WO1995014373A1 - Procede de reproduction de coniferes par embryogenese somatique a l'aide d'un milieu d'entretien enrichi au maltose - Google Patents

Procede de reproduction de coniferes par embryogenese somatique a l'aide d'un milieu d'entretien enrichi au maltose Download PDF

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WO1995014373A1
WO1995014373A1 PCT/US1994/013532 US9413532W WO9514373A1 WO 1995014373 A1 WO1995014373 A1 WO 1995014373A1 US 9413532 W US9413532 W US 9413532W WO 9514373 A1 WO9514373 A1 WO 9514373A1
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embryos
medium
maltose
early stage
stage
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PCT/US1994/013532
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English (en)
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Pramod K. Gupta
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Weyerhaeuser Company
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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

Definitions

  • the present invention is a method for reproducing coniferous plants by somatic embryogenesis using the techniques of plant tissue culture. More specifical- ly, it relates to the use of a selected sugar as energy source in the culture media used during specific stages of somatic embryo development.
  • the invention is especially suited for producing large numbers of clones of superior selections useful for reforestation.
  • the callus was placed on a budding medium where adventitious buds formed. These, in turn, were separated, elongated, and rooted to ultimately form plantlets.
  • a plantlet has the na ⁇ ture of a seedling but is genetically identical to the explant donor plant. Gymnosperms in general, and most forest tree species in particular, proved to be much more difficult to reproduce by tissue culture. It was not until about 1975 that Douglas-fir was successfully reproduced by organogenesis. Loblolly pine was successfully reproduced about two years later.
  • compositions of the media used to initiate embryogenesis and induce embryo maturation are critical to success, regardless of the species being propagated.
  • the type and level of the nitrogen source in the media and the presence or absence, composition, level, and timing of availability of growth hormones have been the key to success. It is also these very factors, particularly the hormones, that have proved to be so unpredictable.
  • Ammirato (1977) conducted a study examining the effects of zeatin (a cytokinin), ABA, and gibberellic acid (GA 3 ) on the yield and morphology of caraway (Carum carv ⁇ ) somatic embryos. These hor ⁇ mones were present singly and in all possible combinations in the media used for the later stages of embryo development.
  • Uddin in U.S. Patent 5,187,092, describes somatic embryogenesis of loblolly pine using glucose or maltose in combination with abscisic acid in the mature embryo development medium.
  • the data available in the Uddin patent are very lim ⁇ ited.
  • a two stage treatment in which the ABA level is in ⁇ creased and the auxin indolebutryic acid is added to the second stage is necessary if the claimed improvements are to be achieved.
  • the high level of ABA and the step- wise increase are at odds with others in the field who have found that ABA is needed at relatively low levels which should preferably be decreased during the development period; e.g. as taught in U.S. Patents 5,034,326 and 5,236,841.
  • the present invention is directed to the use of a particular sugar as the carbon and energy source in the media used at the different stages of conifer embryo- genesis. It is especially directed to the use of the sugar maltose in the maintenance media used following embryo initiation by somatic embryogenesis.
  • the replacement of the sucrose or glucose normally used at this stage of culture by maltose results in larger and more robust advanced early stage embryos of generally improved morphology.
  • Maltose is also advantageously used in place of sucrose in the medium when a singulation step is found useful between the early stage embryo development and cotyledonary embryo development stages. This step is preferably used with Douglas-fir where the early stage embryos tend to form in clumps, some of which may persist throughout the rest of the culturing procedure.
  • the present method is especially suitable for reproducing woody gym- nosperms of the order Coniferales. It is particularly well suited for generating large clones of superior forest trees for reforestation, including species within the families Pinaceae, Cupressaceae, and Taxodiaceae. Most or all species within the genera Abies, Pinus, Picea, Tsuga, Pseudotsuga, Thuja, Junipe ⁇ s, Larix, Taxus and Sequoia are believed to be amenable to multiplication by the present method.
  • the method is particularly advantageous in that it ultimately enables more robust somatic embryos to be produced. These have a high degree of similarity to the natural zygotic embryos produced within the seed.
  • Cytokinins are plant growth hormones that affect the organization of dividing cells.
  • Callus is generally considered to be a growth of unorganized and ei- ther unconnected or loosely connected plant cells generally produced from culturing an explant.
  • Embryogenic callus is a translucent white mucilaginous mass that contains early stage embryos attached to suspensors. This is also referred to as an “embryonal-suspensor mass” or “ESM” by some investigators.
  • An "early stage embryo”, also sometimes referred to as a proembryo before elongation of suspensor, is a small mass of cells with dense cytoplasm and large nuclei that have the potential of forming a plant. The early stage embryo is normally found as a head having a relatively small number of undifferentiated dense cells with large nuclei associated at the end of one or more long thin-walled suspen- sor cells.
  • an "advanced early stage embryo” is larger than an early stage embryo and has a smooth embryonal head associated with multiple suspensor cells.
  • the ad ⁇ vanced early stage embryo is much more robust than an early stage embryo.
  • Many investigators refer to these as "globular embryos".
  • Advanced early stage embryos generally show no or only the initial stages of internal cell differentiation when sectioned.
  • a "cotyledonary embryo”, sometimes simply referred to as an "embry- o" has a well defined elongated bipolar structure with latent meristematic centers having clearly visible cotyledonary primordia and an apical dome at one end and a la- tent radicle at the opposite end.
