Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR IN VITRO SELECTION OF POTATO CLONES RESISTANT
TO BLACKSPOT BRUISING AND THE POTATOES
PRODUCED THEREFROM
FIELD OF THE INVENTION
The present invention relates generally to plant
tissue culturing for desirable characteristics. More
specifically, this invention relates to in vitro selection of
Lemhi and Russet Burbank potato clones resistant to blackspot
bruising.
BACKGROUNQ QF~ THE INVENTION
Blackspot is a physiological (non-infectious)
disorder affecting potato tubers damaged during handling. It
is also known as blue discoloration, blue spotting, bluing,
bruise, internal blackspot, internal bruising, internal
grayspot and stem-end blackening. The disorder appears as an
internal discoloration and blackening that can be seen when
injured tubers are peeled or sliced. The blackening is
usually restricted to the outer 1%4"' to 1/2~' of ' tuber tissue
between the skin and the vascular ring. The color of the spot
can vary from a light gray to a blue-gray to an intense coal
black. The size and intensity of the spot usually reaches a
maximum within 24 hours of bruising and once formed, the
blackened area will not disappear.
Blackspot is normally caused by impacts (bumping,
dropping, etc.) to the tubers during handling, transportation,
storage or packaging but may also be associated to varying
degrees with other physical damage such as pressure bruising
and/or shatter cracking. The force required to initiate a
blackspot need not be severe, particularly to tubers of
susceptible cultivars.
The disorder was first reported and described in
England in 1912 and since then it has become a serious problem,
throughout Europe. It was identified in the United States in
1940 and can now be found in all the potato growing areas in
this country as Wen .
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Although affected potatoes can be eaten, they are of
limited commercial value because ~ of their appearance. The
disorder is particularly serious because affected tubers may
show no external damage, even after the tuber is washed.
Blackspot bruise often results in serious economic losses in
both fresh market and processed potatoes, including chips and
fries.
Tuber susceptibility and an impact of sufficient
magnitude to rupture cells are the two most important factors
responsible for initiation and development of blackspot.
These conditions activate a series of four biochemical
conversions of phenolic compounds (beginning with tyrosine) to
conjugated quinones. Intermediate compounds include caffeic
acid and p-coumaric acid. This sequence, which is mediated by
the action of polyphenyloxidase enzymes, is followed by the
polymerization of the quinones to the black pigment melanin.
In healthy, non-damaged tissue these phenolic compounds and
the polyphenyloxidase enzymes are normally compartmentalized
separately and do not come into contact. However, cell
rupture causes the contents to mix and the blackening reaction
occurs. Although tyrosine and polyphenyloxidase enzymes play
a major role in the development of blackspot, the total amount
of these compounds present in tubers usually does not
correlate with tuber susceptibility or explain differences in
blackspot reaction between tubers and cultivars. The most
recent work on the biochemistry of blackspot phenomenon
indicates that reduction of the free tyrosine pool within the
cell increases tuber resistance to blackening. (Corsini et
al., Evidence for highly conserved tuber tyrosine levels among
potato genotypes and implications for blackspot resistance.
Am. Potato J. 66:511-512 (1989)). These investigators found
total tyrosine content of many potato cultivars to be
remarkably similar though these cultivars ranged widely in
their susceptibilities to blackspot. Those cultivars with the
greatest resistance to blackspot had a large proportion of the
tyrosine bound into protein with very little free tyrosine
available for melanin formation. The opposite was found to be
true in the susceptible cultivars.
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In addition to the biochemical factors discussed
above, there are numerous environmental and cultural factors
that can contribute to the manifestation of this disorder.
Tuber turgor pressure, temperature, specific gravity, mineral
nutrition, date of planting, . soil moisture, and soil
temperature can all influence blackspot development (Hiller et
al., Physiological disorders of potato tubers. Potato
Physiology 389-455, Academic Press, New York (1985)).
Even when all the, predisposing factors are
considered, potato cultivars vary markedly in their response
to impact damage. Some cultivars may be highly resistant to
blackspot while others may be ,highly susceptible. Tubers from
a single plant may differ ,in their blackening responses.
Susceptibility may also vary from the stem end to bud end of
an individual tuber.
Losses due to blackspot can be managed to a certain
extent by production practices employed during the growing
season. Practices currently employed to control blackspot
bruising are to keep ,the plants as healthy as possible by
providing adequate disease and pest control, and good soil
moisture and soil fertility (particularly potassium). Soil
should not be allowed to dry out prior to harvest and vines
should be killed early to reduce water loss from' the tubers.
The most important means of controlling the extent of
blackspot formation is reducing tuber injury both during and
after harvest. This can be implemented by not harvesting
tubers when the soil temperature is low (8°C) and by adjusting
operation speeds, drop ,lengths and padding on all potato
handling equipment. Selection of cultivars that are more
resistant to blackspot i,s also an important consideration.
However, this is not always possible because of production
restrictions. Some cultivars demonstrate great potential for
commercial production but suffer because of blackspot
bruising. Such is the case with the cultivar Lemhi Russet and
to a lesser extent, Russet Burbank.
'Lemhi~ and Russet Burbank have many characteristics
that make them suitable and important cultivars for the
processing industry, including low reducing sugar levels, good
storability, excellent processing and dormancy. However, they
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are extremely susceptible to blackspot. Naturally resistant
Lemhi and Russet Burbank have not been found. This limits
their acceptability because the quality of processed products
(e.g. chips and fries) obtained from bruise-damaged potatoes
is lowered substantially. , Although resistance to blackspot
may be attainable through traditional breeding techniques,
other characters which make the cultivar commercially
acceptable; such as sugar levels, shape, specific gravity, or
yield, could also change. It. is difficult to maintain every
desirable character while breeding for cultivar improvement
and such an approach would involve several years of crossing
and testing.
As an alternative to traditional breeding techniques,
plant cell culture provides the opportunity to evaluate large
quantities of cells (literally millions), having the potential
of regeneration into valuable somaclonal variants. Normally,
a large population of regenerated plants is required in order
to identify somaclones with the desired traits. Increasing
and testing such populations is labor intensive and requires a
tremendous amount of greenhouse and field space. This problem
is usually addressed by developing techniques that will allow
the somaclones to be screened for the required
characteristics) while in tissue culture, prior to being
regenerated into plants. Evaluation at the cell culture level
greatly reduces space involved and increases the number of
somaclonal lines that can be examined. In vitro screening
procedures essentially increase the likelihood of identifying
clones with desirable traits by eliminating unwanted material.
