Title of Invention	A METHOD FOR PRODUCING PROTOPLASTS OF CASSAVE OR A CLOSELY RELATED SPECIES
Abstract	ABSTRACT 1065/MAS/97 A method for producing protoplasts of cassave or a closely related species The present invention relates to a method for producing protoplasts of cassave or a closely related species, which protoplasts are capable of regeneration into plants, comprising producing friable embryogenic callus from explants of cassave or a closely related species and isolating protoplasts from said friable embryogenic callus.

Title of Invention

A METHOD FOR PRODUCING PROTOPLASTS OF CASSAVE OR A CLOSELY RELATED SPECIES

Abstract

ABSTRACT 1065/MAS/97 A method for producing protoplasts of cassave or a closely related species The present invention relates to a method for producing protoplasts of cassave or a closely related species, which protoplasts are capable of regeneration into plants, comprising producing friable embryogenic callus from explants of cassave or a closely related species and isolating protoplasts from said friable embryogenic callus.

Full Text	A method for producing protoplasts of cassave or a closely related species. Genetic modification or transformation is the technique whereby one or a few 96(18(8) are added to a commercial interasting genotype or done. In principle a succesful transformation system requires an efficient system where new plants are formed from specific plant parts (stem, leaf, node and so on) or from protoplasts (single cells without cell wal) derived of these parts, a system to transfer DNA molecules to the plant' s parts or protoplasts and a system to select tissue and plants which contain and express the introduced gwie(s). In principle protoplasts are the most ideal system for DNA delivery. They can be cultured as single cells that produce multicellular colonies from which plants develt^. Plants derived from protoplasts are gwierally clonal in origin. This provides a useful tool for any transformation system, because it will eliminate chimerism in transgenic plants. Cassava is very recalcitrant for plant regeneration of protoplasts. There is only one repffll of shoot regeneration from protoplasts of cassava (Shahin and Shephard, 1980), They used well expanded leaves for the isolation of protoplasts. Despite considerable effort?, plant regeneration from protoplasts (isolated ^m leaves, stems, and roots) has never been repeated since then (Anonymus, 1985; Nzoghe, 1991; Anthony etal-, 1995, Sofiari, 1996). A logical approach was to use tissues which contain embryogenic cells. Such cells are found in the apical meristems, young leaves or somatic embryos cultured on auxin supplemented media (Stamp and Henshaw, 1987a; Raemakers et al,, 1993a). However, protoplasts isolated from these tissues gave in the best case green callus and adventitious roots (Sofiari, 1996). Recently, a new type of somatic embryogenesis was developed. In this in vitro system the embryos do not develop beyond the (pre-)gl(rt)ular stage and the embryogenic callus is highly friable (Taylor et al,, 1995), Transfer of this friable embryogenic callus (FEC) to liquid medium resulted in a suspension-like culture. In leek (Buitenveld and Creamers, 1994), petunia (Power et al„ 1979), rice (Kyozuka et al,, 1988), sugarcane (Chen, et al., 1988), and wheat (Chang et al., 1991) such cultures were an excellent source for protoplast regeneration. We have now found that in cassava FEC is the only tissue from vvhich protoplasts can be isolated which are abia to regerierate into plants aofar. Thus the present invention provides a method for producing protoplasts of cassave or a closely related species, which protoplasts are capable of regeneration into plants, comprising producing friable embryogenic callus from explants of cassave or a Closely related species and isolating protoplasts from said friable embryogenic callus. It appears, as will be described below, that for obtaining suitable protoplasts the culture in solution of the FEC is quite important. Therefore the present invention further provides a method vwhereby the friable embryogenic callus is subjeclMl to culture in a liquid medium. Accordingly the present invention provides a method for producing protoplasts of cassave or a closely related species, which protoplasts are capable of regeneration into plants, comprising producing friable embryogenic callus from explants of cassave or a closely related species and isolating protoplasts from said friable embryogenic callus. Protoplasts are preferably produced by subjecting ptant cells to enzymatic breakdown of the cell wads. The invention thus provides a method whereby a mixture of cell wall degrading enzymes, such as a cetlulase. a pectolyase and/or a macerozyme are used to produce protoplasts, It also appears that the method according to the invention works best when the plants from which expiantsare to be taken are pretreated. Therefore the invention provides a method whereby the plants from which explants are taken are pretreated with an auxin as described below. On the explants preferably embryogenesis is induced resulting in an invented method whereby the friable embryogenic callus is produced from torpedo shaped primary or mature embryos. The reason is explained in the detailed description. Protoplasts obtainable by a method as disclosed above are also part of the invention. An important reason for wanting to have protoplasts which can be regenerErted into plants is of course that protoplasts can be easily tranformed or transduced or provided with additional genetic information by any other suitable method. Thus one Is now able to provide cassave plants or closely related species with genetic material of Interest. The invention thus also provides a method for transforming (defined as providing with In any suitable manner) a protoplast of a cassave or a closely related species by providing said protoplast with additional genetic infonnation through infection by a bacterium comprising said additional genetic infomiation such as Agrobaderium tumefaciens, by electroporation or chemical poration providing a vector comprising said additional genetic infomiation or by paricle bombardment whereby the particles are coated with the additional genetic information, whereby a protoplast obtainable from friable embryogenic callus is transfomned. The invention also encompasses transformed protoplasts obtainable by such a method. Below a short introduction is given on the usefulness of transforming plants, such as cassave. Application of plant gene technology encompasses a multitude of different techniques ranging from isolation of useful genes, their characterization and manipulation, to the reintroduction of modified constructs into the plants (Lonsdale, 1967). Plant gene technology will catalyze progress in plant breeding, as is exemplified by a few examples of transgenic crops like rice (Chen et al., 1987; Shimamotoetal., 1989), maize (Gordon-Kamm et a(., 1990; Vain etal-, 1993), wheal (Marks et a)., 1989), and potato (De Block, 1988; Visser et al., 1989). Rapid progress in gene technology has allowed insight Into the complex molecular mechanism of plant pathogen recognition and the natural defence strategies of host plants. This technology can also be used for controlled and efficient identification of desirable genotypes, far beyond the possibilities of classical breeding. For instance electroporation of protoplasts derived from suspension cultures led to the transformation of maize (Rhodes et at., 1988), rice (Toriyama et al., 1988) and orchardgrass (Horn et al., 1988). Successful attempts have been made to improve resistance against pathogenic viruses like tobamovirus in tobacco (Powel Abel et al., 1986), potexvirus in potato (Hoekemaetal., 1989) and in papaya (Fitch etal., 1992). In the above examples the introduced trait was based on expressing of single genes that are coding for the coat protein. In cassava, African cassava mosaic virus (ACMV) and cassava common mosaic virus (CCMV) may be controlled by the coat protein-mediated resistance technique {Fauquet et al., 1992). The genes encoding key enzymes of cyanogenesis have been cloned (Hughes et al., 1994) which makes manipulation of cassava cyanogenesis by genetic transformation using the antlsense approach feasible. Another embodiment of the invention is the manipulation of starch in the cassava tubers. Thus the present invention provides a transformed protoplast whereby the additional genetic infonmation comprises an antisense construct, particularly one wtiereby the antisense construct is capable of inhibiting the amylose synthesis pathway. A protoplast cannot grow on the field, nor can it be harvested. Though protoplasts are necessary for transformation, it must be possible to regenerate said protoplasts into embryos and/or plants. This is a very important embodiment of the invention, because cassave has been shown to be difficult to regenerate from protoplasts. The detailed description explains how this may be achieved. For further infonmation reference is made to the thesis written by E.Sofiari titled Regeneration and Transformation of Cassava (Manihot Esculenta Crantz.), a copy of which is enclosed with the present application, which is as yet unpublished and which is incorporated herein by reference. Thus the invention provides a method for regenerating plants from protoplasts whereby a protoplast according to the invention is induced to produce an embryo, which embryo is consequently induced to produce a plant. The plants obtainable by said method are aslo part of the invention, in particular plants whereby the tubers contain essentially no amyiose. Detailed description. Initiation of fee The procedure to obtain FEC is outlined in Figure 1. It starts with the induction of primary embryos. Primary embryos are formed In a two step procedure. In the first step explants are cultured on medium supplemented with salts and vitamins (preferably Murashlge and Skoog (1962)), a carbo hydrate source (for example 20 g/l sucrose) and an auxin (e.g. 1-8 mg/1 picloram, or dicamba or 2,4-D) for the initiation of embryos. After 10 to 15 days on this first medium bipolar torpedo shaped embryos are formed. Torpedo shaped embryos posess a clear hypocotyl and cotyledon primordia. After transfer of the explants with torpedo shaped embryos to a step 2 medium (the same medium as step 1 but \Mthout a auxin) the torpedo shaped embryos become mature. Mature embryos possess large green cotyledons. Zygotic embryos (Stamp and Henshaw 1982; Konanetal., 1994), young leaf explants or apical meristems (Stamp and Henshaw, 1987a; Szabados et al., 1987; Mroginsky and Scocchi, 1993; Raemakers 1993a; Narayanaswamy et al., 1995) and floral tissue (Mukherjee 1995) can be used to obtain primary embryos. In this way many different genotypes were evaluated for their ability to form primary embryos. In this protocol primary somatic embryos were only formed after culture on solid medium and never after culture in liquid medium. Furthermore, somatic embryos (primary) were only observed if the auxins Picloram, Dicamba or 2,4-D were used and not with lAA, IBA or NAA. In the presently used protocol there is genotypic variation in the number of mature embryos formed per cultured explant. The genotypes M.Coll505, M.Col22 and Gading gave the highest numbers of mature embryos per cultured leaf explant (ME/CLE). However, the number of mature embryos formed was low. In M.Col22 a maximum of 22 % of the leaf explants isolated from in vitro grown plants and cultured on a step 1 medium with 4 mg/12,4-D, fonned ME with a maximum numtier of 0.8 ME/CLE, On a step 1 medium with 8 mg/1 2,4-D a maximum of 49 % of the leaf explants formed ME with a maximum number of 3.5 ME/CLE. Higher 2,4-D concentrations did not further improve the embryogenic capacity of explants. In an attempt to improve the capacity of leaf explants to produce primary somatic embryos, donor plants were grown under different conditions. Growth of in vitro donor plants under different light regimes (8, 12, 16 or 24 hours) had no influence on the embryogenic response. However, a reduction of the fight intensity had a positive effect. The best results were obtained with leaf explants isolated of donor plants grown at 8 uEm'^s'^ and cultured on a step 1 medium. Other investigators have shown that in certain genotypes, Dicamba (1-66 mg/l) and Picloram (1-12 mg/l) are superior to 2,4D for inducing primary embryogenesis (Ng 1992; Sudarmonowati and Henshaw, 1993; Taylorand