somatic cotyledonary explants from step (i) with the Agrobacterium for 1 to 5 days, (b) selecting the transformed plant material derived from step (a) by transferring the somatic cotyledonary explants of step (a) to a medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3mg/L CuS04 and appropriate agents to kill the Agrobacterium and incubating for at least 7 days, (c) transferring the somatic cotyledonary explants from step (b) to selection medium comprising of atleast 0.1 mg/L BAP and atleast 0.3 mg/L CuS04 and the appropriate selection agents to obtain transformed shoot clusters, and (d) transferring the transformed shoot clusters oi' step (c) to selection medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 and the appropriate selection agents to obtain elongated shoots.

Another embodiment of the present invention provides a process of producing transgenic cassava plant, wherein said process comprises (a) transforming immature leaf lobe explant of the cassava plant with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by: (i) wounding the leaf lobe explant and transferring the wounded leaf lobe explant into a suspension of Agrobaclerium, (ii) transferring the leaf lobe explant from step (i) to a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus; (iii) co-cultivating the leaf lobe of step (ii) with Agrobacterium in suspension for a period of 1 to 5 days (b) selecting the transformed plant material derived from step (a), by transferring the leaf lobe explant to a selection medium comprising at least 4 mg/L 2,4-D, at least 0.3 mg/L CuS04 and appropriate selection agent to kill the Agrobacterium to obtain transformed callus (c) culturing the transformed callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IBA, at least 0.3 mg/L CuS04 and the appropriate selection agent to obtain transformed somatic embryo (d) culturing the transformed somatic embryo on a nutrient medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3 mg/L CuS04 and the appropriate selection agent for 15 to 30 days to obtain matured transformed somatic embryo, and (e) culturing the matured transformed somatic embryo on a medium comprising at least 0.1 mg/L BAP, at least 0.3mg/L CuS04 and the appropriate selection agents for 10 to 30 days to obtain transformed cassava shoots, wherein the process further comprises culturing the shoot on a rooting medium comprising half strength N6 medium and the appropriate selection agents until a root system is developed thereon.

Another embodiment of the present invention provides a process of transforming a cassava plant, wherein said process comprises (a) transforming somatic cotyledonary explants obtained from somatic embryo of cassava with DNA sequences via a vector or
direct gene transfer to produce transformed plant material, wherein transformation is
achieved by: (i) culturing the somatic cotyledonary explants on a culture medium comprising at least 1 mg/L BAP at least 150 mg/L Myo-inositol and at least 0.3 mg/L CuS04 for 1-3 days, (ii) co-cultivating the somatic cotyledonary explants from step (i) with the Agrobacterium for 1 to 5 days, (b) selecting the transformed plant material derived from step (a) by transferring the somatic cotyledonary explants of step (a) to a medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3mg/L CuS04 and appropriate agents to kill the Agrobacterium and incubating for at least 7 days, (c) transferring the somatic cotyledonary explants from step (b) to selection medium comprising of atleast 0.1 mg/L BAP and atleast 0.3 mg/L CuS04 and the appropriate selection agents to obtain transformed shoot clusters, and (d) transferring the transformed shoot clusters 0.1 step (c) to selection medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 and the appropriate selection agents to obtain elongated shoots, wherein the process further comprises culturing the shoot on a rooting medium comprising half strength N6 medium and the appropriate selection agents until a root system is developed thereon.