Accordingly, there is a need to provide an alternative to a
traditional breeding approach for potato cultivar improvement
and development. There is also a need for increasing the
likelihood of identifying blackspot resistant 'Lemhi' and
Russet Burbank potato clones. There is further a need for
techniques which increase the ease and efficiency of
identifying and selecting prospective blackspot resistant
'Lemhi' and Russet Burbank somaclones. ' The present invention
fulfills these needs and provides other related advantages.
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SUMMARY OF THE INVENTION
The present invention resides in methods for in vitro
selection of 'Lemhi' and. Russet Burbank for blackspot
resistance using plant tissue culturing techniques and using
at least one melanin precursor, such as tyrosine or caffeic
acid, added to the tissue culturing media. The present
invention also resides in the blackspot resistant tubers
produced from the in .vitro selection. The methods are based
upon in vitro somatic cell isolation, culture, screening,
selection, and regeneration, not involving sexual crossing.
The methods comprise, generally, culturing tissue
obtained from a potato plant in cell layer and associated
reservoir media, subculturing the tissue on callus
proliferation medium to obtain callus formation, subculturing
the callus on shoot induction medium to obtain shoot formation
and subculturing the shoot on a rooting medium to ensure root
formation, whereby potato plants are regenerated from which
blackspot resistant tubers are produced. At least one melanin
precursor may be added to the reservoir, callus proliferation
and rooting medium to further increase the likelihood of
identifying blackspot resistant clones. When the melanin
precursor screening method is used, potato ' plants are
regenerated from the calli and roots which show no blackening
response when the melanin precursor is present in the various
media.
Other features and advantages of the present
invention will become apparent from the following more
detailed description.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention provides meth ods for increasing the
quantity of regenerated Lemhi and Russet Burbank potato clones
having increased resistance to blackspot and the potato tubers
produced therefrom.
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prnre~~mAa ~aer ~rhe Collection, Culture And
~p~g~grat;e" Of Potato Mesophyll Protoplasts
The following procedures should be viewed with
reference to the abbreviations and compositions that follow:
List of Abbreviations
BAP 6-benzylaminopurine
~A3 gibberellic acid
NAA naphthaleneacetic acid
MES 2[Nrmorpholino]ethanesulfonic acid
PVP-l0 polyvinylpyrrvlidone (10,000 MW)
EDTA ethylenediaminetetraacetic acid (sodium salt)
KN03 19.0 g/1
CaCl2 * 2H20 4.4
MgS04 * 7H20 3.7
KH2P04 1.7,
____________._r_rrr_rrrrr_r__,~_____________________________..
~~aos;t;on Q,~ Minor Elements lStoc x1
HaB04 0.620 g/1
MnCl2 * 4H20 1.980
ZnS04 * 7H20 0.920
_________r_~rrrr-rrrrrrrrrrrr.rrrrrrr_rrrr~rrrrr______________
composition o! Minor Elements [Mock II)
KI 0.083 g/1
Na2Mo04 * 2~0 0.025
CuS04 * 5H20 0.0025
CoS04 * 7H20 0.003
________._r_r_.._r_r_________________________________________
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Composition of Orcranics Stock
myo-Inositol 20.0 g/1
Thiamine * HCl 0.1
Glycine 0.4
Nicotinic Acid 1.0
Pyridoxine * HCl 0.1'
Folic Acid 0.1
Biotin ~ 0.01
Composition Of Fe-EDTA StOCk
Na2 EDTA 0.373 g/1
FeS04 * 7H20 0.278
Comaosition of Miscellaneous Stocks
Casein Hydrolysate ~~ 10.0 mg/ml
NH4C1 0.1 M
MES 0.5 M
Comt~osition of Plant Growth Regulator Stocks
BAP, GA3, NAA and Zeatin stocks at 0.1 mg/ml.
Growth of Source Plants
Potato tubers should be maintained at room temperature to
break dormancy. This usually occurs within_7 to 14 days. After
///
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sprouting is initiated, the tubers are planted individually in
20 cm clay pots containing an artificial soil mix such as
Jiffy-Mix (JPA, 1400 Harvester Rd., West Chicago, IL 60185)
or Sunshine Mix (Fisons Western Corporation, Vancouver, BC
Canada V6N 3V1) . The soil should be kept moist with distilled
water but overwatering should be avoided. The sprouts are
excised from the mother tuber when they are 6 - 10 cm high and
planted in individual 20 cm clay. pots containing the soil mix.
The plants should be grown at 70 - 80% relative humidity in a
controlled environment chamber illuminated with cool white
fluorescent light under a 12 hour photoperiod. The light
regime should consist of 5 hr illumination at 90uE m-2
sec-1 and 2 hr at 325 uE m-2 sec-l, followed by 5 hr at
90 uE m 2 sec-1. The temperature is maintained .at 16°C
except for hours two through six of the light period when it
is increased to 22°C. Plants are fertilized biweekly as
described by Shepard, J.F., Mutant selection and plant
regeneration from potato mesophyll protoplasts, Genetic
Improvement of Crops: Emergent Techniques. University of
Minnesota Press, pp.185-219 (1980).
Protoplast source plants can also be produced from
plants initiated from meristem cultures and increased via
nodal cuttings. They can be grown' and maintained' on medium E
under the conditions described later in the Plant Regeneration
Sequence section dealing with shoot elongation/root
initiation.
grcyl-pplact Isolation (deaf harvest)
Protoplast yields are fairly consistent when leaflets
are collected from plants 4 - 8 weeks after transplanting.
The leaves are excised from nodal position 3 - 7 (from top of
plant). The use of very young leaves or very old leaves should
be avoided. Leaves with large terminal leaflets (7 5 cm)
generally yield large quantities of protoplasts. The largest
leaflets are selected. A portion of the petiole attached to
each leaflet is left there to facilitate handling.
Approximately 3.5 - 4.5 g of leaf tissue (5 - 6 leaflets) will
usually provide enough cells to work with. After the fresh
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weight is determined, the leaflets are ready to be
preconditioned for enzyme isolation.
rotoplast Isolation (leaflet ~reconditioning~~
Utmost care must be taken to assure that conditions
of absolute sterility are maintained throughout all of the in
vitro manipulations described below. The leaflets must be
sterilized before they are placed in the conditioning
solutions. Each leaflet is dipped in 70% ethyl alcohol (1 - 2
sec), then transferred to a beaker containing 1000 ml of a 10%
bleach (Chlorox, Hilex, etc.) solution. The leaflets are kept
submerged in the hypochlorite solution for 2 - 3 minutes and
then transferred to a beaker containing 500 ml of sterile,
distilled water. Each leaflet is dipped in the water
approximately 10 times to rinse and then transferred to a
second beaker containing sterile, distilled water and the
rinse procedure repeated. After the second rinse, the
leaflets are dipped in 70% ethyl alcohol (1 - 2 sec). The
leaflets are now ready for the conditioning solutions. 250 ml
of sterile float solution is aseptically transferred to a
large (20 cm), covered, glass wash bowl (sterile).
