MX2008008730A - Resistance to physiological disorders in lettuce - Google Patents

Abstract

The invention relates to a method for screening a population of plants for the presence therein of individuals that show a reduced susceptibility to ethylene and physiological disorders, in particular Russet Spotting and Yellowing, as compared to a control plant, wherein a population of seeds is germinated in darkness and in the presence of ethylene to obtain seedlings that, when having a longer hypocotyl as compared to the original ethylene- sensitive control under ethylene are selected as plants showing a reduced susceptibility to ethylene and physiological disorders, in particular Russet Spotting or Yellowing. The invention also relates to the plants thus selected.

Description

RESISTANCE TO PHYSIOLOGICAL DISORDERS IN THE LETTUCE FIELD OF THE INVENTION The invention relates to a method for the systematic identification of a population of plants to detect the presence of individuals that have been altered with respect to their mode of response to ethylene. The invention also relates to plants and parts of plants, in particular those identified as leafy vegetables. More particularly, this invention relates to lettuce (Lactuca sativa L.) which shows a modified response to ethylene which leads to a reduction in the susceptibility of this type of vegetable to physiological disorders such as brown stippling and yellowing . The invention also relates to the seeds and progeny of these plants and their parts.

BACKGROUND OF THE INVENTION The reproduction of varieties of leafy vegetables such as lettuce is intended to produce commercial varieties adapted optimally to the market and the production of salable products. Many characteristics must be taken into account during the selection in relation to the entry and exit features. One of the most important features in this respect refers to post-harvest quality; especially Ref .: 194512 the shelf life. Avoiding physiological disturbances and in particular brown stippling and yellowing are important elements that can lengthen the shelf life of a lettuce or its parts. Ethylene is a plant hormone known to stimulate physiological processes related to aging. In lettuce this stimulation becomes visible through the formation of symptoms such as brown stippling and yellowing. The brown spotting disorder is characterized by the appearance of brown spots along the middle rib of the leaves, while yellowing is a general bleaching of the leaves that occurs as a result of the decomposition of chlorophyll. Although mature lettuce heads are known to produce minimal amounts of ethylene, plants are highly sensitive to this plant hormone. Therefore, physiological disorders associated with ethylene sensitivity that reduce the post-harvest quality of lettuce are mainly caused by external sources of ethylene. Exposure to such external sources may occur during harvesting, processing and storage of the product. For example, when lettuce is transported or stored in the vicinity of fruits that produce ethylene such as apples, pears or peaches, severe deterioration can occur.

In addition, when lettuce is processed and used in fresh packaged mixtures there may be limitations with respect to the ingredients that can be used due to the release of ethylene by one or several ingredients. Brown stippling is a physiological disorder that is manifested by the appearance of numerous brown spots along the central rib of the leaf. Symptoms of brown discoloration can spread throughout the leaf during the progressive stages of the disorder. Brown stippling occurs especially when mature lettuce heads are stored at low temperatures (5 SC) in the presence of low concentrations of ethylene (ppm levels). The formation of symptoms can be counteracted by applying the plant hormone auxin or calcium. In addition, modified atmospheres with low oxygen levels reduce the rate of development of symptoms. At a biochemical level, brown stippling may develop as a consequence of a local stimulation of lignin biosynthesis, which causes lignification and thickening of the cell wall around the area of the leaf where the visual symptoms will appear. The brown discoloration is produced by the stimulation of phenolic metabolism. The enzyme phenylalanine ammonia lyase (PAL) that has been proven to be induced by ethylene, catalyzes the first step of the propanoid phenyl path.

