Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01C—PLANTING; SOWING; FERTILISING
  • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
• • 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 to synthetic seed products and methods for their production.
• the present invention derived in part from work performed under Grant 82-CRCR-1-1086 from the U.S. Department of Agriculture. Prior Art
• seed is the primary means of planting most agronomic crops, asexual embryogenesis or the use of genetically uniform tissue culture- derived propagules, engineered to possess similar efficient handling qualities, would be advantageous for production of certain crops or cultivars.
• Potential applications of such "synthetic seed” include: (a) large-scale planting of outstanding genotypes for self-incompatible species that are difficult to propagate vegetatively, (b) production of genetically uniform seed for highly heterozygous crops that are currently propagated only from vegetative material, (c) propagation of novel genotypes produced by genetic engineering that are not meiotically stable, (d) commercialization and maintenance of proprietary germplasm containing intentionally introduced meiotic instability, and (e) maintenance of parental inbred lines.
• Somatic cell embryogenesis is the best prospect for synthetic seed production because somatic embryos are nearly identical to zygotic embryos, and the labor required for their production is low compared to other clonal propagation systems.
• somatic and zygotic embryos typically cease growth, becoming quiescent or dormant as water is lost, storage tissues mature, and the seed coat hardens.
• This arrested growth phase is the major factor accounting for the efficient storage and handling qualities of seed.
• a similar arrested growth phase induced during somatic embryo development would be essential to match the efficiency of seed propagation and would be a pivotal step in the development of synthetic seed technology.
• the above and other objects are achieved by the present invention which provides a synthetic seed product comprising dehydrated somatic plant embryos produced by the method described below which, upon rehydration and growth, yields plants which are essentially identical to the plant from which the somatic plant embryo is developed.
• the method of the invention for producing the synthetic seed product comprises inducing growth quiescence in plant somatic evmbryos by maintaining the somatic embryos in an environment having a relative humidity or storage water content of from about 30% to about 85% for a period of time sufficient to reduce the moisture content of the embryos to from about 85-65% to about 4-15%, depending on plant species, and for the embryos to cease growth.
• the embryos are then capable of being stored for prolonged time periods (e.g., up to about 1 year) at this moisture range.
• the invention further includes a method of developing plants by somatic embryogenesis employing the above-described dehydrated somatic plant embryos.
• the present invention is predicated on the discovery that quiescence may be induced in somatic plant embryos by dehydrating the embryos at a certain critical relative humidity or storage water content range. Attempts to achieve quiescence or dormancy by drying the somatic embryos in atmospheres or environments having a water content outside this range result in products which cannot be stored and/or will not regenerate plants upon rehydration.
• relative humidity and “storage water content” refer to the water content of the atmosphere or environment, to which the somatic embryos are exposed during dehydration.
• the embryo may be maintained in an atmosphere having a relative humidity of 30% to 85% or they may be placed in solutions or coated with compositions which are "osmotically active", i.e., materials which promote the osmotic transfer of moisture from the embryos to,the surrounding medium.
• FIG. 1 depicts the rate of water loss by somatic embryos.
• the solid line shows weight stabilization at about the 24 h mark. After 48 h the embryos were subjected to 60°C for 24 h to remove the remaining water (broken line for determination of fresh and stored water content).
• the method of the invention reversibly arrests the characteristically rapid growth of plant somatic embryos.
• the quiescent embryos may then be stored at room temperature and growth leading to plant development is stimulated by rehydration when desired. This is a crucial stage in synthetic seed technology where fragile, metabolically active somatic embryos must be engineered to mimic durable, quiescent seed.
• the invention will find utility in all instances where clonal plant production with planting efficiencies of conventional seed is necessary.
• plant somatic embryos are isolated from callus or suspension cultures and placed in a controlled, critical relative humidity or storage water content, environment which causes dehydration. This results in a controlled final moisture content. Such embryos cease growth, decrease in size and cellular collapse occurs. The embryos are then stored at standard room temperatures. Upon rehydration, the embryos swell, resume growth and germinate into plants. Dehydrated embryos have been stored for up to 21 days at 23°C.
• the important parameters of the invention include: 1) developmental stage of somatic embryos prior to dehydration, 2) storage water content, 3) storage time, 4) rehydration conditions. Only well developed somatic embryos possessing a morphology consistent with that of zygotic embryos survive the procedure.
• the embryos are placed on solidified medium containing mineral salts, vitamins, and sugars but no growth regulators.
• the dehydrated embryos may be encapsulated in a coating which will preserve the critical moisture content therein.
• the present invention which provides synthetic seeds in an efficient and inexpensive manner will have a significant impact on crop production in the future.
• Commercial applications exist for two broad crop categories. Firstly, synthetic seeds could be used to produce self-incompatible crops that normally must be vegetatively propagated, for example, potato as well as certain fruit and nut trees. In such crops, this would allow for direct planting of non-grafted varieties and would provide a significant alternative for germplasm preservation that presently depends on perpetual maintenance of living plants. Secondly, utilization of elite germplasm for crops that are economically grown only from seeds would be facilitated.
• Examples include forest trees, some forage grasses and vegetables, most major agronomic crops and important sources of oil such as coconut and oil palm.
