Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
2000-04-14
2004-02-10
Campell, Bruce R. (Department: 1661)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C047S057600, C504S116100
Reexamination Certificate
active
06689609
ABSTRACT:
TECHNICAL FIELD
This invention relates to the germination of plant somatic embryos. More particularly, the invention relates to a process of treating plant somatic embryos with a nutripriming step to enhance subsequent germination of such embryos.
BACKGROUND OF THE INVENTION
A plant seed is a complete self-contained reproductive unit generally consisting of a zygotic embryo, storage reserves of nutrients in structures referred to as cotyledons, endosperm or megagametophytes, and a protective seed coat encompassing the storage reserves and embryo. In nature, maturation of plant seeds is usually accompanied by gradual loss of water over a period of time to levels between 10-35% moisture content. Once these low moisture levels are achieved, plant seeds can be stored for extended periods.
Germination of zygotic plant seeds is generally triggered by one or more environmental cues such as the presence of water, oxygen, optimal temperature, and light. Seeds germinate by means of a series of events which commence with the uptake of water by a quiescent dry seed and then subsequently proceed through various biophysical, biochemical and physiological events which ultimately result in the elongation of the embryo along its axis.
For the purpose of simplifying discussion of the present invention, the continuous process of seed germination is divided into three phases. Phase one is referred to as imbibition and is characterized by a rapid initial influx of water into the seed. Other significant events occurring in Phase one are the initiation of repair to damage to DNA and mitochondria which may have occurred during seed desiccation and/or the maturation process, and subsequent commencement of protein synthesis facilitated by existing mRNA.
Phase two is characterized by a significant reduction in the rate of water uptake (i.e., imbibition has been completed). This is accompanied by activation or de novo synthesis of enzymes that specialize in hydrolyzing the complex storage reserves of carbohydrates, proteins, and lipids in the embryo and the cotyledons or megagametophytes. The hydrolysis of these complex storage reserves provides the substrates required for the respiration and growth of the zygotic embryos.
Phase three is characterized by a second rapid increase in the rate of water uptake. Water absorbed during Phase three is used primarily for the initiation of meristomatic cell division at the root and shoot apices of the embryo, and for uptake into the cells along the embryonal axis. Water taken up by the axial cells of the embryo applies turgor pressure which results in axial cell elongation. The net effect is that the embryo elongates to the point of protrusion through the seed coat. Protusion of a shoot or root radicle through the seed coat signifies the completion of germination and the onset of seedling growth and development.
The speed and success for germination of zygotic seeds can fluctuate considerably depending on various factors such as the residual influence of environmental conditions in which the seed developed and maturated, the amount of storage reserve compounds synthesized during the seed maturation process, the duration of storage, and the quality of the storage environment (e.g., temperature and humidity). From a commercial perspective, it is desirable to reduce the risk of germination failure and to ensure that seeds germinate rapidly and uniformly.
The commercial need for optimum seed germination performance has led to the development of processes known in the art for zygotic seeds as “seed priming”. This term may be defined as the “uptake of water to initiate the early events of germination, but not sufficient to permit radicle protrusion, preferably followed by drying” (McDonald, 2000). Four techniques are currently used commercially to accomplish seed priming. These are hydropriming, osmopriming, matripriming and pregermination. However, regardless of the method used, the fundamental principles of seed priming are that: (1) the preliminary stages of germination are activated specifically and exclusively through controlling the availability of water to the seeds, and (2) the germination processes initated through an external priming process are subsequently arrested by a desiccation step.
One of the problems with commercializing somatic embryo technologies has been a relatively low rate of conversion to seedlings and low seedling vigor when conversion takes place. It would clearly be advantageous to improve such rates of conversion and levels of seedling vigor.
However, a significant additional problem is the current inability to use conventional horticultural ex vitro techniques, practices and environments for the sowing and germinating of plant somatic embryos. The main reason for the difficulties in successfully germinating plant somatic embryos in non-sterile commercial growing environments using conventional propagation practices is that during the intitial stages of germination, matured plant somatic embryos cannot produce their own carbon compounds or derive energy from photosynthesis. Furthermore, they lack the presence of their own energy and nutrient sources that are equivalent to storage reserves contained within cotyledons or endosperm or megagametophyte tissues in zygotic seeds. Consequently, an exogenous source of energy in the form of a selected sugar within a culture medium and other nutrients, must be supplied to the plant somatic embryos for successful germination to occur. Such culture media are highly susceptible to invasion by microorganisms which inevitably result in death or interfere with embryo survival and germination. Consequently, sowing and successful germination of plant somatic embryos on culture media must be conducted under strict aseptic conditions.
Although numerous protocols are known for the sowing and germination of somatic embryos and growing them into intact functional seedlings, all of these protocols are dependent on the use of aseptic techniques combined with in vitro systems that must be kept in biological isolation from contaminating microorgansisms and fungi until the plant somatic embryos have successfully completed germination and have achieved autotrophy. Consequently, none of these protocols has demonstrated compatibility with conventional horticultural equipment and practices.
Generally, the known protocols for germinating somatic embryos fall into two categories. The first is a category of protocols based on various in vitro methods which generally are comprised of sowing naked, i.e., uncoated, somatic embryos using aseptic techniques, onto sterilized semi-solid or liquid media contained within a solid-support such as a petri dish or a phytatray to facilitate germination under biologically isolated sterile conditions (e.g., U.S. Pat. Nos. 5,183,757; 5,294,549; 5,413,930; 5,464,769; 5,506,136 all of which are herein incorporated by reference) and subsequently, transplanting the germinants into conventional growing systems. The most significant disadvantages of such in vitro protocols for sowing naked somatic embryos are that (a) each embryo typically must be handled and manipulated by hand for the germination and transplanting steps, and (b) aseptic techniques and culture conditions must be rigorously maintained through to the step of transplanting of somatic germinants out of the in vitro germination media into horticultural growing media. Although various automation options, including robotics and machine vision, have been assessed for their usefulness in cost-effective reduction or elimination of the extensive hand-handling currently necessary to sow naked embryos (Roberts et al., 1995), no commercial equipment currently exists which can reliably, aseptically, and cost-effectively perform the in vitro protocols for germination of naked somatic embryos and subsequent transplanting into conventional propagation systems.
The second category of protocols teach encapsulation (generally gel-encapsulation) of somatic embryos (e.g., U.S. Pat. Nos. 4,777,762; 4,957,866; 5,183,757; 5,482,857 all of which are herein
Fan Shihe
Janic Vesna
Campell Bruce R.
Cellfor Inc.
Para Annette
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