U.S. patent application number 13/397237 was filed with the patent office on 2012-08-16 for red lettuce.
Invention is credited to Adrianus Martinus Jozeph Ammerlaan, Cornelis Marinus MOOR, Kornelius Reinink, Johannes Wilhelmus Schut, Egbert Carolus Johannes Smits.
Application Number | 20120210470 13/397237 |
Document ID | / |
Family ID | 35532762 |
Filed Date | 2012-08-16 |
United States Patent
Application |
20120210470 |
Kind Code |
A1 |
MOOR; Cornelis Marinus ; et
al. |
August 16, 2012 |
RED LETTUCE
Abstract
The present invention relates to a head-forming lettuce plant of
the species Lactuca sativa having red leaves throughout the head,
including the heart. The red leaves in the heart are red even in
the absence of radiation with wavelengths shorter than 400 nm,
wherein the absence of radiation with wavelengths shorter than 400
nm is in the growing environment during the complete period from
sowing until observation. The ratio between anthocyanin and
chlorophyll is between 4 and 50, preferably between 9 and 27. The
invention also relates to progeny of the plant.
Inventors: |
MOOR; Cornelis Marinus;
(Monster, NL) ; Smits; Egbert Carolus Johannes;
(Zevenbergen, NL) ; Ammerlaan; Adrianus Martinus
Jozeph; (Aramon, FR) ; Schut; Johannes Wilhelmus;
(Wouw, NL) ; Reinink; Kornelius; (Delft,
NL) |
Family ID: |
35532762 |
Appl. No.: |
13/397237 |
Filed: |
February 15, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12052690 |
Mar 20, 2008 |
8143487 |
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13397237 |
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PCT/EP2006/009217 |
Sep 20, 2006 |
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12052690 |
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Current U.S.
Class: |
800/305 ;
536/23.6 |
Current CPC
Class: |
A01H 5/12 20130101 |
Class at
Publication: |
800/305 ;
536/23.6 |
International
Class: |
A01H 5/00 20060101
A01H005/00; A01H 5/10 20060101 A01H005/10; C12N 15/29 20060101
C12N015/29 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 20, 2005 |
EP |
05077135.1 |
Claims
1. A head-forming lettuce plant of the species Lactuca sativa
having red leaves throughout the head, including the heart.
2. The lettuce plant of claim 1 having red leaves in the heart,
even in the absence of radiation with wavelengths shorter than 400
nm.
3. The lettuce plant as claimed in claim 2, wherein the absence of
radiation with wavelengths shorter than 400 nm is in the growing
environment during the complete period from sowing until
observation.
4. The lettuce plant as claimed in claim 1, wherein the absorbance
ratio A523/A665 between anthocyanin and chlorophyll of the heart
leaves is between 4 and 50.
5. The lettuce plant as claimed in claim 4, wherein the absorbance
ratio A523/A665 between anthocyanin and chlorophyll of the heart
leaves is between 9 and 27.
6. The lettuce plant as claimed in claim 1, wherein the absorbance
ratio A523/A665 between anthocyanin and chlorophyll of the ten
youngest heart leaves larger than 1 cm is between 4 and 50.
7. The lettuce plant as claimed in claim 6, wherein the absorbance
ratio A523/A665 between anthocyanin and chlorophyll of the ten
youngest heart leaves larger than 1 cm is between 9 and 27.
8. The lettuce plant as claimed in any claim 1 having a genetic
constitution that leads to red colouring of the leaves in the heart
and which comprises a gene locus for anthocyanin expression, at
least two gene loci that are involved in the red colouring in the
heart of the head and optionally at least two gene loci that are
involved in the intensity of red colouring.
9. The lettuce plant as claimed in claim 1, obtainable from seeds
as deposited on Jul. 18, 2005 with the NCIMB under accession
numbers NCIMB 41337, NCIMB 41338 and NCIMB 41339.
10. The lettuce plant as claimed in claim 1 having the genetic
constitution of the seeds as deposited on Jul. 18, 2005 with the
NCIMB under accession numbers NCIMB 41337, NCIMB 41338 and NCIMB
41339 that leads to red colouring of the leaves in the heart of the
head.
11. The lettuce plant of the species Lactuca sativa having red
leaves of claim 1, even in the absence of radiation with
wavelengths shorter than 400 nm in the growing environment during
the complete period from sowing until observation.
12. The lettuce plant as claimed in claim 11, wherein the
absorbance ratio A523/A665 between anthocyanin and chlorophyll is
between 4 and 50.
13. The lettuce plant as claimed in claim 11, wherein the
absorbance ratio A523/A665 between anthocyanin and chlorophyll is
between 9 and 27.
14. The lettuce plant as claimed claim 11, wherein the absorbance
ratio A523/A665 between anthocyanin and chlorophyll of the ten
youngest heart leaves larger than 1 cm is between 4 and 50.
15. The lettuce plant as claimed in claim 14, wherein the
absorbance ratio A523/A665 between anthocyanin and chlorophyll of
the ten youngest heart leaves larger than 1 cm is between 9 and
27.
16. Progeny of lettuce plants as claimed in claim 1.
17. Seeds of the lettuce plants as claimed in claim 1.
18. A head of the lettuce plants as claimed in claim 1.
19. A gene complex which in a lettuce plant of the species Lactuca
sativa leads to red coloration of the leaves throughout the head,
including the heart which comprises a gene locus for anthocyanin
expression, at least two gene loci that are involved in the red
colouring in the heart of the head and optionally at least two gene
loci that are involved in the intensity of red colouring.
