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(11)Patent Number: KE 434
(45) Date of grant: 13/04/2011
(51) Int.C1.7: A 01B 49/04, 51/00, 75/00, A 01C 0/0
(21) Application Number: KE/P/ 2007/ 000593
(22) Filing Date: 17/04/2007
(30) Priority Date:
(73) Owner: DR. BENARD AGGREY NYENDE of P 0 BOX 31382 -00600, NAIROBI., Kenya
(72) Inventor: DR. BENARD AGGREY NYENDE of P 0 BOX 31382 -00600, NAIROBI, Kenya.
(74) Agent/address for correspondence:
(54) Title: SYNTHETIC SEED TECHNIQUE FOR ENCAPSULATION IN CALCIUM ALGINATE HOLLOW BEADS
The present invention relates to synthetic (artificial) seed technique for encapsulation in calcium alginate hollow beads. In particular, the invention relates to the use of artificialseeds in potato and other root and tuber crops to avoid all the described disadvantages in their propagation as seed material. The synthetic seed production is carried out under sterile conditions in the lab providing disease free material. It offers no competition between seed material and the consumable yield. Since the synthetic seeds are small (0.8mm in diameter) they can be stored in small cool boxes that take insignificant space. This technique though labour intensive and technical, is relatively simple and with a short familiarization and availed facilities the local community can he able to perform it. Using the "Novel" artificial seed technique for encapsulation in caicium alginate (hollow beads), synthetic seeds of potato cultivars were produced from unipolar shoot tips (3-4mm) of in-vitro produced plantlets. The shoot tips of potato cultivars were encapsulated in calcium alginate to produce synthetic seeds.
1. Synthetic (artificial) seed technique: For encapsulation in calcium alginate hollow beads.
Calcium alginate-encapsulated shoot tips of potato (Solanum tuberosum L.)
2. Background of the invention
Farming in developing countries with high population densities like tropical Africa and especially Kenya is predominantly small scale and rural based. Its structurally small scale since the farmers are financially unable to afford good quaility, healthy and certified seed material. In certain cases supplied certified seed material is used as food instead, especially for root and tuber crops. The farmers continually use their own poor quality seed material which leads to progressive accumulation of diseases (particuilarly viral diseases) and eventually yield and cultivar losses. In addition a substantial part of the harvest (1/3rd) is stored for next seasons planting and is not utilised as food. This stored seed material is also vulnerable to storage losses (inadequate cool storage facilities, pathogens etc) , is bulky and requires large space. The use of artificial seeds in potato (root and tuber crops) avoids all these described disadvantages The synthetic seed production is carried out under sterile conditions in the lab providing disease free material. It offers no competition between seed material and the consumable yield. Since the synthetic seeds are small ( 0.8mm in diameter) they can be stored in small cool boxes that take insignificant space. This technique though labour intensive and technical, is relatively simple and with a short familiarization and availed facilities the local community can be able to perform it. Using the "Novel " artificial seed technique for encapsulation in calcium alginate (hollow beads), synthetic seeds of potato cultivars were produced from unipolar shoottips (3-4mm) of in-vitro produced plantlets. The Shoot tips of potato cultivars were encapsulated in calcium alginate to produce synthetic seeds.
Key words: Potato; shoot tips; synthetic seeds; encapsulation; glycerine; in vitro
3. Details of the invention
3a. Field of invention and description
Synthetic seed technology offers many useful advantages on a commercial scale for the propagation of a variety of crop plants, especially crops for which true seeds are not used or readily available for multiplication (e.g. potato) or the true seeds are expensive to small scale farmers (e.g. cucumber and geraniums), hybrid plants (e.g. hybrid rice) and other vegetatively propagated crops which are prone to seed bourne diseases (e.g. sugarcane, potato, lily, sweet potato, grape, and mango).