  • the cotyledonary structure frequently appears as a small “crown” at one end of the embryo.
  • a cotyledonary somatic embryo is analo ⁇ gous to a zygotic embryo.
  • a “mature embryo” is a cotyledonary embryo with adequate storage material (proteins, lipids, and carbohydrates) so as to be tolerant to desiccation.
  • An “explant” is a piece of tissue taken from a donor plant for culturing.
  • a “meristem” or “meristematic center” is a group of tissue forming cells capable of further development into plant organs; e.g., shoots and roots.
  • An "osmoticant” or “osmoticum” is a chemical material used for con ⁇ trolling the osmotic potential of a solution. In the present context the solution would be a culture medium.
  • a "plantlet” is a plant asexually reproduced by tissue culture.
  • a “converted embryo” is an embryo that has germinated and been es ⁇ tablished as a plant growing in soil.
  • Somatic embryogenesis is the process using tissue culture techniques for generating multiple embryos from an explant.
  • the embryos generated from a given tissue source are believed to be genetically identical.
  • the present method as a whole comprises a multistage culturing pro ⁇ cess.
  • a suitable explant is first placed on an induction or initiation culture medium. This will usually contain relatively high quantities of growth hormones including at least one auxin and frequently one or more cytokinins. However, with some species growth hormones at this initial stage may not always be necessary or desirable for in- duction of early stage embryos. A number of sources of explants have in the past proved to be satisfactory for culturing.
  • tissue from cotyledons include, but are not limited to, tissue from cotyledons, hypocotyls, epicotyls, buds, meristematic centers for buds or roots, and seed embryos.
  • Zygotic embryos removed from seeds are presently preferred. These may or may not include the surrounding gametophyte. In particular, for spe- cies which before have proved to be very difficult or impossible to propagate by so ⁇ matic embryogenesis, the embryos from immature seeds may be preferred.
  • the first stage induction or initiation medium will normally be one of those well known from past work which contain a balanced concentration of inorgan ⁇ ic salts and organic nutrient materials, with plant growth hormones included as noted above.
  • Auxins are normally present in concentrations which may initially be as high as about 600 ⁇ M/L, more typically not exceeding about 500 ⁇ M/L.
  • Cytokinins if present, may initially be as high as 500 ⁇ M/L.
  • the plant growth hormones may in ⁇ clude at least one auxin and one cytokinin in a combined initial concentration not ex ⁇ ceeding about 1100 ⁇ M/L, more typically not exceeding about 900 ⁇ M/L.
  • auxins and cytokinins used and their exact concentrations, or whether they are used at all, will depend somewhat on the species being cultured and even on the particular genotype within that species. This is something that cannot be easily pre ⁇ dicted but can be readily determined experimentally.
  • These very high levels of growth hormones assume the presence in the medium of an adsorbent material, such as activated charcoal. Where charcoal is not present the levels of growth hormones would normally be much lower; e.g., a full order of magnitude, than those just noted.
  • Culturing during the induction or initiation stage may be carried out in the dark, under very low light conditions, or in full light until an embryogenic mass forms. Lighting conditions will depend in large part on the composition of the par ⁇ ticular medium selected. In general, initiation in full dark is preferred.
  • This em- bryogenic mass has been described by various other names by researchers who have reported it in the past; e.g., embryogenic callus (Hakman and von Arnold 1985) or embryonal-suspensor mass (Durzan and Gupta 1987). It has the appearance of a whitish, translucent, mucilaginous mass containing very small early stage embryos which are readily apparent by low power light microscopy (FIG. 1).
  • the preferred induction medium for Douglas-fir will preferably contain an auxin or auxins in amounts of about 400-600 ⁇ M/L and a cytokinin or cy ⁇ tokinins in the amount of about 240-500 ⁇ M/L in combination with 0.05-1.0% acti- vated charcoal.
  • the osmotic potential of the maintenance medium should be significantly increased over that of the induction medium.
  • the osmotic potential will most usually exceed about 160 mM/kg and will more typically be above about 180-200 mM/kg.
  • the optimum osmoticant levels at each stage will usually differ for each species and often for individual geno ⁇ types within a species.
  • the osmotic level should typically be of the magnitude of at least 200 mM/kg and preferably about 240 mM/kg or even higher. However, lower levels of about 170 mM/kg minimum will suffice for most genotypes of Douglas-fir.
  • This osmotic "pulse” contributes to em ⁇ bryo quality and size with the development of advanced early stage embryos (FIG. 2).
  • Some species such as Norway spruce, which are relatively easy to reproduce, may not require this raised osmotic level, or it may only be necessary for some geno- types.
  • advanced early stage embryo development may usually be achieved without a change in medium composition other than reduced hormone con ⁇ centrations.
  • weekly subcultures are made when the embryos are on mainte ⁇ nance medium.
  • sucrose has been employed as the carbon or energy source in the maintenance medium.
  • maltose is much to be preferred to sucrose.
  • Advanced early stage embryos produced using maltose in the maintenance medium have significantly larger embryonal heads than those produced using sucrose. These are both longer and of greater diameter.
  • the associated suspensor cells are also elongated more. This re- suits in stronger embryos that, in turn, produce more robust cotyledonary somatic embryos having close similarity to zygotic embryos.