Henshaw, 1993). Mathews et al. (1993) improved the efficiency of primary embryogenesis in the genotype M. Col 1505 by transfen"ing explants after 15 days of step 1 medium to a grov^rth regulator-free medium supplemented with 0.5% charcoal. On this medium maturation was improved and as a result the number of mature embryos increased from 0.4 in the control to 3.4 ME/CLE. The best results were obtained if donor plants were pretreated with auxins as 2,4-D or picloram or Dicamba. For this, plants were grown in liquid MS20 medium and suppUed with the auxin (final concentration 8 mg/l) after 12 days of growth. Two days later leaf explants were isolated of the donor plants and cultured on step 1 medium with 8 mg/l 2,4-D, picloram or Dicamba. In the clone M.Col22 this resulted in a production of 9.4 ME/CLE. This was significantly higher than in the HjO-treated control-plants, where 3.5 ME/CLE were produced (Table 3). The general applicability of the auxin pretreatment was tested on several different genotypes. Without a pretreatment of donor plants two genotypes formed ME and at low frequency. After a pretreatment of donor plants, leaf explants of allmost all genotypes formed ME. Eventually we were abte to obtain mature primary somatic embryos from 18 of the 22 tested genotypes (Table 1, except TMS30221, TMS30001, TMS30572 and Sao Paolo). Primary somatic embryos derived from zygotic embryos and from leaves have been used as explants to initiate secondary embryos (Stamp and Henshaw, 1987b; Szabados et al„ 1987; Mathews etal., 1993; Raemakerset al., 1993bc; Luong et al., 1995). Continuous culture of somatic embryos on auxin supplemented medium resulted in a cyclic system of somatic embryogenesis. The way of subculturing somatic embryos for secondary embryogenesis seemed to influence the morphology of the embryogenic tissue. Clumps of somatic embryos recultured monthly on solid 2,4D containing medium in the dark deveioped into finger-like embryo initials formed on the top of older embryos. The embryos did not pass the torpedo-shaped stage. Further development oaajrred if the clumps with embryos were transferred to step 2 medium in the light (Szabados 1987). Normally mature somatic embryos were cultured in step 1 medium in the light and twenty days later the explants were transferred to step 2 medium for maturation. In this system embryos developed to maturity and mature embryos with large green cotyledons were used to start a new cycle of embryogenesis whereas in the system of others torpedo shaped embryos were used to start a new cycle of secondary somatic embryogenesis. This system of multiplication of mature embryos has been tested In 14 of the in Table 1 mentioned genotypes. Despite the fact that in most genotypes only a few mature primary embryos were available, all genotypes, except one, gave new mature embryos after culture on 2,4-D supplemented medium, in a much higher frequency as observed for primary somatic embryogenesis. Embryogenicity was maintained by regular subculture of mature embryos for more than one year {Sza-badosetal., 1987; Mathews etal., 1993; Raemakers, 1993). New somatic embryos were formed both in liquid and solid medium. In all the genotypes it was observed that in liquid medium more embryos were formed than in solid medium and that fragmentation of embryos before the start of a new cycle of secondary somatic embryogenesis increased the production compared to whole embryos. In for example M.Cpl22 whole embryos cultured on solid medium produced 8 embryos per cultured embryo, whereas fragmented embryos cultured in liquid medium produced 32 embryos per cultured embryo (Raemakers et al., 1993c). Not only, 2,4-D, Picloram and Dicamba, but also NAA had the capacity to induce secondary embryogenesis. IBA and lAA did not induce secondary embryogenesis. NAA has been used successfully in Adira 1, Adtra 4, Gading, Line 11, M.Col22, M.CoH 505, TMS90853 and Gading. In general more mature embryos were produced in NAA supplemented medium than in 2,4-D, Picloram or Dicamba supplemented medium. Furthermore, the development of NAA induced embryos was faster than with 2,4-D, Dicamba or Picloram. Shortening the culture duration has a beneficial effect, particularly, when operating on a large scale. Histologically, the by 2,4-D newly induced secondary embryos were attached vertically to the explants whereas those by NAA were horizontally. There is still a problem in obtaining embryogenic cultures of cassava (Mroginski and Scocchi, 1992; Taylor etal., 1992; Narayanaswamy et al., 1995; Sudarmonowati and Bachtiar, 1995). The main problem is not that embryogenic tissue from primary explants can be obtained, but the large scale multiplication of this tissue by secondary embryogenesis. For this purpose, either tissue consisting of torpedo shaped embryos or mature embryos can be used. Multiplication of torpedo shaped embryos is highly genotype dependent, while multiplication of mature embryos is largely genotype independent (Raemakers, 1993). Both primary and secondary somatic embryogenesis are characterized by the formation of propagules with a bipolar structure. These bipolar torpedo shaped embryos are already formed on the auxin supplemented step 1 medium. Therefore, Taylor et al., (1995) proposed the term organized embryogenesis. Organized cells are defined as a group of actively dividing cells, having the tissues and organs formed into a characteristic unified whole {Walker, 1989), A less organized type of somatic embryogenesis was developed by Taylor et al. (1995). With continuous selection, organized embryogenic tissue cultured on a Gresshoff and Doy (1972) medium salts and vitamins supplemented with 10 mg/l Picloram (GD2) converted gradually into a less organized tissue. This tissue consisted of a caltus-)ike mass of (pro-)globuIar embryos which was very friable. Therefore, this tissue was called friable embryogenic callus (FEC). The cells in FEC are continuously in a state where they break away from group control and because of that they are not organized into a unified structure. FEC is maintained on a medium consisting of Gresshoff and Doy (1972) vitamins and salts, 7 g/l Daichin agar, 20 g^ suaose and 10 mg/l Pidoram (solid GD2). Every three weeks the friable embryos were subcultured on the above mentioned medium. In order to initiate liquid suspension cultures 0.5 g of friable embryos was transferred in a flask of 200 ml with 50 mi of liquid medium supplemented with Schenk and Hildebrandt (1972) salts and vitamins, 60 g/l sucrose and 10 mg/l Picloram (liquid SH6). The medium was refreshed every 2 days and after 14 days the content of each flask was divided over 5 new flasks. The pH was adjusted to 5.7 before autoclaving. The temperature in the growth chamber was 30°C, the photoperiod 12 hours and the irradiance 40 pmolm"V\ Suspension cultures were initiated by culturing FEC in Schenk and Hildebrandt (1972) medium supplemented with 6 % (w/v) sucrose and 10 mg/l Picloram (SH6). Every 2-3 days this medium was refreshed. To keep a culture in a highly friable state the FEC has to be sieved once in two monts. In practice the part of the FEC which will go through a sieve with a mesh of 1 mm^ vwll be used for subculture. FEC will allmost never form torpedo shaped embryos on the GD2 or in SH6 medium. Torpedo shaped and subsequent mature embryos are formed if FEC is cultured on maturation medium. Maturation medium consist of Murashige and Skoog (1962) salts and vitamins, 0.1 g/l myo-inositol, 20 g/l sucrose, 18.2 g/l mannitol, 0.48 g/l MES, 0.1 g/l caseinhydrolysate, 0.08 g/l adenine sulphate, 0.5 mg/l d-calcium-panthotenate, 0.1 mg/l choline chloride, 0.5 mg/l ascorbic acid, 2. mg/t nicotinic acid, 1 mg/l pyridoxine-HCI, 10 mg/l thiamine HCI, 0.5 mg/l folic acid, 0.05 mg/l biotin, 0.5 mg/l glycine, 0.1 mg/l L-cysteine, 0.25 mg/l riboflavine and 1 mg/l picloram. TTiis maturation medium was refreshed every 3 weeks. Mature embryos could be induced into secondary somatic embryogenesis by culturing on MS20 medium supplemented with 2,4-D, picloram, Dicamba or N/VA. Primary and secondary somatic embryogenesis are relatively easy to establish in a vwde range of genotypes (see table 1), while FEC is for the time being restricted to a few genotypes. The prospect of FEC for a new system of somatic embryogenesis and genetic transformation is promising, although further research Is needed to make this system applicable to more genotypes. Essential for this process is the availability of high quality organized tissue and the ability of this tissue to convert into FEC. Tayior et al. (1995) "used organized tissues" which were multiplied at the torpedo shaped state to initiate FEC. In this case two steps (initiation of organized tissue and conversion into unorganized tissue) are determinative for the successful initiation of FEC. Both steps are genotype dependent. If organized tissue is multiplied in the mature state as described by Raemakers (1993) then only the ability of this tissue to convert into FEC is a determinative step to initiate FEC. It remains to be investigated whether or not organized tissue can be used as starting material. If organized tissue cannot be used, then this tissue should be first multiplied in the immature state before it can be used to initiate FEC. This is readily accomplished by, either culturing explants at a high density or by reducing the cyclic duration. Regeneration of plants from protoplasts Isolation of protoplasts For protoplast isolation both FEC cultured on solid GD2 or liquid SH6 can be used. However, the highest yield of protoplasts was obtained from FEC which has been cultured for 1 to 3 weeks in liquid SH6. Two gram of FEC was placed in Petri dishes ($ 9 cm) containing 10 ml of cell wall digestion solution. Cell wall digestion solution consisted of a mixture of cell wall degrading enzymes;growth regulators (NAA 1 mg/l, 2,4-D 1 mg/l, Zeatin 1 mg/l); major salts ( 368 mg/l CaClj; 34 mg/l KHjPO^; 740 mg/KNOg; 492 mg/l MgSo^.THjO); minor salts (19.2 mg/l Na-EDTA; 14 mg/l FeSO^.THjO) and osmoticum (91 g/l D-mannitol) and 0.5 g/l MES. The cell wall degradading enzymes cellulase (1-10 g/l) plus Macerozyme (200 mg/l) were succesfull for protoplast isolation. The extra addition of Pectolyase (0.001 -0.01 g/l) and/or Driselase (0.02 g/l) increased the yield of protoplasts. After 18 h of incubation, 10 ml of washing medium was added to the solution. Washing medium with an osmolarity 0.530 mOsm/kg consisted of major salts (see cell wall digestion solution), 45.5 g/l mannitol and 7.3 g/l NaCI. The digested tissue was filtered through a 73 pM pore size filter (PA 55/34 rjybott - Svwtzeriand) into a 250 ml beaker glass. The filtrate was divided equally over two 12 ml conical screw cap tubes, and centrifuged at 600 rpm for 3 min (Mistral 2000). The washing procedure was repeated once after removal of the supernatant. The protoplast solution was resuspended by floating on 9.5 ml solution containing major and minor salts (see cell wall digestion solution) and 105 g/l sucrose. The pH was 5.8 and the osmolarity 0.650 mOsm. The solution with protoplasts was allowed to equilibrate for 5 minutes before 0.5 ml of washing medi¬um was gently added on the top. After centrifugation at 700 rpm for 15 min {Mistral 2000), the protoplasts were concentrated in a band between the sucrose and washing medium. The protoplast layer was harvested with a pasteur pipette and the yield was counted in a standard haemocytometer chamber. Protoplast culture Protoplasts were cultured in media solidified with agarose 0.2 % w/v (Dons en Bouwer, 1986) in petri dishes containing 10 ml of the same liquid medium. The following media resulted in the formation of micro callus: -7M2G medium (Welters et al., 1991) supplemented with only auxins (0.1-10 mg/l NAA or 0.1-10 mg/IPicloram, or 0.1-10 mg/l lAA, or 0.1-10 mg/l 2,4-D, or 0.1-10 mg/l Dicamba, or0.1-10mg/l, or 0.1-10 mg/I) or auxins plus cytokinins (0.01-1 mg/l zeattn, 0.01-1 mg/l 2-iP. 0.01-1 mg/l BA, 0.01-1 mg/l TDZ, 0.01-1 mg/l krnetin). -medium A (Murashige and Skoog (1962) salts and vitamins, 4.5 g/l myo-inositol, 4.55 g/l mannitol, 3.8 g/l xylitol, 4.55 g/l sorbitol, 0.098 g/l MES, 40 mg/I adeninsulp-hate and 150 mg/l caseinhydrolysate, 0.5 mg/l d-calcium-panthoter^ate, 0.1 mg/l choline-chloride, 0.5 mg/ ascorbic acid, 2.5 mg/l nicotinic acid, 1 mg/l pyridoxine-HCI, 10 mg/l thiamine-HCI, 0.5 mg/l folic acid, 0.05 mg/l biotine, 0.5 mg/l glycine, 0.1 mg/l L-cysteine and 0.25 mg/l riboflavine and 59.40 g/l