• synthetic seeds would be essential for utilization of future genetically engineered cultivars containing me,iotically unstable foreign genes. Intentionally introduced meiotic instability would then become an available tool for commercialization of proprietary germplasm. Maintenance of parenteral inbred lines as an adjunct to conventional breeding.would be facilitated by s-ynthetic seeds. In breeding programs, the ability to commercially propagate a new hybrid without "trueing up" the seed and/or producing parental inbreds would become possible.
• Dehydration by controlling the relative humidity of the atmosphere to which the embryos are exposed may be effected according to a variety of systems.
• air or controlled oxygen and/or CO2 mixtures having the requisite relative humidity are passed into and out of a chamber containing the embryos in petri dishes.
• Humidities are generated utilizing either aqueous inorganic salt mistures, aqueous glycerol mixtures or by mixing high humidity air or gas (bubbled through water) with low humidity air or gas (passed through desiccant) to achieve a specific final relative humidity.
• the salt or glycerol mixture is placed in the bottom of the container.
• air or gas is bubbled through the salt or glycerol mixture before passing through the chamber containing the embryos.
• Suitable salt solutions for generating atmospheres having requisite relative humidities include:
• Table 1 sets forth glycerol/water (v/v concentrations) mixtures and the relative humidities generated by each.
• Dehydration may also be achieved by exposing the embryos to "osmotically active" materials which promote the transfer of the requisite amount of water from the embryos to the material.
• the embryos may be either treated with an osmotically active solution prior to encapsulation in a synthetic seed coat or directly encapsulated in an osmotically active seed coat matrial that causes dehydration to a desired level.
• Osmotically active materials include:
• oils lipids
• Basal portions of young orchardgrass leaves were cultured on Schenk and Hildebrandt (SH) medium containing 7 g/1 agar and 30 ⁇ M 3, 6-dichloro- 0-anisic acid (dicamba, Velsicol Chemical Corp., Chicago, IL) as described by Hanning et al, L. Theor. Appl. Genet., Vol. 63, pp. 155-159 (1982). Friable, embrogenic calli were isolated from leaf cultures after 4 wk and were maintained by monthly subcultures on identical medium.
• SH Hildebrandt
• Embryos were manually selected from callus cultures 4 wk after subculture and approximately 30 were placed in each empty 100 x 15-mm sterile plastic petri dish. Only morphologically normal embryos were chosen. Abnormal embryos, i.e., those fused with others or with misshapen scutellar tissues, were excluded. Specific details of selection criteria are provided in Table 1. The dishes were kept at 23°C in the dark at a controlled, relative humidity of 70% ⁇ 5%. Dehydration commenced immediately under these conditions.
• the dehydrated embryos were rehydrated in the dark for 48 h on SH medium lacking dicamba, then placed in a 16-8 h diffuse cool white fluorescent light (30 ⁇ ol photons s ⁇ 1 m ⁇ 2 )-dark cycle at 25°-10°C. Non-dehydrated embryos were included as controls.
• Root hair growth was the first morphologic evidence of germination and was apparent as early as 6 d after placement of dehydrated embryos on fresh medium. Development of root hairs was previously documented for, germinating orchard- grass somatic embryos with time-lapse photomicrography [Gray et al, Trans. Am. Microsc. Soc. Vol. 104, pp.
• Somatic embryos were initated and developed continuously in callus cultures so that several ontogenetic stages were present at a given time [Gray et al, Protoplasma, Vol. 122, pp. 196-202
• Embryogenic cultures of grape were prepared from anthers, ovules or young leaves using methods as described by Gray and Mortensen, Plant Cell Tissue and Organ Culture, Vol. 9, pp. 73-89 (1987). Perennial cultures of Vitis longii ' icrosperma 1 , Vitis rupestris 'St. George', Vitis vinifera 'Thompson Seedless', experimental hybrids B-l, B-2, B-3 and B-4 were established and successfully dehydrated, stored and germinated as described below.
• grape somatic embryos were carefully separated from proliferating cultures. Only embryos with well-developed hypocotyl-radical axes and discrete, separated cotyledons were selected. Pluricotyly, which was characteristic of many embryos, was tolerated in experimental samples. Grape embryos were subjected to the same dehydration procedures as orchardgrass in Example 1 and the morphological changes were generally similar (i.e., decreased size with yellowing, brittleness and cellular collapse). Imbibition was rapid and restored the white, opaque nature of the embryos. However, criteria to assess germination in grape differed from those set for orchardgrass.
• Embryos that produced only a root and/or became yellow to green were considered to have survived dehydration. Embryos that, in addition to the above responses, produced shoots with green leaves were scored as viable.
• White opaque embryos with well-developed embryo axes and discrete unfused cotyledons were maintained in a dehydrated state at 23°C for 0 to 21 days and then allowed to imbibe water. Those that produced roots and/or became yellow to green with no further growth were scored as germinated. Embryos with roots and shoots with green leaves were viable.
• embryos are mixed with a solution of glycerol (87%) and water (13%). They are stored in this solution and are rehydrated by rinsing in a solution of 100% water or liquid culture medium.
• Embryos are mixed with synthetic resin formulated so as to remove embryo water osmotically down to desired level.
• the resin is formed into drops around individual embryos and then polymerized by either heat, UV light or chemical hardener depending on the resin. This results in dehydrated, quiescent synthetic seeds.
• water is added, solubilizing the resin, rehydrating the embryo and causing germination.
• the resin could be formed into a continuous polymerized strand containing evenly-spaced embryos. Such a "synthetic seed string" would make planting crops less labor- and machinery-intensive.

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

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• 1988
  • 1988-11-15 EP EP88910372A patent/EP0397665A1/de not_active Withdrawn
  • 1988-11-15 WO PCT/US1988/003934 patent/WO1989005575A1/en not_active Application Discontinuation
  • 1988-11-21 IL IL88437A patent/IL88437A0/xx unknown

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