Another embodiment of the present invention provides a process of producing transgenic cassava plant, wherein said process comprises (a) transforming immature leaf lobe explant of the cassava plant with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by: (i) wounding the leaf lobe explant and transferring the wounded leaf lobe explant into a suspension of Agrobacterium, (ii) transferring the leaf lobe explant from step (i) to a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus; (iii) co-cultivating the leaf lobe of step (ii) with Agrobacterium in suspension for a period of 1 to 5 days (b) selecting the transformed plant material derived from step (a), by transferring the leaf lobe explant to a selection medium comprising at least 4 mg/L 2,4-D, at least 0.3 mg/L CuS04 and appropriate selection agent to kill the Agrobacterium to obtain transformed callus (c) culturing the transformed callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IBA, at least 0.3 mg/L CuS04 and the appropriate selection agent to obtain transformed somatic embryo (d) culturing the transformed somatic embryo on a nutrient medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3 mg/L CuS04 and the appropriate selection agent for 15 to 30 days to obtain matured transformed somatic embryo, and (c) culturing the matured transformed somatic embryo on a medium comprising at least 1 mg/L BAP, at least 0.3mg/L CuS04 and the appropriate selection agents for 10 to 30 days to obtain transformed cassava shoots, wherein the DNA sequences comprises at least one gene selected from a group consisting of selectable marker gene, reporter gene, insecticidal gene, herbicide resistance gene, disease resistant gene, and agronomically important gene.

Another embodiment of the present invention provides a process of transforming a cassava plant, wherein said process comprises (a) transforming somatic cotyledonary explants obtained from somatic embryo of cassava with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by: (i) culturing the somatic cotyledonary explants on a culture medium comprising at least 1 mg/L BAP at least 150 mg/L Myo-inositol and at least 0.3 mg/L CuS04 for 1 -3 days, (ii) co-cultivating the somatic cotyledonary explants from step (i) with the Agrobacterium for 1 to 5 days, (b) selecting the transformed plant material derived from step (a) by transferring the somatic cotyledonary explants of step (a) to a medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3mg/L CuS04 and appropriate agents to kill the Agrobacterium and incubating for at least 7 days, (c) transferring the somatic cotyledonary explants from step (b) to selection medium comprising of atleast 0.lmg/L BAP and atleast 0.3mg/L CuS04 and the appropriate selection agents to obtain transformed shoot clusters, and (d) transferring the transformed shoot clusters of step (c) to selection medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 and the appropriate selection agents to obtain elongated shoots, wherein the DNA sequences comprises at least one gene selected from a group consisting of selectable marker gene, reporter gene, insecticidal gene, herbicide resistance gene, disease resistant gene, and agronomically important gene.

Another embodiment of the present invention provides a process of producing transgenic cassava plant, wherein said process comprises (a) transforming immature leal" lobe explant of the cassava plant with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by: (i) wounding the leaf lobe explant and transferring the wounded leaf lobe explant into a suspension of Agrobacterium, (ii) transferring the leaf lobe explant from step (i) to a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus; (iii) co-cultivating the leaf lobe explant of step (ii) with Agrobacterium in suspension for a period of 1 to 5 days (b) selecting the transformed plant material derived from step (a), by transferring the leaf lobe explant to a selection medium comprising at least 4 mg/L 2,4-D, at least 0.3 mg/L CuS04 and appropriate selection agent to kill the Agrobacterium to obtain transformed callus (c) culturing the transformed callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IBA, at least 0.3 mg/L CuS04 and the appropriate selection agent to obtain transformed somatic embryo (d) culturing the transformed somatic embryo on a nutrient medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3 mg/L CuS04 and the appropriate selection agent for 15 to 30 days to obtain matured transformed somatic embryo, and (e) culturing the matured transformed somatic embryo on a medium comprising at least 0.1 mg/L BAP, at least 0.3mg/L CuS04 and the appropriate selection agents for 10 to 30 days to obtain transformed cassava shoots, wherein the DNA sequence comprising the selectable marker gene is an antibiotic resistance gene selected from a group consisting of nptll, or hptll.