***********************************************************
FLOAT SOLUTION
***********************************************************
NH4N03 . . . . . . . . . . . . . . 80 mg
CaCl2 * 2H20 . . . . . . . . . . . 147 mg
NAA (2 mg/1) . . . . . . . . . 20 ml stock
BAP (1 mg/1) . . . . . . . . . 10 ml stock
volume to 1000 ml
Autoclave
***********************************************************
The leaflets are floated abaxial (top) side down on
the surface of the float solution. The bowl is covered with
foil and incubated at 24'C for 48 hours. After incubation,
the leaflets are removed and the surface sterilization
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procedure described above is repeated. After the final ?0%
ethyl alcohol dip, the leaflets are allowed to air dry on
sterile paper towels in a laminar flow hood.
A nylon bristled artist's brush is sterili2ed by
immersing it in 95%~ ethyl alcohol for 5 - 10 minutes. The
brush is allowed to air dry, and the adaxial (lower) surface
of each leaflet is carefully brushed. The brushing motion
should be toward the leaflet tip and should be continued until
the epidermis has been stripped from the leaflet. The
stripping procedure is completed when the tissue changes from
light to dark green. The midvein from each leaflet is removed
and discarded. The brushed tissue is aseptically cut into
narrow strips (1 - 3 mm wide) and transferred to a 500 ml
sidearm flask containing 200 ml of sterile soak solution. The
flask is swirled to uniformly distribute tissue slices
throughout the solution. The flask is covered with foil and
incubated at 4'C to 10'C for Z~ hours.
_. **************************************************************
SOAR SOLUTTON
**************************************************************
Major Salts . . . . . . . . . . 2.5
. . . ml
stock
Fe-EDTA . . . . . . . . . . . . 2.5 ml
. . . stock
organics. . . . . . . . . . . . 0.25 ml stock
. . .
Minors X. . . . . . . . . . . . 0.25 ml stock
. . . ~
Minors II . . . . . . . . . . . 0.25 ml stock
. . .
NH4Cl . . . . . . . . . . . . ... 1.0 ml stock
. .
NAA . . . . . . . . . . . . . . 2.0 ml stock
. . .
HAP . . . . . . . . . . . . . . 1.0 ml stock
. . .
Volume to 200 m1
pH 5.6
Autoclave
**************************************************************
After the soak stage is completed, the liquid is
carefully decanted from the flask and replaced with 100 ml of
pnlyme Sclvtio~.
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**************************************************************
ENZYME SOLUTION
**************************************************************
Yakult Macerozyme R-10. . . . . . ° . 0.1 g
Yakult Cellulase RS . . . . . . . . . 0.5 g
Major Salts . . . . . . . . . . . . . 5.0 ml stock
MES . . . . . . . . . . . . . . . . . 1.0 ml stock
PVP-10. . . . . . . . . . . . . . . . 1.0 g
Casein Hydrolysate. . . . . . . . . . 1.0 mg
Sucrose . . . . . . . . . . . . . . ° 10.27 g
NH4C1 . . . . . . . . . . . . . . . 0.5 ml stock
Volume to 100 ml
pH 5.6
Filter Sterilize
*;~**********************************************************
The leaflet section is vacuum infiltrated (25 - 27
inches Hg) for 7 minutes and the flask is placed on a gyratory
shaker (100 RPM) and incubated at room temperature (27°C) until
the tissue has disassociated (3 - 5 hours) . The digestion time
varies with cultivar, age of tissue, temperature, shaker speed,
and specific gravity of the enzyme solution.
~rotoplast Isolation (protoplast harvest)
The protoplasts (cells with the cell wall removed) are
collected by centrifugal flotation in sterile Babcock (milk
test) bottles. The contents of the digestion flask are gently
poured into a sterile funnel containing several layers of
cheesecloth. The funnel tip is fitted with a 5 1/4 inch
Pasteur pipet via a piece of plastic tubing. The pipet tip
should rest against the inside of the Babcock bottle neck about
1/2 to 1/3 of the distance from the mouth. ' Protoplasts are
extremely fragile so the protoplast suspension should flow down
the sides and not splash directly to the bottom of the bottle.
Freshly isolated protoplasts are also light sensitive so the
collection procedure should be carried out under subdued
light. A sterile rinse solution is poured through the debris
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lodged in the cheesecloth filter while gently shaking the
filter with the tip of a pipet.
**************************************************************
RINSE SOLUTION
**************************************************************
Major Salts . . . . . . ... . . . 10.0 ml stock
Casein Hydrolysate. . . , ' . . . . 1.0 ml stock
Sucrose . . . . . . . . . . . . . 20.54 g
NH4C1 . . . . . . . . . . . . . 1.0 ml stock
Volume to 200 ml
pH 5.6
Filter Sterilize
**************************************************************
The debris is . rinsed until 3 Babcock bottles are
filled. A fourth bottle is filled with sterile rinse
solution. / This bottle is used to balance the centrifuge. The
bottles are centrifuged at 500 RPM for 10 minutes (IEC HN-SII
centrifuge With rotor 215 and carriers 367A). The debris is
spun to the bottom of the ~ bottles and intact protoplasts float
to the top. 5 - 10 ml of sterile rinse is removed from the
balance bottle. The protoplast layer from each of the other
bottles is transferred to the rinse bottle with a sterile 9
inch Pasteur pipet. The pipet tip is submerged in the rinse
solution then the protoplasts are slowly and gently forced
into the solution. The bottle is tilted at approximately a 45
degree angle and rotated for several minutes to distribute the
protoplasts evenly throughout the rinse solution. Fresh rinse
solution is added to bring the volume up to the top gradation
on the Babcock bottle neck (#8) and centrifuged again. The
resultant floating protoplasts may now be plated if 1x salts
are used in the CL medium. If 4X salts are included in the CL
medium, the protoplasts must be acclimated in a holding
solution for at least 1 hour prior to plating.