The phenolic compounds that are formed include mainly caffeic acid derivatives as well as a variety of flavonoids such as (+) catechin and (-) epicatechin. The subsequent oxidation of these compounds by polyphenol oxidase (PPO) leads to the typical brown discoloration observed in brown stippling. Finally, symptoms may become more severe due to tissue collapse and cell death. Aging is a process of natural occurrence developed at the end of the life cycle of a plant or a plant organ during which the metabolism is reprogrammed to remobilize resources to reproductive structures such as seeds. Although aging is a developmental process caused by endogenous factors such as physiological age, there are many exogenous factors that can modulate aging. The yellowing of the leaves is the most visible symptom of aging, and is a consequence of the decomposition of chlorophyll during a late stage of aging and that can be enhanced by ethylene once the leaf is receptive. Some other well-known factors that produce aging are bruising, darkness and nutrient deficiency. Although ethylene is the most important plant hormone to stimulate aging, other hormones such as jasmonate can also contribute to this process. From the moment the lettuce is harvested until its consumption, the product can be exposed to various exogenous factors that contribute to its aging. These can be bruising during harvest and processing, darkness and deficiency of nutrients during storage and ethylene during processing and storage. These factors strongly stimulate post-harvest disorders that may become visible in the form of brown stippling and yellowing. Although these effects are basically cosmetic the product becomes much less attractive and therefore not marketable. To counteract the effects of deterioration many post-harvest measures can be taken to reduce these effects. For example, harvested lettuce can be stored at low temperatures to retard aging. Although this may reduce the yellowing speed, brown dotting may be increased. In addition, certain logistical measures can be implemented to reduce the time of transport necessary from the field to the consumer or prevent the storage of lettuce in the vicinity of an ethylene source. In addition, certain chemical treatments can be applied to prevent post-harvest spoilage although the safety of the food and consumer acceptance, in this case, become an important issue.

Many of the post-harvest measures are successful to some degree, but there is certainly room to improve them. In addition, the costs involved can be important, which gives another reason to investigate alternatives that reduce the need to apply post-harvest treatments. Preferably a genetic solution should be found that reduces or eliminates the need to use the costly preventive measures currently used to maintain post-harvest quality at high levels.

BRIEF DESCRIPTION OF THE INVENTION It is the object of the present invention to provide an evaluation method that allows the identification of plants insensitive to ethylene. It is also an object of the invention to provide plants that can be obtained by this method.

This objective is achieved according to the invention by a method of evaluating a population of plants to find the presence in it of individuals showing a lower susceptibility to ethylene • and physiological disorders, in particular brown stippling and yellowing, in comparison to the control plant; the method comprises the following: a) providing a population of seeds b) germinating the seeds in the dark and in the presence of ethylene to obtain seedlings; c) select seedlings that show hypocotyls longer than the hypocotyls of the ethylene-sensitive control; d) choose the selected seedlings to produce seeds; e) germinate a plant of the seeds produced by each seedling selected in darkness and in the presence of ethylene and another part of the seeds of each selected seedling in the dark and air; and f) measure the relative growth of the hypocotyls of the seedlings germinated under ethylene against the growth of the hypocotyls of the plants germinated in air to distinguish those plants that have a longer hypocotyl compared to the ethylene-sensitive control, both in ethylene and in air; of plants that have a longer hypocotyl compared to the original ethylene sensitive control only under ethylene, where plants that have longer hypocotyl in comparison sensitive to ethylene only under ethylene are identified as plants that show a susceptibility to ethylene and physiological disorders , in particular to brown stippling and yellowing. The invention is based on the hypothesis that plants insensitive to ethylene, in particular lettuce, would be resistant to post-harvest physiological disorders such as brown stippling and yellowing. In a preferred embodiment the method of invention includes the additional step of testing the plants that have been identified in the previous phases as plants with a lower susceptibility to ethylene for their resistance to brown stippling and / or yellowing. The length of the hypocotyl and the length of the root can be observed in comparison to the standard and qualified from 1, which means equal to the sensitive ethylene standard under ethylene, up to 3, which means equal to the ethylene sensitive standard under air. If desired, the measurements in millimeters can be used. Using one of these measures, simple statistical analyzes such as a t-test, well known to those skilled in the art, can be used to establish whether a plant or group of plants is significantly less sensitive to ethylene than the ethylene sensitive standard as cv. . "Troubadour" (Rijk Zwaan, De Lier, NL) The applied significance level of a one-sided test is 0.001. For mutants, the statistical comparison is preferably made between the hypocotyl lengths and optionally the root lengths of the original variety that was used for the mutagenesis treatment, which is the best possible standard, and the lengths of the hypocotyl and the root of the individual mutants and / or their offspring.

