Note: Descriptions are shown in the official language in which they were submitted.
SPECIFICATION
This invention relates to a chemical that is used in plant
tissue, organ or cell culture media to induce the development of
embryos from gametic or somatic-plant cells.
Natural and synthetic auxins, which are a class of plant
growth regulators, have been used to induce the development of
gametic and somatic embryos from plant cells. However, there are
only a few instances reported in the scientific literature where
large numbers of high quality somatic or gametic embryos have been
produced (Cell Culture and Somatic Cell Genetics, ed. I.K. Vasil,
Academic Press, Orlando, 1984). In most cases, somatic or gametic
embryogenesis was a rare event and the quality of the embryos
either was not determined or Was shown to be poor~in relation to
zygotic embryos produced in vivo.
Gametic embryos from microspores and ovules are valuable in
pure line production for plant breeding and as a vehicle for plant
mutagenesis, in vitro selection, and transformation. Somatic
embryos could be produced from a variety of somatic plant cells
including protoplasts and are useful in producing clones or
"artificial seeds" as well as for mutagenesis, in vitro selection
and plant transformation studies.
We have found that Phenylacetic Acid (PAA), which is a
naturally occurring auxin found in plants, when added to plant
tissue, organ or cell culture media at appropriate concentrations
and used with the proper procedures, induces the development of
large numbers of high quality somatic or gametic embryos. We have
used PAA in a range of plant cell culture systems ranging from the
culture of barley microspores to produce gametic embryos, ~to the
culture of geranium hypocotyls to produce somatic embryos. In all
instances, when PAA was compared with the best avai~able protocols,
PAA induced the development of larger numbers of high quality
embryos.
PAA has been used to produce callus cultures in plants (Lee
and Skoog, Physiol. Plant. 18:386-402, 1965; Milborrow et al., Ann.
Bot. 39:1143-1146, 1975). However, there has been no reported
instance of it's use for inducing the development of gametic or
somatic embryos. The use of PAA is not obvious because: 1) for
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more than 30 years, somatic embryos have been produced from plant
cells and there is no published report of the use of PAA for this
purpose; 2) even though PAA is naturally occurring, it's role and
function in plants is at present poorly understood.
The following examples illustrate the ways in which PAA has
been used to induce the development of gametic or somatic embryos.
Example 1: Wheat Anther Culture, Gametic Embryogenesls Using PAA
Materials and Methods:
Plants of the spring wheat cv. Veery `S' were grown in a
greenhouse under cool conditions. The culture medium was BACl
(Marsolais and Kasha, Can. J. Bot. 63:2209-2212, 1985) containing
2000 mg/l myo-inositol, 6% sucrose, 1.75% glucose and 100 g/l
Ficoll 400. A 2x concentration of the liquid medium without Ficoll
was prepared and filter sterilized. The Ficoll was made as a 2x
concentration by heating in distilled water and sterilization by
autoclaving. Equal volumes of the filtered medium and the Ficoll
solution were combined to make the final medium.
The two treatments were: PAA at 100 mg/l and 2,4-D at 8 mg/l
in the induction medium. The 2,4-D treatment and concentration was
chosen because it has given the best results in previous studies.
The auxin stock solutions were made by dissolving lg of PAA in loo
ml of hot distilled water. 2,4-D stock solutions were made by
dissolving 50 mg of 2,4-D in 5 ml of methanol, then bringing the
solution up to 100 ml with distilled water. PAA stock solutions
were made fresh before addition to the medium, and culture media
were used within 2 days. Spikes were harvested when the
microspores were in the mid-uninucleate stage (Wheatley et al.,
Plant Cell Rep. 5:47-49 1986), and 80 to 90 anthers were inoculated
into each 60x15 mm petri dish containing 3 ml of medium. There
were 5 replications of each treatment. Anthers were cultured at
30C in the dark for 17 days, then 1 ml of BACl replenishment
medium ( BACl medium with 30 g/l sucrose, 17.5 g/l glucose, 1 mg/l
IAA, and lOOg/l Ficoll 400) was added to each dish. The anthers
and embryos were cultured for a further 10 days at 27C, then
observations were made. Embryos were transferred to BACl
regeneration medium ( BACl + 3% sucrose + 0.25 mg/l BAP + 100 mg/l
myo-inositol + 0.8% Gibco Phytagar, no Ficoll, no glucose) when
they were well developed ( 28-35 days after culture initiation )
and cultured at 23-25C in the light at 40-80 umol m2s~1 for a 16
hour day. Developing embryos that had not regenerated to plants
within 2 weeks were subcultured to fresh regeneration medium.