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**************************************************************
HOLDING SOLUTION
**************************************************************
Major Salts . . . . . . . . . . . . . 20.0 ml stock
Fe-EDTA . . . . . . . . . . . . . . . 5.0 ml stock
Minors I. . . . . . . . . . . . . . . 0.5 ml stock
Minors II . . . . . . . . . . . . . . 0.5 ml stock
Casein Hydrolysate. . . . . . . . . . 0.1 ml stock
Sucrose . . . . . . . . . . . . . . . 6.8 g
Inositol. . . . . . . . . . . . . . . 0.45 g
Xylitol . . . . . . . . . . . . . . . 0.45 g
Sorbitol. . . . . . . . . . . . . . . ~ 0.455 g
Mannitol. . . . . . . . . . . . . . . 0.455 g
Volume to 100 ml
pH 5.6
Filter Sterilize
**************************************************************
The number of divisions occupied by the protoplast
layer is read off the bottle neck 'to estimate the number of
cells collected (one large division contains approximately 2.5
million protoplasts) . The inside ~of a sterile Pasteur pipet
is coated with holding solution by rinsing several times and
drawing off the protoplast layer and then diluting the
protoplasts with holding solution to a concentration of 1
million cells/ml. The protoplasts are maintained in holding
solution at 22 - 24 C'for a minimum of 1 hour.
Method for In Vitro Se~ection of Hlack Soot Resistant
Protonlast-derived Potato Clones
a t' a ce roto las culture
Protoplasts are cultured in plastic quadrant plates.
The plates are prepared by making a slit with an electric
soldering gun along the base of each divider. Each of two
opposite sectors are filled with 10 ml of R medium.
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*************************************************'*************
R M E DIUM
**************************************************************
Major Salts . . . . . . . . . . 50.0 ml stock
. . .
NH4Cl . . . . . . . . . . . . . 5.0 ml stock
.
Fe-EDTA . .. . . . ... . . .: . 50.0 ml stock
. .
Minors I. . . . . . . . . . : . 5.0 ml stock
. . .
Minors II . . . . . . . . . . . 5.0 m1 stock
. .
Organics . . . . . . . . . . . . 5.0 ml stdck
.
Casein Hydrolysate. . . . . . . 100.0 mg
. . .
Mannitol . . . . . . . . . . . . 18.2 g
. .
Sucrose.. . . . . . . . . . . . 34.2 g
. . .
NAA . . . . . . . . . . . 10.0 ml stock
HAP . . . . . . . . . . . . . 4.0 ml stock
.
Difco Purified Agar . . . . . . 6.0 g
. . .
Volume to 1000 ml
pH 5.6
Autoclave
**********************************************,~*************
The working concentrations of the R Medium are as
follow s:
NN4C1 27 mg/1
Major Salts
KN03 950 mg/1
CaCl2 * 2HZ0 220
MgS04 *, 7Ii20 185
KH2P04 85
Iron and Minor Elements
Na2 * EDTA 18.5 mg/1
FeS04 * 7H20 13.9
H3g04 3.1
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M1tC12 4H20 9.9
*
ZnS04 7H20 4.6
*
RI 0.4 2
Na2Mo04* 2H20 0.13
CuS04 5H20 0.013
*
CoS04 7H20 0.015
*
Organics
Thiamine * HC1 0.5 mg/1
Glycine 2. 0
Nicotinic Acid 5.0
Pyridoxine * HCl 0.5
Folic Acid 0.5
Biotin 0.05
Casein Hydrolysate 100.0
Osmoticum
Sucrose 0.1 M
Mannitol 0.1
Other
NAA 1.0 mg/1
BAp 0.4 mg/1
Agar 0.6 %
PH 5.6
The protoplast cell layer medium (CL medium) is
prepared by mixing 4 parts SLLX solution with one part of the
CL component.
**************************************************************
SLLX
**************************************************************
Major Salts . . . . . . . . . . , , 50.0 ml stock
Sucrose . . . . . . , , . . . , , , 0.56 g
Inositol. . . . . . . , , . . , , , 0.56 g
Xylitol . . . . . . . . . . . . . . 0.56 g
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S orbitol. . . . . . . . . . . . . . 0:5'
Mannitol. . . . . . . . . . . . . . 0.56 g
Volume to 100 ml
pH 5.6
Filter Sterilize
**************************************************************
CL
**************************************************************
Fe-EDTA . . . . . . . , . . . 6.25 ml stock
. . .
Minors I. . . . . . . . . . . 0.625 ml stock
. . .
Minors II . . . . . . . . . . 0.625 ml stock
. . .
Casein Hydrolysate. . . . . . 0.625 ml stock
. . .
NAA . . . . . . . . . . . . . 1.25 ml stock
. . .
BAP . . . . . . . . . . . . . 0.5 ml stock
. . .
Agarose (Type VII) . . . . . 0.56 g
. . . .
Volume to 25 ml
pH 5.6
Autoclave
**************************************************************
The working concentrations of the CL medium are set
forth below:
CL Medium - Working Concentrations
Major Salts
KN03 7600 mg/1
CaCl2 * 2H20 1?60
MgS04 * 7H20 1480
IiH2P04 680
Iron and Minor Elements
Na2 * EDTA 18.5 mg/1
FeS04 * 7H20 13.9
H3B04 3.1
MnCl2 * 4H20 9.9
ZnS04 * 7H20 4.6
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KI 0.42
Na2Mo04 * 2H20 0.13
CuS04 * 5H20 0.013
CoS04 * 7H20 0.015
Organics
Thiamine * HCl 0.5 mg/1
Glycine 2.0
Nicotinic Acid 5.0
Pyridoxine * HCl 0.5
Folic Aeid 0.5
Biotin o.05
Casein Hydrolysate 50.0
Osmoticum
Sucrose 0.200 M
M annitol~ 0.02 5
myo-Inositol . 0.025
S orbitol 0. 02 5
Xylitol 0.025
other
NAA 1:0 mg/1
BAP ~ 0.4 mg/1
Agarose (type VII) 0.45%
pH . 5.6
The mixed CL/SLLX medium is allowed to cool to room
temperature (10 - 15 minutes). Then, the protoplast
suspension is diluted to the desired concentration (20,000 -
40,000 cells/ml). Using a sterile pipet tip, the protoplasts
are gently mixed to a uniform suspension in the medium. 3 ml
of the suspension is transferred to each of the empty quadrant
plate sectors for a total of 6 ml per plate. The plates are
sealed with Parafilm~ and incubated at 24~C for 10 - 14 days
under constant illumination (cool, white fluorescent light) .