To find plants insensitive to ethylene in existing plant material, one or more representative samples of the varieties, reproduction lines and / or accesses to a gene bank are selected. The statistical comparison is made between the lengths of the hypocotyl and the root of the individual accesses under investigation and the rest of the population. When statistical tests are performed on individuals to find significantly longer roots and / or hypocotyls, it may be necessary to perform multiple comparison tests to maintain adequate levels of significance, for example, Dunnett's multiple comparison test with a standard (Dunnett, CW). , J. Americ, Statist. Assoc. 50: 1096-1121 (1955)). The plants to be sampled are generally leafy vegetables, in particular, plants belonging to the genus Lactuca, and in particular, to the species Lactuca sa tiva. The population of plants is preferably a population of mutant plants, since the chances of finding a plant of the invention in a variant population are higher. However, any other plant population that differs in its genetic constitution can be sampled according to the invention. The population of mutant plants is preferably obtained by a mutagenesis treatment using chemicals and / or irradiation. Mutagenic treatments are well known and will be described in more detail later. The ethylene concentration in step b) is at least 10 μg / liter, preferably between 11 and 25 μg / liter. The concentration of ethylene in step d) is between 4 and 5 μg / liter. The selection step in the method of systematic identification of the invention is based on the elongation of the hypocotyls. Exposure to ethylene from seedlings that germinate in the dark causes radial swelling of the hypocotyl and inhibition of root and hypocotyl growth. This phenomenon is generally described as the triple response (see for example Guzmán, P and Ecker, J, Plant Cell 2: 513-523 (1990)). The reproducibility of this response allows the selection of mutants that show an altered triple response in the presence or absence of ethylene. In addition to or instead of measuring the elongation of the hypocotyl, the selection according to the invention can be based on one or more of the other elements of the triple response test. According to a further aspect thereof, the invention provides plants that show reduced susceptibility to ethylene and to physiological disorders, in particular brown stippling and yellowing, compared to the control plant; such plants are obtained by subjecting a population of seeds to a method of systematic identification of the invention and selecting plants from the population that show longer hypocotyls compared to the ethylene sensitive control as plants showing a lower sensitivity to ethylene and physiological disorders and in particular brown stippling or yellowing. The plant of the invention is preferably a leafy vegetable, in particular a plant belonging to the genus Lactuca and in particular to the species Lactuca sa tiva. With the method of systematic identification of the invention, the ethylene-insensitive mutants were identified and selected. The seeds of these mutants were deposited at NCIMB Ltd., Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA UK, on January 3, 2007 and possess the access numbers listed in table 1. Details about the offspring of the seeds of the deposits are shown in example 3 and example 4. These deposits are made because they have the unique specific characteristics of having a significantly lower susceptibility to ethylene. They were not tested for DUS criteria for variety records eg distinction, uniformity and stability in all registration characteristics and are not expected to meet these criteria Table 1.

The invention therefore relates to plants exhibiting a lower susceptibility to ethylene and to physiological disorders, in particular, to brown stippling or yellowing and which are obtained by crossing a plant of the invention with another plant of the same species. The trait "reduced susceptibility to ethylene and to physiological disturbances, in particular brown spotting or yellowing" can be induced to other plants that originally do not have it. If the plants resulting from such a cross are in fact plants of the invention can be tested by subjecting them to the evaluation method of the invention. The invention also relates to the progeny of a parent plant of the invention which shows reduced susceptibility to ethylene and to physiological disorders, in particular brown stippling or yellowing. Said progeny may have been separated through several generations of the mother. As long as the trait of "reduced susceptibility to ethylene and to physiological disturbances, particularly brown stippling or yellowing" is present, the plant of the progeny is a plant of the invention. The invention also relates to parts of plants of the invention. Plant parts are, for example, heads of lettuce or leaves, such as tender leaves, processed heads or cut leaves. The plant parts of the invention can be used in tissue cultures to regenerate plants having reduced susceptibility to ethylene and physiological disorders, in particular brown stippling or yellowing such as that found in the plant from which the tissue used for the treatment was obtained. culture. Said regenerated plants are also part of this invention. The invention further relates to the seed of a plant of the invention. From the seeds can be obtained plants that have the trait of "reduced susceptibility to ethylene and physiological disorders, in particular punteado brown or yellowing." The fact that the seeds and plants they produce have that feature or not can be proven by the method of systematic identification of the invention. The invention also relates to the subsequent generation of seeds that maintain reduced susceptibility to ethylene and to physiological disorders, in particular brown stippling or yellowing, such as that found in the original seeds. According to a further aspect, the invention relates to a plant product comprising a plant or a part thereof of the invention. Preferably the vegetable is a leafy vegetable, and in particular the vegetable is a lettuce. When sampled with the method of the invention, the plant product exhibits reduced susceptibility to ethylene and physiological disturbances, in particular brown stippling or yellowing. It should be noted that the evaluation method of the invention is a relatively simple method. The plants of the invention which have a reduced susceptibility to ethylene and to physiological disorders, in particular brown stippling or yellowing can be identified in any variant population that is sufficiently large. Excessive experimentation is not required to reproduce the invention and the plants of the invention should not be construed as limited to those that are deposited.