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0 1 7 4 1 7
Results
There was no significant difference between PAA and 2,4-D in
percent anther response (31~ vs 27~) and the number of embryos
produced per 100 anthers plated (173 vs 145) (Table 1). Embryos
produced on PAA medium regenerated into plants more readily than
those produced on 2,4-D medium (145 vs 57 per 100 anthers plated).
Typically, embryos from the PAA medium were more similar to zygotic
embryos than those produced on 2,4-D medium. The higher rate of
regeneration probably was due to the quality of the embryos
produced on PAA medium. The number of albino plants regenerated
was not influenced significantly by the treatments.
Table 1. ~-~
PAA vs 2,4-D in anther culture of the spring wheat cv. Veery 'S'.
No. anthers % anther No. embryos No. GP No. AP
Auxin plated res~onse / 100 anthers /100 anthers ~ ~-
PAA 436 31a 173a 145a la
lOOmg/l ~;
2,4-D 443 27a 145a 57b 9a
8mg/1
Means within each column followed by the same letter are not ; -
significantly different at the .05 level of probability according
to the t-test.
GP = green plants -~
AP = albino plants
- "..'.`'''
Example 2: Wheat Anther Culture, Gametic Embryogenesis Using PAA
Materials and Methods
Twelve different spring wheat Fl hybrids were grown in a
greenhouse under cool conditions. The experiment was designed as
a paired comparison with two treatments: ( 100 mg/l PAA, 8 mg/l
2,4-D) in the induction medium. Anther staging, media preparation, -~
inoculation, culture conditions and plant regeneration procedures ~-
were the same as described in Example 1.
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Results
This study with 12 spring wheat F1 hybrids confirms the
results of the previous experiment in which one cultivar was used.
PAA produced approximately the same number of embryos and
significantly more green plants per 100 anthers plated (Table
2)(Figure 1,2). Very few albino plants were regenerated.
Table 2.
Comparison of PAA and 2,4-D for anther culture of 12 different
spring wheat Fl hybrids.
No. anthers % anther No. embryos No. GP No. AP
Auxin plated response ~100 anthers /100 anthers
PAA 2011 46a 379a 332a 2a
lOOmg/l
2,4-D 2038 39a 307a 97b 8a
8mg/1
Means within each column followed by the same letter are not
significantly different at the .05 level of probability according
to the t-test.
Example 3: Barley Microspore Culture, Gametic Embryogenesis using
PAA
Materials and methods
Seeds of the winter barley cv. Igri were germinated in Turface
for 1 week and then transferred for vernalization (4C) for 8
weeks. The plants were then grown in a growth room with day/night
temperatures of 12C/lO~C for 7 weeks. Spikes containing
microspores at the mid-uninucleate stage were harvested and surface
sterilized with 70% ethanol. All subsequent procedures were done
under sterile conditions.
The anthers were cultured at a density 20 anthers/ml in 0.3
M mannitol. These plates were incubated in the dark for 3 days at
25C. For mechanical isolation of microspores, the 400 anthers in
mannitol were placed in a glass homogenizer and macerated with a
teflon rod to rupture the anther wall and release the microspores.
The suspensions were centrifuged at 500 r.p.m. for 5.0 min. and the
supernatant containing the anther wall debris was removed and
discarded. The microspore pellets were rinsed with mannitol and
centrifuged three more times.
Liquid FHG Media (Hunter, 1988 Ph.D. Thesis, Univ. of London)
was added to the microspore pellet to bring the volume to 0.6- 0.8
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ml. Approximately 0.2 ml of the suspension was plated on top of
each plate containing the same media (3 ml) but solidified with
0.8% sea Plaque agarose. A total of three replications were used
per treatment. The plates were wrapped with Parafilm and incubated
at 2sc in the dark for 40 days.