Another embodiment of the present invention provides a process of transforming a cassava plant, wherein said process comprises (a) transforming somatic cotyledonary explants obtained from somatic embryo of cassava with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by: (i) culturing the somatic cotyledonary explants on a culture medium comprising at least 1 mg/L BAP at least 150 mg/L Myo-inositol and at least 0.3 mg/L CuS04 for 1 -3 days, (ii) co-cultivating the somatic cotyledonary explants from step (i) with the Agrobacterium for 1 to 5 days, (b) selecting the transformed plant material derived from step (a) by transferring the somatic cotyledonary explants of step (a) to a medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3mg/L CuS04 and appropriate agents to kill the Agrobacterium and incubating for at least 7 days, (c) transferring the somatic cotyledonary explants from step (b) to selection medium comprising of atleast 0.1 mg/L BAP and atleast 0.3mg/L CuS04 and the appropriate selection agents to obtain transformed shoot clusters, and (d) transferring the transformed shoot clusters of step (c) to selection medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 and the appropriate selection agents to obtain elongated shoots, wherein the DNA sequence comprising the selectable marker gene is an antibiotic resistance gene selected from a group consisting of nplll, or hptll.

Another embodiment of the present invention provides a process of producing transgenic cassava plant, wherein said process comprises (a) transforming immature leaf lobe explant of the cassava plant with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by: (i) wounding the leaf lobe explant and transferring the wounded leaf lobe explant into a suspension of Agrobacterium, (ii) transferring the leaf lobe explant from step (i) to a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus; (iii) co-cultivating the leaf lobe of step (ii) with Agrobacterium in suspension for a period of 1 to 5 days (b) selecting the transformed plant material derived from step (a), by transferring the leaf lobe explant to a selection medium comprising at least 4 mg/L 2,4-D, at least 0.3 mg/L CuS04 and appropriate selection agent to kill the Agrobacterium to obtain transformed callus (c) culturing the transformed callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IB A, at least 0.3 mg/L CuS04 and the appropriate selection agent to obtain transformed somatic embryo (d) culturing the transformed somatic embryo on a nutrient medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3 mg/L CuS04 and the appropriate selection agent for 15 to 30 days to obtain matured transformed somatic embryo, and (e) culturing the matured transformed somatic embryo on a medium comprising at least 0.1 mg/L BAP, at least 0.3mg/L CuS04 and the appropriate selection agents for 10 to 30 days to obtain transformed cassava shoots, wherein the DNA sequence comprising the reporter gene encodes GFP or GUS.
Another embodiment of the present invention provides a process of transforming a cassava plant, wherein said process comprises (a) transforming somatic cotyledonary
explants obtained from somatic embryo of cassava with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by: (i) culturing the somatic cotyledonary explants on a culture medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol and at least 0.3 mg/L CuS04 for 1 -3 days, (ii) co-cultivating the somatic cotyledonary explants from step (i) with the Agrobacterium for 1 to 5 days, (b) selecting the transformed plant material derived from step (a) by transferring the somatic cotyledonary explants of step (a) to a medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3mg/L CuS04 and appropriate agents to kill the Agrobacterium and incubating for at least 7 days, (c) transferring the somatic cotyledonary explants from step (b) to selection medium comprising of atleast 0.1 mg/L BAP and atleast 0.3mg/L CuS04 and the appropriate selection agents to obtain transformed shoot clusters, and (d) transferring the transformed shoot clusters o\' step (c) to selection medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 and the appropriate selection agents to obtain elongated shoots, wherein the DNA sequence comprising the reporter gene encodes GFP or GUS.

Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible. As such, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiment contained therein.

EXAMPLES

The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

Example 1

Somatic embryogenesis of cassava plant Nodal explants were collected from field (5-months old plants) and washed with water for 10 minutes. The nodal cuttings were soaked with distilled water containing two drops of Tween-20 for 5 minutes followed by two times washing with water. Commercial fungicide (2% Bavistin) was used to wash the cuttings for 10 minutes. The nodal cuttings were again washed two times with distilled water. Finally, the nodal cuttings were surface sterilized using distilled water containing two drops of Tween-20 and 4% sodium hypochlorite solution for 20 minutes. The nodal cuttings were thoroughly washed four times with sterile distilled water. The nodal cuttings were placed on half strength N6 basal medium for three weeks in photoperiod cycles (16 hours light/8 hours dark) at 26°C. Buds emerged from nodal regions and developed as in vitro mother plants.