  • Incubation at this stage is usually carried out in the dark or in greatly reduced light until robust advanced early stage embryos have formed. Subcultures are usually carried out on a weekly basis at this stage. The embryos may then be transferred to a cotyledonary embryo development medium which usually lacks aux ⁇ ins and cytokinins entirely.
  • Douglas-fir should generally have an intermediate culturing step be ⁇ tween the advanced early stage embryo growth stage and the final cotyledonary em ⁇ bryo development stage. With this species many of the embryos form in tight clumps or clusters. These are first preferably singulated before going to the development stage. Singulation is carried out in a series of liquid shake cultures lacking auxins and cytokinins but which have exogenous abscisic acid added as a necessary new hor ⁇ mone.
  • ABA will usually initially be within the range of 5-15 mg/L (20-60 ⁇ M/L) with osmotic potential levels in the range of 130-160 mM/kg.
  • singula ⁇ tion process will encompass two or three transfers at weekly intervals following the initial singulation treatment.
  • a preferred procedure uses an initial treatment with ABA at a 10 mg/L level followed by two treatments .at weekly intervals with ABA at a 5 mg/L concentration.
  • the present invention should be considered sufficiently broad so that the terms "singulation” or “singulation stage” are fully equivalent to "maintenance culture” or “maintenance stage”.
  • the singulation stage may be considered a special ⁇ ized type of maintenance stage.
  • species other than Douglas-fir can be advantageously cultured by beginning early cotyledonary embryo development in a series of media
  • the advanced early stage embryos are then placed on a cotyledonary embryo development medium.
  • the final development stage or stages it is most desirable for the final development stage or stages to be carried out on either solid medium or with liquid medium using a pad system.
  • the osmotic potential of the later stage cotyledonary development medium should be sharply raised above that of any of the
  • Initially levels may be in the 300-350 mM/kg range but these should be increased to levels of at least about 400 mM/kg as development proceeds. If development is started at levels around 300-350 mM/kg, the osmotic level may be increased during development by a complete medium change, a partial change in which some old medium is replaced, or by adding an appropriate form, such as a
  • the osmotic levels at the end of the development period should be at least about 450 mM/kg although with some genotypes lower lev ⁇ els are acceptable. With some Douglas-fir genotypes final osmotic levels as high as
  • Osmotic potential in the later stages of cotyledonary development is best controlled by a combination of osmoticants.
  • One of these should be a readily metabolized carbohydrate energy source, preferably a sugar such as sucrose, glucose, fructose, maltose, or galactose.
  • Sucrose is a preferred ingredient and may be present in amounts in the range of 2-6% .
  • the other is a poorly metabolized osmoticant of which sorbitol, lactose, or a polyalkylene glycol would be examples.
  • sorbitol, lactose and polyethylene glycol has proved very effective.
  • the mo ⁇ lecular weight of the PEG is not critical and may fall in the range of several hundred to several thousand. While the salts and organic components of the medium make a small contribution to the osmolality, the osmotic potential is primarily controlled by the energy-providing sugar and the other osmoticants. It is sometimes advantageous to use one combination of osmoticants at the beginning of development and transfer to a medium having a different combination at some point during the development stage.
  • the penultimate media should have osmotic potentials of at least about 350 mM/kg, preferably about 400 mM/kg or higher.
  • abscis ⁇ ic acid with the adsorbent usually required a higher initial concentration of abscisic acid than was the case if no adsorbent was present in the medium.
  • ABA may be reduced in stepwise fashion as detailed in U.S. Patent 5,236,841. Acti ⁇ vated charcoal or other adsorbents are not necessary using the procedure of this pat- ent.
  • the level of exogenous abscisic acid should be generally continuously low ⁇ ered over time from the 5-15 mg/L normally found necessary at the beginning of the singulation step or cotyledonary embryo development stage to a level perhaps of about 1-2 mg L, or even to zero, at the end of the development stage.
  • Accurate mea- surements of abscisic acid present in the development stage have not yet been made due to the extreme difficulties of analyzing the medium. It is possible in some cases to produce cotyledonary embryos without exogenous ABA in the development me ⁇ dium. However, the embryos so produced are usually of inferior quality.
  • the embryos may be placed directly on a germination medium for conversion into plantlets (FIG. 4). Al ⁇ ternatively, they may be converted into artificial seeds by any of a number of pub ⁇ lished processes.
  • the germination medium has no exogenous hormones, a lowered or ⁇ ganic nitrogen content, and a reduced level of osmoticants.
  • the coty ⁇ ledonary embryos will have developed into plantlets. Douglas-fir does not require an initial dark period although an initial four day dark period is usually more satisfacto- ry. A one week dark period is useful for Norway spruce.
  • the time period for ger ⁇ mination will be about 1-2 months.
  • the resulting plantlets will have a well developed radicle and cotyledonary structure with a growing epicotyl and are ready for planting in soil.
  • the present invention is primarily concerned with the composition of the embryo maintenance and multiplication media and the method of their use.
  • the composition of the embryo singulation medium is also a con ⁇ cern.
  • the replacement of sucrose by maltose as the carbon and energy source gives improved size and vigor of advanced early stage embryos and further improves the morphology of subsequently cultured cotyledonary embryos. This im- provement is manifested in an improved conversion rate.