Low light intensity (approximately 600 lux) is required during
ti',~s ir~tial growth pY~ase.
SUBSTITUTE SHEET (RULE 26)
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Plant Rggeneration Seauence (callus culturel
The cell layer in gel form is cut into sections with
a sterile spatula and these gel slabs are transferred to Petri
plates, each containing 20 ml of C medium (=C1) (callus
proliferation medium). The entire contents of one quadrant
should be layered on each C medium plate. The plates are
sealed with Parafilm* and incubated at 24°C under 'constant
illumination of approximately 3000 lux (cool, white
fluorescent light). After 2 - 3 weeks, the calli (small
aggregates of cells) should be light green and about 1 - 2 mm
in diameter. Individual calli are picked from the gel with
the tip of a sterile scalpel blade and transferred to a plate
containing fresh C medium (25 - 50 calli/plate) . These C
medium. plates (=C2) are incubated as directed above.
**************************************************************
C M E DIUM
**************************************************************
Major Salts. . . . . . . . . . . . . 100.0 ml stock
.
NH4C1. . . . . . . . . . . . . . . . 20.0 ml stock
Fe-EDTA. . . . . . . . . . . . . .~. 50.0 ml stock
. .
Minors- I . . . . . . . . . . . . . 5.0 ml stock
. .
Minors II. . . . . . . . . . . . . . 5.0 ml stock
.
organics . . . . . . . . . . . . . . 5.o ml stock
.
Casein Hydrolysate . . . . . . . . . 400.0 mg
:
Adenine Sulfate. . . . . . . . . . . 40.0 mg
.
Mannitol . . . . . . . . . . . . . . 54.6 g
.
Sucrose. . . . . . . . . . . . . . . 2.5 g
.
NAA. . . . . . . . . . . . . . . . . 1.0 ml stock
.
BAP. . . . . . . . . . . . . . . . . 5.0 ml stock
.
MES. . . . . . . . . . . . . . . . . 10.0 ml stock
.
Difco Purified Agar. . . . . . . . . 9.0 g
.
Volume to 1000 ml
pH 5.6
Autoclave
**************************************************************
SUBSTITUTE SHEET (RULE 26)
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The Working concentrations of the C medium are set
forth below:
NH4C1 107 mg/1
Major Salts
KN03 1900 mg/1
CaCl2 * 2HZ0 440
MgS04 * 7H20 370
KH2P04 170
Iron and
Minor Elements
Na2 * EDTA 18.5 mg/1
FeS04 * 7H20 13.9
H3 B04 3.1
MnCl2 * 4H20 9.9
ZnS04 * 7HZ0 4.6
KI 0.42
NaZMo04 * 2H20 0.13
CuS04 * 5H20 0.013
CoSo4 * 7H20 0.015
Organics
Myo-Inositol. 100.0 mg/1
Thiamine * HCl 0.5
Glycine 2.0
Nicotinic Acid 5.0
Pyridoxine * HC1 0.5
Folic Acid 0.5
Biotin o.05
casein Hydrolysate loo.o
Adenine Sulfate 40.0
osmoticum
Sucrose 0.25 %
Mannitol 0.3 M
SUBSTITUTE SHEET (RULE 26)
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ether
-20-
NAA 0.1 mg/1
BAp 0.5 mg/1
MES 5.0 mM
Agar 0.9 %
pH 5.6
plant Reaenerati_or~ Sequence (callus differentiation)
The calls from the C medium plates are transferred to
plates containing shoot induction medium (6D medium) (10 - 25
calli/plate) when the calls become dark green (3-5 weeks) .
The plates are sealed with Parafilm~ and incubated at 24°C
under cool, white fluorescent light (3000 - 4000 lux) with a
diurnal cycle of 16 hours light / 8 hours dark. Shoot
induction usually occurs after 6 - 8 weeks. However, some
cultivars only produce shoots after long term culture (6 - 8
months) on shoot regeneration medium.
**************************************************************
6D MEDIUM
**************************************************************
Ma~or Salts. . . . . , . , , , , , , 100.0 ml stock
,
NH4C1. . . . . . . , , , , , , , , , 50.0 ml stock
Fe-EDTA. . . . . , , , , , , , , , , 50.0 ml stock
,
M snore I . . . . . . . . . , , , , , 5. 0 ml stock
,
Minors II. . . . . . , . , , , , , , 5.0 ml stock
,
Drganics . . . . . . . , . . . , , , 5.0 ml stock
,
Casein Hydrolysate . . . . . . . . . 100.0 mg
.
Adenine Sulfate. . . . . . . . . . . 80.0 mg
,
Mannitol . . . . . . . . . . . , , , 36.4 g
,
Sucrose. . . . . . . . . . . . , , , 2.5 g
,
Zeatin (traps isomer). . . . . . . , 20.0 ml stock
.
NAA. . . . . . . , , , , , , , , , , 0.1 ml stock
,
GA3. . . . . . , , , , , , , , , , , 0.1 ml stock
MES. . . . . , . , , , , , , , , , , 10.0 ml stock
,
Difco Purified Agar. . . . . . . , . 10.0 g
.
SUBSTITUTE SHEET (RULE 26)
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Volume to 1000 ml
pH 5.6
Autoclave
Zeatin & GA3- added after autoclave
**************************************************************
The working concentrations of the 6D medium are set
forth below:
6D Medium - Working Concentrations
NH4C1 267.5 mg/1
Major Salts
KN03 1900 mg/1
CaCl2 * 2H20 440
MgS04 * 7H20 370
KH2P04. 170
Iron and Minor Elements
Na2 * EDTA ~ 18.5 mg/1
FeS04 * 7H20 13.9
H3H04 3.1
MnCl2 * 4HZ0 9.9
ZnS04 * 7H20 4.6
KI 0.42
Na2Mo04 * 2H20 0.13
CuS04 * 5H20 0.013
CoS04 * 7H20 0.015
organics
Myo-Inositol 100.0 mg/1
Thiamine * HCl 0.5
Glycine 2.0
N icotinic Acid , 5. o
Pyridoxine * HCI 0.5
Folic Acid 0.5
Biotin 0.05
Casein Hydrolysate 100.0
lldQnino Sulfate 80.0
SUBSTITUTE SHEET (RULE 26)
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Osmoticum
Sucrose 0,25 %
Mannitol 0.2 M
Other
Zeatin (traps isomer) 2.0 mg/1
NAA 0.01
GAS 0.01
MES 5.0 mM
Agar 1.0 %
pH 5.6
Pant Reaangrat~pn Sequence
jshoot e~onc~ation~oot initiation)
The shoots from individual calls are excised when they are
2 - 10 mm long and transferred to glass test tubes (25 X 150
mmj containing 15 ml of E medium. The base of each shoot is
pushed into the medium, the tubes capped and sealed with
filter tape. The tubes are incubated at 24'C under cool,
white fluorescent light (1500 - 2000 lux with a daily 8 hour
dark period) . Root initiation should occur within 2 - 5 weeks.