Nodal cuttings from in vitro mother plants were placed on cassava shoot induction medium to develop into fresh shoots. The plates were incubated in photoperiod cycles at 26°C for 15 days. The nodal cuttings were used for further in vitro plant development after explant collection.

Immature leaf lobes (1-6 mm in length) were excised as explants from in vitro plants and placed on callus induction medium (CCIM) and incubated in dark at 26°C for 30 days (Figure 2, A and B). The developed calli were transferred into somatic embryo induction medium (SEIM) and incubated at 26°C for 15 days in photoperiod cycles (Figure 2C).
The developed greenish globular shaped somatic embryos were transferred into somatic embryo maturation medium (SEMM) and incubated in photoperiod cycles at 26°C for 30 days for maturation (Figure 2D). Somatic cotyledon pieces were cut off from matured somatic embryos and transferred into shoot induction medium (SIM). This was incubated at 26°C for 60 days in photoperiod cycles (Figure 2E). Well elongated shoots were placed in rooting medium (CRM) and incubated in photoperiod cycles at 26°C for 15 days to develop roots (Figure 2F). After rooting, well developed plants were acclimatized in sand: soil: coir pith (1:1:1) mixture in pots in the green house.

The regeneration frequency of the three cassava cultivars was found to be 1.196% for H-226, 1: 0.952% for MVD-and 0.870% for Kungumarose.

Example 2

In-vitro Regeneration of cassava plants from somatic cotyledonary embryo Somatic cotyledon pieces obtained from matured somatic embryos were used as explants for in-vitro regeneration. The explants were transferred into shoot induction medium (SIM) comprising MS salts, B5 vitamins, 0.3 mg/L CuS04, 0.1 mg/L BAP and phytagel 3 g/L (pH 5.6-5.8) and incubated at 26°C ±2 for 20 days in photoperiod of 16 hours light/8hours dark cycles. The selected explants further transferred to fresh SIM medium every 20 days once until shoots are developed. Well elongated shoots were placed in rooting medium (CRM) and incubated in photoperiod of 16 hours light/8hours dark cycles at 26°C for 15 days to develop roots. After rooting, well developed plants were acclimatized in sand: soil: coir pith (1:1:1) mixture in pots in the green house.
The regeneration frequency of the three cassava cultivars was found to drastically increase. A single cotyledonary explant was able to produce minimum three shoots in H-226 and a minimum of 2 shoots in MVD-1 and Kungumarose varieties.

Example 3

Agrobacterium mediated transformation of cassava plant

1. Explant: somatic embryo cotyledon

Nodal explants from field grown plants (5-months old) were collected and washed
thoroughly with running tap water for 10 minutes. The nodal cuttings were soaked with
sterile distilled water containing two drops of Tween-20 for 5 minutes and then washed two times with sterile water. Commercial fungicide (2% Bavistin) was used to wash the cuttings for 10 minutes. The cuttings were again washed two times with distilled water. Finally, the nodal cuttings were surface sterilized using Tween-20 (two drops) and 4% sodium hypochlorite solution for 20 minutes. The nodal explants were thoroughly washed four times with sterile distilled water, then placed on half strength N6 basal medium (Table 2) for three weeks in photoperiod cycles (16 hours light/8 hour dark) at 26°C. Buds emerged from nodal regions and developed as in vitro plants.

Explant Initiation

Young nodal regions are necessary to develop immature leaf lobes. For the same, the nodal cutting from in vitro mother plants were excised and placed on cassava shoot induction medium (CSIM ) in petri plates to develop into fresh shoots. The plates were incubated in photoperiod cycles (16 hours light/8 hours dark) at 26°C for 15 days. Leaf lobes were formed in the in vitro plants.