This synthetic seed technology could also be useful for multiplying genetically engineered plants (transgenic -GM-plants), somatic and cytoplasmic hybrids (obtained via proptoplast isolation and fusion technology), sterile and unstable genotypes. Further, synthetic seeds or artificial seeds could be usefull for genetic preservation of genetic resources (cryopreservation). Besisdes, synthetic seeds can also be a usefull experimental research tool to understand various processes of germination. Including zygotic embrogenesis and understanding the role of endosperm in normal embryo development.
3b The concept of synthetic seeds
Originating in 1977 from an idea proposal by Murashige (1977; 1978) the concept of synthetic seeds (synseeds) or artificial seeds has evolved from a futuristic idea into a real field of experimentation and research. Other early efforts for development of artificial seeds were by Keith Walker, at Monsanto Company, working with alfalfa (Medicago sativa L.) and by Robert Lawrence at Union carbide working with Celery and lettuce (Redenbaugh et al, 1991). His (Murashige) original definition was that of "an encapsulated single somatic embryo", a clonal product that could be handled and used as a real seed for storage and sowing and that therefore would eventually convert either in vivo or ex vivo, into a plantlet and grow. This limited artificial seeds to the encapsulation of somatic embryos (Datta et al, 1999). It was Kamada (1985) who broadened the scope of the technique by suggesting that an artificial seed comprises a capsule prepared by coating a cultured matter, like a tissue piece or an organ, which can grow into a complete plant, alongside with nutrients and in an artificial covering. Apart from Kamada et al., (1988) and Redenbaugh (1991), no consistent embryo to plant conversion had occurred from encapsulated embryos then. Reports on employing apical or axial shoot buds followed and Bapat et al.,1987 subsequently proposed the making of synthetic seeds through encapsulation of in vitro-derived propagules, different from somatic embryos especially in non-embryonic species. In mulberry (Morus indica), for instance they proposed the use of encapsulated axillary buds.
A more current definition is the one given by Aitken-Christie et al., 1995 which considers synthetic seed as, "artificially encapsulated somatic embryos, shoots or other tissues which can be used for sowing under in vitro or ex vitro conditions", and hence extends the concept of synthetic seeds to any type of vegetative propagule, characterising it only as a seed like structure. According to Piccioni (1997), he emphasised that the propagule must be able to grow into a plantlet after sowing (conversion). Redenbaugh (1993), described conversion as the production of a green plant with a normal phenotype. Encapsulation can be considered an important application of micropropagation to improve the success of in vitro-derived plant delivery into field or greenhouse or to contribute to synthetic seed technology (Piccioni and Standardi, 1995). Less attention has been given to the possibility of encapsulating non-embryonic in vitro-derived vegetative propagules. Nonetheless some researchers have tried to encapsulate shoot tips or axillary buds of different species with promising results (Mathur et al., 1989; Ganapathi et al., 1992; Bapat, 1993). These kind of capsules could be useful in the exchange of sterile material between laboratories, due to the small size and relative ease in handling of the structures, or in the germplasm conservation, with proper preservation techniques (Fabre and Dereuddree, 1990; Niino et al., 1992; Accart et al., 1994; Na and Kondo, 1996) or even in plant propagation and nurseries, if the development of the plant could be properly directed towards proliferation, rooting and elongation (Mathur et al., 1989; Bapat, 1993).
The prospect of using plant somatic embryos produced in tissue culture as synthetic or artificial seeds has been a subject of increasing interests (Rogers, 1983; Herman, 1985) and somatic embryogenesis has been obtained by more than 150 species of important agricultural crops (Tissert et al., 1979) and is a routine procedure in crops like soybean (Ranch et al., 1985; Barwale et al., 1986), grasses and cereals (Gray and Conger, 1984: Vasil and Vasil, 1984).