  • Maltose has been found to be advantageous on concentrations as high as 6% w/v (60,000 mg/L) in the mainte ⁇ nance media. Preferred concentrations are on the 2-4% range.
  • FIGURE 1 shows early stage embryos.
  • FIGURE 2 shows advanced early stage embryos.
  • FIGURE 3 depicts cotyledonary stage embryos.
  • FIGURE 4 shows a plantlet ready for transfer to soil.
  • FIGURES 5 and 6 respectively show changes over time in pH and os- molality of maintenance media made using sucrose and maltose.
  • FIGURES 7 and 8 are microphotographs showing early stage and ad ⁇ vanced early stage Douglas-fir embryos maintained respectively on sucrose and maltose-containing media.
  • FIGURES 9 and 10 are low power microphotographs of loblolly pine cotyledonary embryos cultured using sucrose and maltose respectively in the mainte ⁇ nance stage.
  • the process of the present invention is not limited to any single basal culture medium or to the use of specific growth hormones other than those defined in the claims.
  • Any of a number of well known basal media such as that of Murashige and Skoog (1962), may be used.
  • the present inventors have found the bas- al media described in Table 1 to give excellent results, particularly when used for culturing Douglas-fir (Pseudotsuga menziesif).
  • the basal media are modified for each of the various culturing stages as shown in Table 2. Similar media particularly preferred for Norway spruce (Picea abies) are given in Tables 9 and 10, and for Lo ⁇ blolly pine (Pinus taeda) in Tables 11 and 12.
  • a number of abbreviations are used in the following text. These are in com ⁇ mon use in the field of tissue culture.
  • BAP N*-benzylaminopurine (or N*-benzyladenine), a cytokinin.
  • KIN kinetin (6-furfurylaminopurine), also a cytokinin 2,4-D — 2,4-dichlorophenoxyacetic acid, an auxin NAA - 2-naphthylacetic acid (naphthalene-2-acetic acid), also an auxin.
  • ABA abscisic acid (5-(l-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohex- en-l-yl)-3-methyl-2,4-pentadienoic acid), a maturation promoter.
  • IAA indole-3-acetic acid
  • IBA indole-3-butyric acid
  • NAA naphthalene-2-acetic acid
  • 2-IP N 6 -isopentenylaminopurine
  • zeatin are frequently used as cytoldnins.
  • the fol ⁇ lowing table of conversions from weight to molar concentrations might be useful.
  • U.S. Patent 4,957,866 pointed out the importance of the control of osmotic potential of the media used in the various culturing stages.
  • a large group of chemical materials are suitable as os ⁇ moticants. In general these are highly water soluble polyhydroxylated molecules that include either simple or complex sugars, hexitols, and cyclitols.
  • the cyclitols are normally six carbon ring compounds that are hexahydroxylated. The most readily available cyclitol is my ⁇ -inositol but any of the other eight stereoisomeric forms, such as --ry-./o-inositol are believed to be quite suitable.
  • sucrose and glucose are known to be very effective and have been widely used in the past.
  • V 2 embryonic heads present but no organization of suspensor cells around heads.
  • 1 embryonic heads formed but rough or irregular. Suspensor cells partly organized around heads.
  • a basal culture medium has been developed by the present inventors specifically to give more successful initiation and multiplication of Douglas-fir.
  • Pre ⁇ ferred media compositions are given in Tables 1 and 2.
  • a number of ingredients may be varied in quantity, such as those that affect the level and balance between organic and inorganic nitrogen, depending on the response of individual genotypes. This re- sponse cannot be readily predicted and media optimization must largely be achieved by a combination of intuition and trial and error.
  • Sorbitol (D-glucitol), D-mannitol, and galactitol (dulcitol) are straight chain sugar alcohols suitable as osmoticants. Lactose is a sugar effective as an osmoticant. Other materials suitable as osmoticants may include glycol ethers such as poly(ethylene glycol) and poly(propylene glycol) and their respective monomers.
  • /ny ⁇ -Inositol 1000 1000 1000-30,000 100 100 100
  • Kinetin 43 0.22 0.22 ⁇ — ⁇
  • sucrose is the sugar used in Stage 1 and Stages 5 and 6.
  • sucrose or maltose is used as shown in the specific examples. Malt ⁇ ose has proved to give superior results.
  • a raised osmotic level following initiation is desirable for good quality advanced early stage embryo development. This level will differ somewhat between genotypes within each species as it does between species.
  • the level of abscisic acid present should be gradually reduced during the singulation stage and also during the cotyledonary embryo development period, if exogenous ABA is added in that stage. This may be done either by the inclusion of activated charcoal in the medium or by a stepwise reduction effected by multiple transfers to media of successively lower ABA concentration.
  • the exogenous ABA level is preferably gradually reduced from that present at the beginning of the singulation stage so that little or none is available at the end of the development period.
  • a preferred explant for Douglas-fir is an immature zygotic embryo with the gametophyte still attached. Best results have been realized with embryos selected in the interval just prior to the development of an apical dome up to the time just before cotyledon primordia become visible.