Callus induction

Immature leaf lobes (1 to 6 mm in length) formed were excised from in vitro plants,
and as explants, placed on callus induction medium (CCIM) and incubated in dark at 26°C for 30 days for callus induction. The developed calli were transferred into somatic embryo induction medium (SEIM) and somatic embryos were formed after incubation in
photoperiod cycles (16 hours light / 8 hours dark) at 26°C for 15 days. The developed
somatic embryos (greenish globular shaped) were transferred into somatic embryo
'"N maturation medium (SEMM) and incubated in photoperiod cycles (16 hours light/8 hours dark) at 26°C for 30 days.

Explant Pre-incubation

Somatic cotyledonary explants obtained from mature somatic embryos preferably from 10 days-old somatic embryos were cultured on Pre-induction medium (SEMM) and incubated in photoperiod cycles (16 hours light/8 hours dark) at 26°C for 3 days.

Binary vector

pCAMBIA 1305.1 comprises a GUS plus region driven by CaMV 35S promoter and hpt region for plant selection marker. The vector is depicted in Figure 1.

Agrobacterium culture

Single colony of A. tumefaciens strain LBA4404 harboring a binary vector pCAMBIAl305.1 was picked up from an AB agar plate (AB media, Table 3) using an inoculation loop and inoculated into a test tube containing 5 ml of fresh AB broth added with 10 mg/L Rifampicin and 50 mg/L Kanamycin as a bacterial selection markers. The test tube was incubated overnight on an orbital shaker (120 rpm at 27°C). Aliquots of 200^1 of bacterial suspensions were transferred to 50 ml of fresh AB broth with the same antibiotics mentioned above in 250 ml conical flasks and cultured for 12 hours to 20 hours to attain the optical density (OD) of 0.7 at 660 nm (checked using Spectrophotometer).

Co-cultivation

Well grown bacterial culture were centrifuged at 5400 rpm at 4°C for 12 minutes in 50 ml centrifuge tubes and the pellets were re-suspended in 20 ml of full strength MS liquid medium (pH-5.2) supplemented with lOOum concentration of Acetosyringonc. Agrobacterium culture was incubated in conical flask for one hour at 27°C in an orbital shaker at 120 rpm. The somatic cotyledonary explants obtained from matured somatic embryos which were pre-incubated on SEMM for 3 days were used for co-cultivation. The explants were added in a conical flask containing 20 ml of bacterial suspension and the flask was kept in a shaker at 120 rpm for 3 hours. Bacterial suspension was decanted with a pipette and the treated explants were placed on the sterile filter paper for a minute to dry excess of bacterial culture. Subsequently the explants were transferred to SEMM plates supplemented with 100 uM concentration of Acetosyringone and co-cultivated in dark at 26°C for 4 days.

Co-cultivated explants were washed three times with sterile distilled water for 3 minutes each to eliminate the bacterial culture and then washed twice with MS liquid (pi I 5.6) for 5 minutes. Finally, Cefotaxime (300mg/L) was added into full strength MS liquid and allowed for 4 minutes to prevent further bacterial growth. Explants were blotted well on sterile tissue paper to draw excess liquid. Selection and regeneration of transgenic cassava plants Blot dried explants were placed onto SEMM medium (30 explants/petri dish) added with 300 mg/L of Cefotaxime to prevent bacterial growth. The explants were incubated for 10 days of photoperiod cycles (16 hours light / 8 hours dark) at 26°C.
After 10 days, healthy explants were transferred to the selection medium, SIM with 20 mg/L Hygromycin (Sigma) as a plant selection marker and 300 mg/L Cefotaxime (Sigma) and incubated for 15 days in photoperiod cycles (16 hours light / 8 hours dark) at 26°C.