glucose) supplemented with only auxins (0.1-10 mg/l NAA or 0.1-10 mg/IPicloram, or 0.1-10 mg/l lAA, or 0.1-10 mg/l 2,4-D, or 0.1-10 mg/l Dicamba plus cytokinins {0.01-1 mg/l zeatin, 0.01-1 mg/l 2-iP, 0.01-1 mg/l BA, 0.01-1 mg/l TDZ, 0.01-1 mg/l kinetin). The media were refreshed every 10 days, by replacing 9 ml with fresh medium. After two months of culture in the first medium, high quality FEC was selected and either cultured for further proliferation or for maturation. For proliferation FEC was transferred to Gresshoff and Doy (1974) medium supplemented with 40 g/l sucrose, 7 g/l Daichin agar and 2 mg/l picloram (GD4). After 3 weeks the FEC was transferred to a Gresshoff and Doy medium supplemented with 20 g/l sucrose, 7 g/l agar and 10 mg/l Picloram (GD2). Suspension cultures were initiated by transferring 1.0 g of FEC to liquid SH6% medium supplemented with 10 mgfl Picloram. Two weeks later the suspension was divided over new flasks with an initial packed cell volume of 1.0 ml. After 2 months of culture, 10^ protoplasts cultured in TM2G supplemented with 0.5 mg/l NAA and 1 mg/l Zeatin at a density of 10^/ml produced 1058 micro-calli, v\iereas 10^ protoplasts cultured at a density of 10^/ml only produced 64 micro-calli. Replacing TM2G medium with medium A reduced at both densities the number of micro-calli significantly. At this stage at least three types of calli could be distinguished. One type consisted of globular shaped embryos which were mostly observed in protoplasts cultured at a density of 10^. Some of them developed cotyledon like structures, light green in colour. However, these embryos could not be germinated properly. Another type was fast growing and consisted of a large compact callus, they were observed in protoplast cultures of both densities. This callus never developed embryos. The third type was highly friable callus and was observed at both densities. At a density of 2-5 x 10^ (medium TM2G) about 60 % of the calli were friable and embryogenic. The FEC was either subcultured for further proliferation or for maturation. Proliferation of FEC derived from protoplasts Following selection of FEC, 0.1 g of it cultured for three weeks on GD 4 plus 2 mg/l Picloram inaeased into 0.7 g of tissue. More than 95 % of the tissue consisted of high quality FEC. Subsequently, this tissue was maintained by subcultures of three weeks on GD2 medium supplemented with 10 mg/l Picloram. To initiate suspension cultures FEC was transfe^ed to liquid medium. The increase in packed cell volume (PCV) of this material was slightly higher than that of the original material (data not shown). Maturation of FEC derived from protoplasts In an attempt to induce maturation of embryos, FEC isolated after two months of culture in TM2G was cultured on maturation medium. Maturation medium consisted of Murashige and Skoog (1962) salts and vitamins, 0.1 g/l myo-inositol, 20 g/l sucrose, 18.2 g/l mannitol, 0.48 g/t MES, 0,1 g/l caseinhydrolysate, 0.08 g/l adenine sulphate, 0.5 mg/l d-calcium-panthotenate, 0.1 mg/l choline chloride, 0.5 mg/l ascorbic acid, 2. mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/t thiamine HCl, 0.5 mg/l folic acid, 0.05 mg/l biotin, 0.5 mg/l glycine, 0.1 mg/l L-cysteine, 0.25 mg/l riboflavine and 1 mg/l picloram. This maturation medium was refreshed every 3 weeks. On this medium there is a gradual shift from proliferation to maturation. As a result the packed eel) volume had increased with a factor 4 after two weeks of culture in liquid maturation medium. Also after transfer to solid maturation medium there is proliferation. After two weeks on solid medium most of the embryos had reached a globular shape and only a few of these globular embryos developed further. The first torpedo shaped embryos became visible after one month of culture on solid maturation medium. The number of mature and torpedo shaped embryos was not correlated with the plating efficiency but with the density of the initially cultured protoplasts. No such embryos were obtained if protoplasts were cultured on TM2G without growth regulators. The highest number of mature and torpedo shaped embryos was formed from protoplasts cultured on TM2G supplemented with 0.5 mg/l NAA and 1 mg/l Zeatin. If NAA was replaced by Picloram than the number of torpedo shaped and mature embryos was significantly lower (Table 2). From the tested Picloram concentrations 2 mg/l gave the best results. After 3 months of culture between 60 and 200 torpedo shaped and mature embryos were isolated per agarose drop. Torpedo shaped embryos became mature at high frequency if they were cultured on fresh maturation medium or on MS2 plus 0.1 mg/l BAP. Secondary somatic embryogenesis and germination of mature embryos derived from protoplasts Only a few torpedo shaped embryos formed secondary embryos if cultured on liquid or solid MS2 medium supplemented with 10 mg/l NAA or 8 mg/l 2,4-D (data not shown). Mature embryos were better explants for secondary embryogenesis. In both liquid and solid medium 2,4-D was superior for induction of secondary embryogenesis as compared to NAA. If mature embryos were first cultured in 2,4-D and than in liquid NAA the response was comparable with culture in 2,4-D alone. Also embryos which first had undergone a cycle of secondary somatic embryogenesis in medium with 2,4-D, produced highly efficient secondary embryos In MS20 supplemented with 10 mg/l NAA. The germination of cyclic or secondary somatic embryos, induced In liquid medium by the auxins 2,4-dichlorophenoxyacetic acid (2,4-0) or naphthalene acetic acid (NAA), was compared. In all genotypes desiccation stimulated normal genmination of NAA Induced embryos. However, the desiccated embryos, required a medium supplemented with cytoklnins such as benzylaminopurine (BAP) for high frequency germination. The morphology of the resulting seedling was dependent on the concentration of BAP. With 1 mg/l BAP plants with thick and short taproots and branched shoots vwth short internodes were formed. With 0.1 mg/l BAP the taproots vrere thin and slender and the shoot had only one or two apical meristems. If the embryos vrere desiccated sub-optimally, higher concentrations of BAP were needed than if the embryos were optimally desiccated to stimulate germination. Also desiccated embryos which were cultured in the dark required a lower concentration of BAP and, furthermore, these embryos germinated faster than embryos cultured in the light. Complete plants were obtained four weeks after the start of somatic embryo induction. 2,4-D induced embryos showed a different response. In only one genotype desiccation enhanced germination of 2,4-D induced embryos and in three other genotypes it did not. In all genotypes desiccation stimulated root formation. Embryos cultured in the dark formed predominantly adventitious roots, whereas embryos cultured in the light formed predominantly taproots. 4.0 Gene transfer systems Over the past years several transfer techniques of DNA to plant protoplasts have been developed such as silicon fibers {Kaeppleret al., 1990), microinjection (DeLaat and Blaas, 1987) and electrophoresis (Griesbach and Hammond, 1993), The most commonly used and potentially-applicable ones are Agrobacterium-mediated gene delivery, microprojectile/particle bombardment and protoplast electroporation. The Agrobacterium tumefadens DNA delivery system is the most commonly used technique. It probably relates to the first invention of DNA delivery in plants by this method. Initially it was limited to Kalanchoe and Solanaceae, particularly tobacco. Nowadays, the use of Agrobacterium-mediatedi transformation has changed dramatically, it is possible to transform a wide range of plants with a limitation in monocots (reviewed by Wordragen and Dons, 1992). Although cassava is a host for Agrobacterium it has proven to be not highly amenable to it. In principle protoplasts are the most ideal explants for DNA delivery. They can be cultured as single cells that produce multicellular colonies from wtiich plants develop. Plants derived firom protoplasts are generally clonal in origin. This provides a useful tool for any transformation system, because it will eliminate chimerism in transgenic plants. The use of protoplasts is, however, hampered by the regeneration system which is highly species dependent. For transformation, protoplasts can be used in conjunction with PEG to alter the plasma membrane which causes reversible permeabilization that enables the DNA to enter the cytoplasm as was demonstrated, for example, in Lolium multiform {Potrykus et al., 1985) and Triticum monococct/m (Lorzetal., 1985). Another technique to inaease the permeability of plasma membranes and even cell walls to DNA is by electroporation (for review see Jones et al., 1987). In this method electrical pulses enable the DNA to enter the cells. Rice was the first crop In which fertile transgenic plants resulted from protoplast electroporation (Shimamoto et al., 1989). The use of particle bombardment or biolistics to deliver foreign DNA provides an alternative method in cassava transformation. Particle bombardment is the only procedure capable of delivering DNA into cells almost in any tissue. The first transgenic plant obtained by using this method was in tobacco (Klein et al., 1989). Follovwng this successful transformation method, particle bombardment is widely used in plants which are less amenable to Agrobacterium infection, particulariy monocots. Improvement of several DNA delivery devices to accelerate the particle (microprojectile) has resulted in the most recent model the Biolistic ™ PDS-1000 {Bio-Rad Laboratories, Richmond, Ca). Those devices are available commercially, however the price is relatively high at present. Tungsten or gold particles, coated with DNA, are commonly used as microprojectiles to deliver DNA into the target tissue (recently reviewed by Songstad et al., 1995). 5. Selection and reporter genes used in genetic modifications To be able to identify transformed cells, the gene of interest is coupled to a selectable marker gene. This mariner gene is necessary to select transformed cells. Selection can be based on a visual characteristic of the transformed cellftissue. An example is the luciferase gene isolated from the firefly. Plant cells expressing this gene and supllied with substrate (luciferin) will emit light which can be detected with special equipment (Ow et al., 1986). Another way to select transfomied tissue is the introduction of a gene* encodes resistance to antibiotics or herbicides (Thompson et al., 1987; Gordon-Kamm et al., 1990). *which A number of antibiotics and herbicides has been used as selective agent in plant transformation. In cereals resistance to the herbicide phosphinothricin (PPT) was chosen for the selection of transgenic plants {Cao et a!., 1990). In Carica papaya (Fitch et ai., 1994), Vitis vinifera (Nakano et al., 1994; Scorza et al., 1995), maize (Rhodes et al., 1988) and rice (Chen et al., 1987) the neomycine phosphothansferase (NPTll) gene, which confers resistance to kanamycin and related antibiotics (Fratey et al., 1986), was used as a selectable marker. In cassava all above- mentioned systems of selection can be used, however PPT based selection has as advantage that it improves the ability of FEC to form mature embryos and in this way increase plant regeneration. legend to figure 1 Figure 1: Schematic representation of somatic embryogenesis in cassava, including primary, secondary somatic embryogenesis, selection of friable embryogenic callus, maturation and desiccation follo>A«d by germination. gd2= medium supplemented with Gresshoff and Doy salts (1974) and vitamins plus 20 g/l sucrose. gd4= medium supplemented with Gresshoff and Doy salts (1974) and vitamins plus 40 g/l suaose. ms2= medium supplemented vwth Murashige and Skoog salts and vitamins plus 20 g/l sucrose. pic= 10 mg/l Picloram, N/\A=10 mg/l naphthalene acetic acid, 2,4-D=8 mg/l, 2,4- dichlorophenoxy acetic acid. sh6= medium supplemented with Schenk and Hildebrandt (1972) salts and vitamins plus 60 g/l sucrose. Table 1. Genotypes of cassava used for somatic embryogenesis. Table 2. Influence of light intensity during growth of donor plants in vitro on the number of leaf explants responding with the formation of mature embryos and the number of mature embryos per cultured leaf explant (#ME/CLE)- Table 3. Influence of 2,4-D pretreatment on production of primary mature embryos (# mature embryos per cultured leaf explant isolated from in vitro plants), followed by the multiplication of mature embryos by secondary somatic embryogenesis in 11 Nigerian cassava genotypes and in M.Col22. Cited literature Anonymus, 1985, ClAT: Annual report: Centro International de Agricultura Tropical, Cali, Columbia, Pp: 197-217. Anthony, P., Davey, MR., Power, J.B., and Lowe, K.C. 1995. An improved protocol for the culture of cassava leaf protoplasts. Plant Cell Tissue and Organ Culture. 