  • the cones are split longitudinally and seeds iso ⁇ lated from young ovuliferous scales. Seeds are sterilized by first being agitated in 10% Liqui-Nox laboratory cleaner (Alconox, Inc, New York, New York) with a small addi ⁇ tional amount of liquid surfactant for about 10 minutes. They are then rinsed in running tap water for 30 minutes.
  • the embryonal-suspensor masses containing early stage embryos are transferred to a solid Stage II maintenance and multiplication medium containing greatly reduced plant growth hormones and preferably a somewhat raised osmotic level. Again, culturing is carried out in the dark with subcultures made at no greater than about two week intervals. The clone can be maintained at this stage for long pe ⁇ riods of time.
  • liquid mainte- nance media maltose is substituted for the sucrose used in the initiation culture on an equal weight basis unless otherwise indicated in the following examples.
  • Stage III second maintenance medium having a sig ⁇ nificantly raised osmotic level.
  • An osmotic level of at least about 170 mM/kg will usually suffice for Douglas-fir although some genotypes may require levels as high as 240 mM/kg.
  • a y ⁇ -inositol which will normally be around 5000 mg/L, may need to be adjusted somewhat depending on the needs of the particular genotype in order to obtain optimum results.
  • Culture is carried out in the dark and is periodically subcul- tured, usually weekly. Robust advanced early stage embryos estimated to have 100 or more cells will develop during this time, normally 5-6 weeks.
  • Stage IV liquid medium for the singulation step referred to earlier.
  • the singulation medium has a reduced osmotic level and is free of auxins and cytokinins.
  • Abscisic acid is a newly added hormone in an initial amount in the range of about 5-15 mg/L, more usually about 5-10 mg/L. Cultures are again carried out in the dark. From two to four sub ⁇ cultures are made on a weekly basis. The level of exogenous abscisic acid will drop somewhat during each subculture.
  • the level of abscisic acid at the beginning of a new subculture should not be significantly higher than the level used in the previous subculture.
  • a preferred schedule is one week on a medium containing 10 mg/L ABA, a second week on a medium containing 5 mg/L ABA, and a third week on a medium also with 5 mg/L ABA. This gradual decrease in ABA level will continue through the development period.
  • the embryos are rinsed with a fresh singulation medium in which ABA is reduced to 2.5 mg/L, before transfer to the cotyledonary development medium.
  • the embryos are ready to complete their development to cotyledonary embryos on a Stage V medium. They are transferred to either a solid medium or supported on a pad or bridge of filter paper using a liquid medium.
  • This will normally contain exogenous ABA which may be present up to about 50 mg/L. More typically, ABA will not generally exceed about 10 mg/L and most usually will not initially exceed 5 mg/L and may be considerably lower. In some cases it is not necessary to add any exogenous ABA to the develop ⁇ ment medium since a sufficient amount will be carried over with the residual singula ⁇ tion or rinse medium accompanying the embryos when the transfer is made from the last singulation stage.
  • the development medium may also contain from 0.5-50 mg/L of a selected gibberellin.
  • the cotyledonary embryos may be placed on a Stage VI germination medium for production of plantlets. Alternatively, they may be placed in artificial seeds for sowing in soil or other medium.
  • Example 2 An experiment was carried out using cultures of three Douglas-fir ge ⁇ notypes with four different maintenance media. These were made using 3% and 5% sucrose and 3% and 5% maltose. These concentrations of sugars were used in both the Stage II and Stage III maintenance media. Cultures were repUcated three times. The first cultures in the Stage III liquid media were made using the entire culture of embryonic cells from the Stage II solid media using 20-25 mL of medium in a 250 mL Erlenmeyer flask. Thereafter subcultures were made using 5 mL settled cells and 45 mL of medium. Four to five subcultures were made on a weekly basis. Quality rating of the advanced early stage embryos is shown in the following table. Table 3
  • Example 3 The above experiment was repeated using Genotype 995/36 from the previous example and three new genotypes of Douglas-fir. Embryo quality was ob- served as follows after 4-5 Stage III subcultures:
  • the improved embryo quality resulting from the use of maltose in the maintenance medium is again readily apparent.
  • both 3% and 5% maltose were superior to either of the sucrose containing media.
  • the results using a medium with 3% maltose were superior to the medium using 5% maltose.
  • Average osmolalities of the media containing sucrose were noted to in ⁇ crease after each one week culture period.
  • the medium with 3% sucrose increased from 190 to 260 mM/kg while that with 5% sucrose went from 300 to 359 mM/kg.
  • the 3% maltose medium showed only an insignificant change from an initial 189 to 5 193 mM/kg while the 5% maltose medium increased from 260 to 261 mM/kg.
  • Example 4 To further investigate the effect of osmotic change during the weekly subculturing periods, in this example 3% filter sterilized maltose was used in side-by- 0 side comparison with the 3% sucrose normally used in the Stage II and III Douglas- fir maintenance media.
  • the Stage 3 liquid shake culture was carried out using 270 mL of medium and 30 mL of settled cells in 1 L Erlenmeyer flasks.
  • fructose, a hydrolysis product of su ⁇ crose may be toxic or is otherwise a poorly metabolized or inefficient energy source.
  • FIGS. 7 and 8 show typical embryos. These are photomicrographs at 2.5 X in which FIG. 7 is representative of the early stage embryos cultured on the sucrose-containing medium and FIG. 8 representative of the embryos cultured on maltose-containing medium. The improved head size and morphology of the maltose treated embryos shown in FIG. 8 is immediately evident.