As a result of its insensitivity to ethylene, the plant material of lettuce and the seed thereof derived from the invention show a strong increase with respect to its post-harvest quality, especially in relation to the phenomena associated with aging, such as brown stippling. and yellowing.

BRIEF DESCRIPTION OF THE FIGURE Figures 1A-1B: The phenotype of mature heads of lettuce after exposure to ethylene at 8 ° C in the dark for 9 days. The left panel shows a representative sample of ethylene resistance (Ethylene R) and ethylene-sensitive (control) control heads of the Troubadour variety. The panel on the right shows a similar image of the mutants derived from the Apache variety.

DETAILED DESCRIPTION OF THE INVENTION Since ethylene is the most important plant hormone to stimulate aging and since brown stippling and yellowing are associated with aging, a genetic approach was taken in which genetic variants were produced and selected for ethylene insensitivity. It was found that this indirectly leads to the identification of mutants that are affected in their post-harvest aging responses.

Detailed studies using plant model species such as Arabidopsis thaliana as well as crop species provide information on the biochemical trajectories involved in the biosynthesis and perception of ethylene. Many allelic forms of genes have been characterized for their role with respect to providing an intricate image of the function of ethylene in plants. Ethylene, like other plant hormones, plays an important role in many physiological processes placed in complicated interactive regulatory networks. The spatial and temporal activity of the hormone is determined inter alia at the level of ethylene biosynthesis fundamental gene expression, hormone perception, signal transduction and the activation of effector proteins in the 3 'direction. Allelic variants of genes involved in different levels can determine the strength of the response as well as the level of interference to other signaling paths. Since ethylene biosynthesis and perception are poorly characterized in lettuce and in order to allow for the identification of different response forces and their fundamental allelic variants, it was reasoned that an unbiased approach can be more successful in this regard compared to a of target gene modification. 7 Such unbiased approach preferably encompasses a random chemical or physical mutagenesis procedure combined with an efficient phenotypic systematic selection and identification procedure based on a response of discolored seedlings to ethylene. Such a seedling-based selection system is far more efficient in terms of the number of plants that can be evaluated per man-hour when compared to the use of mature lettuce heads. Additionally, the time to produce the plant material for systematic identification is obviously much reduced in the case of seedlings when compared to mature heads. An additional advantage of this approach is the use of selection conditions in the seedling stage as a productive phenotypic marker for the post-harvest aspect in consecutive generations once a successful event has been identified. There is a clear risk in the fact that selection in the seedling stage may not lead to genetic variants expressed by the selected trial at the post-harvest, mature level. Additionally, it is recognized that ethylene plant-type hormones are involved in many physiological processes that can lead to pleiotropic effects. These can be either positive or negative depending on the crop and its growing conditions. The approach taken therefore preferably comprises the following steps: 1. Generation of a mutant population by treatment of seeds or plant tissues with mutagenic agents such as ethane methylsulfonate (ems) or gamma radiation. 2. The placement of an efficient phenotypic systematic identification in which the selection is based on a response of discolored seedlings of plants, in particular lettuce, to ethylene characterized by a strong reduction of the hypocotyl elongate, a thickened, shortened root and an exaggerated apical hook curvature. This response is called the triple response that is typical for discolored seedlings and is found to occur in several grades in many plant species when exposed to ethylene-containing atmospheres (see for example Ecker J, Science 268 (5211): 667-675 (1995)). 3. Characterization of the modified mutants in at least one test of their triple response with respect to postharvest deterioration comprising brown stippling and yellowing. Optionally, the determination of pleiotropic effects of the modification to exclude negative pleiotropic effects that would affect the value of the plants. In the method of systematic identification of the invention the seed population of the plant to be tested is thus preferably a population of mutant seeds obtainable by a method comprising: a) treating seed MO of a plant species to be modified with a mutagenic agent to obtain seeds Mi; b) growing the plants of the Mi seeds thus obtained to obtain Mi plants; c) produce M2 seeds by self-fertilization of the Mi plant; and d) optionally repeating step b) and e) n times to obtain seeds Ml + n. The seeds Ml + n thus obtained are then germinated in the dark and in the presence of a high concentration of ethylene to obtain seedlings. Subsequently, the seedlings are selected so that they do not show a response to ethylene. The response to be measured is the development of hypocotyls that elongate in comparison with the hypocotyls of a seedling sensitive to ethylene. Then, the progeny of each selected plant is separated and grown in the dark: half of the progeny under ethylene, half of the progeny under air. The relative growth of the hypocotyl under ethylene against air is measured for each progeny under both conditions. These observations are used to distinguish progenies that have a longer hypocotyl than the control sensitive to the original ethylene under ethylene, but also under air, and which can therefore be concluded to be ethylene sensitive and unwanted, of the progenies that certainly they have a reduced susceptibility to ethylene. An example of a well-known mutagen is ems. G residues primarily alkylated ems from a strand of DNA that during DNA replication causes pairing with T instead of C. Therefore, GC base pairs change to AT base pairs at a frequency that is determined by the effective dose of ems and the activity of the plant error repair system. The effective dose of ems depends on the concentration used, the size of seed and other physical properties and the incubation time of the seeds in the ems solution. Seeds that have been treated with ems are typically called Mi seeds. As a consequence of the