42:229-302 Buiteveld, J., and Creemers-Molenaar, J, 1994. Plant regeneration from protoplasts isolated from suspension cultures of leek (Allium ampeloprasum L) Plant Science. 100:203-210, Cao, J., Duan, X., McEh-oy, D,, and Wu, R. 1990. Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Ceil Rep, 11: 586-591. Chang, Y.F., Wang, W.C, CoUeen, Y.W., Nguyen, H.T., and Wong, J.R. 1991. Plant regeneration from protoplasts isolated from long-term cell cultures of wheat {Triticum aestivumy Plant Cell Rep. 9:611-614. Chen, W H, Davey, MR., Power, J.B., and Cocking, EC. 1988. Sugarcane protoplasts: factors affecting division and plant regeneration. Plant Cell Rep. 7:344-347. Chen, W.H., Gartland, K.M.A., Davey, MR., Sotak, R., Gartland, J,S., MuUigan, B.J,, Power, J.B., and Cocking, E.C, 1987. Transformation of sugarcane protoplasts by direct uptake of a selectable chimeric gene. Plant Cell Rep. 6:297-301. DeLaat, A., and Blaas, J., 1987. An improved method for protoplast microinjection suitable for transfer of entire plant chromosomes. Plant Sci 50:161-169. Fitch, M.M.M., Pang, S,Z., Slightom, Lius. S., Tennant, P., Manshardt, R.M., and GonsaJves, D. 1994. Genetic transformation in Caricapapaya (Papaya). In: B^^ (Eds). Biotechnology in Agriculture and Forestry Vol 29, Plant protoplasts and genetic engineering V. Springer-Verlag,BerUn.P:237-255. Dons, J.J.M., and Bouwer, R. 1986. Improving the culture of cucumber protoplasts by using an agarose-disc procedure. Proceedings of an international symposmm on nuclear techniques and in vitro culture for plant improvement. Jointly organized by the International Atomic Energy Agency and the Food and Agriculture Organization of the United Nations. Held in Vienna, 19-23 August 1985. P:498-504. Fraley R.T., Rogers S.G., and Horsch, R.B. 1986. Genetic transformation in higher plants. CRC Critical reviews in Plant Sciences 4(1): 1-46. Gordon-Kamm, W J., Spencer, T.M., Mangano, MR., Adams, T.R., Daines, R-J, William, as., O'Brien, J V., Chambers, S.A. Adams, Jr W.R., Willetts, N G, Rice, T.B., Mackey C J, Krueger, R.G., K^sch, A.P,, and Lemaux P.G. 1990, Transformation of maize cells and regeneration of fertile transgenic plants. The Plant Cell. 2:603-618. Gresshoff, P.M , and Doy, C,H, 1974 Development and difFerentiation of haploid Lycopersicon esailentum (tomato). Planta 107:161-170. Griesbach, R.J,, and Hammond, J. 1993 Incorporation of the GUS gene into orchids via embryo electrophoresis. Acta, Hon. 336:165-169. Horn, M.E., Shilhto, R.D., Conger, B.V., and Harms, C,T, 1988, Transgenic plants of orchardgrass {Dactytis glomeratoL.) from protoplasts. Plant Cell Rep. 7:469-472. Jones H., TempeJaar M,/., and Jones, M.G.K. 1987. Recent advances in plant eiectroporation. Oxford Surveys of Plant Mol. and Cell Biol. 4:347-357. Kaeppler, H.F,, Gu, W., Somres, D.A., Rines, H.W., Cockbum, A.F. 1990. SUicon carbide fiber-mediated DNA dehvery into plant cells. Plant Cell Rep. 8:415-418. Klein T.M., Komstein L., Sanfords J.C, and Fromm M.E, 1989, Genetic transformation ofmaize cells by particle bombardment. Plant Physiol. 91:440-444. Konan N.K., Sangwan R.S., and Sangwan-Norrell, 1994. Nodal axillary meristems as target tissue for shoot production and genetic transformation in cassava {Manihot esculenta Crantz). Second International Scientific Meeting of Cassava Biotechnology Network 11. Bogor. Indonesia. p:276-288. Kyozuka, I, Otoo, E , and Shimamoto, K. 1988. Plant regeneration fi^Om protoplasts of indica rice; genotypic differences in culture respond. Theor. Appl. Genet. 76:887-890, Lorz, H., Baker, B., and Schell, J, 1985. Gene transfer to cereal cells mediated by protoplast transformation. Mol. Gen. Genetic 199: 178-182. Luong, H,T., Shewry, PR., and Lazzeri, PA. 1994, Gene transfer to cassava somatic embryos via tissue eiectroporation and particle bombardment. In: Second International Scientific Meeting ofCassava Biotechnology Network 11. Bogor. Indonesia. p:303-314. Mathews, H., Carcamo, R., Chavarriaga, Schfipke, C.P., Fauquet, C. and Beachy, R.N., 1993. Improvement of somatic embryogenesis and plant recovery in cassava. Plant Cell Rep. 12:328-333. Mroginski and Scocchi, 1992. Somatic embryogenesis of Argentine cassava varieties. In: Roca, W.M., and Thro, A.M. (Eds). Proceedings First Scientific Meetingg of the Cassava Biotechnolgy Network. Cartagena, Colombia 25-28 August 1992. P: 175-179. Mukherjee, A. 1994. Embryogenesis and regeneration from cassava calli of anther and leaf. The Cassava Biotechnology Network. Proceeding of the Second International Scientific Meeting. Bogor.Indonesia, 22-26 August 1994. P:375-377. Murashige, T,, and Skoog, F 1962. A revised medium for rapid growth and bioassay with tobacco cultures. Phvsiol Plantarum. 15:473-497. Nakano, M,, Hoshino, Y., and Mil, M, 1994, Regeneration of transgenic plants of grapevine (Vitis vim/era L.) via Agrobacterium rhizogenesis-mediated transformation of embryogenic calli. J. of Exp. Bot, 45 (274); 649-656. Narayanaswamy, T.C., Ramaswamy, N.M,, and Sree Rangaswamy, S.R. 1995. Somatic embryogenesis and plant regeneration in cassava. The Cassava Biotechnology Network. Proceeding of the Second International Scientific Meeting. Bogor,Indonesia, 22-26 August 1994. P:324-329. Ng SYC (1992) Tissue culture of root and tuber crops at IITA. In: ThottappUly G, Monti LM, Mohan Raj DR, Moore AW (eds). Biotechnology: enhancing research on tropical crops in Afiica CTA/ntA co-pubUcation, IITA, Ibadan, Nigeria, pp 135-141. Nzoghe, D. 1989. Recherche de conditions permettant I'obtention neoformations chez differents genotypes de manioc (Manihot esculenta Crantz). Extension a la culture de protoplastes. These. Universite De Paris Sud Centre D'Orsay. P : 119. Ow, D.W., Wood, K.V., DeLuca, M„ De Wet, JR., Hehnski, DR., and Howell, S.H. 1986. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234:856-859. Potrykus, I., Saul, M., Paskowski, J., and Shillito, R,D. 1985. Direct gene transfer into protoplasts of a graminaciousmonocot. Mol. Gen.Genet. 199:183-188. Power, J.B., Bery, S.F., Chapman J.V., and Cocking, E.G. 1979. Somatic hybrids between unilateral cross-incompatible Peft/ma species. Theor. Appl. Genet. 55: 97-99. Raemakers, C.J.J.M. 1993. Primary and cycUc somatic embryogenesis in cassava A/oni/io/ esculenta Crantz. PhD thesis Agricultural University Wageningen, The Netherlands. P: 119. Raemakers, C.J.J.M., Bessembinder, J., Staritsky, G,, Jacobsen, E., and Visser, R.G.F. 1993a. Induction, germination and shoot development of somatic embryos in cassava. Plant Cell Tissue and Organ Culture 33:151-156. Raemakers, C.J.J.M., Amari, M., Staritsky, G, Jacobsen, E., and Visser, R.G.F. 1993b Cyclic somatic embryogenesis and plant regeneration in cassava. Annals ofBot. 71:289-294. Raemakers, C.J.J.M., Schavemaker, CM,, Jacobsen, E., and Visser, R.G.F. 1993c. Improvements of cyclic somatic embryogenesis of cassava {Manihot esculenta Crantz). Plant Cell Rep, 12:226-229. Rhodes, C.A., Pierce, DA., Metier, I.J., Mascarenhas, D., and Detmer, J.J. 1988. Genetically transformed maize plants from protoplasts. Science 240:204-207, Scorza, R., Cordts, J.M., Ramming, D.W,, and Emershad, R.L. 1995. Transformation of grape {Vitis vinifera L.) zygotic-derived somatic embryos and regeneration of transgenic plants. Plant Cell Rep. 14:589-592. Shahin, E.A,, and Shepard, JF 1980. Cassava mesophyll protoplasts: isoJalion, proliferation and shoot formation. Plant Science Letters 17:459-465, Shenk, R.U., and Hjidebrandt, AC. 1972. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J.Bot. 50:99. Shimamoto, K., Terada, R., Izawa, T., and Fujimoto, H. 1989. Fratile transgenic rice plants regenerated from transformed protoplasts. Nature 338:274-276. Sofiari, E, 1996. Regoiration and transformation in cassavaMmito/ esculenta Crantz. PhD thesis Agricultural University Wageningen, The Netherlands. P: 136, Songstad, D.D., Somers, DA., and Griesbach, R.J. 1995. Advances in alternative DNA delivery techniques. Plant Cell Tissue and Organ Culture 40:1-15, Stamp, J.A., and Henshaw, G.G. l9S7a. Somatic embryogenesis from clonal leaf tissue of cassava. Annals of Bot. 59:445-450. Stamp, J.A, 1987. Somatic embryogenesis in cassava: the anatomy and morphology of the regeneration process. Annals ofBot. 59: 451-459. Stamp, J.A., and Hen^w, G.G. 1987b. Secondary somatic embryogenesis and plant regener-arion in cassava. Plant Cell Tissue and Organ Culture 10:227-233. Stamp, J.A., and Henshaw, G.G. 1982. Somatic embryogenesis in cassava. Zeitschrift fur Pflanzenphysiologje. 105:183-187. Sudarmonowati and Bachtiar, 1995. Induction of somatic embryogenesis in Indonesian cassava genotypes. The Cassava Biotechnology Network. Proceeding of the Second International Scientific Meeting. Bogor.Indonesia, 22-26 August 1994. P.364-374) Sudarmonowati, E., and G.G. Henshaw, 1992, The induction of somatic embryogenesis of recalcitrant cultivars using picloram and dicamba. In: Roca, W.M., and Thro, A.M, (Eds), Proceedings First Scientific Meetingg of the Cassava Biotechnology Network. Carugena, Colombia 25-28 August 1992, P: 128-133. Szabados L,, Hoyos R. and Roca W. 1987. In vitro somatic embryogenesis and plant regeneration of cassava. Plant Cell Rep, 6:248-251. Taylor, N,J., Clarke, M,, and Henshaw, G,G. 1992. The induction of somatic embryogenesis in fifteen African cassava cultivars. In; Roca, W.M„ and Thro, A.M, (Eds), Proceedings First Scientific Meeting of the Cassava Biotechnology Network. Cartagena, Colombia 25-28 August 1992. P: 134-137. Taylor, N.J,, Edwards, M., and Henshaw, G.G. 1995. Production of fiiable embryogenic calli and suspension culture system in two genotypes of cassava. Second International Scientific Meeting of Cassava Biotechnology Network 11, Bogor, Indonesia. P:229-240, Thompson, J.C, Mowa, N R , Tizard, R., Crameri, R , Davies, J.E., Lauwereys, M., and Bottemian, J. 1987. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus The EMBO J,6 (9):2519-2523. Toriyama, K., Arimoto, Y., Uchimiya, H., and Hinata, K. 1988. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technology. 6:1072-1074, Walker, P.M.B. 1989, Chambers Biology Dictionary. W&R Chamber Ltd, Clay Ltd, St, Ives Pic, England, P:205. Wolters, A.M,A,, Schoenmakers, H.C.H,, van der Meulen-Muiser, J,J,M,, van der Knaap, E,, Derks, F,H.M.,Koomneef, M,,andZelcer, A, 1991, Limited DNA elunination from the irradiated potato parent in fiision products of albino Lycopersicon esculentum and Solanum tuberosum. Theor i^pl. Genet. 83:225-232, Wordragen M,F.,,and Dons, H,J,M, 1992, Agrohacterium tumefaciens mediated transformation of recalcitrant crops. Plant Mol. Biol. Reporter 10:12-36. WE CLAIM: 1. A method for producing protoplasts of cassave or a closely related species, which protoplasts are capable of regeneration into plants, comprising producing friable embryogenic callus from explants of cassave or a closely related species and isolating protoplasts from said friable embryogenic callus. 2. The method according to claim 1 whereby the friable embryogenic callus is subjected to culture in a liquid medium. 3. The method according to claim 1 or 2 whereby a mixture of cell wall degrading enzymes, such as a cellulase, a pectolyase and/or a macerozyme are used to produce protoplasts. 4. The method according to any one of the aforegoing claims whereby the plants from which explants are taken are pretreated with an auxin. 5. The method according to anyone of the aforegoing claims whereby the friable embryogenic callus is produced from torpedo shaped primary or mature embryos. 6. The method according to claim 5 whereby the embryos are induced on primary explants. 7. The method according to claim 1 wherein the protoplast of cassave or a closely related species is transformed by providing with additional genetic information through infection by a bacterium comprising said additional genetic information such as Agrobacterium tumefaciens, by electroporation or chemical poration providing a vector comprising said additional genetic information or by particle bombardment whereby the particles are coated with the additional genetic information.