The resistant shoot clusters or clusters with shoot primordia were selected and further placed onto the same media (SIM) and selection conditions mentioned above, for 20 days of photoperiod cycles (16 hours light / 8 hours dark) at 26°C. Resistant shoot clumps were split out and placed in same culture medium mentioned previously for shoot elongation (SIM) for 15 days of photoperiod cycles (16 hours light / 8 hours dark) at 26°C. Emerging shoots were transferred to SIM with 20 mg/L Hygromycin and 300 mg/L Cefotaxime for 15 days of photoperiod cycles (16 hours light / 8 hours dark) at 26°C and allowed for shoot elongation. The well elongated shoots were placed onto CRM with lOmg/L Hygromycin for rooting for 20 days of photoperiod cycles (16 hours light / 8 hours dark) at 26°C. Well rooted putative transgenic plants were further taken for pre hardening stage. Well rooted putative transgenic plants were taken for pre-hardening stage in full strength MS liquid for a week. The putative transgenic cassava plants were transferred and acclimatized in sand: soil: coir pith (1:1:1) mixture in mud pots in the green house and allowed for establishment.

The putative transgenic cassava plants were subjected to Polymerase chain reaction (PCR) analysis for amplification of hptll gene. Genomic DNA from 20 putative transgenic plants was isolated and subjected to PCR amplification of hptll gene using the hptll gene specific primers. All 20 putative transgenic plants showed amplification of 820 bp fragment (Figure 3A).

2. Explant: leaf lobes Binary vector pCAMBIA 1305.1 comprises a GUS plus region driven by CaMV 35S promoter and hpt region for plant selection marker. The vector is depicted in Figure 1. Agrobacterium culture Single colony of A. tumefaciens strain LBA4404 harboring a binary vector pCAMBIA 1305.1 was picked up from an AB agar plate (AB media, Table 3) using an inoculation loop and inoculated into a test tube containing 5 ml of fresh AB broth added with 10 mg/L Rifampicin and 50 mg/L Kanamycin as a bacterial selection markers. The test tube was incubated overnight on an orbital shaker (120 rpm at 27°C). Aliquots of 200ul of bacterial suspensions were transferred to 50 ml of fresh AB broth with the same antibiotics mentioned above in 250 ml conical flasks and cultured for 12 hours to 20 hours to attain the optical density (OD) of 0.7 at 660 nm (checked using Spectrophotometer).

Co-cultivation

Well grown bacterial culture were centrifuged at 5400 rpm at 4°C for 12 minutes in 50 ml centrifuge tubes and the pellets were re-suspended in 20 ml of full strength MS liquid medium (pi 1-5.2) supplemented with lOOum concentration of Acetosyringone. Agrobacterium culture was incubated in conical flask for one hour at 27°C in an orbital shaker at 120 rpm.

Immature leaf lobes of 1-6 mm in length excised from in vitro grown cassava plants were wounded and the explants were added in a conical flask containing 20 ml of bacterial suspension and the flask was kept in a shaker at 120 rpm for 3 hours. Bacterial suspension was decanted with a pipette and the treated explants were placed on the sterile filter paper for a minute to dry excess of bacterial culture. Subsequently the explants were transferred to callus induction medium (CCIM) (Table 1) comprising MS salts, 100 mg/L myo-inositol, 4 mg/L 2,4-D, 0.3 mg/L CuS04 and phytagel (pH 5.6-5.8) and 100 uM Acetosyringonc and co-cultivated in dark at 26°C for 4 days.

Co-cultivated explants were washed three times with sterile distilled water for 3 minutes each to eliminate the bacterial culture and then washed twice with MS liquid (pH 5.6) for 5 minutes. Finally, Cefotaxime (300mg/L) was added into full strength MS liquid and allowed for 4 minutes to prevent further bacterial growth. Explants were blotted well on sterile tissue paper to draw excess liquid.

Selection and regeneration of transgenic cassava plants

Blot dried explants were placed onto callus induction medium (CCIM) (30 explants/petri dish) added with 300 mg/L of Cefotaxime to prevent bacterial growth. The explants were incubated for 30 days of photoperiod cycles (16 hours light / 8 hours dark) at 26°C. The developed calli were transferred into somatic embryo induction medium (SEIM) supplemented with 300 mg/L of Cefotaxime and 20mg/L Hygromycin and incubated at 26°C for 15 days in photoperiod cycles. The developed greenish globular shaped transformed somatic embryos were transferred into somatic embryo maturation medium (SEMM) supplemented with 300 mg/L of Cefotaxime and 20mg/L Hygromycin and incubated in photoperiod cycles at 26°C for 30 days for maturation. The transformed and matured somatic embryo were cultured onto the shoot induction medium (SIM, Table 1) supplemented with 300 mg/L Cefotaxime and 20 mg/L Hygromycin, at 26°C for 15 days in photoperiod cycles (16 hours light / 8 hours dark) to obtain transformed cassava shoots.

The resistant shoot clusters or clusters with shoot primordia were selected and further placed onto the SIM supplemented with 300 mg/L Cefotaxime and 20 mg/L Hygromycin for 20 days of photoperiod cycles of 16 hours light / 8 hours dark, at 26°C. Resistant shoot clumps were split out and placed in SIM media supplemented with 300 mg/L Cefotaxime and 20 mg/L Hygromycin for 15 days of photoperiod cycles (16 hours light / 8 hours dark) at 26°C. Emerging shoots were transferred to SIM supplemented with 20 mg/L Hygromycin and 300 mg/L Cefotaxime for 15 days of photoperiod cycles (16 hours light / 8 hours dark) at 26°C and allowed for shoot elongation.

Alternatively, the transformed somatic cotyledon pieces were cut off from matured transformed somatic embryos and transferred into shoot induction medium (SIM) supplemented with 300 mg/L of Cefotaxime and 20 mg/L Hygromycin. This was incubated at 26°C for 60 days in photoperiod cycles to obtain elongated transformed shoots.

The well elongated shoots thus obtained were placed onto Cassava Rooting Medium (CRM) with lOmg/L Hygromycin for rooting for 15-20 days of photoperiod cycles of 16 hours light / 8 hours dark, at 26°C. Well rooted putative transgenic plants were taken for pre-hardening stage in full strength MS liquid for a week. The putative transgenic cassava plants were transferred and acclimatized in sand: soil: coir pith (1:1:1) mixture in mud pots in the green house and allowed for establishment.

The putative transgenic cassava plants were subjected to Polymerase chain reaction (PCR) analysis for amplification of hptll gene to confirm the successful transformation oi' the plants.

Southern Hybridization

Genomic DNA of the transgenic cassava plants that showed positive results in a PCR assay were isolated and used for hybridization experiments according to standard molecular biology protocols (Sambrook J, Fritsch EF and Maniatis T, 1989, Molecular Cloning: A Laboratory Manual. 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Southern hybridization was performed on 10 transgenic cassava plants that were positive in the PCR assay. 10 jig of DNA was digested with the restriction enzyme, Hindlll. Hybridization of the digested DNA was carried out using a [a-32P] dCTP-labelled hptll gene region as a probe of 820 bp. Out of 10 plants tested, only 7 plants showed the transgene integration (Figure 3B).

I/We Claim:

1. A process of somatic embryogenesis of a cassava plant, wherein said process comprises

a. culturing an explant from cassava plants on a culture medium comprising at least 4mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus;

b. transferring the callus on a somatic embryo induction medium comprising at least 1mg/L BAP,- at least 1 mg/L Kinetin, at least 0.05 mg/L IBA and at least 0.3 mg/L
CuS04 to obtain somatic embryo;

c. culturing the somatic embryo for 15 to 30 days on a nutrient medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol and at least 0.3 mg/L CuS04 to
obtain matured somatic embryo; and

d. culturing the matured somatic embryo for 10 to 30 days on a medium comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 to obtain shoot.

2. The process as claimed in claim 1, wherein the explant is selected from a group consisting of immature leaf lobe and mature leaf lobe.

3. A process of regeneration of cassava plant, wherein said process comprises

a. culturing a somatic embryo cotyledon obtained from somatic embryo of cassava
plant on a nutrient medium comprising, at least 0.1 mg/L BAP and at least 0.3 mg/L
CuS04 for 10 to 20 days to obtain shoot clusters; and

b. separating the shoot clusters followed by culturing the shoot clusters onto a nutrient medium comprising at least 0.1 mg/L BAP and at least 0.3 m£/'L CuS04, for 10-30 days to obtain elongated shoots.

4. The process as claimed in claim 1 or 3, wherein the process further comprises culturing the shoot on a rooting medium comprising half strength N6 medium until a root system is developed thereon.

5. A process of producing transgenic cassava plant, wherein said process comprises

a. transforming immature leaf lobe explant of the cassava plant with DNA sequences via a vector or direct gene transfer to produce transformed plant material, wherein transformation is achieved by: (i) wounding the leaf lobe explant and transferring the wounded leaf lobe explant into a suspension of Agrobacterium, (ii) transferring the leaf lobe explant from step (i) to a culture medium comprising at least 4 mg/L 2,4-D and at least 0.3 mg/L CuS04 to obtain callus; (iii) co-cultivating the leaf lobe
explant of step (ii) with Agrobacterium in suspension for a period of 1 to 5 days;

b. selecting the transformed plant material derived from step (a), by transferring the
leaf lobe explant to a selection medium comprising at least 4 mg/L 2,4-D, at least
0.3 mg/L CuS04 and appropriate selection agent to kill the Agrobacterium to obtain
transformed callus;

c. culturing the transformed callus on a somatic embryo induction medium comprising at least 1 mg/L BAP, at least 1 mg/L Kinetin, at least 0.05 mg/L IBA, at least 0.3 mg/L CuS04 and the appropriate selection agent to obtain transformed somatic
embryo;

d. culturing the transformed somatic embryo on a nutrient medium comprising at least 1 mg/L BAP, at least 150 mg/L Myo-inositol, at least 0.3 mg/L CuS04 and the
appropriate selection agent for 15 to 30 days to obtain matured transformed somatic
embryo; and,

e. culturing the matured transformed somatic embryo on a medium comprising at least 0.1 mg/L BAP, at least 0.3mg/L CuS04 and the appropriate selection agents for 10 to 30 days to obtain transformed cassava shoots.

6. A process of transforming a cassava plant, wherein said process comprises

a. transforming somatic cotyledonary explants obtained from somatic embryo of
cassava with DNA sequences via a vector or direct gene transfer to produce
transformed plant material, wherein transformation is achieved by: (i) culturing the
somatic cotyledonary explants on a culture medium comprising at least 1 mg/L
BAP, at least 150 mg/L Myo-inositol and at least 0.3 mg/L CuS04 for 1-3 days (ii)
co-cultivating the somatic cotyledonary explants from step (i) with the
Agrobacterium for 1 to 5 days;

b. selecting the transformed plant material derived from step (a) by transferring the
somatic cotyledonary explants of step (a) to a medium comprising at least 1 mg/L
BAP, at least 150 mg/L Myo-inositol, at least 0.3mg/L CuS04 and appropriate
agents to kill the Agrobacterium and incubating for at least 7 days,

c. transferring the somatic cotyledonary explants from step (b) to selection medium
comprising of atleast 0.1 mg/L BAP and atleast 0.3 mg/L CuS04 and the appropriate
selection agents to obtain transformed shoot clusters,

d. transferring the transformed shoot clusters of step (c) to selection medium
comprising at least 0.1 mg/L BAP and at least 0.3 mg/L CuS04 and the appropriate
selection agents to obtain elongated shoots.

7. The process as claimed in claim 5 or 6, wherein the process further comprises culturing the shoot on a rooting medium comprising half strength N6 medium and the appropriate selection agents until a root system is developed thereon.

8. The process as claimed in claim 5 or 6, wherein the DNA sequences comprises at least one gene selected from a group consisting of selectable marker gene, reporter gene, insecticidal gene, herbicide resistance gene, disease resistant gene, and agronomically important gene.

9. The process as claimed in claim 8, wherein the selectable marker gene is an antibiotic resistance gene selected from a group consisting of nptll, or hptll.

10. The process as claimed in claim 8, wherein the reporter gene encodes GFP or GUS.