treatment, the tissues of the Mi seeds contain random point mutations in the genomes of their cells and those present in the subpopulation of cells that are formed from the germline tissue (germ cells) are transferred to the next generation that is call M2. Mutations or combinations thereof that are insufficient in haplo thereby causing sterility or inducing the mortality of the embryo are not transferred to the M2 generation. A similar procedure as described above for the use of ems applies to other mutagenic agents. The M2 population can be used in systematic identification procedures directed at a reduced triple response of discolored seedlings. Other mutagenic agents, in particular mutagenic alkylating agents, are diethyl sulfate (des), ethylene imine (ei), propane sultone, N-methyl-N-nitrosourethane (mnu), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea (enu), sodium azide. Alternatively, the mutatiare induced by means of radiation, which is selected, for example, from X-rays, fast neutr UV radiation. In another embodiment of the invention, the mutatiare induced by genetic engineering, such as by means of using chimeric oligonucleotides, homologous recombination, introduction of modified target genes that compete with the endogenous product, down-regulation through RNA interference, etc. The technology that allows modifying target genes residing in the genome of a plant in a specific manner is known to the person skilled in the art. For example, chimeric oligonucleotides have been shown to be effective mutagenes with a specific mode of action. Another approach is to modify the gene's targets through homologous recombination or gene targeting. Using this approach, a fragment of a gene is exchanged for an introduced DNA fragment containing a desired modification. Transgenic approaches are also possible in which modified target genes are introduced that compete with the endogenous product. This can lead to dominant negative effects. On the other hand, the specific down-regulation of gene expression is possible through the interference of RN. In the case of mutagenic oligonucleotides, gene targeting or transgenic approaches are used to modify a genetic factor involved in the ethylene function, obviously, the primary structure of the relevant genes should be known. However, currently for lettuce the knowledge in such genes is limited. Preferably, the invention further comprises pyramidal alleles of reduced susceptibility to ethylene. The production of seeds Mi and Ml + n is carried out appropriately by means of self-pollination. The invention also relates to plants or plant parts, which have genetic information in their genome that is respble for the reduced susceptibility to physiological disorders such as brown stippling and yellowing and is found in the genome of a lettuce plant as listed in Table 1. The progeny of the plants as claimed are also part of this invention. The "progeny" as used herein is intended to encompass all plants that have the same or a similar reduction in susceptibility to ethylene and physiological disorders, in particular brown stippling or yellowing, as the original plants described herein and it releases it in any way, such as by sexual reproduction, self-fertilization type or cross-fertilization with another plant of the same genus, or vegetative reproduction such as cutting, tissue culture, haploid culture, protoplast culture, protoplast fusion or other techniques Such a progeny is thus the first generation of the plants as identified according to the invention, as well as the first generation of the plants derived by one or more of these techniques, but also each additional generation of the plants derived by one or more of these techniques, providing that the derived plants have reduced susceptibility. In order to determine the resp of discolored lettuce seedlings to ethylene, the use is made of a specially designated plastic container in which the lettuce seedlings were grown on filter paper under an atmosphere in which the ethylene levels they may vary. This is indeed the fact that the lettuce seedling responds to the presence of ethylene by a reduced extension of the hypocotyl in which, in principle, it should be possible to select for the ethylene-insensitive mutants in case of such mutant residues in the available population and in the case insensitivity is expressed phenotypically at the level of the seedling under the experimental conditiin which it is applied. By the growth of a large number of discolored lettuce seedlings from a population that contains randomly induced mutatiunder an atmosphere containing ethylene, it was found that seedlings showing reduced ethylene sensitivity when compared to the ethylene sensitivity of the starting population can be obtained and selected. The selected seedlings show hypocotyl elongation to a degree comparable to a situation in which the atmosphere is composed of air without ethylene. The seedlings identified in this way are qualified as ethylene insensitive. In order to confirm that the ethylene-insensitive mutants are resistant to brown stippling and yellowing, the mutants identified in the systematic identification are tested for resistance to brown stippling and yellowing. A brown spot test adequately comprises storing mature heads harvested in a closed vessel at a temperature of 8 ° C in the dark, and exposing them to ethylene gas at a concentration between 6 and 7 vpm (volume in parts per million). ) and is tested in the presence of the symptoms of brown stippling after 7 days, 5 preferably after 9 days. Suitably a control head sensitive to ethylene is incubated together with the plants to be tested and the brown stippling of the plants to be tested is compared to it. A yellowing test comprises storage of mature heads of the lettuce plants to be tested in an ethylene-free storage chamber at 8 ° C and the yellowing of the base sheets is evaluated after 10 days, preferably after 14 days. Plants resistant to yellowing are plants that do not show yellowing of the base leaves after 14 days. Suitably an ethylene sensitive control head is incubated together with the plants to be tested and the response to yellowing of the plants to be treated is compared independently. As illustrated in the examples, it has been shown for the first time that ethylene insensitivity leads to brown dot resistance and resistance to yellowing that is not induced by ethylene. Surprisingly, the seedlings which show an elongation response which is reduced when compared to the control conditions but which elongate when compared to the sensitive controls, are also found which are considered insensitive to ethylene as well as although partial .