Full Text

A method for producing protoplasts of cassave or a closely related species.
Genetic modification or transformation is the technique whereby one or a few
96(18(8) are added to a commercial interasting genotype or done. In principle a
succesful transformation system requires an efficient system where new plants are
formed from specific plant parts (stem, leaf, node and so on) or from protoplasts
(single cells without cell wal) derived of these parts, a system to transfer DNA
molecules to the plant' s parts or protoplasts and a system to select tissue and
plants which contain and express the introduced gwie(s).
In principle protoplasts are the most ideal system for DNA delivery. They can be
cultured as single cells that produce multicellular colonies from which plants
develt^. Plants derived from protoplasts are gwierally clonal in origin. This provides
a useful tool for any transformation system, because it will eliminate chimerism in
transgenic plants.
Cassava is very recalcitrant for plant regeneration of protoplasts. There is only one
repffll of shoot regeneration from protoplasts of cassava (Shahin and Shephard,
1980), They used well expanded leaves for the isolation of protoplasts. Despite
considerable effort?, plant regeneration from protoplasts (isolated ^m leaves,
stems, and roots) has never been repeated since then (Anonymus, 1985; Nzoghe,
1991; Anthony etal-, 1995, Sofiari, 1996). A logical approach was to use tissues
which contain embryogenic cells. Such cells are found in the apical meristems,
young leaves or somatic embryos cultured on auxin supplemented media (Stamp
and Henshaw, 1987a; Raemakers et al,, 1993a). However, protoplasts isolated from
these tissues gave in the best case green callus and adventitious roots (Sofiari,
1996). Recently, a new type of somatic embryogenesis was developed. In this in
vitro system the embryos do not develop beyond the (pre-)gl(rt)ular stage and the
embryogenic callus is highly friable (Taylor et al,, 1995), Transfer of this friable
embryogenic callus (FEC) to liquid medium resulted in a suspension-like culture. In
leek (Buitenveld and Creamers, 1994), petunia (Power et al„ 1979), rice (Kyozuka
et al,, 1988), sugarcane (Chen, et al., 1988), and wheat (Chang et al., 1991) such
cultures were an excellent source for protoplast regeneration.
We have now found that in cassava FEC is the only tissue from vvhich protoplasts
can be isolated which are abia to regerierate into plants aofar.
Thus the present invention provides a method for producing protoplasts of cassave
or a closely related species, which protoplasts are capable of regeneration into
plants, comprising producing friable embryogenic callus from explants of cassave or
a Closely related species and isolating protoplasts from said friable embryogenic
callus. It appears, as will be described below, that for obtaining suitable protoplasts
the culture in solution of the FEC is quite important. Therefore the present invention
further provides a method vwhereby the friable embryogenic callus is subjeclMl to
culture in a liquid medium.

Accordingly the present invention provides a method for producing protoplasts of cassave or a closely related species, which protoplasts are capable of regeneration into plants, comprising producing friable embryogenic callus from explants of cassave or a closely related species and isolating protoplasts from said friable embryogenic callus.
Protoplasts are preferably produced by subjecting ptant cells to enzymatic breakdown of the cell wads. The invention thus provides a method whereby a mixture of cell wall degrading enzymes, such as a cetlulase. a pectolyase and/or a macerozyme are used to produce protoplasts,
It also appears that the method according to the invention works best when the plants from which expiantsare to be taken are pretreated. Therefore the invention provides a method whereby the plants from which explants are taken are pretreated with an auxin as described below.
On the explants preferably embryogenesis is induced resulting in an invented method whereby the friable embryogenic callus is produced from torpedo shaped primary or mature embryos. The reason is explained in the detailed description. Protoplasts obtainable by a method as disclosed above are also part of the invention.
An important reason for wanting to have protoplasts which can be regenerErted into plants is of course that protoplasts can be easily tranformed or transduced or provided with additional genetic information by any other suitable method. Thus one Is now able to provide cassave plants or closely related species with genetic material of Interest. The invention thus also provides a method for transforming (defined as providing with In any suitable manner) a protoplast of a cassave or a closely related species by providing said protoplast with additional genetic infonnation through infection by a bacterium comprising said additional genetic infomiation such as Agrobaderium tumefaciens, by electroporation or chemical poration providing a vector comprising said additional genetic infomiation or by paricle bombardment whereby the particles are coated with the additional genetic information, whereby a protoplast obtainable from friable embryogenic callus is transfomned. The invention also encompasses transformed protoplasts obtainable by such a method. Below a short introduction is given on the usefulness of transforming plants, such as
cassave.
Application of plant gene technology encompasses a multitude of different techniques ranging from isolation of useful genes, their characterization and manipulation, to the reintroduction of modified constructs into the plants (Lonsdale, 1967). Plant gene technology will catalyze progress in plant breeding, as is

exemplified by a few examples of transgenic crops like rice (Chen et al., 1987; Shimamotoetal., 1989), maize (Gordon-Kamm et a(., 1990; Vain etal-, 1993), wheal (Marks et a)., 1989), and potato (De Block, 1988; Visser et al., 1989). Rapid progress in gene technology has allowed insight Into the complex molecular mechanism of plant pathogen recognition and the natural defence strategies of host plants. This technology can also be used for controlled and efficient identification of desirable genotypes, far beyond the possibilities of classical breeding. For instance electroporation of protoplasts derived from suspension cultures led to the transformation of maize (Rhodes et at., 1988), rice (Toriyama et al., 1988) and orchardgrass (Horn et al., 1988).
Successful attempts have been made to improve resistance against pathogenic viruses like tobamovirus in tobacco (Powel Abel et al., 1986), potexvirus in potato (Hoekemaetal., 1989) and in papaya (Fitch etal., 1992). In the above examples the introduced trait was based on expressing of single genes that are coding for the coat protein. In cassava, African cassava mosaic virus (ACMV) and cassava common mosaic virus (CCMV) may be controlled by the coat protein-mediated resistance technique {Fauquet et al., 1992). The genes encoding key enzymes of cyanogenesis have been cloned (Hughes et al., 1994) which makes manipulation of cassava cyanogenesis by genetic transformation using the antlsense approach feasible. Another embodiment of the invention is the manipulation of starch in the cassava tubers.
Thus the present invention provides a transformed protoplast whereby the additional genetic infonmation comprises an antisense construct, particularly one wtiereby the antisense construct is capable of inhibiting the amylose synthesis pathway.
A protoplast cannot grow on the field, nor can it be harvested. Though protoplasts are necessary for transformation, it must be possible to regenerate said protoplasts into embryos and/or plants. This is a very important embodiment of the invention, because cassave has been shown to be difficult to regenerate from protoplasts. The detailed description explains how this may be achieved. For further infonmation reference is made to the thesis written by E.Sofiari titled Regeneration and Transformation of Cassava (Manihot Esculenta Crantz.), a copy of which is enclosed with the present application, which is as yet unpublished and which is incorporated herein by reference. Thus the invention provides a method for regenerating plants from protoplasts whereby a protoplast according to the invention is induced to produce an embryo, which embryo is consequently induced to produce a plant. The plants obtainable by said method are aslo part of the invention, in particular

plants
whereby the tubers contain essentially no amyiose.
Detailed description.
Initiation of fee
The procedure to obtain FEC is outlined in Figure 1. It starts with the induction of
primary embryos. Primary embryos are formed In a two step procedure. In the first
step explants are cultured on medium supplemented with salts and vitamins
(preferably Murashlge and Skoog (1962)), a carbo hydrate source (for example 20
g/l sucrose) and an auxin (e.g. 1-8 mg/1 picloram, or dicamba or 2,4-D) for the
initiation of embryos. After 10 to 15 days on this first medium bipolar torpedo shaped
embryos are formed. Torpedo shaped embryos posess a clear hypocotyl and
cotyledon primordia. After transfer of the explants with torpedo shaped embryos to a
step 2 medium (the same medium as step 1 but \Mthout a auxin) the torpedo shaped
embryos become mature. Mature embryos possess large green cotyledons.
Zygotic embryos (Stamp and Henshaw 1982; Konanetal., 1994), young leaf
explants or apical meristems (Stamp and Henshaw, 1987a; Szabados et al., 1987;
Mroginsky and Scocchi, 1993; Raemakers 1993a; Narayanaswamy et al., 1995) and
floral tissue (Mukherjee 1995) can be used to obtain primary embryos. In this way
many different genotypes were evaluated for their ability to form primary embryos.
In this protocol primary somatic embryos were only formed after culture on solid
medium and never after culture in liquid medium. Furthermore, somatic embryos (primary)
were only observed if the auxins Picloram, Dicamba or 2,4-D were used and not with
lAA, IBA or NAA.
In the presently used protocol there is genotypic variation in the number of mature
embryos formed per cultured explant. The genotypes M.Coll505, M.Col22 and
Gading gave the highest numbers of mature embryos per cultured leaf explant
(ME/CLE). However, the number of mature embryos formed was low. In M.Col22 a
maximum of 22 % of the leaf explants isolated from in vitro grown plants and
cultured on a step 1 medium with 4 mg/12,4-D, fonned ME with a maximum numtier
of 0.8 ME/CLE, On a step 1 medium with 8 mg/1 2,4-D a maximum of 49 % of the
leaf explants formed ME with a maximum number of 3.5 ME/CLE. Higher 2,4-D
concentrations did not further improve the embryogenic capacity of explants.
In an attempt to improve the capacity of leaf explants to produce primary somatic
embryos, donor plants were grown under different conditions. Growth of in vitro
donor plants under different light regimes (8, 12, 16 or 24 hours) had no influence
on the embryogenic response. However, a reduction of the fight intensity had a

positive effect. The best results were obtained with leaf explants isolated of donor plants grown at 8 uEm'^s'^ and cultured on a step 1 medium. Other investigators have shown that in certain genotypes, Dicamba (1-66 mg/l) and Picloram (1-12 mg/l) are superior to 2,4D for inducing primary embryogenesis (Ng 1992; Sudarmonowati and Henshaw, 1993; Taylorand Henshaw, 1993). Mathews et al. (1993) improved the efficiency of primary embryogenesis in the genotype M. Col 1505 by transfen"ing explants after 15 days of step 1 medium to a grov^rth regulator-free medium supplemented with 0.5% charcoal. On this medium maturation was improved and as a result the number of mature embryos increased from 0.4 in the control to 3.4 ME/CLE.
The best results were obtained if donor plants were pretreated with auxins as 2,4-D or picloram or Dicamba. For this, plants were grown in liquid MS20 medium and suppUed with the auxin (final concentration 8 mg/l) after 12 days of growth. Two days later leaf explants were isolated of the donor plants and cultured on step 1 medium with 8 mg/l 2,4-D, picloram or Dicamba. In the clone M.Col22 this resulted in a production of 9.4 ME/CLE. This was significantly higher than in the HjO-treated control-plants, where 3.5 ME/CLE were produced (Table 3). The general applicability of the auxin pretreatment was tested on several different genotypes. Without a pretreatment of donor plants two genotypes formed ME and at low frequency. After a pretreatment of donor plants, leaf explants of allmost all genotypes formed ME.
Eventually we were abte to obtain mature primary somatic embryos from 18 of the 22 tested genotypes (Table 1, except TMS30221, TMS30001, TMS30572 and Sao Paolo).
Primary somatic embryos derived from zygotic embryos and from leaves have been used as explants to initiate secondary embryos (Stamp and Henshaw, 1987b; Szabados et al„ 1987; Mathews etal., 1993; Raemakerset al., 1993bc; Luong et al., 1995). Continuous culture of somatic embryos on auxin supplemented medium resulted in a cyclic system of somatic embryogenesis. The way of subculturing somatic embryos for secondary embryogenesis seemed to influence the morphology of the embryogenic tissue. Clumps of somatic embryos recultured monthly on solid 2,4D containing medium in the dark deveioped into finger-like embryo initials formed on the top of older embryos. The embryos did not pass the torpedo-shaped stage. Further development oaajrred if the clumps with embryos were transferred to step 2 medium in the light (Szabados 1987). Normally mature somatic embryos were cultured in step 1 medium in the light and twenty days later the explants were transferred to step 2 medium for maturation. In this system embryos developed to maturity and mature embryos with large green cotyledons were used to start a new