  • sucrose was used as the sugar in the Stage II medium.
  • No sub- cultures were made at Stage II and the cultures were transferred to Stage 3 after two weeks.
  • Four genotypes were used with each condition being replicated three times. Subcultures were carried out in 250 mL Erlenmeyer flasks using 5 mL of settled cells and 45 mL of the medium being tested. Embryo quality measurements after 4-5 sub ⁇ cultures are given in Table 7 .
  • Example 6 It was noted earlier that maltose was beneficial when used as the car ⁇ bon and energy source for the Stage IV Douglas-fir singulation cultures following the maintenance stages. The following experiment was designed to show this effect.
  • Two batches of Stage IV singulation medium (from Table 2) was made up, one using 2% sucrose and the other 2% maltose.
  • the singulation treatment was started using 5 mL of settled cells from Stage III and 45 mL of medium in 250 mL Erlenmeyer flasks. A singlation schedule of 10/5/5 mg/L ABA was used.
  • the initial singulation medium contained 10 mg/L ABA. After one week the embryos were transferred to a medium of similar composition except that ABA was reduced to 5 mg/L.
  • the embryos were transferred to a third medium identical to the second one; i.e., with 5 mg/L ABA, for a third week of treatment.
  • the embryos were rinsed with the Stage IV shake medium having 2.5 mg/L ABA prior to transfer to a Stage V cotyledonary develop- ment medium
  • Three genotypes of Douglas-fir were used in the present experiment. Table 7 shows embryo quality ratings after the first and second ABA shake treatments.
  • the yield was markedly higher with those on the maltose media averaging 45+7 compared with 23+6 embry- os per plate on the all sucrose media.
  • Cotyledonary embryos grown on both maltose containing maintenance and singulation were elongated even more with a yield per plate of 42+5 embryos per plate.
  • Morphology of cotyledonary embryos grown on either maltose regimen was markedly more like zygotic embryos than those on the su ⁇ crose regimen. They tended to be more evenly tapered and smoother, with far fewer wart-like protuberances or callusing on the surface.
  • KNO j reduced to 1170 mg/L
  • myo-Inositol reduced to 100 mg/L
  • Sucrose reduced to 20.0 g/L
  • L-Glutamine and Casamino acids removed.
  • Agar are added.
  • Basal medium A from Table 4 ⁇ 2
  • Basic medium B from Table 4 ⁇ 3
  • 2-Naphthylacetic acid Naphthalene-2-acetic acid
  • Explants were the female gametophytes containing the zygotic embryos which had been removed from seeds 4 to 5 weeks after fertilization. The seed coat was removed but the embryo was not further dissected out of the surrounding gametophyte. Seeds were obtained from cones supplied by a Weyerhaeuser Company seed orchard located at Washington, North Carolina. The cones were stored at 4 ° C until used. Immedi ⁇ ately before removal of the immature embryos the seeds were sterilized using a modi- fied method of Gupta and Durzan (1985). Briefly, this involves an initial washing and detergent treatment followed by a first sterilization in 30% H 2 O 2 and a second in diluted 10% v/v household bleach. The additional HgCi. treatment used by Gupta and Durzan was not found to be necessary to ensure sterility. The explants were thoroughly washed with sterile distilled water after each treatment. Tables 11 and 12 give media compositions for loblolly pine embryogenesis.
  • Maltose is substituted for sucrose on an equal weight basis as indicvated in the examples.
  • the following amino acid mixture is add- ed: L-proline - 100 mg/L, L-asparagine - 100 mg/L, L- arginine - 50 mg/L, L-alanine 20 mg/L, and L-serine - 20 mg/L.
  • BM Germination Medium BM modified by reducing sucrose to 20,000 mg/L, myo-inositol to
  • Stage I - Induction Sterile dissected embryos were placed on a solid BM, culture medium and held in an environment at 22-25C with a 24 hour dark pho- toperiod for a time of 3-5 weeks. The length of time depended on the particular ge ⁇ notype being cultured. At the end of this time a white mucilagenous mass had formed in association with the original explants. This appears to be identical with that described by Gupta and Durzan (1987). Microscopic examination revealed nu- merous early stage embryos associated with the mass. These are generally character ⁇ ized as having a long thin-walled suspensor associated with a small head with dense cytoplasm and large nuclei. Typical early stage embryos are illustrated in FIG. 1.
  • Osmolality of the induction medium may in some instances be as high as 170 mM/kg. Normally it will be about 160 mM/kg or even lower.
  • the osmolal- ity of the medium described above was 150 mM/kg.
  • Stage II - Maintenance and Multiplication Early stage embryos re ⁇ moved from the masses generated in the induction stage were first placed on a BM 2 gelled maintanance and multiplication medium. This differs from the induction me ⁇ dium in that the growth hormones (both auxins and cytokinins) were reduced by a full order of magnitude. Osmolality of this medium will typically be raised from that of the induction medium to about 190 mM/kg or higher by increasing the concentra ⁇ tion of myo- inositol to 0.5% w/v. The temperature and photoperiod were again 22 ° -25 ° C witii 24 hours in the dark.
