Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Involving particle-mediated transfecion
Reexamination Certificate
2000-10-30
2003-05-27
Campell, Bruce R. (Department: 1661)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Involving particle-mediated transfecion
C800S278000, C800S300000, C800S300100, C800S320100, C435S470000, C435S440000, C435S419000, C435S430000, C435S431000
Reexamination Certificate
active
06570068
ABSTRACT:
BACKGROUND OF THE INVENTION
Introduction
Recent advances in genetic engineering have given new impetus to crop improvement. Major breakthroughs have been achieved via development of genetic transformation techniques that facilitate introduction of heritable material such as deoxyribonucleic acid (DNA) into living organisms. Genetic transformation is a process which simply involves the uptake of foreign DNA by somatic cells of an organism. It is an unique mechanism by which foreign DNA of any origin (bacterial, animal, plant, etc.) is stably incorporated into the host genome. As a result, the introduced DNA becomes a part of the parent genome inflicting a permanent genetic change and can be inherited by the subsequent progeny, Chibbar and Kartha, 1994. Such human-engineered organisms harboring additional genetic information are called “transgenic organisms”, whether they are plants or animals, Dekeyser et al., 1990.
Plant transformation techniques involving different systems of gene transfer have been a boon to plant breeders to introduce variability at the molecular level, Cocking and Davey, 1987, and thereby increase genetic diversity, Barton and Brill, 1983. It has helped in the creation of genotypes with novel traits. The recently released, extended shelf-life Flavr-Savr™ tomatoes, Redenbaugh et al., 1992, transgenic Bt-cotton containing resistance to the cotton boll worm, Perlak et al., 1990, and many other such examples, have helped mankind save on costs due to fruit rot and insect control, to name a few. Therefore, transgenic plants could potentially provide an economic edge over conventionally bred crop species in terms of being environmentally friendly, in reducing the risks from using hazardous pesticides and herbicides, in addition to bolstering yields either directly or indirectly, Gasser and Fraley, 1992.
Although more is known about its genetics than many other crops, and despite extensive breeding efforts, maize (
Zea mays
L., Poaceae) as a crop, still requires continual genetic improvement. Examples include developing resistance (or tolerance) to diseases, pests, and herbicides, and improving the protein quality, all of which contribute to an increased net economic gain. Great emphasis has been placed on maize breeding for crop improvement due to its extreme value as a cereal crop worldwide, Chassan, 1992. In fact, maize ranks as the world's third largest crop, trailing only behind wheat and rice, Langer and Hill, 1991. In the United States, it is the leading cereal (grain) crop and in 1995, 3,351,762 metric tonnes were produced on 26,314 hectares and was valued at greater than $23 billion,
U.S. Department of Agriculture-National Agricultural Statistics Service
, 1995-96. Due to the importance of maize in the U.S. and worldwide, many maize improvement programs are currently utilizing genetic engineering protocols to complement/enhance classical breeding efforts which are limited to working with genes (traits) already present in the maize germplasm.
The biolistic system has certain unique advantages over other gene transfer systems. It is very simple to operate and has universal applications because it can be used for gene transfer into cells of plants, animals, or microbes, Klein et al., 1992. Since its advent, it has been a very useful method commonly employed to transform the world's cereal crops like rice, barley, sorghum, oats, wheat and maize, Christou et al. 1991; Klein et al., 1989; King et al., 1994; Ritala et al., 1994; Somers et al., 1992; Vasil et al., 1994. The biolistic process has since proven to be the best available system for transforming monocots, Batty and Evans, 1992.
Regeneration of maize involves the use of juvenile tissues by employing explants or tissues derived from seeds either pre- or post-germination. Successes have been limited to the use of embryogenic calli maintained in suspension cell culture (e.g. BMS and other elite inbred lines) initiated from immature embryos, and whole immature embryos as targets for developing biolistics-based transformation systems for maize, Gordon-Kamm et al., 1990; Fromm et al., 1990; Walters et al., 1992; Lowe et al., 1995. Ideal target tissues would be capable of accepting foreign DNA at high frequencies and regenerating plants (hopefully transgenic) efficiently.