cycle of embryogenesis whereas in the system of others torpedo shaped embryos were used to start a new cycle of secondary somatic embryogenesis. This system of multiplication of mature embryos has been tested In 14 of the in Table 1 mentioned genotypes. Despite the fact that in most genotypes only a few mature primary embryos were available, all genotypes, except one, gave new mature embryos after culture on 2,4-D supplemented medium, in a much higher frequency as observed for primary somatic embryogenesis. Embryogenicity was maintained by regular subculture of mature embryos for more than one year {Sza-badosetal., 1987; Mathews etal., 1993; Raemakers, 1993). New somatic embryos were formed both in liquid and solid medium. In all the genotypes it was observed that in liquid medium more embryos were formed than in solid medium and that fragmentation of embryos before the start of a new cycle of secondary somatic embryogenesis increased the production compared to whole embryos. In for example M.Cpl22 whole embryos cultured on solid medium produced 8 embryos per cultured embryo, whereas fragmented embryos cultured in liquid medium produced 32 embryos per cultured embryo (Raemakers et al., 1993c). Not only, 2,4-D, Picloram and Dicamba, but also NAA had the capacity to induce secondary embryogenesis. IBA and lAA did not induce secondary embryogenesis. NAA has been used successfully in Adira 1, Adtra 4, Gading, Line 11, M.Col22, M.CoH 505, TMS90853 and Gading. In general more mature embryos were produced in NAA supplemented medium than in 2,4-D, Picloram or Dicamba supplemented medium. Furthermore, the development of NAA induced embryos was faster than with 2,4-D, Dicamba or Picloram. Shortening the culture duration has a beneficial effect, particularly, when operating on a large scale.
Histologically, the by 2,4-D newly induced secondary embryos were attached vertically to the explants whereas those by NAA were horizontally. There is still a problem in obtaining embryogenic cultures of cassava (Mroginski and Scocchi, 1992; Taylor etal., 1992; Narayanaswamy et al., 1995; Sudarmonowati and Bachtiar, 1995). The main problem is not that embryogenic tissue from primary explants can be obtained, but the large scale multiplication of this tissue by secondary embryogenesis. For this purpose, either tissue consisting of torpedo shaped embryos or mature embryos can be used. Multiplication of torpedo shaped embryos is highly genotype dependent, while multiplication of mature embryos is largely genotype independent (Raemakers, 1993). Both primary and secondary somatic embryogenesis are characterized by the formation of propagules with a bipolar structure. These bipolar torpedo shaped embryos are already formed on the auxin supplemented step 1 medium. Therefore, Taylor et al., (1995) proposed the term organized embryogenesis. Organized cells are defined as a group of actively

dividing cells, having the tissues and organs formed into a characteristic unified whole {Walker, 1989),
A less organized type of somatic embryogenesis was developed by Taylor et al. (1995). With continuous selection, organized embryogenic tissue cultured on a Gresshoff and Doy (1972) medium salts and vitamins supplemented with 10 mg/l Picloram (GD2) converted gradually into a less organized tissue. This tissue consisted of a caltus-)ike mass of (pro-)globuIar embryos which was very friable. Therefore, this tissue was called friable embryogenic callus (FEC). The cells in FEC are continuously in a state where they break away from group control and because of that they are not organized into a unified structure. FEC is maintained on a medium consisting of Gresshoff and Doy (1972) vitamins and salts, 7 g/l Daichin agar, 20 g^ suaose and 10 mg/l Pidoram (solid GD2). Every three weeks the friable embryos were subcultured on the above mentioned medium. In order to initiate liquid suspension cultures 0.5 g of friable embryos was transferred in a flask of 200 ml with 50 mi of liquid medium supplemented with Schenk and Hildebrandt (1972) salts and vitamins, 60 g/l sucrose and 10 mg/l Picloram (liquid SH6). The medium was refreshed every 2 days and after 14 days the content of each flask was divided over 5 new flasks. The pH was adjusted to 5.7 before autoclaving. The temperature in the growth chamber was 30°C, the photoperiod 12 hours and the irradiance 40 pmolm"V\ Suspension cultures were initiated by culturing FEC in Schenk and Hildebrandt (1972) medium supplemented with 6 % (w/v) sucrose and 10 mg/l Picloram (SH6). Every 2-3 days this medium was refreshed. To keep a culture in a highly friable state the FEC has to be sieved once in two monts. In practice the part of the FEC which will go through a sieve with a mesh of 1 mm^ vwll be used for subculture.
FEC will allmost never form torpedo shaped embryos on the GD2 or in SH6 medium. Torpedo shaped and subsequent mature embryos are formed if FEC is cultured on maturation medium. Maturation medium consist of Murashige and Skoog (1962) salts and vitamins, 0.1 g/l myo-inositol, 20 g/l sucrose, 18.2 g/l mannitol, 0.48 g/l MES, 0.1 g/l caseinhydrolysate, 0.08 g/l adenine sulphate, 0.5 mg/l d-calcium-panthotenate, 0.1 mg/l choline chloride, 0.5 mg/l ascorbic acid, 2. mg/t nicotinic acid, 1 mg/l pyridoxine-HCI, 10 mg/l thiamine HCI, 0.5 mg/l folic acid, 0.05 mg/l biotin, 0.5 mg/l glycine, 0.1 mg/l L-cysteine, 0.25 mg/l riboflavine and 1 mg/l picloram. TTiis maturation medium was refreshed every 3 weeks.
Mature embryos could be induced into secondary somatic embryogenesis by culturing on MS20 medium supplemented with 2,4-D, picloram, Dicamba or N/VA. Primary and secondary somatic embryogenesis are relatively easy to establish in a vwde range of genotypes (see table 1), while FEC is for the time being restricted to a

few genotypes. The prospect of FEC for a new system of somatic embryogenesis and genetic transformation is promising, although further research Is needed to make this system applicable to more genotypes. Essential for this process is the availability of high quality organized tissue and the ability of this tissue to convert into FEC. Tayior et al. (1995) "used organized tissues" which were multiplied at the torpedo shaped state to initiate FEC. In this case two steps (initiation of organized tissue and conversion into unorganized tissue) are determinative for the successful initiation of FEC. Both steps are genotype dependent. If organized tissue is multiplied in the mature state as described by Raemakers (1993) then only the ability of this tissue to convert into FEC is a determinative step to initiate FEC. It remains to be investigated whether or not organized tissue can be used as starting material. If organized tissue cannot be used, then this tissue should be first multiplied in the immature state before it can be used to initiate FEC. This is readily accomplished by, either culturing explants at a high density or by reducing the cyclic duration.
Regeneration of plants from protoplasts
Isolation of protoplasts
For protoplast isolation both FEC cultured on solid GD2 or liquid SH6 can be used. However, the highest yield of protoplasts was obtained from FEC which has been cultured for 1 to 3 weeks in liquid SH6.
Two gram of FEC was placed in Petri dishes ($ 9 cm) containing 10 ml of cell wall digestion solution. Cell wall digestion solution consisted of a mixture of cell wall degrading enzymes;*growth regulators (NAA 1 mg/l, 2,4-D 1 mg/l, Zeatin 1 mg/l); major salts ( 368 mg/l CaClj; 34 mg/l KHjPO^; 740 mg/KNOg; 492 mg/l MgSo^.THjO); minor salts (19.2 mg/l Na-EDTA; 14 mg/l FeSO^.THjO) and osmoticum (91 g/l D-mannitol) and 0.5 g/l MES. The cell wall degradading enzymes cellulase (1-10 g/l) plus Macerozyme (200 mg/l) were succesfull for protoplast isolation. The extra addition of Pectolyase (0.001 -0.01 g/l) and/or Driselase (0.02 g/l) increased the yield of protoplasts. After 18 h of incubation, 10 ml of washing medium was added to the solution. Washing medium with an osmolarity 0.530 mOsm/kg consisted of major salts (see cell wall digestion solution), 45.5 g/l mannitol and 7.3 g/l NaCI. The digested tissue was filtered through a 73 pM pore size filter (PA 55/34 rjybott - Svwtzeriand) into a 250 ml beaker glass. The filtrate was divided equally over two 12 ml conical screw cap tubes, and centrifuged at 600 rpm for 3 min (Mistral 2000). The washing procedure was repeated once after removal of the supernatant. The protoplast solution was resuspended by floating on 9.5 ml solution

containing major and minor salts (see cell wall digestion solution) and 105 g/l sucrose. The pH was 5.8 and the osmolarity 0.650 mOsm. The solution with protoplasts was allowed to equilibrate for 5 minutes before 0.5 ml of washing medi¬um was gently added on the top. After centrifugation at 700 rpm for 15 min {Mistral 2000), the protoplasts were concentrated in a band between the sucrose and washing medium. The protoplast layer was harvested with a pasteur pipette and the yield was counted in a standard haemocytometer chamber.
Protoplast culture
Protoplasts were cultured in media solidified with agarose 0.2 % w/v (Dons en Bouwer, 1986) in petri dishes containing 10 ml of the same liquid medium. The following media resulted in the formation of micro callus:
-7M2G medium (Welters et al., 1991) supplemented with only auxins (0.1-10 mg/l NAA or 0.1-10 mg/IPicloram, or 0.1-10 mg/l lAA, or 0.1-10 mg/l 2,4-D, or 0.1-10 mg/l Dicamba, or0.1-10mg/l, or 0.1-10 mg/I) or auxins plus cytokinins (0.01-1 mg/l zeattn, 0.01-1 mg/l 2-iP. 0.01-1 mg/l BA, 0.01-1 mg/l TDZ, 0.01-1 mg/l krnetin). -medium A (Murashige and Skoog (1962) salts and vitamins, 4.5 g/l myo-inositol, 4.55 g/l mannitol, 3.8 g/l xylitol, 4.55 g/l sorbitol, 0.098 g/l MES, 40 mg/I adeninsulp-hate and 150 mg/l caseinhydrolysate, 0.5 mg/l d-calcium-panthoter^ate, 0.1 mg/l choline-chloride, 0.5 mg/ ascorbic acid, 2.5 mg/l nicotinic acid, 1 mg/l pyridoxine-HCI, 10 mg/l thiamine-HCI, 0.5 mg/l folic acid, 0.05 mg/l biotine, 0.5 mg/l glycine, 0.1 mg/l L-cysteine and 0.25 mg/l riboflavine and 59.40 g/l glucose) supplemented with only auxins (0.1-10 mg/l NAA or 0.1-10 mg/IPicloram, or 0.1-10 mg/l lAA, or 0.1-10 mg/l 2,4-D, or 0.1-10 mg/l Dicamba plus cytokinins {0.01-1 mg/l zeatin, 0.01-1 mg/l 2-iP, 0.01-1 mg/l BA, 0.01-1 mg/l TDZ, 0.01-1 mg/l kinetin). The media were refreshed every 10 days, by replacing 9 ml with fresh medium. After two months of culture in the first medium, high quality FEC was selected and either cultured for further proliferation or for maturation. For proliferation FEC was transferred to Gresshoff and Doy (1974) medium supplemented with 40 g/l sucrose, 7 g/l Daichin agar and 2 mg/l picloram (GD4). After 3 weeks the FEC was transferred to a Gresshoff and Doy medium supplemented with 20 g/l sucrose, 7 g/l agar and 10 mg/l Picloram (GD2). Suspension cultures were initiated by transferring 1.0 g of FEC to liquid SH6% medium supplemented with 10 mgfl Picloram. Two weeks later the suspension was divided over new flasks with an initial packed cell volume of 1.0 ml.
After 2 months of culture, 10^ protoplasts cultured in TM2G supplemented with 0.5 mg/l NAA and 1 mg/l Zeatin at a density of 10^/ml produced 1058 micro-calli, v\*iereas 10^ protoplasts cultured at a density of 10^/ml only produced 64 micro-calli.