The objective of the development of synthetic seeds is to produce a propagule that is genetically, developmentally, and morphologically as close as possible to the
seed of the plant from which it is derived. According to Standardi and Piccioni (1998), germination which is a property of plant seeds, zygotic and apomictic seeds, or plantlet formation which can generally be related to any way of producing a small rooted plant from micropropagated units (Debergh and Read, 1991) should be avoided when talking about synthetic seeds. They proposed that the general term re-growth should be used to indicate any vegetative development that is performed by an encapsulated propagule after sowing and that the re-growth should be specified as sprouting, rooting or shoot elongation accordingly. Conversion in itself would be considered a type of re-growth. The synthetic seeds could be considered a compendium of advantages of micropropagation, high productive efficiency, perfect sanitary conditions, reduced space requirements and the use as of plant seeds with handling and sowing ease, storability, reduced size of the propagules, transportability and mechanisation potential. Artificial seeds can also have particular relevance in the propagation of hand pollinated hybrids, elite germplasms and genetically engineered hybrids with sterility or unstable genotype complications (Mathur and Ahuja 1985). As an academic tool, artificial seeds offer comparative aid for better understanding of zygotic embrogeny, role of endosperm during embryo conversion, and seed coat formation.
Artificial seed technology can also be a promising alternative to microtuber production, since in vitro tuberisation is a time consuming, labour intensive process and the potato genotypes differ widely in terms of microtuber size, number, storage potential and dormancy (Debebrata and Naik 1997; Naik and Sarkar 1997). For vegetatively produced crops like potato, some fruit and nut trees, synthetic seeds would allow direct planting of non-grafted cultivars and provide an important alternative to the perpetual maintenance of living specimens for germplasm conservation. Synthetic seeds would be essential for genetically engineered crop plants that do not breed true due to the incorporation of meiotically unstable foreign genes. The intentionally introduced meiotic instability then would become an alternative to hybrid seed for commercialising propriety germplasm (Desai et al., 1997).
Synthetic or artificial seeds are basically of two types, embryonic or non-embryonic, hydrated or desiccated. Hydrated artificial seeds consist of propagules individually entrapped in a hydrogel. A variety of hydrogels are being employed (Table 1), however the most widely used is sodium alginate because it complexes easily with calcium chloride through an ionic exchange reaction, is biologically non-damaging, bio-degradable, universally available and is cheap.
Table 1. Hydrogels for artificial seed production
Working Complexing Working
Hydrogel concentration agent concentration
(WN %) (mM)
Sodium Alginate 0.5-6.0 Calcium chloride 30-100 Sodium alginate 2.0-4.0 Calcium chloride 30-100 + gelatin 5.0-6.0 Carrageenan 0.2-0.8 Potassium chloride 500 + or
Locust bean gum 0.4-0.8 Ammonium chloride Gelrite 0.25-0.40 Lowering of
Source: Redenbaugh el al. (1986); Redenbaugh et al. (1987)
Desiccated artificial seeds, on the other hand are produced by mixing propagules with 2.5% polyoxyethylene glycol and the dispensing 0.2ml of this mixture on a teflon surface in the form of wafers. These are left to dry for several hours under sterile conditions (Mathur and Ahuja, 1985).The encapsulate is a bio-degradable synthetic polymer coating that acts as an artificial seed coat.
The use of somatic embryos as synthetic seeds was long felt as a promising alternative to conventional potato propagule (Sarkar and Naik, 1998) who also showed for the first time that in vitro-derived potato nodal segments encapsulated in alginate-MS solution can be used for potato propagule production. As high as 57% encapsulated nodal segments survived in the soil when not covered and only 3% when covered on soil in the greenhouse after incubation under light for three days and treatment with a rooting hormone at planting time. Embryonic capacity was first observed by Lam (1975), and to date, few studies have involved potato somatic embryogenesis. Petrova and Dedicova (1992), showed somatic embryogenesis in
potato cultivar Désiréé from immature, zygotic-embryo sections. Garcia and Martinez (1995), reported the formation of somatic embryos from potato cells which become incorporated into mature tissue. However, somatic embryogenesis has not been common in potato tissue culture and as a result little has been accomplished to develop synthetic seeds in potato, coupled to this, the problems of abnormal somatic embryos, the long periods required to induce the embryos and non ability to synchronise the embryo maturity has greatly discouraged many researchers. Synthetic seeds offer the potential advantages of virus free, genetically identical materials, easy to handle, transport and increased efficiency of in vitro propagation in terms of space, time, labour and overall cost. Although most scientists have carried out encapsulation in sodium-alginate and with an increased research in the area of synthetic seed production, little attention has been directed towards storage of encapsulated propagules. Furthermore, storage of synthetic seeds using an alginate-encapsulation protocol has been attempted in only a few species with various degrees of success (Redenbaugh et al., 1987; Ganapathi et al., 1992; Muruyama et al., 1997a; 1997b). These hydrated encapsulated embryos could only be stored using low temperatures for a few weeks (Redenbaugh et al., 1986; Fujii et al., 1989; 1992). Here we present results of optimised storability studies of encapsulated shoot tips in calcium alginate hollow beads of potato.
3c. Disclosure of invention: Encapsulation of shoot tips in calcium alginate hollow beads (figure 1).
Synthetic seeds produced from shoot tips.
The shoots of in vitro propagated potato plantlets are dissected and trimmed to 3-4 mm in size using hypodermic needles, under a stereomicroscope placed in the laminar flow hood (Nyende, 2003; Schaefer-Menuhr et al., 1996). The trimmed, naked mersitematic shoot tips are then transferred to already autoclaved Petri dishes containing sterile Murashige and Skoogs media (Murashige and Skoog, 1962) containing 10 g/I sucrose, 3.4 g/I gelrite at pH 5.8, to avoid desiccation.
After which the shoot tips are mixed with 10 ml of 5% calcium chloride solution containing 2.5% carboxvmethylecellulose and the suspension dropped with a 10 ml hypodermic syringe into 500 ml of 0.5% sodium alginate water solution, stirred in a beaker (Nyende, 2003). As descrribed by Fiegert (1996), at the drop surface a calcium alginate laver is formed, developing and growing from inside to outside. After
very rare cases will the developing plantlets remain in the capsules. Root development may also be observed while the developing shoot tips re still in the capsules.
Figure 2. Developing shoot tips from alginate hollow beads
A B C D E
Synthetic seeds derived from somatic embryogenesis.
Shoot tip meristems are dissected from the 3-4 weeks old in vitro propagated potato (Solanum tuberosum ), using a microscope under sterile conditions. The excised shoot tips are then first cultivated in a liquid cullus induction medium containing growth hormones NAA and BAP (Fiegert et al., 2001) for a maximum of 25-33 days. Hard callus should be formed approximately 20 days after culture. Some calli may turn brown and fail to prolifereate. The shoot tip meristems are then placed in a 100 ml Erlenmeyer flask filled with 25 ml liquid callus induction medium and incubated on a shaker with 75 movements per minute under 12 hours of light with intensity of 850 Lm/m2. Temperature is maintained at 25 C. The formed calli are transferred to a liquid callus multiplication medium for multiplication under the same environmental conditions for 15 to 20 days. Approximately 10 % of the shoot tip meristems can be still vital after cultivation on this medium. Finally the calli are transferred to an embryo formation medium. On this medium calli differentiate after 30-35 days in to embyos upto torpedo stage When embryos are visible, the calli are transferred to solid MS-medium without phytohormones. Proliferation and production of embyos continue for more than 60 days after transfer on this medium. Each piece of calli can produce up to 26 embryos with bipolar structure that are loosely connected to the calli. On testing, the isolated embryos develop shoots and roots when germinated on hormone free MS medium. Unfortunately this method takes long, up to 80 days to produce embryos in liquid culture. This method still requires to be optimised in order to prevent to prevent the early death of shoot tips, to obtain a higher yield of mature embryos, and to synchronise the development stage of the embryos. Another alternative source of embryos would would be to use sterile shoots cut into 3 mm pieces, and or leaves cut into 1 mm size (Patel et al. 2000)
Chemicals: Sodium alginate (protanol LF20/60) obtained from Protan, Norderstedt, Germany and carboxymethylcellulose (Blanose 7MXF) from Aqualon, Dusseldorf, Germany. Gelrite from Roth, Karlsruhe, Germany. Carbendazim obtained from BASF, Germany. These materials could also be outsourced elsewhere.