**************************************************************
E MEDIUM
**************************************************************
Major Salts. . . . . . . . . 100.0 ml stock
.
NH4C1. . . . . . . . 50.0 ml stock
Fe-EDTA. . . . . . . . . . 50.0 ml stock
.
Minors I . . . . . . . . . . . 5.0 ml stock
. . .
Minors II. . . . . . . . . . . 5.0 ml stock
Organics . . . . . . . . . . . . 5.0 ml stock
. . .
Adenine Sulfate. . . . . . . . . 40.0 mg
. . .
Sucrose. . . . . . . . . . . . . 6.0 g
. . . .
MES. . . . . . . . . . . . . . 10.0 ml stock
. .
Difco Purified Agar. . . . . . . 5.0 g
. . .
SUBSTITUTE SHEET (RULE 26)
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Volume to 1000 ml
pH 5.6
Autoclave
**************************************************~***********
The working concentrations of the E Medium are as
follow s:
E Medium - ~jorkinq Concentrations
NH4C1 267.5 mg/1
Major Salts
KN03 1900 mg/1
CaCl2 * 2H20 440
MgS04 * 7H20 370
KH2PO4 170
Iron and
Minor Elements
Na2 * EDTA 18.5 mg/1
FeS04 * 7H20 13.9
H3B04 3.1
MnCl2 * 4H20 9.9
ZnS04 * 7H20 4.6
KI 0.42
Na2Mo04 * 2H20 0.13
CuS04 * 5H20 0.013
CoS04 * 7H20 0.015
Organics
Myo-Inositol 100.0 mg/1
Thiamine * HCl 0.5
Glycine 2.0
Nicotinic Acid 5.0
pyridoxine * HC1 0.5
Folic Acid 0.5
Biotin 0.05
lldvniQ Sulfate ~0.0
SUBSTITUTE SHEET (RULE 26)
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osmoticum
Sucrose 1.0 %
Mannitol 0.1 M
Other
Agar 0.5 %
p H ~5.6
______________________._______________________________~____
Plant Rec,Leneration Sequence
jg~rowth of regenerated plantletsi
The plantlets are removed from the tubes and the
roots washed free of agar with distilled water. Each plantlet
is transferred to an 8 cm clay pot containing Jiffy-Mix. The
pots are watered with approximately 100 ml of Peters 20-20-20
fertilizer (0.5 g/1). Each pot is sealed inside a clear
plastic bag. Bagged pots are incubated under flu orescent
lights at room temperature (22 - 27'C). After one week, the
tops of the bags are opened. The openings are increased over
the next 7 to 10 days, after which the bags can be removed
entirely. The plants are allowed to grow for an additional 2
- 4 weeks. When the plants are 15 - 25 cm tall, they should
be transplanted to larger clay pots (20 - 30 cm) containing
Jiffy-Mix. The plants are then grown in the greenhouse until
tuber set is complete (2 - 4 months). About one week before
harvesting, the vines are cut and the soil in the pots allowed
to dry. The tubers from each protoplast-derived clone are
collected in paper bags and stored under refrigeration for
field increase.
The techniques described above are based upon the
procedures described by Shepard, J.F. Mutant selection and
plant regeneration from potato mesophyll protoplasts, Genetic
Improvement of Crops: Emergent Techniques. University of
Minnesota Press, pp. 185-219 (1980) as modified by Taylor,
R.J, and Secor, G.A., A shoot induction procedure altered for
increased shoot efficiency of potato protoplast - derived
calli, Potato Research 31:651-658 (1988), and incorporated by
TQfQ~encP H CTP~n.
SUBSTITUTE SHEET (RULE 26)
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In a preferred embodiment, potato mesophyll
protoplasts are collected, cultured, and regenerated according
to the procedures described above except that at least one
melanin precursor such as tyrosine or caffeic acid is included
in some of the tissue culture media. This method allows for
additional increases in the number of clones resistant to
blackspot. The culture induced screening method without at
least one melanin precursor, as described above itself results
in some blackspot resistant clones.
At.Stage 1 of.the melanin precursor screening method,
protoplasts are diluted (40,000 cells/ml) in normal CL medium
(melanin precursor-free) and then transferred to culture
plates containing R medium (reservoir medium) with tyrosine or
caffiec acid at 0.25 mM. After the normal incubation period,
the protoplasts and protoplast-derived micro calli axe
transferred, along with the cell layer medium (CL), to C
medium containing tyrosine at 0.5 mM. This first transfer to
C medium is Stage 2 of the process. After the standard
incubation period, only individual calli showing no blackening
response are selected and transferred to fresh C medium
containing tyrosine or caffeic acid at 1.0 mM for Stage 3
screening. The calli axe incubated for an additional 2 - 4
weeks and those that remain green (no blackening 'evident) are
wounded by cutting with a sterile scalpel blade. After 24
hours the cut calli are evaluated for blackening in the
wounded area. After this Stage 4 screen, only the surviving
green calli are transferred to normal 6D medium (shoot
induction medium) (without melanin precursor) for plantlet
initiation. Regenerated shoots are transferred to E medium
(rooting medium) containing tyrosine or caffeic acid at 0.5 mM
for the final (Stage 5) screen. All shoots that develop roots
which do not exhibit blackening in the medium are rescued as
potential blackspot resistant clones. These plants are
transplanted to individual pots and grown in the greenhouse
for tuber production. Each plant normally produces from 6 -
24 tubers which are maintained until the following field
growing season. Tubers of the selected clones are
subsequently increased 1 or 2 field generations. Anatomical
characteristics of the clones are examined in both the
SUBSTITUTE SHEET (RULE 26)
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greenhouse and the field and harvested tubers are tested for
blackspot bruising. It may be advantageous to add tyrosine or
caffeic acid at only some of the stages as the protoplasts and
calli may otherwise become damaged and thus unusable.
TABLE 1: Summary of the minimum time involved in screening and
regeneration sequences.