Optimization must be conducted for each explant/tissue type and is usually based on the transient expression of the introduced genes. Transient expression refers to the expression of gene sequences that may or may not be integrated into the host genome and are usually conducted after a brief (42-72 h) post-bombardment period. Transient expression frequencies provide rapid and useful information as to whether foreign DNA was or was not introduced. In addition, correlations can be made to stable transformation frequencies. Researchers estimate the stable transformation frequency to be from less than 1% up to 5% of the transient expression frequencies, Finer and McMullen, 1990; Klein et al., 1988b. Thus, transient expression has proven to be a very useful indicator of DNA delivery and is routinely used in investigating the optimum conditions required to deliver DNA into different explant/tissue types via biolistics. For many crop species, optimization of bombardment (DNA delivery) is conducted by measuring the transient expression of the reporter gene, &bgr;-glucuronidase (GUS), by a histochemical assay, 48-72 h post-bombardment, Jefferson et al., 1987.
It is evident that development of regeneration protocols for various target tissues and optimization of critical biolistic parameters utilizing these tissues assume great importance in developing a biolistics-based transformation system for maize.
Literature Review
Maize Tissue Culture
Maize has been regenerated in vitro by following different systems of regeneration. These include regeneration via somatic embryogenesis and organogenesis with both utilizing adventitious (including de novo) regeneration protocols, Vasil, 1986.
Maize Regeneration From Different Explant Sources
Immature Zygotic Embryos As Explants For Callus Cultures
The successful induction of somatic embryogenesis, maturation and germination of embryoids into plantlets are dependent on a number of variables. These variables include: genotype, age of the extracted immature zygotic embryos (days post-pollination; dpp), sucrose concentration in the media, the plant growth regulators (PGRs) utilized along with other media additives and associated culture conditions. Table 1 lists the genotypes successful in plant regeneration utilizing immature zygotic embryos as explants. Regeneration was a result of scutellar tissue proliferation which lead to the formation of embryogenic callus from which mature bipolar somatic embryos emerged and were subsequently regenerated into whole plantlets.
TABLE 1
Plant regeneration via somatic embryogenesis from the scutellum of
immature zygotic embryos.
Plant Growth Regulators (PGRs)
Genotype/
Maturation
Germination
Cultivar
Induction Medium
Medium
Medium
Reference
A188
2,4-D
x
: 2.0 mg/l
2,4-D: 0.25 mg/l
PGR-free
Green & Phillips,
A188 X R-njR-nj
1975
B73
2,4-D: 0.5 mg/l
PGR-free
y
PGR-free
Lowe et al. 1985
Silver Queen
2,4-D: 0.5 mg/l
PGR-free
GA
3
x
: 1.0 mg/l
Lu et al. 1982
Asgrow Rx112
2,4-D: 0.25-1.0 mg/l
PGR-free
PGR-free
Lu et al. 1983
Coker 16
Coker 22
Dekalb XL80
Dekalb XL82
Florida Stay
Sweet
Funk G4864
Funk G4507A
Pioneer 3030
Pioneer 3320
Silver Queen
Dekalb XL 82
2,4-D: 0.5-1.0 mg/l
ABA
s
: 0.02 mg/l
Vasil et al. 1985
A188
2,4-D: 1.0 mg/l
PGR-free
PGR-free
Armstrong & Green,
L-proline: 690 mg/l
1985
Mo17
2,4-D: 1.0 mg/l
BAP
w
: 3.5 mg/l
PGR-free
Duncan & Widholm,
Pa91
L-proline: 1.38 g/l
AgNO
3
r
: 34 mg/l
1988
R99
A188
2,4-D: 1.0 mg/l
NAA
v
: 1.0 mg/l
Torne et al. 1984
H-fl
2
& 2iP
u
: 0.05 mg/l
H-113
V444
W64A
0202
A188
2,4-D: 1.0 mg/l
PGR-free
PGR-free
Duncan et al. 1985
A658
B79
H60
H97
H99
L317
Oh7
2,4-D: 1.0 mg/l
PGR-free
PGR-free
Duncan et al. 1985
Pa91
R806
Wf9
W64A
A634 X A188
2,4-D: 0.5 mg/l
PGR-free
PGR-free
Hodges et al. 1986
A632 X A188
B73 X A188
B14 X A188
B68 X
Reichert Nancy A.
Young Margaret M.
Campell Bruce R.
Grunberg Anne Marie
Kelber Steven B.
Mississippi State University
Piper Rudnick LLP
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