Replacing TM2G medium with medium A reduced at both densities the number of micro-calli significantly. At this stage at least three types of calli could be distinguished. One type consisted of globular shaped embryos which were mostly observed in protoplasts cultured at a density of 10^. Some of them developed cotyledon like structures, light green in colour. However, these embryos could not be germinated properly. Another type was fast growing and consisted of a large compact callus, they were observed in protoplast cultures of both densities. This callus never developed embryos. The third type was highly friable callus and was observed at both densities. At a density of 2-5 x 10^ (medium TM2G) about 60 % of the calli were friable and embryogenic. The FEC was either subcultured for further proliferation or for maturation.
Proliferation of FEC derived from protoplasts
Following selection of FEC, 0.1 g of it cultured for three weeks on GD 4 plus 2 mg/l Picloram inaeased into 0.7 g of tissue. More than 95 % of the tissue consisted of high quality FEC. Subsequently, this tissue was maintained by subcultures of three weeks on GD2 medium supplemented with 10 mg/l Picloram. To initiate suspension cultures FEC was transfe^ed to liquid medium. The increase in packed cell volume (PCV) of this material was slightly higher than that of the original material (data not shown).
Maturation of FEC derived from protoplasts
In an attempt to induce maturation of embryos, FEC isolated after two months of culture in TM2G was cultured on maturation medium. Maturation medium consisted of Murashige and Skoog (1962) salts and vitamins, 0.1 g/l myo-inositol, 20 g/l sucrose, 18.2 g/l mannitol, 0.48 g/t MES, 0,1 g/l caseinhydrolysate, 0.08 g/l adenine sulphate, 0.5 mg/l d-calcium-panthotenate, 0.1 mg/l choline chloride, 0.5 mg/l ascorbic acid, 2. mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/t thiamine HCl, 0.5 mg/l folic acid, 0.05 mg/l biotin, 0.5 mg/l glycine, 0.1 mg/l L-cysteine, 0.25 mg/l riboflavine and 1 mg/l picloram. This maturation medium was refreshed every 3 weeks.
On this medium there is a gradual shift from proliferation to maturation. As a result the packed eel) volume had increased with a factor 4 after two weeks of culture in liquid maturation medium. Also after transfer to solid maturation medium there is proliferation. After two weeks on solid medium most of the embryos had reached a globular shape and only a few of these globular embryos developed further. The first torpedo shaped embryos became visible after one month of culture on solid maturation medium. The number of mature and torpedo shaped embryos was not

correlated with the plating efficiency but with the density of the initially cultured protoplasts. No such embryos were obtained if protoplasts were cultured on TM2G without growth regulators. The highest number of mature and torpedo shaped embryos was formed from protoplasts cultured on TM2G supplemented with 0.5 mg/l NAA and 1 mg/l Zeatin. If NAA was replaced by Picloram than the number of torpedo shaped and mature embryos was significantly lower (Table 2). From the tested Picloram concentrations 2 mg/l gave the best results. After 3 months of culture between 60 and 200 torpedo shaped and mature embryos were isolated per agarose drop. Torpedo shaped embryos became mature at high frequency if they were cultured on fresh maturation medium or on MS2 plus 0.1 mg/l BAP.
Secondary somatic embryogenesis and germination of mature embryos
derived from protoplasts
Only a few torpedo shaped embryos formed secondary embryos if cultured on liquid or solid MS2 medium supplemented with 10 mg/l NAA or 8 mg/l 2,4-D (data not shown). Mature embryos were better explants for secondary embryogenesis. In both liquid and solid medium 2,4-D was superior for induction of secondary embryogenesis as compared to NAA. If mature embryos were first cultured in 2,4-D and than in liquid NAA the response was comparable with culture in 2,4-D alone. Also embryos which first had undergone a cycle of secondary somatic embryogenesis in medium with 2,4-D, produced highly efficient secondary embryos In MS20 supplemented with 10 mg/l NAA.
The germination of cyclic or secondary somatic embryos, induced In liquid medium by the auxins 2,4-dichlorophenoxyacetic acid (2,4-0) or naphthalene acetic acid (NAA), was compared. In all genotypes desiccation stimulated normal genmination of NAA Induced embryos. However, the desiccated embryos, required a medium supplemented with cytoklnins such as benzylaminopurine (BAP) for high frequency germination. The morphology of the resulting seedling was dependent on the concentration of BAP. With 1 mg/l BAP plants with thick and short taproots and branched shoots vwth short internodes were formed. With 0.1 mg/l BAP the taproots vrere thin and slender and the shoot had only one or two apical meristems. If the embryos vrere desiccated sub-optimally, higher concentrations of BAP were needed than if the embryos were optimally desiccated to stimulate germination. Also desiccated embryos which were cultured in the dark required a lower concentration of BAP and, furthermore, these embryos germinated faster than embryos cultured in the light. Complete plants were obtained four weeks after the start of somatic embryo induction. 2,4-D induced embryos showed a different response. In only one genotype desiccation enhanced germination of 2,4-D induced embryos and in three other genotypes it did not. In all genotypes desiccation stimulated root formation. Embryos cultured in the dark formed predominantly adventitious roots, whereas embryos cultured in the light formed predominantly taproots.
4.0 Gene transfer systems

Over the past years several transfer techniques of DNA to plant protoplasts have been developed such as silicon fibers {Kaeppleret al., 1990), microinjection (DeLaat and Blaas, 1987) and electrophoresis (Griesbach and Hammond, 1993), The most commonly used and potentially-applicable ones are Agrobacterium-mediated gene delivery, microprojectile/particle bombardment and protoplast electroporation.
The Agrobacterium tumefadens DNA delivery system is the most commonly used technique. It probably relates to the first invention of DNA delivery in plants by this method. Initially it was limited to Kalanchoe and Solanaceae, particularly tobacco. Nowadays, the use of Agrobacterium-mediatedi transformation has changed dramatically, it is possible to transform a wide range of plants with a limitation in monocots (reviewed by Wordragen and Dons, 1992). Although cassava is a host for Agrobacterium it has proven to be not highly amenable to it. In principle protoplasts are the most ideal explants for DNA delivery. They can be cultured as single cells that produce multicellular colonies from wtiich plants develop. Plants derived firom protoplasts are generally clonal in origin. This provides a useful tool for any transformation system, because it will eliminate chimerism in transgenic plants. The use of protoplasts is, however, hampered by the regeneration system which is highly species dependent. For transformation, protoplasts can be used in conjunction with PEG to alter the plasma membrane which causes reversible permeabilization that enables the DNA to enter the cytoplasm as was demonstrated, for example, in Lolium multiform {Potrykus et al., 1985) and Triticum monococct/m (Lorzetal., 1985). Another technique to inaease the permeability of plasma membranes and even cell walls to DNA is by electroporation (for review see Jones et al., 1987). In this method electrical pulses enable the DNA to enter the cells. Rice was the first crop In which fertile transgenic plants resulted from protoplast electroporation (Shimamoto et al., 1989).
The use of particle bombardment or biolistics to deliver foreign DNA provides an alternative method in cassava transformation. Particle bombardment is the only procedure capable of delivering DNA into cells almost in any tissue. The first transgenic plant obtained by using this method was in tobacco (Klein et al., 1989). Follovwng this successful transformation method, particle bombardment is widely used in plants which are less amenable to Agrobacterium infection, particulariy monocots. Improvement of several DNA delivery devices to accelerate the particle (microprojectile) has resulted in the most recent model the Biolistic ™ PDS-1000 {Bio-Rad Laboratories, Richmond, Ca). Those devices are available commercially, however the price is relatively high at present. Tungsten or gold particles, coated with DNA, are commonly used as microprojectiles to deliver DNA into the target tissue (recently reviewed by Songstad et al., 1995).
5. Selection and reporter genes used in genetic modifications
To be able to identify transformed cells, the gene of interest is coupled to a selectable marker gene. This mariner gene is necessary to select transformed cells. Selection can be based on a visual characteristic of the transformed cellftissue. An example is the luciferase gene isolated from the firefly. Plant cells expressing this gene and supllied with substrate (luciferin) will emit light which can be detected with special equipment (Ow et al., 1986). Another way to select transfomied tissue is the introduction of a gene* encodes resistance to antibiotics or herbicides (Thompson
et al., 1987; Gordon-Kamm et al., 1990).
*which

A number of antibiotics and herbicides has been used as selective agent in plant transformation. In cereals resistance to the herbicide phosphinothricin (PPT) was chosen for the selection of transgenic plants {Cao et a!., 1990). In Carica papaya (Fitch et ai., 1994), Vitis vinifera (Nakano et al., 1994; Scorza et al., 1995), maize (Rhodes et al., 1988) and rice (Chen et al., 1987) the neomycine phosphothansferase (NPTll) gene, which confers resistance to kanamycin and related antibiotics (Fratey et al., 1986), was used as a selectable marker.
In cassava all above- mentioned systems of selection can be used, however PPT based selection has as advantage that it improves the ability of FEC to form mature embryos and in this way increase plant regeneration.
legend to figure 1
Figure 1: Schematic representation of somatic embryogenesis in cassava, including primary, secondary somatic embryogenesis, selection of friable embryogenic callus, maturation and desiccation follo>A«d by germination.
gd2= medium supplemented with Gresshoff and Doy salts (1974) and vitamins plus
20 g/l sucrose.
gd4= medium supplemented with Gresshoff and Doy salts (1974) and vitamins plus
40 g/l suaose.
ms2= medium supplemented vwth Murashige and Skoog salts and vitamins plus 20
g/l sucrose.
pic= 10 mg/l Picloram, N/\A=10 mg/l naphthalene acetic acid, 2,4-D=8 mg/l, 2,4-
dichlorophenoxy acetic acid. sh6= medium supplemented with Schenk and
Hildebrandt (1972) salts and vitamins plus 60 g/l sucrose.

Table 1. Genotypes of cassava used for somatic embryogenesis.

Table 2. Influence of light intensity during growth of donor plants in vitro on the number of leaf explants responding with the formation of mature embryos and the number of mature embryos per cultured leaf explant (#ME/CLE)-

Table 3. Influence of 2,4-D pretreatment on production of primary mature embryos (# mature embryos per cultured leaf explant isolated from in vitro plants), followed by the multiplication of mature embryos by secondary somatic embryogenesis in 11 Nigerian cassava genotypes and in M.Col22.

Cited literature
Anonymus, 1985, ClAT: Annual report: Centro International de Agricultura Tropical, Cali, Columbia, Pp: 197-217.
Anthony, P., Davey, MR., Power, J.B., and Lowe, K.C. 1995. An improved protocol for the culture of cassava leaf protoplasts. Plant Cell Tissue and Organ Culture. 42:229-302
Buiteveld, J., and Creemers-Molenaar, J, 1994. Plant regeneration from protoplasts isolated from suspension cultures of leek (Allium ampeloprasum L) Plant Science. 100:203-210,
Cao, J., Duan, X., McEh-oy, D,, and Wu, R. 1990. Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Ceil Rep, 11: 586-591.
Chang, Y.F., Wang, W.C, CoUeen, Y.W., Nguyen, H.T., and Wong, J.R. 1991. Plant regeneration from protoplasts isolated from long-term cell cultures of wheat {Triticum aestivumy Plant Cell Rep. 9:611-614.
Chen, W H, Davey, MR., Power, J.B., and Cocking, EC. 1988. Sugarcane protoplasts: factors affecting division and plant regeneration. Plant Cell Rep. 7:344-347.
Chen, W.H., Gartland, K.M.A., Davey, MR., Sotak, R., Gartland, J,S., MuUigan, B.J,, Power, J.B., and Cocking, E.C, 1987. Transformation of sugarcane protoplasts by direct uptake of a selectable chimeric gene. Plant Cell Rep. 6:297-301.
DeLaat, A., and Blaas, J., 1987. An improved method for protoplast microinjection suitable for transfer of entire plant chromosomes. Plant Sci 50:161-169.
Fitch, M.M.M., Pang, S,Z., Slightom, Lius. S., Tennant, P., Manshardt, R.M., and GonsaJves, D. 1994. Genetic transformation in Caricapapaya (Papaya). In: B^^ (Eds). Biotechnology in Agriculture and Forestry Vol 29, Plant protoplasts and genetic engineering V. Springer-Verlag,BerUn.P:237-255.
Dons, J.J.M., and Bouwer, R. 1986. Improving the culture of cucumber protoplasts by using an agarose-disc procedure. Proceedings of an international symposmm on nuclear techniques and in vitro culture for plant improvement. Jointly organized by the International Atomic Energy Agency and the Food and Agriculture Organization of the United Nations. Held in Vienna, 19-23 August 1985. P:498-504.
Fraley R.T., Rogers S.G., and Horsch, R.B. 1986. Genetic transformation in higher plants. CRC Critical reviews in Plant Sciences 4(1): 1-46.
Gordon-Kamm, W J., Spencer, T.M., Mangano, MR., Adams, T.R., Daines, R-J, William, as., O'Brien, J V., Chambers, S.A. Adams, Jr W.R., Willetts, N G, Rice, T.B., Mackey C J, Krueger, R.G., K^sch, A.P,, and Lemaux P.G. 1990, Transformation of maize cells and regeneration of fertile transgenic plants. The Plant Cell. 2:603-618.