Results
A major improvement in barley anther and microspore culture -
was the replacement of sucrose with maltose (Hunter, 1988). His
medium (FHG) is a modified MS medium but success still required a
28 day cold pretreatment (4C) of harvested spikes. We have -~
observed that the cold pretreatment requirement could be replaced
by using ovary conditioned FHG culture medium (Ziauddin et al.,
1990 Plant Cell Rep. (accepted)). We now have evidence (Table 3; ~-~
Fig. 3) that the use of PAA (coded NX in Fig. 3) in FHG medium
provides very good results with freshly harvested microspores, -
similar to results using 28 day cold pretreatment or ovary
conditioned media. Thus, PAA can make microspore culture a much
more efficient process by replacing cold pretreatments or
conditioning of media.
Table 3: Response from Igri microspore culture. ;~
Treatment Replicate # of embrvos # of calli #of shoots** ;~
FHG (no auxin)* 1 o o o
2 o 0 o
3 0 0
FHG + 100mg/1 PAA 1 623 186 600
2 283 198 300
3 260 200 320
'' ,. ;~.,
* only multicellular microspores formed but these did not form
structures. `
** spontaneous development of shoots in original media.
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Example 4. Somatic Embryogenesis Using PAA
Materials and methods `
Seeds of the geranium cultivar 'Scarlet Orbit Improved' were
surface sterilized by agitating them for 20 minutes in a 1.4%
solution of sodium hypochlorite containing 1 drop of Tween 20 per
200 ml of solution. Then the seeds were rinsed five times in
sterile distilled water and placed on the surface of sterile
~`- distilled water solidified with 8 g/l Gibco Phytagar in a 100 x 15
mm plastic petri dish. The plates were sealed with Parafilm, -
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wrapped in aluminum foil and incubated for 5 days,in a dark growth
room set at 22C. The length of the hypocotyls were 0.5 to 1.5 cm
after this culture period. The hypocotyls were cut into 0.5 cm
transverse sections and plated side-down on the medium. Ten
hypocotyl sections were plated per 100 x 15 mm plastic petri dish
containing 25 ml of solidified media. The medium consisted of MS
salts and vitamins (Murashige and Skoog, Physiol. Plant. 15:473-
497, 1962), 2 x l06M 6-benzylaminopurine, 30 g/l sucrose, 1500 mg/l
L-glutamine, 10 g/l Gibco Phytagar, various concentrations of
auxins described below, and at pH 5Ø
Two auxins (IAA (indole3-acetic acid), PAA) were each tested
at two concentrations ( 106M, 105M,) in the culture medium. The
culture medium, IAA treatments and culture conditions were selected
based on previous research (Marsolais et, al., 1990 Can. J. Bot.
submitted). Auxin stock solutions were made according to the
description in Example 1 and appropriate amounts were added before
sterilization of the media in an autoclave. There were five
replications of each auxin concentration treatment combination.
The petri dishes containing the hypocotyl sections were
incubated at 24C in the light at 40-60 umol m~s~' for a 16 hour
day. The number of somatic embryos were counted 28 days after
culture initiation. Somatic embryos were removed from the explant
tissue and germinated on MS basal medium with vitamins and 30 g/l
sucrose, and at pH 5.6. The number of germinating somatic embryos
was recorded 14 days later.
Results
PAA at either l06M or 105M gave the same number of somatic
embryos as IAA at 106M, which was the best IAA treatment (Table 4).
The quality of the PAA induced somatic embryos was superior to
somatic embryos produced on IAA. This improvement in quality was
reflected in the higher percent qermination scores achieved with
PAA compared to IAA.
Table 4: Somatic embryogenesis from hypocotyl sections of Scarlet
Orbit Improved using IAA and PAA. Means + standard error.
No. Somatic Embryos Percent
Auxin Concentration per 100 Hv~ocotvl Sections Germination
IAA 106N 3643 + 760 71 + 5
105M 2056 + 445 56 + 9
PAA 106M 3717 + 832 86 + 3
105M 3288 + 671 82 + 5
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