20. The gene complex as claimed in claim 19 as present in the seeds
as deposited on Jul. 18, 2005 with the NCIMB under accession
numbers NCIMB 41337, NCIMB 41338 and NCIMB 41339.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/052,690 filed Mar. 20, 2008 which is a continuation-in-part
of International application no. PCT/EP2006/009217, filed Sep. 20,
2006, published as WO 2007/039137 on Apr. 12, 2007, and claiming
priority to EP 05077135.1, filed Sep. 20, 2005.
[0002] All of the foregoing applications, as well as all documents
cited in the foregoing applications ("application documents") and
all documents cited or referenced in the application documents are
incorporated herein by reference. Also, all documents cited in this
application ("herein-cited documents") and all documents cited or
referenced in herein-cited documents are incorporated herein by
reference. In addition, any manufacturer's instructions or
catalogues for any products cited or mentioned in each of the
application documents or herein-cited documents are incorporated by
reference. Documents incorporated by reference into this text or
any teachings therein can be used in the practice of this
invention. Documents incorporated by reference into this text are
not admitted to be prior art.
FIELD OF THE INVENTION
[0003] The present invention relates to a red lettuce plant, to the
head of lettuce that can be obtained therefrom and to the gene
complex that is responsible for the red colour of the lettuce.
BACKGROUND OF THE INVENTION
[0004] Lifestyles change and the demand from restaurants and
catering firms for colourful and interesting garnish and even from
the housewife for ready-to-use processed salads continue to rise.
As a result, the breeding companies are looking for varieties with
prominent colour, better taste and a wide variety of texture. The
lettuce market can be divided into three groups, namely entire
whole heads, pre-cut whole heads and baby leaves.
[0005] At present the attractive red colour in pre-cut lettuce
mixtures is often provided by the presently available "red" lettuce
(Lactuca sativa), or by radicchio rosso (Cichorium intybus), red
cabbage or red chard. The "red" lettuce that has been known so far
is not really red throughout the leaves. In particular the head
forming types are either only red along the leaf edges or speckled
with red. They are never completely red in the heart. This is
caused by the fact that the red colour is only expressed in those
parts of the head that are exposed to daylight, more specifically
to UV-radiation. Leaves of the so-called "red" lettuces are
therefore for the main part green and their red does hardly
contribute to the red colour impression of a pre-cut lettuce
mixture.
[0006] Often the transitional stage between the red part and the
green part of the leaf is brown-coloured. This brown colour is
considered as visually unattractive. Red speckled lettuces are
associated with plant diseases or bloodstains. At present plant
breeders select against the brown colour as well as against the
speckled red colouring. In addition, it is even found that speckled
or brown coloured lettuce leaves are often discarded, both in the
lettuce packaging industry and by the consumer.
[0007] The disadvantage of using leaves from other vegetables than
lettuce is that the different taste of these other vegetables is
often experienced as undesirable. Radicchio rosso is for example a
Cichorium inlybus that has a bitter taste. The texture of red
cabbage is entirely different from the much softer texture of
lettuce.
[0008] Anthocyanin synthesis in lettuce is induced by UV-radiation
(see for example Voipio & Autio, 1995, Responses of red-leaved
lettuce to light intensity, UV-A radiation and root zone
temperature. In: Acta Horticulturae 399. Greenhouse environmental
control and automation. Eds. B J Bailey, T Takakura. Kyoto, Japan.
p 183-187; Benoit, et al., 1998, Effect of a photoselective
greenhouse film on a few vegetable crops in the Belgian North Sea
climate. In: 14th International congress on plastics in
agriculture, Tel Aviv, Israel, March 1997. Laser Pages Publishing,
Jerusalem, Israel. p 81-92; Krizek et al., 1998, Inhibitory effects
of ambient levels of solar UV-A and UV-B radiation on growth of cv.
New Red Fire lettuce. Physiologia Plantarum 103(1), p 1-7;
Kleinhenz et al., 2003, Variety, shading, and growth stage effects
on pigment concentrations in lettuce grown under contrasting
temperature regimens. Hortechnology 13(4), p 677-683.). The
development of a red colour in many plant species is dependent on
the production of anthocyanin.
[0009] The many different varieties of lettuce are often grouped
into three types. The most common is head lettuce of which there
are the crisp head (or iceberg) and butter head. Romaine lettuce
(or cos lettuce) forms a loose upright head. The "leaf" lettuce
types are non-heading and loose leafed.
[0010] In head-forming lettuce types the heart of the head is to a
more or lesser extent closed and cannot be reached by light. It is
thus not possible to have anthocyanin production in closed heads
such as iceberg lettuce and butter lettuce or in romaine lettuce,
which has a loosely closed head.
[0011] A related problem is the lack of red colouration of red
lettuce grown in glasshouses, plastic tunnels, or in closed
containers. Due to the lack of UV-radiation, which is reflected by
the glass or plastic covering or which is lacking in the artificial
assimilation light spectrum, the expression of anthocyanin is much
less than in outdoor conditions. This reduces the possibilities to
produce red lettuce under indoor conditions, and therefore the
possibility to produce red lettuce protected from adverse weather
conditions, like cold, heat, and all sorts of precipitation.
SUMMARY OF THE INVENTION
[0012] The present invention now provides a red lettuce, having red
leaves throughout the head, including the heart. The heart leaves
of red lettuce of the invention are in essence completely red,
which means that they contain a uniquely high
anthocyanin/chlorophyll-ratio. This is very surprising because
light, more specifically UV-light, which is deemed necessary for
the synthesis of anthocyanin is not able to penetrate into the
heart of the head. This demonstrates that in the lettuce of the
invention a light-independent mechanism is responsible for the
production of anthocyanin.
[0013] This light-independency also allows for the cultivation of
red lettuce, headed or non-headed, under glass or plastic or in
closed containers. The latter uses artificial assimilation lighting
which usually gives problems with the development of red colour.
This is due to the fact that the UV-wavelengths (280-400 nm) which
are normally essential for induction of anthocyanin synthesis, are
more or less lacking in the light spectrum.
[0014] Under glass or plastic a similar lack of UV-radiation is
commonly caused by strong UV-interception of the covering. However,
due to its UV-independent anthocyanin expression the red lettuce of
the invention is suitable for glasshouse, plastic house, and closed
container cultivation. This is due to the light-independent
mechanism of the present invention, which will also be indicated as
"UV-independent anthocyanin expression", or "UV-independent red
colouration".
Deposits
[0015] The Deposits with DEPOSITORY, under deposit accession number
NCIMB 41337, NCIMB 41338 and NCIMB 41339 were made pursuant to the
terms of the Budapest Treaty. Upon issuance of a patent, all
restrictions upon the deposit will be removed, and the deposit is
intended to meet the requirements of 37 CFR
.sctn..sctn.1.801-1.809. The deposit will be maintained in the
depository for a period of 30 years, or 5 years after the last
request, or for the effective life of the patent, whichever is
longer, and will be replaced if necessary during that period.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The present invention will be further illustrated in the
non-limiting examples that follow and that refer to the following
figures:
[0017] FIG. 1 is a graph that shows the distribution of within-line
segregation of red colouration of outer and inner leaves for
population of 212 F3-lines obtained from cross `99P.30637.times.cv.
`Sharp Shooter`.
[0018] FIG. 2 is a diagram that shows the genetic model for
anthocyanin expression based on segregation in cross
`99P.30637`.times.cv. `Sharp Shooter`.
[0019] FIGS. 3A-3F show representative examples of young plants
from the lines 03P82421, Pierrot, Pippo, Gringo, Apache and Bijou,
respectively.
[0020] FIG. 4 shows a comparison of representative examples of a
young leaf from a plant of the present invention with young leaves
from varieties Pippo, Pierrot and Gringo.
[0021] FIGS. 5A-5D show a comparison of representative examples of
a plant of the invention (01P80164) and the variety Darkland
(99R10044).
[0022] FIG. 6A-6D show a comparison of representative examples of a
plant of the invention (01P80164) and the variety Pierrot
(95G2237).
[0023] FIG. 7A-7D show a comparison of representative examples of a
plant of the invention (01P80164) and the variety Roxy
(99R10283).
[0024] FIG. 8A-8D show a comparison of representative examples of a
plant of the invention (01P80164) and the variety Sierra
(95G1986).
DETAILED DESCRIPTION
[0025] In a particular embodiment of the invention, the absorbance
ratio A523/A665 between anthocyanin and chlorophyll is between 4
and 50, preferably between 9 and 27.
[0026] Whether the expression of anthocyanin in the present
invention is completely independent of UV-radiation, or that it is
triggered by much lower levels of UV-radiation than normally
required for anthocyanin expression in regular red lettuce plants,
is not completely known yet. However, for practical usefulness of
the invention this difference does not matter.
[0027] Although the invention is particularly useful in
head-forming lettuce types, the light-independent red colouring of
the invention can also be used in other lettuce types such as leaf
lettuce and in baby-leaf production, which is the production of
young lettuce plants for harvesting small, immature leaves.
[0028] The pedigree of a red lettuce of the invention is given in
Example 1.
[0029] The invention relates to all progeny of the original parents
that have red leaves in the heart of the head and that meet one or
more of the other requirements of the invention, such as the ratio
between chlorophyll and anthocyanin of the heart leaves.
[0030] In addition, non-heading, loose leaf or baby lettuce plants
that have the UV independent red colouration of the invention are
also considered progeny of the completely red head-forming lettuce
plants and thus part of this invention.
[0031] The selection of lettuce plants that already are or can lead
to the lettuce plants of the invention can be based on visual
selection of red-coloured young plants (step 1). Growing them under
glass or plastic can subsequently distinguish between UV-dependent
anthocyanin expression, i.e. poor red colouration, and
UV-independent anthocyanin expression, i.e. a relatively strong red
colouration. These latter plants are planted and grown until they
are headed. Then visual selection of plants with red heart leaves
is performed by cutting the top off each plant. The red-hearted
plants are then selected to produce offspring seed.
[0032] Crosses are chosen to increase the level of red colouration,
and/or to obtain the required level of heading of the mature
plant.
[0033] As used herein, a lettuce plant is defined as headed, when
it has got an `open head` or a `closed head`, according to the
guidelines of UPOV for the characteristic `head formation`
('TG/13/9. Lettuce (Lactuca sativa L.)). Guidelines for the conduct
of tests for distinctness, uniformity and stability.` International
Union for the Protection of New Varieties of Plants. Geneva.
2004).
[0034] The lettuce hearts of the present invention have an
absorbance ratio A523/A665 between chlorophyll and anthocyanin
which is preferably higher than 9, more preferably 13 or higher. In
normal red lettuce this ratio was never found to be higher than 3.
The chlorophyll and anthocyanin concentrations used in determining
the ratio of the invention are determined by means of
spectrophotometry. Sample preparation and analysis is presented in
Example 2.
[0035] It is to be noted that anthocyanin levels as high as in the
heart leaves of the present invention can possibly be found in
other lettuce varieties, which are non-heading. Some examples of
such non-heading varieties are 40-0203103-B (Knerr L D, 2005.
Lettuce cultivar 40-0203103-B. US Patent application US
2005/0144672 A1), Galactic, New Red Fire, Rolina (Kleinhenz et al,
2003. Variety, shading and growth stage effects on pigment
concentrations in lettuce grown under contrasting temperature
regimens. Horttechnology 13(4) p. 677-683), Red Salad Bowl, and
Sesam (Voipio I. and Autio J., 1995. Responses of red-leaved
lettuce to light intensity, UV-A radiation and root zone
temperature. Acta horticulturae 399. p. 183-187).
[0036] However, these varieties have heart leaves with very high
chlorophyll levels, in comparison with the heart leaves of the
present invention, as a result of high light exposure due to the
non-heading characteristic. On the other hand, heading varieties
can possibly be found with similarly low chlorophyll levels in the
heart leaves as in the heart leaves of the present invention.
However, the heart leaves of these heading varieties do not show
the high anthocyanin levels in comparison with the heart leaves of
the present invention. Due to the low light exposure of the heart
leaves anthocyanin synthesis is normally poorly induced. In the
present invention, the anthocyanin production is UV-independent.
This explains why the anthocyanin/chlorophyll-ratio of the present
invention is on an unprecedentedly high level.
[0037] This new form of red colouration that is UV-independent is
found and developed in headed lettuce plants and differs from the
red colouration as presented in the prior art. However, the
invention also relates to non-headed lettuce plants, such as loose
leaf or baby leaf lettuce plants or lettuce plants that are grown
in glass houses or under plastic where the amount of UV is lower
that have the feature "UV-independent red colouration" of the
invention.
[0038] Red colouration of the heart leaves was also scored on the
RHS colour chart (The Royal Horticultural Society, London, UK).
Heart leaf lamina colour, especially on the top of leaf, of the
present invention was scored as 183A, 184A, or 187B, all in the
greyed-purple group. The colour of the rest of the leaf lamina was
scored as 180B, 180C, 180D, or 181C, all in the greyed-red
group.
[0039] It is also possible to score the red colouration and
anthocyanin expression on plants grown under glass or plastic, i.e.
conditions with reduced levels of UV-radiation, when compared to
the light in outdoor conditions. Also here the plants of the
present invention can show higher levels of anthocyanin and a
darker red colour than regular red lettuce. Especially newly
appearing leaves of the plants of the present invention are much
more red than the newly appearing leaves of common red lettuce
plants.
[0040] A genetic analysis of the red lettuce of the invention was
performed as described in Example 2. It was found therein that at
least three genes are involved in the red colouring of the heart of
the head.
[0041] The present invention thus relates to lettuce plants that
are capable of expressing anthocyanin and have in addition at least
the three genes that are involved in the red colouring of the heart
of the head. Preferably, a plant of the invention comprises the
complete gene complex described in Example 2.
[0042] It is possible to assess the presence of the loci involved
in red coloration of the present invention by closely linked
DNA-markers, like AFLP, RFLP, RAPD, SCAR, CAPS, SSR, or SNP. For
instance, a marker-trait linkage analysis in the population
mentioned above, and its offspring can provide such closely linked
DNA-markers.
[0043] The presence in a lettuce plant of a similar genetic
constitution of red colouration of the inner leaves as in the
present invention, i.e. assessing whether a lettuce plant is a
plant according to the invention can easily be assessed by
comparison of the phenotype of such potential plant of the
invention with the phenotype of a known plant of the present
invention. The phenotype can be assessed by, for example, the red
colouration of the inner leaves, the anthocyanin/chlorophyll-ratio
of the heart leaves, and/or the red colouration of plants grown
under glass or plastic.
[0044] Another way of assessing the similarity of the genetic
constitution between a potential plant of the invention and a known
plant of the invention is by comparison of the marker genotype of
the potential plant with the marker genotype of a known plant of
the present invention. The marker genotype is defined by a set of
DNA-based markers, like AFLP, RFLP, RAPD, SCAR, CAPS, SSR, or SNP,
which are closely linked to the loci which involved in the red
expression of the present invention.
[0045] Another way of assessing the similarity of the genetic
constitution, is the comparison of the genotype of a potential
plant of the invention with the genotype of a known plant of the
present invention. This genotype comparison is done on an
F2-population, derived by self-fertilising an F1-plant from a cross
between the potential plant and a known plant of the present
invention. The F2-population can be investigated for absence of
segregation for the phenotype, i.e. the red colouration of the
inner leaves. In all comparisons phenotypes can also be assessed
by, for example, the anthocyanin/chlorophyll-ratio of the heart
leaves, or the red colouration in plants grown under glass or
plastic.