Gresshoff, P.M , and Doy, C,H, 1974 Development and difFerentiation of haploid Lycopersicon esailentum (tomato). Planta 107:161-170.
Griesbach, R.J,, and Hammond, J. 1993 Incorporation of the GUS gene into orchids via embryo electrophoresis. Acta, Hon. 336:165-169.
Horn, M.E., Shilhto, R.D., Conger, B.V., and Harms, C,T, 1988, Transgenic plants of orchardgrass {Dactytis glomeratoL.) from protoplasts. Plant Cell Rep. 7:469-472.
Jones H., TempeJaar M,/., and Jones, M.G.K. 1987. Recent advances in plant eiectroporation. Oxford Surveys of Plant Mol. and Cell Biol. 4:347-357.
Kaeppler, H.F,, Gu, W., Somres, D.A., Rines, H.W., Cockbum, A.F. 1990. SUicon carbide fiber-mediated DNA dehvery into plant cells. Plant Cell Rep. 8:415-418.
Klein T.M., Komstein L., Sanfords J.C, and Fromm M.E, 1989,
Genetic transformation ofmaize cells by particle bombardment. Plant Physiol. 91:440-444.
Konan N.K., Sangwan R.S., and Sangwan-Norrell, 1994. Nodal axillary meristems as target tissue for shoot production and genetic transformation in cassava {Manihot esculenta Crantz). Second International Scientific Meeting of Cassava Biotechnology Network 11. Bogor. Indonesia. p:276-288.
Kyozuka, I, Otoo, E , and Shimamoto, K. 1988. Plant regeneration fi^Om protoplasts of indica rice; genotypic differences in culture respond. Theor. Appl. Genet. 76:887-890,
Lorz, H., Baker, B., and Schell, J, 1985. Gene transfer to cereal cells mediated by protoplast transformation. Mol. Gen. Genetic 199: 178-182.
Luong, H,T., Shewry, PR., and Lazzeri, PA. 1994, Gene transfer to cassava somatic embryos via tissue eiectroporation and particle bombardment. In: Second International Scientific Meeting ofCassava Biotechnology Network 11. Bogor. Indonesia. p:303-314.
Mathews, H., Carcamo, R., Chavarriaga, Schfipke, C.P., Fauquet, C. and Beachy, R.N., 1993. Improvement of somatic embryogenesis and plant recovery in cassava. Plant Cell Rep. 12:328-333.
Mroginski and Scocchi, 1992. Somatic embryogenesis of Argentine cassava varieties. In: Roca, W.M., and Thro, A.M. (Eds). Proceedings First Scientific Meetingg of the Cassava Biotechnolgy Network. Cartagena, Colombia 25-28 August 1992. P: 175-179.
Mukherjee, A. 1994. Embryogenesis and regeneration from cassava calli of anther and leaf. The Cassava Biotechnology Network. Proceeding of the Second International Scientific Meeting. Bogor.Indonesia, 22-26 August 1994. P:375-377.
Murashige, T,, and Skoog, F 1962. A revised medium for rapid growth and bioassay with tobacco cultures. Phvsiol Plantarum. 15:473-497.

Nakano, M,, Hoshino, Y., and Mil, M, 1994, Regeneration of transgenic plants of grapevine (Vitis vim/era L.) via Agrobacterium rhizogenesis-mediated transformation of embryogenic calli. J. of Exp. Bot, 45 (274); 649-656.
Narayanaswamy, T.C., Ramaswamy, N.M,, and Sree Rangaswamy, S.R. 1995. Somatic embryogenesis and plant regeneration in cassava. The Cassava Biotechnology Network. Proceeding of the Second International Scientific Meeting. Bogor,Indonesia, 22-26 August 1994. P:324-329.
Ng SYC (1992) Tissue culture of root and tuber crops at IITA. In: ThottappUly G, Monti LM, Mohan Raj DR, Moore AW (eds). Biotechnology: enhancing research on tropical crops in Afiica CTA/ntA co-pubUcation, IITA, Ibadan, Nigeria, pp 135-141.
Nzoghe, D. 1989. Recherche de conditions permettant I'obtention neoformations chez differents genotypes de manioc (Manihot esculenta Crantz). Extension a la culture de protoplastes. These. Universite De Paris Sud Centre D'Orsay. P : 119.
Ow, D.W., Wood, K.V., DeLuca, M„ De Wet, JR., Hehnski, DR., and Howell, S.H. 1986. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234:856-859.
Potrykus, I., Saul, M., Paskowski, J., and Shillito, R,D. 1985. Direct gene transfer into protoplasts of a graminaciousmonocot. Mol. Gen.Genet. 199:183-188.
Power, J.B., Bery, S.F., Chapman J.V., and Cocking, E.G. 1979. Somatic hybrids between unilateral cross-incompatible Peft/ma species. Theor. Appl. Genet. 55: 97-99.
Raemakers, C.J.J.M. 1993. Primary and cycUc somatic embryogenesis in cassava A/oni/io/ esculenta Crantz. PhD thesis Agricultural University Wageningen, The Netherlands. P: 119.
Raemakers, C.J.J.M., Bessembinder, J., Staritsky, G,, Jacobsen, E., and Visser, R.G.F. 1993a. Induction, germination and shoot development of somatic embryos in cassava. Plant Cell Tissue and Organ Culture 33:151-156.
Raemakers, C.J.J.M., Amari, M., Staritsky, G, Jacobsen, E., and Visser, R.G.F. 1993b Cyclic somatic embryogenesis and plant regeneration in cassava. Annals ofBot. 71:289-294.
Raemakers, C.J.J.M., Schavemaker, CM,, Jacobsen, E., and Visser, R.G.F. 1993c. Improvements of cyclic somatic embryogenesis of cassava {Manihot esculenta Crantz). Plant Cell Rep, 12:226-229.
Rhodes, C.A., Pierce, DA., Metier, I.J., Mascarenhas, D., and Detmer, J.J. 1988. Genetically transformed maize plants from protoplasts. Science 240:204-207,
Scorza, R., Cordts, J.M., Ramming, D.W,, and Emershad, R.L. 1995. Transformation of grape {Vitis vinifera L.) zygotic-derived somatic embryos and regeneration of transgenic plants. Plant Cell Rep. 14:589-592.

Shahin, E.A,, and Shepard, JF 1980. Cassava mesophyll protoplasts: isoJalion, proliferation and shoot formation. Plant Science Letters 17:459-465,
Shenk, R.U., and Hjidebrandt, AC. 1972. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J.Bot. 50:99.
Shimamoto, K., Terada, R., Izawa, T., and Fujimoto, H. 1989. Fratile transgenic rice plants regenerated from transformed protoplasts. Nature 338:274-276.
Sofiari, E, 1996. Regoiration and transformation in cassavaMmito/ esculenta Crantz. PhD
thesis Agricultural University Wageningen, The Netherlands. P: 136,
Songstad, D.D., Somers, DA., and Griesbach, R.J. 1995. Advances in alternative DNA delivery techniques. Plant Cell Tissue and Organ Culture 40:1-15,
Stamp, J.A., and Henshaw, G.G. l9S7a. Somatic embryogenesis from clonal leaf tissue of cassava. Annals of Bot. 59:445-450.
Stamp, J.A, 1987. Somatic embryogenesis in cassava: the anatomy and morphology of the regeneration process. Annals ofBot. 59: 451-459.
Stamp, J.A., and Hen^w, G.G. 1987b. Secondary somatic embryogenesis and plant regener-arion in cassava. Plant Cell Tissue and Organ Culture 10:227-233.
Stamp, J.A., and Henshaw, G.G. 1982. Somatic embryogenesis in cassava. Zeitschrift fur Pflanzenphysiologje. 105:183-187.
Sudarmonowati and Bachtiar, 1995. Induction of somatic embryogenesis in Indonesian cassava genotypes. The Cassava Biotechnology Network. Proceeding of the Second International Scientific Meeting. Bogor.Indonesia, 22-26 August 1994. P.364-374)
Sudarmonowati, E., and G.G. Henshaw, 1992, The induction of somatic embryogenesis of recalcitrant cultivars using picloram and dicamba. In: Roca, W.M., and Thro, A.M, (Eds), Proceedings First Scientific Meetingg of the Cassava Biotechnology Network. Carugena, Colombia 25-28 August 1992, P: 128-133.
Szabados L,, Hoyos R. and Roca W. 1987. In vitro somatic embryogenesis and plant regeneration of cassava. Plant Cell Rep, 6:248-251.
Taylor, N,J., Clarke, M,, and Henshaw, G,G. 1992. The induction of somatic embryogenesis in fifteen African cassava cultivars. In; Roca, W.M„ and Thro, A.M, (Eds), Proceedings First Scientific Meeting of the Cassava Biotechnology Network. Cartagena, Colombia 25-28 August 1992. P: 134-137.
Taylor, N.J,, Edwards, M., and Henshaw, G.G. 1995. Production of fiiable embryogenic calli and suspension culture system in two genotypes of cassava. Second International Scientific Meeting of Cassava Biotechnology Network 11, Bogor, Indonesia. P:229-240,

Thompson, J.C, Mowa, N R , Tizard, R., Crameri, R , Davies, J.E., Lauwereys, M., and Bottemian, J. 1987. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus The EMBO J,6 (9):2519-2523.
Toriyama, K., Arimoto, Y., Uchimiya, H., and Hinata, K. 1988. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technology. 6:1072-1074,
Walker, P.M.B. 1989, Chambers Biology Dictionary. W&R Chamber Ltd, Clay Ltd, St, Ives Pic, England, P:205.
Wolters, A.M,A,, Schoenmakers, H.C.H,, van der Meulen-Muiser, J,J,M,, van der Knaap, E,, Derks, F,H.M.,Koomneef, M,,andZelcer, A, 1991, Limited DNA elunination from the irradiated potato parent in fiision products of albino Lycopersicon esculentum and Solanum tuberosum. Theor i^pl. Genet. 83:225-232,
Wordragen M,F.,,and Dons, H,J,M, 1992, Agrohacterium tumefaciens mediated transformation of recalcitrant crops. Plant Mol. Biol. Reporter 10:12-36.

WE CLAIM:
1. A method for producing protoplasts of cassave or a closely related species, which
protoplasts are capable of regeneration into plants, comprising producing friable embryogenic callus from explants of cassave or a closely related species and isolating protoplasts from said friable embryogenic callus.
2. The method according to claim 1 whereby the friable embryogenic callus is subjected to culture in a liquid medium.
3. The method according to claim 1 or 2 whereby a mixture of cell wall degrading enzymes, such as a cellulase, a pectolyase and/or a macerozyme are used to produce protoplasts.
4. The method according to any one of the aforegoing claims whereby the plants from which
explants are taken are pretreated with an auxin.
5. The method according to anyone of the aforegoing claims whereby the friable
embryogenic callus is produced from torpedo shaped primary or mature embryos.
6. The method according to claim 5 whereby the embryos are induced on primary explants.
7. The method according to claim 1 wherein the protoplast of cassave or a closely related species is transformed by providing with additional genetic information through infection by a bacterium comprising said additional genetic information such as Agrobacterium tumefaciens, by electroporation or chemical poration providing a vector comprising said additional genetic information or by particle bombardment whereby the particles are coated with the additional genetic information.

Documents:

1065-mas-1997 abstract-duplicate.pdf

1065-mas-1997 abstract.pdf

1065-mas-1997 claims-duplicate.pdf

1065-mas-1997 claims.pdf

1065-mas-1997 correspondence-others.pdf

1065-mas-1997 correspondence-po.pdf

1065-mas-1997 description (complete)-duplicate.pdf

1065-mas-1997 description (complete).pdf

1065-mas-1997 drawings.pdf

1065-mas-1997 form-1.pdf

1065-mas-1997 form-13.pdf

1065-mas-1997 form-19.pdf

1065-mas-1997 form-26.pdf

1065-mas-1997 form-4.pdf

1065-mas-1997 form-6.pdf

1065-mas-1997 petition.pdf

« Previous Patent

Next Patent »

Patent Number

223523

Indian Patent Application Number

1065/MAS/1997

PG Journal Number

47/2008

Publication Date

21-Nov-2008

Grant Date

12-Sep-2008

Date of Filing

20-May-1997

Name of Patentee

NATIONAL STARCH AND CHEMICAL INVESTMENT HOLDING CORPORATION

Applicant Address

1000 UNIQEMA BLVD. NEW CASTLE, DELAWARE 19720,

Inventors:

#	Inventor's Name	Inventor's Address
1	CHRISTIAAN JOSEF JOHANNES RAEMAKERS	DR. W DREESSINGEL 39, 6836 CN ARNHEM,
2	EVERT JACOBSEN	RITZEMS BOSWEG 77 6706 BD WAGENINGEN,
3	JOHANNA ELISABETH MARIA BERGERVOET-VAN DEELEN	HOGENKAMPSEWEG 119, 6871 JM RENKUM,
4	RICHARD GERARDUS FRANCISUS VISSER	EMMALAAN 17M 6721 ET BENNEKOM,

PCT International Classification Number

A01H1/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA