WO1990000002A1 - Procede de maturation d'embryons somatiques destines a etre plantes dans des environnements naturels - Google Patents

Procede de maturation d'embryons somatiques destines a etre plantes dans des environnements naturels Download PDF

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Publication number
WO1990000002A1
WO1990000002A1 PCT/US1989/002562 US8902562W WO9000002A1 WO 1990000002 A1 WO1990000002 A1 WO 1990000002A1 US 8902562 W US8902562 W US 8902562W WO 9000002 A1 WO9000002 A1 WO 9000002A1
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embryos
medium
somatic embryos
aba
somatic
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Jo Ann Fujii
David Slade
M. Keith Redenbaugh
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Kirin Brewery Co Ltd
Monsanto Co
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Kirin Brewery Co Ltd
Plant Genetics Inc
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A01H4/006—Encapsulated embryos for plant reproduction, e.g. artificial seeds

Definitions

  • the present invention relates generally to the asexual reproduction of plants for agriculture, such as crop, ornamental, and forestry plant production. More specifically, the invention relates to a method for the development of tissue culture-derived propagules that can be planted in a fashion similar to true seed.
  • Vegetative or asexual propagation has been used in agriculture throughout recorded history.
  • the advantage of vegetative propagation over seed propagation is that only mitotic cell division occurs, so that each daughter cell contains the same genetic information as the parent.
  • the progeny are clones of the parent.
  • seed propagation involves eiotic division and genetic recombination, such that the progeny contain genetic information from both parents.
  • Asexual propagation is especially important for heterozygous crops, such as bananas, conifers, figs, oranges, grapes, and many others. See Hartmann, H.T. and D. Kester, Plant Propagation. Prentice-Hall, Englewood Cliffs, New Jersey (1975) .
  • vegetative propagation is usually labor-intensive and slow, involving large areas of greenhouse or field space to maintain the mother plants. Although vegetative propagation is used for many crops, it is limited to those that have a high per-unit value.
  • plant tissue culture has been proposed and used for clonal propagation. Tissue culture methods were developed because of the faster propagation rate and the requirement for significantly less space for maintaining and producing plants. Tissue culture techniques, including somatic embryogenesis and organogenesis, have been used extensively for clonal propagation of plants.
  • the basic tissue culture process involves the following steps: 1) excise pieces of tissue from intact plants, 2) surface sterilize and place the tissue under a sterile environment with supplemental nutrients, carbohydrates such as sucrose, and other necessary growth components, 3) regenerate plants from the original tissue or from callus derived from the tissue via organogenesis or somatic embryogenesis, 4) grow the plants in vitro until sufficient leaves are developed for subsequent autotrophic growth, 5) harden the plants under controlled laboratory, growth chamber, or greenhouse conditions where the humidity can be regulated, and 6) transplant the resultant, hardened plant to a greenhouse or field environment for crop production. See Handbook of Plant Cell Culture, Evans, D.A. et al. editors, Vol. 1, Mac illan Publishing Co., New York (1983).
  • tissue culture technology as with vegetative propagation, has been useful for many plants.
  • the tissue culture process has several limitations; it requires many separate manipulations, the production of plants must be carefully performed in vitro to prevent premature water loss and wilting, and the process, though not as costly as vegetative propagation, is still too expensive for a broad variety of crops, particularly those for which the per-unit value (such as cost of seed) is medium to low.
  • the per-unit value such as cost of seed
  • Somatic embryos after formation, undergo a form of precocious germination when placed on the appropriate culture medium. Consequently, somatic embryos have not been handled and planted naturally under greenhouse conditions, with the exception of a few examples in which a small number of plants were produced. See Redenbaugh, K. , D. Slade, P. Viss, and J. Fujii, "Encapsulation of Somatic Embryos in Synthetic Seed Coats," HortSci. .22:803-809 (1987); Redenbaugh, K. B. Paasch, J. Nichol, M. Kossler, P. Viss, and . Walker, "Somatic Seeds: Encapsulation of Asexual Embryos," Bio/technology 4_:797-801 (1986). No plants are known to have been produced from somatic embryos planted naturally and directly under field conditions for any plant species.
  • somatic embryogeny is and will be analogous to zygotic embryogeny.
  • ABA abscisic acid
  • a high osmotic in vitro environment for example, high levels of mannitol or sucrose in the medium
  • a high osmotic in vitro environment for example, high levels of mannitol or sucrose in the medium
  • ABA and mannitol were compared side-by-side with in vitro culture of zygotic embryos, identical responses were measured in morphology, fresh weight, protein content, and levels of agglutinin in the embryos. See Morris, P.C. et al. , “Changes in the Levels of Wheat and Barley-germ Agglutinin During Embryogenesis In Vivo, In Vitro, and During Germination," Planta 166:407-413 (1985) .
  • ABA and embryo development are not mediated directly by ABA, but through the action of the growth regulator on water uptake.
  • Morris et al. postulated that when immature embryos are cultured upon media of high osmotic potential, they might adjust their endogenous ABA content so that the combined influence of the external osmoticum and endogenous growth substance inhibit precocious germination and allow continued embryo development. Consequently, ABA and osmotic agents would be expected to have a similar effect on embryo development.
  • a further effect of ABA during mid- to late- embryogeny is a selective inhibition of synthesis and translation of specific mRNA's, apparently those involved with embryogenesis.
  • ABA has been reported not to restrict accumulation of proteins, presumably because mRNA's for storage proteins are transcribed during early embryogenesis. Instead, ABA has been implicated in affecting partitioning of photosynthate and in not inhibiting storage protein mRNA's, thereby increasing levels of storage proteins in the zygotic embryos (Ackerson, R.C., "Regulation of Soybean Embryogenesis by Abscisic Acid,” J. Exp. Bot. 3_5:403-413 (1984)).
  • Another aspect of zygotic embryogeny that has been used to enhance true seed germination is to treat seed prior to planting with an osmotic agent, such as polyethylene glycol or mannitol, or with gibberellic acid. Also, simple imbibition of the seeds has been shown to be beneficial for germination. See Bradford, K. , “Manipulation of Seed Water Relations Via Osmotic Priming to Improve Germination Under Stress Conditions," HortSci. .21:1105-1112 (1986) and Bewley, J.D. and M. Black, Physiology and Biochemistry of Seeds. Volume 2, Springer-Verlag, New York (1982) .
  • Yet another object of the invention is to improve somatic embryo maturation.
  • a further object of the invention is to control the germination of somatic embryos so that their shelf life is significantly increased.
  • Still another object is to utilize pregermination of somatic embryos for enhancing conversion of somatic embryos planted directly in the growth chamber, greenhouse, and field.
  • plant somatic embryos at the precocious germination developmental stage are subjected to a maturation treatment wherein the somatic embryos are placed on a medium which contains abscisic acid at a concentration sufficient to induce embryo maturation for the times and conditions employed.
  • the maturation treatment of the present invention is surprisingly effective for somatic embryo germination outside an in vitro environment.
  • Matured somatic embryos produced in accordance with the invention are surprisingly unlike non-treated embryos in appearance as well as germination and conversion ability.
  • the maturation treatments were found to be useful for three plant species and are expected to be equally applicable to other species.
  • a method for maturing somatic embryos such that they can then be planted under growth chamber, greenhouse, and field conditions, without requiring a sterile, in vitro environment or added carbohydrates to sustain growth and viability.
  • Such mature somatic embryos are capable of forming complete plants under environments in which previously only true seeds could germinate and grow.
  • somatic embryos and other meristematic tissue is now a widely occurring phenomenon among many plant genera. Numerous important crop and horticultural species, including alfalfa, celery, carrot and lettuce, have been shown to be capable of propagation through tissue culture and somatic embryogenesis. For recent lists of such species, see Evans, D.A. et aJL. , “Growth and Behavior of Cell Cultures: Embryogenesis and Organogenesis," in Plant Tissue Culture: Methods and Applications in Agriculture, Thorpe, ed. , Academic Press, pg. 45 et seq. (1981); Ammirato, P., "Embryogenesis," In: Handbook of Plant Cell Culture (Evans, D. et aj ⁇ .
  • an in vitro environment is taken to mean one in which the sterility is maintained and a metabolizable carbon source is added for cell nutrition.
  • the temperature, humidity, and/or photoperiod are capable of being regulated in an in vitro environment to prevent their fluctuation with varying ambient conditions.
  • Abscisic acid has been used to improve the quality and appearance of somatic embryos via its addition to the plant cell culture medium during embryo formation.
  • ABA has been reported to permit embryo maturation, inhibit abnormal secondary embryo production, and repress precocious germination. See Ammirato, P., "Embryogenesis," In: Handbook of Plant Cell Culture (Evans, D. et al., eds.), Macmillan Publishing Co, New York, pp. 82-123 (1983) .
  • ABA Abscisic acid
  • ABA treatment in accordance with the present invention provides improved embryo maturation and allows somatic embryos which do not possess leaves, secondary roots or extended shoot apex to be planted naturally, as are true seeds.
  • the somatic embryos prepared in accordance with the present method can be simultaneously or sequentially subjected to a pregermination treatment.
  • pregermination is taken in a generic sense to mean any method to begin the biochemical or physiological processes of germination before planting of the embryos.
  • Other terms which are also used for this process include priming, osmoconditioning, vigorizing, chitting, etc.
  • callus is initiated from surface sterilized explant tissue from a selected plant species capable of undergoing somatic embryogenesis. Typically, leaf petioles will be employed to provide the explant tissue. Other explants which may be used for callus initiation are immature zygotic embryos, unfertilized ovules, anthers, young inflorescence, leaf sheaths, and somatic embryos.
  • the selected explant tissue is plated on a culture medium containing mineral salts, a carbon source, a plant growth regulator and at least one auxin.
  • the culture media which are utilized in various stages of the present method include any nutrient media known and developed for regeneration of whole plants from callus tissue.
  • Culture media useful in the present invention are generally composed of mineral salts, vitamins, carbohydrate, and, in certain stages of somatic embryogenesis, one or more hormones.
  • the mineral salts used in the media of the invention are well-known materials in the art, and are comprised of macroelements and microelements.
  • the mineral salt macroelements and microelements used in the induction medium are well known materials generally selected from the following compounds: ammonium sulfate, potassium nitrate, monopotassium phosphate, magnesium sulfate heptahydrate, manganese sulfate dihydrate, zinc sulfate heptahydrate, boric acid, potassium iodine, calcium chloride dihydrate, ferrous sulfate heptahydrate, ethylenediamine tetraacetic acid (di ⁇ odium salt) .
  • Other combinations of mineral salts may also be used.
  • Representative culture media mineral salts include, but are not limited to: Schenk- Hildebrandt salts (SH) , Schenk, R.U. and A.C. Hildebrandt, Can. J. Bot. .50:199-204 (1972) , or Murashige-Skoog salts (MS) , Murashige, T. and F.K. Skoog, Physiol. Plant. 15:473-497 (1962) .
  • SH Schenk- Hildebrandt salts
  • MS Murashige-Skoog salts
  • SH mineral salts comprise (in milligrams per liter) : potassium nitrate (2500) , calcium chloride dihydrate (200) , magnesium sulfate heptahydrate (400) , ammonium dihydrogen phosphate (300) , potassium iodide (1.0) , boric acid (5.0) , manganese sulfate monohydrate (10) , zinc sulfate heptahydrate (1.0), sodium molybdate dihydrate (0.1) , cupric sulfate pentahydrate (0.2) , cobalt chloride hexahydrate (0.1), ferrous sulfate heptahydrate (15) , disodium ethylenediamine tetraacetic acid (20) .
  • MS mineral salts comprise (in milligrams per liter) : ammonium nitrate (1650) , potassium nitrate (1900) , calcium chloride dihydrate (440) , magnesium sulfate heptahydrate (370) , cupric sulfate pentahydrate (0.025), manganese sulfate monohydrate (16.9), zinc sulfate heptahydrate (8.6), potassium phosphate (170) , boric acid (6.2) , potassium iodine (0.83), sodium molybdate dihydrate (0.25), cobalt chloride hexahydrate (0.025), disodium ethylenediamine tetraacetic acid (37.3), ferrous sulfate heptahydrate (27.8).
  • other combinations of mineral salts comprised of macroelements and microelements may be known or hereafter developed and incorporated into the practice of the invention.
  • Vitamins are also generally included in the medium. Typical vitamins, such as inositol, nicotinic acid, pyridoxine hydrochloride, and thiamine hydrochloride, among others, are included in plant tissue culture medium, in accordance with known techniques.
  • a carbon source generally consisting of readily metabolizable carbohydrate, is also included in the medium.
  • the most commonly used carbon source is the disaccharide sucrose.
  • Other saccharides, including fructose or maltose, can be employed in the medium at, e.g., approximately 5 to lOOg per liter to provide acceptable cell culture production.
  • the carbon source most often employed in the media of the invention is usually sucrose, in a concentration of approximately 30g per liter of medium.
  • An optional plant growth regulator such as a cytokinin is often included in plant growth media at a concentration sufficient to stimulate the growth of the plant tissue.
  • cytokinin Sigma Chemical Co. ; Hoechst
  • kinetin Sigma Chemical Co. ; Hoechst
  • kinetin can be employed as a plant growth regulator and will generally be used at a concentration of approximately 0.1 to 20 ⁇ M, more usually at an optimum range of from approximately 1 to 15 ⁇ M.
  • Hormones such as "auxins,” are known to be useful during somatic embryogenesis and are employed in the media of the present invention including, e.g., 2,4-D (2,4-dichlorophenoxyacetic acid), picloram (4-amino-3,5,6-trichloro picolinic acid), DICAMBA (2,6- dichloro-o-anisic acid) , IAA (indole-3-acetic acid) and NAA (naphthaleneacetic acid) may also be used, either alone or in combination, in the practice of the invention.
  • 2,4-D (2,4-dichlorophenoxyacetic acid) picloram (4-amino-3,5,6-trichloro picolinic acid)
  • DICAMBA 2,6- dichloro-o-anisic acid
  • IAA indole-3-acetic acid
  • NAA naphthaleneacetic acid
  • the auxin employed in the media used in the practice of the invention to produce and grow callus tissue is generally a known auxin such as a- naphthaleneacetic acid, at a concentration of approximately 0.1 to 500 ⁇ M, with an optimum range of from approximately 1 to 50 ⁇ M, either alone or in combination with other auxins.
  • Cells are induced to form embryos by incubation in an induction medium, such as SH medium + 50 ⁇ M 2,4-D, for approximately four days, after which time the cells are transferred to a regeneration medium.
  • an induction medium such as SH medium + 50 ⁇ M 2,4-D
  • the embryo regeneration medium employed in the invention is also generally a medium well-known in the art, consisting of mineral salts and other components, as described above, but generally without auxins.
  • auxins may be present at concentrations much lower than for the induction medium. See Stuart, D. and S. Strickland, "Somatic Embryogenesis From Cell Cultures of Medicago sativa L. I. The Role of Amino
  • the regeneration medium may contain other beneficial substances desirable for plant cell growth and development, such as one or more amino acids.
  • amino acids are grouped generally in accordance with certain characteristics of particular subclasses. Amino acid residues can be generally classified into four major subclasses as follows:
  • Acidic - i.e., the residue has a negative charge due to loss of H ion at physiological pH
  • Neutral/non-polar i.e., the residues are not charged at physiological pH and the residue is repelled by aqueous solution
  • Neutral/polar i.e., the residues are not charged at physiological pH and the residue is attracted by aqueous solution .
  • Acidic Aspartic acid and Glutamic acid
  • Neutral/polar Glycine, Serine, Cysteine, Threonine, Asparagine, Glutamine, Tyrosine;
  • the regeneration medium can contain an addition of one or more neutral/non-polar amino acids.
  • amino acids particularly preferred are L-proline and L-alanine.
  • Plant cells are incubated for approximately three weeks in the regeneration medium, after which the formed embryos are usually aseptically transferred to a solidified conversion medium consisting of half-strength mineral salts, a carbon source such as carbohydrate, preferably maltose at approximately 1.5% (w/v), and with reduced concentration or no auxins. Somatic embryo quality is observed after approximately 30 days, by counting the percent of embryos which ultimately form plants with roots and shoots.
  • a solidified conversion medium consisting of half-strength mineral salts, a carbon source such as carbohydrate, preferably maltose at approximately 1.5% (w/v), and with reduced concentration or no auxins. Somatic embryo quality is observed after approximately 30 days, by counting the percent of embryos which ultimately form plants with roots and shoots.
  • a plant species can be selected and somatic embryos formed having a bipolar structure consisting of a root
  • embryos are at or near the developmental stage where precocious germination can occur. At this stage, the somatic embryos may be able to germinate .in vitro and produce whole plants, but they are unable to produce plants at any consequential frequency in an uncontrolled environment, such as that found in the field.
  • the somatic embryos are then placed on a medium, such as an agar medium, a liquid suspension, or a humid environment, which contains abscisic acid (ABA) at a concentration ranging from 0.001 to lOOO ⁇ M, more usually 0.01 to 100 ⁇ M, and preferably 0.1 to lO ⁇ M.
  • a medium such as an agar medium, a liquid suspension, or a humid environment, which contains abscisic acid (ABA) at a concentration ranging from 0.001 to lOOO ⁇ M, more usually 0.01 to 100 ⁇ M, and preferably 0.1 to lO ⁇ M.
  • the medium may also contain nutrients and carbohydrates as desired to promote the growth and viability of a particular species.
  • the medium will consist of those components necessary for basic growth of tissue cultures, such as Murashige and Skoog medium (MS salts plus the components recited above) or Schenk and
  • ABA is presently considered to be the preferred embodiment for the maturation treatment of the present invention, it will be readily appreciated that other compounds with ABA-like activity may be employed as either alternatives or supplements to ABA.
  • ABA-analog compounds including:
  • PA Phaseic acid
  • DPA Dihydrophaseic acid
  • HM-ABA 6*-hydroxymethyl abscisic acid beta-hydroxy abscisic acid beta-methylglutaryl abscisic acid beta-hydroxy-beta-methylglutarylhydroxy abscisic acid 4'-desoxy abscisic acid abscisic acid beta-D-glucose ester
  • the temperature will be 0-40°C, more usually 10-30°C, and preferably 15-25°C.
  • the embryos will be matured in light or dark conditions, more preferably dark.
  • the embryos After ABA maturation, the embryos will have a hardier, more robust appearance.
  • the embryos can be stored on the ABA medium further, if greenhouse and field conditions are not suitable for planting.
  • the embryos Prior to planting, the embryos are removed from the ABA medium and placed on a medium, such as an agar medium, a liquid suspension, or a humid environment, to promote priming and pregermination of the somatic embryos.
  • a medium such as an agar medium, a liquid suspension, or a humid environment.
  • This treatment also allows for ABA to leach out of the embryos and for the embryos to begin the process of germination away from the inhibitory effects of ABA.
  • the medium will be a basic medium such as SH or MS. Alternatively, the medium may consist solely of water to promote embryo germination.
  • Certain embodiments of the present invention include a pregermination treatment, administered either simultaneously or sequentially with the maturation treatment of the invention.
  • an osmotic agent in an aqueous solution of sufficient concentration to inhibit root and shoot growth is added to the medium at the appropriate time.
  • a typically useful osmotic agent such as mannitol, generally administered at 1 to 15%, will serve to control root emergence.
  • Other known pregermination agents include a monovalent salt. Many monovalent salts are useful, such as potassium nitrate (KNO 3 ) . Potassium nitrate inhibits germination at concentrations between 0.3 and 1.0M, preferably 0.4 to 0.6M. Small molecular weight organic molecules can also serve as an osmoticum.
  • An additional, optional pregermination agent which can be added as a component of the maturation medium of the invention, is one or more gibberellins, preferably GA 3 , GA 4 , or GA 7 , depending on the plant species employed to generate somatic embryos.
  • the gibberellin component is usually added at concentrations of 0.001 to lOOO ⁇ M, more usually 0.01 to 500 ⁇ M, and preferably 0.1 to 300 ⁇ M.
  • the somatic embryos are primed/pregerminated for 1 hour to 1 month, more usually 12 hours to 1 week, and preferably 1 to 5 days.
  • the temperature will be 0-40°C, more usually 10-30°C, and preferably 15-25 ⁇ C.
  • the embryos will be in light or dark, more preferably light.
  • After preparing the somatic embryos in accordance with the invention they will ordinarily be planted in a growth chamber, greenhouse or directly in the field.
  • the present somatic embryos are planted in the greenhouse or field and covered with a translucent moisture barrier, generally in accordance with the teachings of U.S. Patent No. 4,612,725, the relevant portions of which are incorporated herein by this reference.
  • the somatic embryo-derived plantlet will be maintained under the moisture barrier until the plantlet is capable of withstanding the less controlled planting environment.
  • the following demonstrations were carried out with a variety of species. Although it was expected that the effect of ABA and of pregermination might be confined to only one species, alfalfa, it was subsequently found to be a universal effect, useful for all plant species tested. Consequently, the ABA and pregermination effects are not to be construed to be limited only to the examples presented, but to be widely applicable in plant somatic embryogenesis.
  • A.l. Production of Alfalfa Somatic Embryos Callus was produced from alfalfa leaf petioles placed on Schenk and Hildebrandt medium (SH) , supplemented with 25 ⁇ M ⁇ -naphthaleneacetic acid (NAA) and lO ⁇ M kinetin. The callus was then incubated for 3 weeks on SH medium with potassium nitrate replaced by 20mM potassium citrate and 25mM glutamine.
  • SH Schenk and Hildebrandt medium
  • NAA ⁇ M ⁇ -naphthaleneacetic acid
  • lO ⁇ M kinetin The callus was then incubated for 3 weeks on SH medium with potassium nitrate replaced by 20mM potassium citrate and 25mM glutamine.
  • the callus was induced to alter its developmental progression by being cultured on SH medium with 50 ⁇ M 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 ⁇ M kinetin for 3 days.
  • the induced callus was placed on SH medium with lOmM ammonium nitrate and 30mM proline, causing numerous somatic embryos to form after three weeks at 24°C. See Stuart, D. and S. Strickland, "Somatic Embryogenesis From Cell Cultures of Medicago sativa L. I. The Role of Amino Acid Additions to the Regeneration Medium," Plant Sci. Lett. 34:165-174 (1984) ; and Stuart, D. and S.
  • the embryos were placed in shallow furrows, with cotyledons facing upright, and without any potting mix covering.
  • the pan of potting mix was covered by a clear plastic cover, slightly ajar, and placed into a growth chamber at 25°C under fluorescent lights (12-hour days) .
  • the number of plantlets formed from embryos was counted six weeks after planting.
  • Example A.l were stored at 4°C in the dark for one day.
  • Selected embryos were placed on SH medium containing 0, 1, 2.5, 5, 7.5 or lO ⁇ M ABA (maturation medium) and cultured for 14 days at 24°C in the dark.
  • somatic embryos were removed from the media and placed on conversion medium as described in Example A.2.
  • the ABA matured somatic embryos had significantly changed in their color and appearance.
  • the embryos had gone from a green to white color and had become bulkier, heavier and more robust.
  • All treatments that received an ABA treatment formed plantlets in potting mix with plantlet formation ranging from 36-54% (of total embryos planted) .
  • Optimum ABA concentrations under these conditions were between 1 and 7.5 ⁇ M. The embryos that received no ABA exposure did not form any plants.
  • A.3.b Somatic embryos produced as described in Example A.l were placed onto SH + 2.5 ⁇ M ABA medium for various lengths of time at 24°C in the dark to determine the optimum maturation period. The embryos were removed from the medium and placed on conversion medium as described in Example A.2. Maximum conversion of 40-44% was obtained when the ABA exposure length was 20 to 31 days. The lowest conversion (8-14%) was obtained when the ABA exposure was less than 14 days. For maturation periods greater than 31 days, conversion decreased slightly (30-37%) , indicating that a 20-31 day period was optimum. Without ABA treatment, no plants formed.
  • A.3. ⁇ Somatic embryos were produced as described in Example A.l.
  • the embryos were placed into liquid SH + ABA (5, 10, or 20 ⁇ M) in a flask and shaken on a rotary shaker at 24°C in the dark for 20 days.
  • Other embryos were placed onto semi-solid medium SH + ABA (5, 10, or 20 ⁇ M) at 24 ° C in the dark for 20 days.
  • the embryos which were matured in the liquid culture medium had the same high quality appearance as embryos matured on the semi-solid medium, indicating that the ABA exposure is equally effective when done in liquid as in semi-solid medium.
  • Somatic embryos produced as described in Example A.l were matured under one of two in vitro environments: SH + 5 ⁇ M ABA at room temperature or on the original regeneration medium at 4°C for 30 days. Embryos were subsequently removed from the culture treatment and some of the embryos were examined for changes in fresh and dry weights, while other embryos were converted as described in Example A.2.
  • ABA-treated embryos displayed an increase of fresh weight from 2mg/embryo to 6mg/embryo and a dry weight increase of 0.3mg/embryo to 2mg/embryo, while cold-treated embryos showed no increase in fresh or dry weights.
  • ABA-treated embryos increased in average percent dry matter from 14% to 31%, while cold-treated embryos had no increase. Finally, ABA-treated embryos had 47% conversion while cold-treated embryos had 0% conversion.
  • ABA-matured embryos displayed an increase in starch content from 10 ⁇ g starch/embryo (or 4 ⁇ g starch/g fresh weight) to 164 ⁇ g starch/embryo (or 22 ⁇ g starch/g fresh weight)
  • cold-matured embryos increased starch content from lO ⁇ g starch/embryo (or 4 ⁇ g starch/g fresh weight) to 16 ⁇ g starch/embryo (or 6 ⁇ g starch/g fresh weight) .
  • the somatic embryos were converted as per Example A.2. ABA-matured embryos had 83% conversion, whereas cold-matured embryos had 5% conversion.
  • A.3.f Somatic embryos produced as described in Example A.l were matured on SH + 5 ⁇ M ABA for 3-12 weeks. The embryos were removed from the maturation medium and converted to plants as described in Example A.2. The conversion frequency remained unchanged over the maturation period at 56-66%.
  • A.4.a Somatic embryos produced as described in Example A.l, except that 50 ⁇ M 2,4-D was replaced with lOO ⁇ M 2-(2,4-dichlorophenoxy) propanoic acid. The embryos were placed onto SH + 5 ⁇ M ABA for 21 days at 24°C.
  • the embryos were then transferred to SH, containing various levels of GA 3 (0, 25, 50, 100, 150 ⁇ M) for 2 days at 24°C (pregermination treatment).
  • the embryos were planted in potting mix as described in Example A.2, except the embryos were covered by a thin layer of potting mix. This planting method was harsher than in previous examples, as the embryos were required to germinate and grow through a covering of potting mix.
  • the highest conversion (19%) was obtained when the pregermination treatment consisted of SH + lOO ⁇ M GA3.
  • somatic embryos that had received higher or lower levels of GA 3 also converted to plants, but at a lower frequency (5-10%) than embryos that received the 100 ⁇ M GA 3 treatment.
  • A.4.b Somatic embryos produced as described in Example A.4.a and treated with SH medium + 5 ⁇ M ABA for 21 days were placed into liquid SH medium + lOO ⁇ M GA3, either shaking on a rotary shaker or without shaking, or on semi-solid medium containing agar. Conversion of all the treatments was done as described in Example A.2 and the frequencies were similar (64-78%) , indicating that the pregermination treatment can be used equally well with semi-solid medium as well as liquid medium.
  • A.5. Effect of Sequence of Exposure to ABA and GA 3 on Conversion in the Growth Chamber.
  • Somatic embryos were produced as described in
  • A.6 Effect of Osmotica on Conversion in Growth Chamber.
  • A.6.a Somatic embryos produced as described in Example A.l were placed onto SH media containing various levels of ABA and 8% mannitol for 16 days at 24°C in the dark. Subsequently, the somatic embryos were converted as described in Example A.2.
  • A.6.b Somatic embryos produced as described in Example A.l were placed onto SH media containing l ⁇ M ABA with 11% sucrose or 5 ⁇ M ABA without sucrose for 21 days at 24°C in the dark. Subsequently, the somatic embryos were converted as described in Example A.2.
  • sucrose with ABA gave a low conversion frequency of 4%, while with ABA alone, the conversion was 48%. Sucrose was not sufficient as a substitute for ABA during embryo maturation.
  • Somatic embryos produced as described in Example A.l were placed onto SH medium + 5 ⁇ M ABA for 21 days at 24°C in the dark. The embryos were removed from the medium and placed on SH medium + 100 ⁇ M GA 3 for 2-4 days at 24°C in the light. The embryos were then planted directly in the field using a natural process common to planting true seed. The embryos were sown and then left uncovered, covered by a styrofoam cup, covered with a white plastic sheet, or covered with a cloth sheet (white polyester from McCalif Growers Supplies, Inc., San Jose, CA) . After 6 weeks, conversion was measured.
  • the callus was further maintained on SH medium containing l.O ⁇ M 2,4-D and 0.5 ⁇ M kinetin at 25°C in the dark. Somatic embryos were formed after 3 weeks incubation at 25°C under fluorescent lights by placing the callus onto SH medium with the ammonium level reduced to 5mM, the sucrose reduced to 1%, and with the addition of 8% mannitol.
  • Somatic embryos produced as described in Example B.l were planted in a potting mix (10:9:1 ratio of peat:vermiculite:perlite; obtained from McCalif Growers Supplies, Inc., San Jose, CA) , without sucrose and without efforts to maintain a sterile environment, using a process similar to that used for true seed.
  • the embryos were placed in shallow furrows, with cotyledons facing upright, and without any potting mix covering.
  • the pan of potting mix was covered by a clear plastic cover, slightly ajar, and placed into a growth chamber at 25°C under fluorescent lights (12-hour days) .
  • the number of plantlets formed from embryos was counted six weeks after planting.
  • Somatic embryos produced as described in Example B.l were matured at 4°C in the dark for three days on the original regeneration petri dishes. Selected embryos were placed on SH media containing 1, 10 or lOO ⁇ M ABA for 5 days at 24"C in the dark. The somatic embryos were then converted as described in Example B.3. All ABA-treated embryos formed plants in potting mix (at frequencies of from 7-40%) with the greatest frequency of plant formation when the embryos were matured on SH + l ⁇ M ABA (40%) . Somatic embryos that had not been treated with ABA had no conversion in the potting mix.
  • Somatic embryos produced as described in Example B.l were matured at 4°C in the dark for three days on the original regeneration petri dishes. Selected embryos were placed on SH medium containing 10, 20, 30, 40 or 50 ⁇ M ABA for 5 days at 24°C in the dark.
  • Example B.l were placed onto SH medium + 40 ⁇ M ABA for 21 days at 24°C. The embryos were then transferred to SH medium containing various levels of GA 3 (0, 50, 100,
  • Example B.l were placed onto SH medium + 40 ⁇ M ABA for 21 days at 24°C. The embryos were then transferred to SH medium containing various levels of GA 3 (0, 25, 50, 100,
  • Embryos that did not receive the pregermination treatment of GA 3 converted at a frequency of 5%. The highest conversion frequency (56%) was achieved using SH medium with no GA 3 . All of the other embryos that had been pregerminated had significantly higher conversion frequencies than embryos that did not receive any pregermination treatment.
  • Somatic embryos produced as described in Example B.l were placed onto SH medium + 30 ⁇ M ABA, containing various levels of mannitol (0, 2, 4, or 6%), for 10 days at 24°C in the dark. The embryos were then pregerminated on SH for 2 days at 24°C in the dark. Subsequently, the somatic embryos were converted as described in Example B.3. The highest conversion frequency of 52% was obtained in the treatment using the ABA medium containing 4% mannitol. Embryos matured on the ABA medium without the addition of mannitol converted at a frequency of 32%.
  • Carrot seeds were surface sterilized and plated onto MS medium for ten days at 25°C in the dark.
  • Petioles were excised and placed into static liquid culture in Lin & Staba medium (LS) (Fuji ura, T. and A. Komamine, "Effects of Various Growth Regulators on the Embryogenesis in Carrot Cell Suspension Culture," Plant Science Lett. .5:359-354 (1975)), containing lpp 2,4-D, for one month at 25°C in the dark.
  • Cell clusters were sieved and fractions under 300 ⁇ m in size were cultured in liquid LS medium containing O.lppm 2,4-D for one month at 25° C, in the dark, on a rotary shaker at 80 rpm.
  • Somatic embryos produced as described in Example C.l were planted in a potting mix (5:2:1 ratio of peat:vermiculite:perlite; obtained from McCalif Growers Supplies, Inc., San Jose, CA) , without sucrose and without efforts to maintain a sterile environment, using a process similar to that used for true seed.
  • the embryos were placed in shallow furrows, with cotyledons facing upright, and without any potting mix covering.
  • the pan of potting mix was covered by a clear plastic cover, slightly ajar, and placed into an growth chamber at 25°C under fluorescent lights (12-hour days) . The number of plantlets formed from the embryos was counted six weeks after planting.
  • C.3. ABA Effect on Embryo Conversion in Growth Chamber.
  • C.3.a Somatic embryos produced as described in Example C.l were matured at 4°C in the dark for eight days. Selected embryos were placed on SH medium containing 1 or 10 ⁇ M ABA for 7 days at 24°C in the dark. These and other embryos that had not been matured with ABA were planted in potting mix as described in Example C.2.

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  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

Le nouveau procédé décrit sert à produire des plantes à partir d'embryons somatiques qu'il n'est pas nécessaire de planter dans un environnement in vitro. On produit ainsi des embryons somatiques mûrs obtenus par culture tissulaire, qui présentent une robustesse similaire à des embryons de vraies graines (zygotiques) et réagissent de la même façon lorsqu'ils sont plantés dans des environnements d'ensemencement types.
PCT/US1989/002562 1988-06-14 1989-06-13 Procede de maturation d'embryons somatiques destines a etre plantes dans des environnements naturels Ceased WO1990000002A1 (fr)

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WO1991001629A1 (fr) * 1989-08-01 1991-02-21 British Columbia Research Corporation Procede de fabrication, de sechage et de germination des embryons somatiques de coniferes
US5334530A (en) * 1992-03-10 1994-08-02 Woods Susan H Method and media for the somatic embryogenesis and regeneration of bamboo
US6340594B1 (en) 1991-12-19 2002-01-22 Cellfor, Inc. Production of desiccation-tolerant gymnosperm embryos
US6372496B1 (en) 1991-12-19 2002-04-16 Cellfor, Inc. Desiccation-tolerant gymnosperm embryos

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001629A1 (fr) * 1989-08-01 1991-02-21 British Columbia Research Corporation Procede de fabrication, de sechage et de germination des embryons somatiques de coniferes
GB2250674A (en) * 1989-08-01 1992-06-17 British Columbia Res Corp A process for the production, drying and germination of conifer somatic embryos
GB2250674B (en) * 1989-08-01 1994-03-02 British Columbia Res Corp A process for the propagation of conifer somatic embryos
US6340594B1 (en) 1991-12-19 2002-01-22 Cellfor, Inc. Production of desiccation-tolerant gymnosperm embryos
US6372496B1 (en) 1991-12-19 2002-04-16 Cellfor, Inc. Desiccation-tolerant gymnosperm embryos
US5334530A (en) * 1992-03-10 1994-08-02 Woods Susan H Method and media for the somatic embryogenesis and regeneration of bamboo

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AU3842589A (en) 1990-01-23

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