  • Embryos were cultured 12-14 days on the BM 2 solid medium before transferring to a liquid medium for further subculturing. This liquid medium was of similar composition but lacked the gellant. The embryos at the end of die solid maintenance stage were similar in appearance to those from Stage 1. After 5 to 6 weekly subcultures advanced early stage embryos had formed. These are characterized by smooth embryonal heads estimated to have over 100 individual cells with multiple suspensors, as exemplified in FIG. 2.
  • Osmotic potential of the maintenance medium should typically fall within the range of about 190-400 mM/kg for Pinus taeda. Most typically it should be in the neighborhood of about 1.5 times higher than that of the induction or multi- pliction media. As was noted earlier, the requirements for elevation of osmotic po- tential at this stage will vary for different species and may vary somewhat even for differing genotypes within a given species.
  • Stage IIII - Embryo Development The advanced early stage embryos from Stage II culture were transferred to a solid BM 3 medium.
  • devel ⁇ opment may be on a saturated pad or similar support on liquid medium.
  • This me- dium either lacks growth hormones entirely or has them present only at very low levels and has the same lower level of osmoticants as Stages I and II.
  • abscisic acid (5-(l-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-l-yl)-3-methyl-2,4- pentadienoic acid) appears to be a necessary material for further development.
  • abscisic acid (5-(l-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-l-yl)-3-methyl-2,4- pentadienoic acid) appears to be a necessary material for further development.
  • an adsorbent material in this medium is highly advantageous.
  • the adsorbent may be chosen from a number of chemical ma ⁇ terials having extremely high surface area and/or controlled pore size such as acti ⁇ vated charcoal, soluble and insoluble forms of poly (vinyl pyrrolidone), activated alumina, silica gel, molecular sieves, etc.
  • the adsorbent will normally be present in a concentration of about 0.1-5 g/L, more generally about 0.25-2.5 g/L.
  • the osmotic potential of this medium may be raised substantially over that of the maintenance medium. It has been found advantageous to have an osmolality as high as 300 mM/kg or even higher.
  • development is preferably carried out in complete darkness at a temperature of 22 ° -25 ° C. Development time was 5-6 weeks after which elongated cotyledonary embryos 4-5 mm long were present. These appeared as represented in FIG. 3.
  • Stage IV - Germination Cotyledonary embryos from Stage III were placed on solid BM 4 medium for germination. This is a basal medium lacking growth hormones which has been modified by reducing sucrose, wy ⁇ -inositol and organic ni ⁇ trogen. After about 6-8 weeks under environmental conditions of 23 ° -25 ° C and a 16 hour light/8 hour dark photoperiod the resulting plantlets were approximately 20 mm in length and had a well developed radicle and hypocotyl and green cotyledonary structure and epicotyl. Alternatively, the cotyledonary embryos may be made into artificial seeds as was noted earlier. The young plantlets are shown in FIG. 4.
  • the osmotic poten ⁇ tial of the germination medium is further reduced below that of the development me ⁇ dium. It will normally be below about 150 mM/kg and was, in the present example, about 100 mM/kg.
  • Stage V Conversion Plantlets from Stage IV were removed from the culture medium and planted in a soil comprising equal parts of peat and fine perlite. Rooting percentage was excellent and the resulting plants showed good growth and vigor.
  • Example 8 In order to see whether the advantageous effects of using maltose in the maintenance medium observed with Douglas-fir and Norway spruce also held true for loblolly pine, the following tests were made. One set of solid and liquid mainte ⁇ nance media was made using 3% sucrose while a similar set of media were made with 3% maltose. Early stage embryos from initiation were placed on each solid maintenance medium for 2 weeks then the resulting mass of embryos was transferred to a corresponding liquid maintenance culture using 20-25 mL of medium in a 250 mL Erlenmeyer flask. After the first liquid culture and thereafter 5 mL of settled cells were transferred to 45 mL of medium After 5-6 weekly subcultures the embry ⁇ os were examined. The advanced early stage embryos cultured on the maltose media were better singulated and more robust than those cultured on sucrose. They had sig ⁇ nificantly larger and smoother heads with more elongated suspensors.
  • the advanced early stage embryos from the maintenance media were tiien placed on BM 3 cotyledonary development medium conbtaining 3% sucrose and otherwise composed as described in Tables 11 and 12 for further development.
  • 1 mL of settled cells was placed on 10 mL of solid cotyledonary develop ⁇ ment medium. After abour six weeks of culturing, the resulting cotyledonary embry ⁇ os were compared.
  • the embryos from the cultures maintained on the maltose containing media were significantly improved over those maintained on the sucrose containing media (FIG. 9).
  • the maltose cultured embryos were morphologically more were like zygotic embryos. They were longer and smoother and had more uni ⁇ form taper, lacking the prominent inflated "waist area" of their sucrose cultured counterparts.
  • the maltose maintained embryos had a greater number of cotyledons. This is believed to be a definite advantage for germination and conversion since the cotyledons rapidly take over the process of manufacturing nutri ⁇ ents after germination. This also points out the importance of having very strong ad ⁇ vanced early stage embryos for subsequent development.