STAGE Duration Cumulative
Time
plantlet Reveneration
CL/R Screen (1) 10 days 10 days
C1 Screen (2) 10 days 20 days
C2 Screen (3) 21 days 41 days
C2 Screen (4) 1 day 42 days
6D Regeneration 45 days 87 days
E Screen (5) 21 days 108 days
Tuber production
(lab preconditioning) 14 days., 122 days
(greenhouse) 90 days ~ ' 212 days
___________________
____ ____ ___________________________________
D iscussion'
The procedure for identifying sells and calli that
could yield plants resistant to blackspot is based upon the
biochemical processes that normally occur in bruised tubers.
Tyrosine, which is. the principle compound converted to the
black pigment melanin through a series of biochemical
reactions, or caffeic acid, an intermediate compound in the
melanin pathway, are included as additional components in the
standard tissue culture media described above. Calli derived
from individual protoplasts are evaluated for the production
of black pigmentation at several stages during the tissue
culture process. Those that demonstrate a blackening response
in the presence of tyrosine or caffeic acid are rejected as
presumed suscepts and those that show no blackening throughout
the growth and regeneration process are retained as potential
rQS i rt a,at c1 onPS. P1 ants produced from such cal ~ i arQ carri pd
SUBSTITUTE SHEET (RULE 26)
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through greenhouse and field increase in order to produce
sufficient quantities of tubers for subsequent bruising and
blackspot evaluation.
Blacksnot Testina (methods and ratincL system used)
Tubers are bruised under controlled conditions using
an apparatus similar to the one described by Kunkel et al.,
Improvements of techniques for blackspot evaluation arid some
errors associated with measurements. Am. Potato J. 63: 13-23
(1986), said description incorporated herein by reference.
Five tubers from each clone were tested by striking them with
a 115 g weight dropped from a height of 45 or 50 cm, with each
tuber receiving 9 impacts (3 stem end, 3 middle, 3 bud end).
The tubers were maintained at 75°C for 24 hours, and then
evaluated. In year 1 of testing, the quantity of available
tubers regenerated was minimal due to environmental
conditions. In years 1 and 3, 5 tubers from each clone were
tested at 9 impacts per tuber for a total of 45 bruise spots.
In year 2, testing was repeated 3 times for each clone (15
tubers/clone) at 9 impacts per tuber for a total of 135 bruise
spots. The results shown below for year 2 reflect the average
from three trials. For some clones, year 2 was the first year
of testing for that clone. ~ Blackspot development was
determined visually by slicing through the impact areas with a
knife. Thin tissue slices were removed parallel to the tuber
surface until maximum diameter and blackening intensity were
attained.
Three criteria are used to quantify blackspot
susceptibility: (1) The severity (S) of bruising at each
impact point was rated according to a 5 point scale where:
0 = no spot development
1 = very small spot with a trace of blackening
2 = small spot with limited blackening
3 = moderate spot with moderate blackening
4 = large black spot
= very large spot with intense blackening
(2) The spots were counted and the percentage (PC) of
impact areas that developed spots is determined from this
SUBSTITUTE SHEET (RULE 26)
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information.(3) A blackspot index (BI) is calculated from
the above
information
according
to the following
formula:
BI = S X PC
Susc eptibility/resistance of a cultivar
is expressed
as an average
blackspot
index calculated
from all
bruised
tubers.
esu is
The results are shown on a percentagebasis below:
Summary Table
Treatment Resistant*/Total % Resistant
Culture-induced
Screening
Year 1 8/22 36.4
Year 2 12/38. 31.6
Year 3 7/26 26.9
Mean: 31.6
Screened Melanin Precursors
with
Year 1 5/7 71.42
Year 2 21/40 52.5
year 3 22/49 44.9'
MEAN: 56.3
Screened Caffeic Acid
with
Year 1 5/7 71.42
Year 2 3/9 33.3
Year 3. 3/9 33.3
MEAN: 46.0
Screend withTyrosine
Year 1 ----
Year 2 18/31 58.1
Year 3 19/40 47.5
MEAN: 52.8
* Those thatwere statistically more resistant
than the mother
clone.
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The following table identifies clones regenerated
from 'Lemhi~ callus lines that were not carried through the
melanin precursor in vitro selection screening procedure.
This population represents the distribution of blackspot
resistant variants one could expect from the in vitro
screening without melanin precursor. It can be seen that the
tissue culture process itself increases the number of
blackspot resistant Lemhi clones since Lemhi in nature
normally shows relatively little, if any, resistance.
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SUBSTITUTE SHEET (RULE 26)
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30-
Blackspot population of
reaction tissue cultured
in a screened
'Lemhi'
clones
CLONE ID BLACKSPOT INDEX
Year 1 Year 2 Year 3
84-39 182.7
92-10 1.2*
84-114 3.5* 5.0* 55.0*
84-2108 5.4* 98.5 102.8
84-196 10.6* 144.2 62.6
84-117 11.4* 60.4 13.0*
84-256 12.1* 78.8 83.1
84-254 16.7* 102.3 47.7*
84-8 20.7*
84-148 42.6 97.9 126.8
84-16' 42.6 113.1 163.3
84-305 47.9 158.6 172.3
92-25 64.1 53.2* 19.2*
84-231 101.0 29.5*
84-155 105.0 36.2*
84-1 106.4 151.4 180.7
84-5 121.7 206.4 218.8
84-143 141.3 255.9 163.3
84-9 160.2 133.6 101.3
84-63 203.3 137.2 188.8
81-1 237.2 135.1 161.3
84-249 236.6
84-329 181.9
84-163 125.5
84-31 99.2
84-184 70.9 98.7 69.8
84-278 79.5 84.8
84-351A 74.6
84~27 73.2 60.5
84-20 69.2
84-314 50.9* 105.7
84-492 46.1* 113.9
84'393 46.0* 105.1
84-459 24.9* 50.5*
84-78 0.4* 1.2*
92-3 125.0
115-3 103.6 103.2
91-64 39.1*
81-8 36.3*
120-11 1.7*
84-402 199.5
84-166 47.3*
Lemhi MC 51.5 124.1 147.4
(protoplastsource)
Lemhi 47.9 94.5 160.5
(state seedmeristem
program)
* = Significantly resistant than
more the cultivar
Lemhi
(p=0.05)
Year 1 databased upon 1 bruise testingexperiment (5 tubers)
Year 2 databased upon 3 bruise testingexperiments (15 tubers)
Year 3 databased upon 1 bruise testingexperiment (5 tubers)
SUBSTITUTE SHEET (RULE 26)
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These results demonstrate that variation in blackspot
susceptibility readily occurs and somaclones with enhanced
resistance to blackspot bruise can be found in a screened
population of 'Lemhi' somaclones.