[0046] Seed of red lettuce plants according to the invention was
deposited with the NCIMB on 18 Jul. 2005 under accession numbers
NCIMB 41337, NCIMB 41338 and NCIMB 41339.
[0047] The invention also relates to progeny of these seeds, as
well as to plants that have obtained the genetic constitution or
gene complex of plants of the invention that leads to the red
colouring according to the invention, either by crossing or by
means of molecular biological techniques. The invention also
relates to the progeny of these plants that have maintained or
acquired the trait of red colouring of the invention.
[0048] To transfer the genes that are responsible for the red
colour to another plant backcross breeding can be used. For this a
desirable homozygous cultivar or inbred is the recurrent parent.
The source of the trait to be transferred is called the donor
parent. The resulting plant is expected to have the attributes of
the recurrent parent (e.g., cultivar) and the desirable trait
transferred from the donor parent. After the initial cross,
individuals possessing the phenotype of the donor parent (red
colouring of the leaves in the heart) are selected and repeatedly
crossed (backcrossed) to the recurrent parent. The resulting plant
is expected to have the attributes of the recurrent parent (e.g.,
cultivar) and the desirable trait transferred from the donor
parent. In case the inheritance of the red colouration is more
complex than the inheritance of the other desired trait, or
combination of traits, the parent with red-coloured heart leaves
can be used as recurrent parent, and the parent with the other
desired trait, or combination of traits, can be used as donor
parent. Descriptions of other breeding methods that are commonly
used for different traits and crops can be found in one of several
reference books (e.g., "Principles of Plant Breeding" John Wiley
and Son, pp. 115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et
al., 1979; Fehr, 1987).
[0049] In greater detail, FIG. 1 shows distribution of within-line
segregation of red colouration of outer and inner leaves for
population of 212 F3-lines obtained from cross
`99P.30637`.times.cv. `Sharp Shooter`. Three classifications of red
colouration were applied on the population: `red outside`, i.e.
within-line frequency of plants with red outer leaves, irrespective
of inner leaf colour; `red outs&red ins`, i.e. within-line
frequency of plants with red outer and red inner leaves, `red
ins/red outs`, i.e. frequency of plants with red inner leaves
within the line-total of plants with red outer leaves. O=observed;
E=expected, according to genetic model.
[0050] FIG. 2 shows the genetic model for anthocyanin expression
based on segregation in cross `99P.30637`.times.cv. `Sharp
Shooter`.
[0051] FIG. 3a shows a plant of the invention. This young plant is
a plant from a seed from the line 03P82421, which was obtained
after three generations of self fertilisation of plant 99P38154.
FIGS. 3b, 3c, and 3d show young plants of parents Pierrot, Pippo,
and Gringo, respectively. FIGS. 3e and 3f show young plants of
comparison varieties Apache and Bijou, respectively. All these
young plants were grown on peat blocks in a growth chamber with 14
h light at 16.degree. C. and 10 h dark at 12.degree. C. Light was
produced by Philips TLD 36 W 840 REFLEX tubes, with 1 tube per 0.24
square m, at 0.6 m distance above the plants.
[0052] FIG. 4 shows a young leaf of a plant of the invention,
indicated by `NEW`, in comparison to young leaves from the
commercial varieties Pippo, Pierrot and Gringo. These young plants
were grown on peat blocks in a growth chamber with 14 h light at
16.degree. C. and 10 h dark at 12.degree. C. Light was produced by
Philips TLD 36 W 840 REFLEX tubes, with 1 tube per 0.24 square m,
at 0.6 m distance above the plants.
[0053] FIGS. 5-8 show comparisons between a plant of the invention
(01P80146) and the commercial varieties Darkland (indicated by
`99R10044`; FIGS. 5a-5d), Pierrot (indicated by `95G2237`; FIGS.
6a-6d), Roxy (indicated by `99R10283`; FIGS. 7a-7d) and Sierra
(indicated by `95G1986`; FIGS. 8a-8d), respectively. The a-figures
(5a, 6a, 7a, 8a) are showing the full harvested heads, The
b-figures (5b, 6b, 7b, 8b) are showing the heart without outer
leaves, The c-figures (5c, 6c, 7c, 8c) are showing a longitudinal
section of the heart. The d-figures (5d, 6d, 7d, 8d) are showing
cut heart leaves. The lettuce plants of the invention are
completely red in the heart of the head whereas the other lettuce
plants are not. All plants are grown in Aramon, France in 2002:
sown on 10 Jan. 2002, transplanted into a confidential open field
on 15 Feb. 2002, harvested on 25 Apr. 2002.
EXAMPLES
Example 1
Pedigree of a Red Lettuce of the Invention
[0054] The lettuce of the invention was obtained according to the
following pedigree: in 1986 a cross was made between a plant of cv.
Pippo (Rijk Zwaan; red-coloured) and a plant of cv. Blonde
Maraichere (Caillard; green-coloured).
[0055] In 1988 a red offspring plant from this cross was used as a
father in a cross with a plant of cv. Gringo (Rijk Zwaan;
red-coloured). In the same year a cross was made between a plant of
cv. Pierrot (Rijk Zwaan; red-coloured) and a plant of cv. Roxette
(Rijk Zwaan; green-coloured).