The basal medium in these experiments comprised the inorganic and organic salts of a Murashige and Skoog (1962), at a pH of 5.8 and was autoclaved for 10 minutes at 128°C or 15 minutes at 121°C.
Source of explants: In vitro potato cultivars of potato (this also applies to other root crops) that have been certified to be disease free.
3e. Culture conditions and experimental medium.
The growth of sterile in vitro plantlets should be closely monitored as they are multiplied and propagated in Magenta jars (8 cm high). The plantlets are raised on MS (1962) medium at 20+/- 2°C and a 16 hour photoperiod provided by warm white fluorescent overhead light system (36-40 ΰmol m2 s2) for growth .
The MS media (40-50 ml in each jar) containing 10g "I sucrose, 3.4g-1 gelrite and is
autoclaved for 10 minutes at 128°C or 15 minutes at 121°C, depending on the autoclave type used. Plants are propagated by the shoot tips and leaf buds. One cycle lasts 3-4. In vitro materials of the same varieties are kept for safety precautions at 10°C under light intensities of 1500-2000 lux for a 16 hour day as described but without growth retardants (Mix, 1984 and Schaefer-Menuhr et al., 1998).
3f. Preparation of shoot tips.
Under a stereo-electron microscope placed on a clean bench (laminar flow hood), the shoot tip buds of the main shoot of approximately 5cm high plantlets are dissected using hypodermic needles according to Schaefer-Menuhr et al., (1996, 1998), Patel et al., 2000, Nyende, 2003 and Nyende et al., 2005).
1. A method of encapsulating plant parts into calcium alginate hollow beads
2. According to 1, the encapsulation involves mixing of the plants parts with 5% calcium chloride into 2.5 % carboxymethylecellulose and this suspension dropped into 0.5% sodium alginate. Ratio of 1:50 (VN) is maintained. This creates the hollow beads. This gelation process takes 30 minutes.
3. That the dropping in 2 above will involve the use of a 10 ml hypodermic needle (or a simulation of it: whether small scale or bio reactor magnitude).
4. That the size of the hollow beads in 1 can be adjusted by changing the gelation period. Either more or less time with respect to 30 minutes.
5. That the hollow beads in 1, can then be used as sowing or plant propagating materials.
6. That the hollow beads in 1, can be used as plant conservation materials either in vitro or under cryo.
7. That the hollow beads in 1, can be used as genetic exchange material between countries; can be used as active material for scientific research, and are guaranteed to be disease free.
Title: Synthetic (artificial) seed technique: For encapsulation in calcium alginate hollow beads
The present invention relates to synthetic (artificial) seed technique for encapsulation in calcium alginate hollow beads. In particular, the invention relates to the use of artificial seeds in potato and other root and tuber crops to avoid all the described disadvantages in their propagation as seed material. The synthetic seed production is carried out under sterile conditions in the lab providing disease free material. It offers no competition between seed material and the consumable yield. Since the synthetic seeds are small (0.8mm in diameter) they can be stored in small cool boxes that take insignificant space. This technique though labour intensive and technical, is relatively simple and with a short familiarization and availed facilities the local community can be able to perform it. Using the "Novel " artificial seed technique for encapsulation in calcium alginate (hollow beads), synthetic seeds of potato cultivars were produced from unipolar shoot tips (3-4mm) of in-vitro produced plantlets. The Shoot tips of potato cultivars were encapsulated in calcium alginate to produce synthetic seeds.
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