The following table identifies clones regenerated
from 'Lemhi' callus lines that were carried through the
melanin precursor in vitro screening procedure with r~o
blackening response. These clones were screened~at various
stages as shown below.
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SUBSTITUTE SHEET (RULE 26)
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Blackspot population of 'Lemhi' clone
reaction
in
a
s screened
in precursors (tyrosine
vitro or caffeic
with acid
melanin
(:-CA).).
CLONE ID SCREENING
STAGES
(Stage at BLACKSPOT
INDEX
which Melanin
Precursor
added) Year 1 Year 2 Year 3
-
L-DB27(CA) 1 2 3 4 0.0* 68.3 - -53.0* -
L-DB23(CA) 1 2 3 4 1.2* 83.6 75.9
L-DB14(CA) 1 2 3 4 3.0* 27.6* 93.0
L-DB18(CA) 1 2 3 4 3.4* 90.6 105.2
L-DB21(CA) 1 2 3 4 4.9* 144.6 170.5
L-DB24(CA) 1 2 3 4 36.8 241.3
92-7 (CA) 3 60.5 109.1 128.6
111-6 2 3 5 203.8 89.6
103-13 2 3 5 174.5 69.4
111-4 2 3 5 157.0 136.7
111-17 2 3 5 107.3 2.5
98-2 2 3 5 104.8
94-1 5 97.4 103.3
111-13 2 3 5 84.4 51.3*
97-19 2 3 5 83.6 50.7*
111-20 2 3 5 77.7 83.2
111-12 2 3 5 75,g
111-14 2 3 5 75.4 134.4
98-1 2 3 5 66.6
103-3 2 3 5 62.6 106.3
91-95 5 55.3* 33.8*
97-18 2 3 5 53.6* 21.0*
97-26 2 3 . 5 46.6* 36.1*
111-3 ~ 2 3 5 45.6* 100.8
91-13 5 42.1* 54.7*
103-2 2 3 5 41.7* 199.4
111-28 2 3 5 38.5* 94.8
92-67 5 35.3* 15.3
L-D84 1 2 3 4 31.8*
91-62 5 23.8* 59.2*
L-D86 1 2 3 4 18.6* 35.1*
91-28 5 15.2*
L-DB25(CA) 1 2 3 4 14.6* 2.5*
114-8 2 3 5 13.4* 83.8*
97-30 2 3 5 9.5* 13Z.9
92-66 5 2.5*
91-7 5 1.9*
L-DB22(CA) 1 2 3 4 0,5*
115-1 2 3 5 0.4* 57.5
111-33 3 5 0.1* 55.8
84-515 5
L-DB19(CA) 1 2 3 4 114.4
114-5 2 3 4 5 102.0
111-38 2 3 5 gg,1
97-43 3 95.5
110-17 3 4 5 72,6
97-17 3 5 63.5
97-44 1 3 5 57,7
SUBSTITUTE SHEET (RULE 26)
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91-14 5 38.9*
103-14 2 3 5 33.1*
91-61 5 32.3*
L-DB15 (CA)1 2 3 4 31.1*
97-12 3 5 27.6*
91-12 5 26.9*
111-37 2 3 5 22.4*
98-5 3 4 5 21.2*
115-2 14.1*
111-29 2 3 4 5 11.8*
Lemhi MC 51.5 147.4
124.1
(protoplastsource)
Lemhi 47 .9 94.5 160.5
(state seedmeristem
source)
* Significantly
mare resistant
than the
cultivar
Lemhi
(p=o.o5)
Year 1 databased upon 1 bruisetesting experiment (5 tubers)
Year 2 databased upon 3 bruisetesting experiments(15 tubers)
Year 3 databased upon 1 bruisetesting experiment (5 tubers)
The following clones were regenerated
from 'Lemhi'
callus linesthat were carried
though
the
melanin
precursor
in
vitro screening a blackening response
procedure in at
with
least one
stage.
CLONE ID SCREENING BLACKS POT TNDEX
STAGES
Year 1 Year 2 Year 3
111-15 2 3 5 133.4
103-1 2 3 5 121.2 30.2*
110-2 3 5 _ 75.4
91-51 5 57.7
111-11 2 3 5 39.7*
97-8 3 5 36.7* 16.1*
115-9 2 3 5 0.2* 7.4*
111-35 2 3 4 .5 58.0
1I1-26 Z 3 5 28.4*
92-125 5 35.6*
Lemhi Mother Clone 51.5 124.1 147.4
(protoplastsource)
Lemhi 47.9 94.5 160.5
(state seedmeristem
source)
* - Significantly
more resistant
than the
cultivar
Lemhi
(p=0.05)
Year 1 databased upon 1 bruisetesting experiment (5 tubers)
Year 2 databased upon 3 bruisetesting experiments
(15 tubers)
Year 3 databased upon 1 bruisetesting experiment (5 tubers)
SUBSTITUTE SHEET (RULE 26)
CA 02404937 2002-10-11
WO 01/83708 PCT/US00/11767
-34-
These results demonstrate that susceptibility to
blackspot bruise can be identified in an in vitro selected
population of somaclones and suggest that the proportion of
clones with resistance to blackspot can be increased by
screening the material while in tissue culture.
Conclusion
Source germplasm 'Lemhi' was generally less
susceptible to bruising than in past years. 'Lemhi' is
normally highly susceptible with BI values of 250 - 30o
expected. In contrast, the average BI of 'Lemhi' tubers grown
in year 1 was 51.1, in year 2, the average BI was 124.1, and
in year 3 147.4. This demonstrates how closely expression of
this disorder can be tied to environmental conditions and
physiology of the plant. The blackspot response of 'Lemhi' is
used as a comparative means of evaluating the somaclones.
It is clear that clones showing elevated resistance
to blackspot bruising can be obtained via in vitro selection.
A functioning in vitro screening system for blackspot
resistance could prove to be a factor in how this disorder is
controlled. Implementation .of .such a system would be
significant because a large proportion of somaclones with
elevated resistance to blackspot could be identified. This
would greatly increase the likelihood of obtaining clones
possessing all the important characteristics of the original
cultivar. The probability of finding somaclones showing
improvements in other traits in addition to blackspot
resistance would be increased as well.
Although a particular embodiment of the invention has
been described in detail for purposes of illustration, various
modifications may be made without departing from the spirit
and scope of the invention. Accordingly, the invention is not
to be limited, except as by the appended claims.
SUBSTITUTE SHEET (RULE 26)