  • Plant Cell Reports 132 199-202. Sondahl, Maro R., T. B. Sereduk, Stephan M. Bellato, and Zhenghua Chen

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Abstract

L'invention concerne un procédé pour reproduire des conifères par embryogénèse somatique à l'aide de techniques de culture de tissus végétaux dans un processus de mise en culture multi-étapes. Un explant approprié, en général l'embryon fertilisé excisé d'une graine immature, est tout d'abord mis en culture sur un milieu induisant de multiples pro-embryons précoces. Ceux-ci se multiplient dans une deuxième culture possédant des hormones de croissance réduites. Un apport de maltose constitue la source de carbone et d'énergie dans cette deuxième culture. Les embryons précoces croissent en taille et en vigueur et deviennent des embryons précoces avancés. Ces embryons sont ensuite transférés à une culture de développement d'embryons cotylédonaires. Après plusieurs semaines, des embryons somatiques présentant l'aspect d'embryons zygotiques se seront formés. Leur germination peut s'effectuer avant ou après stockage et leur transplantation en terre peut intervenir en vue de leur croissance ultérieure. L'emploi de maltose dans le milieu de culture d'entretien et de multiplication permet d'obtenir des embryons précoces avancés, d'une taille et d'une robustesse accrues, qui, à leur tour, donnent des embryons cotylédonaires de morphologie très similaire à des embryons zygotiques naturels. L'utilisation de maltose à des stades plus précoces du développement des embryons est plus importante que son emploi pour leur maturation.
PCT/US1994/013532 1993-11-23 1994-11-23 Procede de reproduction de coniferes par embryogenese somatique a l'aide d'un milieu d'entretien enrichi au maltose WO1995014373A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996037097A1 (fr) * 1995-05-26 1996-11-28 Weyerhaeuser Company Procede permettant de reproduire des coniferes par embryogenese somatique au moyen d'un milieu de conservation enrichi en maltose
US6417001B2 (en) 1995-05-25 2002-07-09 Carter Holt Harvey Limited Embryogenesis process for initiation
EP2332405A1 (fr) * 2003-06-23 2011-06-15 Weyerhaeuser Company Procédés et milieux de culture tissulaires destinés à induire une maturation de l'embryon somatique de conifère
CN103461119A (zh) * 2013-08-20 2013-12-25 中国林业科学研究院林业研究所 粗枝云杉体细胞胚胎发生与植株再生方法
CN104304034A (zh) * 2014-11-11 2015-01-28 新疆林科院造林治沙研究所 天山云杉体细胞胚愈伤组织的诱导培养方法及其专用诱导培养基
US9078427B1 (en) 2014-08-29 2015-07-14 Pioneer Hi Bred International Inc Method of storing plant embryos
US10278345B2 (en) 2014-08-29 2019-05-07 Pioneer Hi-Bred International, Inc. Methods and devices for creating doubled haploid embryos using oil matrices
CN114788496A (zh) * 2022-04-07 2022-07-26 江苏省中国科学院植物研究所 一种固液交替培养诱导落羽杉高效体胚发生的方法

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US4801545A (en) * 1983-05-19 1989-01-31 Plant Genetics, Inc. Enhanced somatic embryogenesis using maltose
US5036007A (en) * 1989-03-09 1991-07-30 Weyerhaeuser Company Method for reproducing coniferous plants by somatic embryogenesis using abscisic acid and osmotic potential variation
US5187092A (en) * 1990-03-22 1993-02-16 Institute Of Paper Science And Technology, Inc. Somatic embryogenesis in gymnosperms

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US4801545A (en) * 1983-05-19 1989-01-31 Plant Genetics, Inc. Enhanced somatic embryogenesis using maltose
US5036007A (en) * 1989-03-09 1991-07-30 Weyerhaeuser Company Method for reproducing coniferous plants by somatic embryogenesis using abscisic acid and osmotic potential variation
US5187092A (en) * 1990-03-22 1993-02-16 Institute Of Paper Science And Technology, Inc. Somatic embryogenesis in gymnosperms

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6417001B2 (en) 1995-05-25 2002-07-09 Carter Holt Harvey Limited Embryogenesis process for initiation
WO1996037097A1 (fr) * 1995-05-26 1996-11-28 Weyerhaeuser Company Procede permettant de reproduire des coniferes par embryogenese somatique au moyen d'un milieu de conservation enrichi en maltose
EP2332405A1 (fr) * 2003-06-23 2011-06-15 Weyerhaeuser Company Procédés et milieux de culture tissulaires destinés à induire une maturation de l'embryon somatique de conifère
CN103461119A (zh) * 2013-08-20 2013-12-25 中国林业科学研究院林业研究所 粗枝云杉体细胞胚胎发生与植株再生方法
US9078427B1 (en) 2014-08-29 2015-07-14 Pioneer Hi Bred International Inc Method of storing plant embryos
US10278345B2 (en) 2014-08-29 2019-05-07 Pioneer Hi-Bred International, Inc. Methods and devices for creating doubled haploid embryos using oil matrices
US10477859B2 (en) 2014-08-29 2019-11-19 Pioneer Hi-Bred International, Inc. Plant embryo storage and manipulation
CN104304034A (zh) * 2014-11-11 2015-01-28 新疆林科院造林治沙研究所 天山云杉体细胞胚愈伤组织的诱导培养方法及其专用诱导培养基
CN114788496A (zh) * 2022-04-07 2022-07-26 江苏省中国科学院植物研究所 一种固液交替培养诱导落羽杉高效体胚发生的方法

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