[0056] In 1989 a red offspring plant from this cross was used as a
father in a cross with a plant of cv. Krizet (Rijk Zwaan;
green-coloured).
[0057] In 1992 a red F3-offspring plant was selected from the
Krizet.times.(Pierrot.times.Roxette)-cross mentioned above and it
was used as a mother in a cross with a father plant, Which was a
selected red offspring plant from the
Gringo.times.(Pippo.times.Blonde Maraichere)-cross mentioned above.
A red F4-plant from this newly obtained 1992-cross was selected in
1995 and used as a mother in a cross with a plant of cv. Roxette
(Rijk Zwaan; green-coloured).
[0058] Selection on type, heading, and colour, which was performed
in the F2-, F3-, and F4-generation from this cross, resulted in a
red F4-plant (98P.31582) in 1998. The F5-line appeared to be
segregating for colour (red vs. green), but selection in the next
generation resulted in three headed F5-plants with completely red
heart leaves (99P.38152; 99P.38154; 99P.30637), which were
multiplied in the years afterwards.
[0059] The offspring showed no segregating green plants, and seeds
were deposited under nrs. 02R.2413, 01R.1439, 02R.2418. These
F5-plants and their offspring have served as a parent source for
further breeding of lettuce with light-independent red
colouration.
[0060] In conclusion, it can be said that none of the used parent
varieties, i.e. Pippo, Blonde Maraichere, Gringo, Pierrot, Roxette,
and Krizet, have the characteristic red leaves in the heart of the
head. The invention comprises the unique and new combination of
genes from these red and green parent varieties, which is providing
the completely red leaves in the heart of the head.
Example 2
Genetic Analysis of the Red Lettuce of the Invention
[0061] As used herein, a locus (plural: loci) is defined as the
specific place on a chromosome where a gene is located. (Griffiths
A J F, Miller J H, Suzuki, D T, Lewontin R C, Gelbart, W M. `An
introduction to genetic analysis.` 6.sup.th edition. 1996. WH
Freeman and Company, New York.) The number of loci responsible for
red colouration in a given plant can be established by a genetic
analysis of the offspring of a cross of this plant with the
green-coloured cv `Sharp Shooter`.
[0062] A genetic analysis of the red lettuce of the invention was
performed as follows: a cross was made between an offspring plant
from F5-plant nr. 99P.30637, i.e. a plant of the present invention,
and a non-brilliant green-coloured iceberg lettuce plant of cv.
Sharp Shooter (S V S, Waycott et. al., 1999: U.S. Pat. No.
5,973,232). From the offspring of this cross 212 random F2-plants
were multiplied into F3-lines.
[0063] These 212 F3-lines were evaluated in a confidential outdoor
trial in Fijnaart, the Netherlands. Seeds were sown on peat blocks
on Jun. 17, 2002, young plants were raised in a glasshouse, and
transplanted in the field on Jul. 5, 2002. The harvest date (time
of observation) was on Aug. 19 until Aug. 23, 2002. The plot size
was 24 plants per F3-line, i.e. 4 rows of 6 plants. Lines were not
replicated, because expression of colour traits is very stable
within one trial. Observations were done per F3-plant.
[0064] Observed traits were: a) colour, and, if red, its intensity
and expression pattern on the outer leaves, b) colour, and, if red,
its intensity and expression pattern on the inner leaves, c) degree
of heading, d) in case of green outer leaves: scoring in 2 classes:
brilliant or non-brilliant.
[0065] The intensity and expression pattern of the red colouration
was scored, in increasing order, as: 1) tinged or blushed, i.e.
light red colouration on the outer and inner leaf edges, or on the
inner leaf base, 2) red spotted, 3) green spotted, i.e. green spots
on red leaf surface, 4) fully red, i.e. intensely red coloured
without spots, on the light-exposed part of the outer leaf parts,
and, in case of inner leaves, throughout the inner leaf.
[0066] Heading was scored between 1) slightly open heading, like
Romaine or cos lettuce, and 2) strong heading with clearly
overlapping leaves, like iceberg lettuce.
[0067] Some plants in the trial died before harvest. Of the
intended 5088 plants, i.e. 212.times.24, in total 5007 F3-plants
reached harvestable stage and were scored (see Table 1).
[0068] All plants showed heading levels, which were at least
comparable to Romaine or cos lettuce. Data showed a segregation of
55 fully green lines out of the total of 212. Out of the 157 lines
that were having at least one plant with red colouration, 28 lines
comprised of plants which were all showing anthocyanin expression
on the outer leaves. Out of these 28 lines, 8 lines comprised of
plants which were all showing anthocyanin expression on the heart
leaves. The red-coloured plants out of 33 of the 157 lines with at
least one red-coloured plant, were showing anthocyanin expression
only in the outer leaves and not in the heart leaves. The
red-coloured plants out of another 33 of the 157 lines were always
showing anthocyanin expression on both the outer leaves as well as
the heart leaves. The red-coloured plants out of the remaining 91
lines all showed anthocyanin expression in the outer leaves.
However, these plants showed a within-line segregation of
anthocyanin expression in the heart leaves. Not a single plant out
of the 5007 plants showed anthocyanin expression in the heart
leaves in combination with green-coloured outer leaves, which had
no anthocyanin expression.
[0069] It is therefore concluded that one or more genes that result
in anthocyanin expression in the outer leaves, are required for
anthocyanin expression in the heart leaves. In the case of
segregation between red and green, segregation ratios between red
and green ranged from 23:1 to 1:22 for colouration of outer leaves.
For inner leaves the segregation ratios between red and green
colouration ranged from 18:1 to 1:23, excluding all plants with
green outer leaves. Table 1 shows the within-line distribution of
plants with red and green colouration of outside and inside leaves
for a population of 212 randomly derived F3-lines from cross
`99P.30637`.times.cv. `Sharp Shooter`.
TABLE-US-00001 TABLE 1 all plants with part of all plants green
outer plants with with red leaves red outer leaves outer leaves all
plants with green 55 29 4 inner leaves part of plants with red 0 75
16 outer leaves have red inner leaves all plants with red outer 0
25 8 leaves have red inner leaves
[0070] These segregation data were used to construct a genetic
model, under the assumption of independent Mendelian segregation
ratios. It was found that at least three loci are involved in
obtaining UV-independent anthocyanin expression in the inner
leaves. The first locus, further indicated as A-a, is also found in
known red lettuce and the dominant allele A is required in all
cases to obtain expression of anthocyanin. Probably this locus is
the C or G-locus (Robinson et al., 1983, The genes of lettuce and
closely related species. In: Plant Breeding Reviews 1. Ed. J.
Janick. p 267-293).
[0071] A second locus is found by fitting the model, further
indicated as B-b, where the homozygous presence of recessive allele
b results in anthocyanin expression of outer leaves in combination
with A. Two more loci, further indicated as C-c and D-d, were found
to be also involved in anthocyanin expression in the outer leaves.
Either presence of at least one copy of the dominant allele C, or
homozygous presence of the recessive allele d results in
anthocyanin expression in the outer leaves, but only in the case
that also at least one copy of allele A is present.
[0072] Three more loci, further indicated as E-e, F-f, and G-g,
were found. To obtain UV-independent anthocyanin expression in the
inner leaves the presence of at least one A-allele, and two
b-alleles is required, in combination with either the presence of
one copy of the dominant E-allele, or the homozygous presence of
the f-allele, or the homozygous presence of the g-allele. So the
presence of alleles for red colouration on at least three loci,
namely A-a, B-b, and either E-e, F-f, or G-g, is required for
UV-independent anthocyanin expression in the inner leaves.
Furthermore, it is assumed that at least three, but probably all
seven loci mentioned above are involved in the intensity of red
colouring of the present invention.
Example 3
Determination of Colour, Anthocyanins and Chlorophylls in
Lettuce
[0073] 1. Sample Preparation and Analyses
[0074] A spectrophotometer UltrospecIII (Pharmacia), with the
following specifications was used: [0075] Monochromator: Czerny
Turner with holographic diffraction grating (1200 lines/mm) [0076]
Wavelength accuracy: .+-.1 nm [0077] Wavelength reproducibility:
.+-.0.5 nm [0078] Detector type: single solid state silicon
photodiode [0079] Bandwidth: 5 nm
[0080] 2. Principle
[0081] The red colour (anthocyanins) and green colour
(chlorophylls) are determined with a biochemical method. Two
extracts are made, one for measuring the absorbance at 523 nm which
is a measure for total anthocyanins and one for measuring the
absorbance at 665 nm which is a measure for total chlorophylls
(chlorophyll a and b).
[0082] 3. Sample Preparation
[0083] The headed and mature lettuce plant is harvested and outer
leaves are taken off, until the heart is left over. The heart of
the lettuce is used for the analysis. The heart leaves should not
have been exposed to direct sunlight before harvest, except for a
small tip of the leaf (maximum 10%) on the top of the plant. A
heart contains at least 10 leaves with a length of 1 cm or longer.
The oldest leaves of the heart should be concave.
[0084] The hearts are put in plastic bags and frozen at
<-70.degree. C. After at least a few days in <-70EC the
frozen hearts are pulverized with a sledgehammer. The fine sample
is grinded in a Grindomix (GM 200, Retsch, 5'' 3000 rpm followed by
5'' 5000 rpm) using the free floating lid in presence of liquid
nitrogen (sample should be kept frozen) to obtain a powder.
[0085] The powder is then placed in a tube and the tube with powder
is chilled in liquid nitrogen and optionally stored at
<-70.degree. C. until analysis.
[0086] 4. Analysis
[0087] Three grams of the powder are weighed into four tubes of 50
ml. Two tubes are used for the analysis of anthocyanins and two
tubes for the analysis of chlorophylls.
[0088] For measurements of anthocyanins 1.0 M HCl in 50% methanol
is immediately added to the two tubes. 5-10 ml/g sample is used
dependent on the colour of the sample and extract. The used volume
(ml) is noted. The sample solution is mixed by hand and put on ice.
A part of the solution is put in a 1.5 ml eppendorf tube and the
tube is centrifuged at 4.degree. C., 13000 rpm for 4 min.
[0089] Spectra are measured with a spectrophotometer with a band
width of 5 nm. A 1 cm cuvette is used.
[0090] The spectrum of 360-900 nm is measured and the absorbance at
523 nm (if necessary after dilution with extraction liquid) and the
maximum wavelength (8.sub.max) are determined. 8.sub.max should be
close to 523 nm.
[0091] Chlorophylls are measured by immediately adding 100%
methanol to the two tubes. 5-10 ml/g sample methanol is used
dependent on the colour of the sample and extract. The volume (ml)
used is noted. The sample solution is mixed by hand and sonificated
in an ultrasonic bath for 5 min at `set degas`. A part of the
solution is put in a 1.5 ml eppendorf tube and centrifuged at
4.degree. C., 13000 rpm for 4 min.
[0092] The spectrum of 360-900 nm is measured and the absorbance at
665 nm (if necessary after dilution with extraction liquid) and the
maximum wavelength (8.sub.max) determined. 8.sub.max should be
close to 665 nm.
[0093] The A523 and A665 are corrected to represent the absorbance
of a solution of 1 g fresh weight in 10 ml extraction volume. The
corrected A523 and A665 are calculated with a correction for
weighted sample (in g), extraction volume (in ml) and if necessary
dilution using the following formula:
[0094] Correction:
A corrected = A measured * extr . volume weight * 10 * dilution
##EQU00001##
[0095] The ratio A523/A665 is calculated.
[0096] Table 2a shows the chlorophyll and anthocyanin absorbance
and anthocyanin/chlorophyll-ratio observed on lettuce in Aramon,
France (sowing 10 Jan. 2002, transplanting 15 Feb. 2002, harvested
25 Apr. 2002). Line 01P.80146 is an offspring line obtained from
plant 99P.38154 by 2 generations of self-fertilisation. Pierrot
(Rijk Zwaan), Darkland (Central Valley), Sierra (Vilmorin), and
Roxy (Enza) are common lettuce varieties.
[0097] Table 2b shows chlorophyll and anthocyanin absorbance and
anthocyanin/chlorophyll-ratio observed on lettuce grown in
Fijnaart, the Netherlands (sowing 21 May 2002, transplanting 10
Jun. 2002, harvested 20 Aug. 2002). Line 01P.80146 is an offspring
line obtained from plant 99P.38154 by 2 generations of
self-fertilisation. Pierrot (Rijk Zwaan), Pippo (Rijk Zwaan), Red
Rosalita (Johnny's Selected & Orsetti), Darkland (Central
Valley), Sierra (Vilmorin), and Roxy (Enza) are common lettuce
varieties.
TABLE-US-00002 TABLE 2a id. nr. (breeding) 01P.80146 Pierrot
Darkland Sierra Roxy Total chlorophyll A665 Mean 0.105 0.396 0.198
0.476 0.228 Stdev 0.028 0.073 0.062 0.038 0.080 Min 0.083 0.321
0.132 0.451 0.155 Max 0.150 0.486 0.279 0.520 0.353 Nr 5 5 4 3 5
Total anthocyanin A523 Mean 1.589 0.883 0.006 0.033 0.087 Stdev
0.195 0.123 0.001 0.006 0.030 Min 1.370 0.725 0.005 0.029 0.063 Max
1.890 1.070 0.006 0.041 0.137 Nr 5 5 4 3 5 Total anthocyanin/ Total
chlorophyll: A523/A665 Mean 16.16 2.27 0.03 0.07 0.38 Stdev 4.88
0.38 0.01 0.01 0.01 Min 9.13 1.82 0.02 0.06 0.36 Max 20.17 2.68
0.04 0.08 0.40 Nr 5 5 4 3 5
TABLE-US-00003 TABLE 2b id. nr. (breeding) Sierra Roxy Darkland
01P.80146 Red Rosalita Pippo Pierrot Total chlorophyll A665 Mean
0.570 0.267 0.343 0.063 0.278 0.456 0.325 Stdev 0.189 0.105 0.101
0.013 0.075 0.244 0.113 Min 0.345 0.180 0.205 0.049 0.205 0.244
0.196 Max 0.836 0.405 0.447 0.082 0.388 0.823 0.505 Nr 5 5 5 5 5 5
5 Total anthocyanin A523 Mean 0.038 0.046 0.010 1.228 0.030 0.693
0.870 Stdev 0.019 0.020 0.004 0.145 0.014 0.437 0.277 Min 0.028
0.027 0.005 1.042 0.017 0.371 0.474 Max 0.072 0.080 0.013 1.422
0.052 1.422 1.231 Nr 5 5 5 5 5 5 5 Total anthocyanin/ Total
chlorophyll: A523/A665 Mean 0.07 0.18 0.03 20.08 0.12 1.53 2.69
Stdev 0.02 0.05 0.01 4.09 0.08 0.46 0.31 Min 0.05 0.11 0.02 15.41
0.06 1.04 2.42 Max 0.09 0.22 0.05 26.18 0.25 2.23 3.10 Nr 5 5 5 5 5
5 5
Deposit Information
[0098] The F5-plants 01R.1439, 02R.2413 and 02R.2418 were deposited
on 18 Jul. 2005 with the NCIMB, Ferguson Building, Craibstone
Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom under the
deposit accession numbers NCIMB 41337, NCIMB 41338 and NCIMB 41339,
respectively.
[0099] Various modifications and variations of the described
products and methods of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in chemistry, biology or related fields
are intended to be within the scope of the following claims.
* * * * *