The varying levels of ethylene insensitivity that are observed may reflect either the presence of different mutant sites or different allelic forms of identical sites that affect this trait in the original population. In the case of the recessive mutation, these two possibilities can easily be distinguished by carrying out the allelism assays which comprise the crossing of the two mutant events and determine the phenotype of the hybrid. In the case of allelism of the mutations, the insensitivity to ethylene should be apparent in the Fl while in the case of the phenotype in the mutants is determined by different regressive places these should not be the case. As random mutagenesis was applied as a preferred means to generate the starting population, mutations in the genetic structure may also contribute to the variation of the seedling phenotype under the experimental conditions. In order to discriminate between the simple mutations of the different forces and a combination effect of the mutation in the genetic structure, the crossing should be carried out to create uniform genetic backups for the different events insensitive to ethylene. Such procedures are also relevant in order to determine whether mutations in specific sites that are involved in ethylene sensitivity display pleiotropic effects. The M2 plants thus selected at the base of a reduced response to ethylene were used to grow the M3 seeds. In some cases the M3 plants that were grown from the M3 seeds were selected to reduce the sensitivity of ethylene in a triple response test and which is pollinated to produce the M4 seeds. This was repeated even up to M5 in some cases. In some cases a growth of the M3 plant of the M3 seed is selected to reduce the sensitivity of ethylene in a triple and cross-response test with an ethylene sensitivity and the sensitive precursor of brown stippling to obtain the seed Fl. A growth of the plant Fl of the seed Fl undergoes pollination to produce the seed F2. A growth of the F2 plant of these seeds F2 is selected to reduce the sensitivity of ethylene in a triple response test and is pollinated to produce the F3 seed. This F3 line was added for the set of M3 and M4 of the innate lines subsequently, the innate descending lines of the insensible ethylene events are re-evaluated for their responses to ethylene. The level of insensibility of each innate line was recorded at the base of the relative growth of the seedling under an atmosphere containing ethylene against air. The basis in these criteria lines 12/54 which register the ethylene insensitivity previously was now classified as sensitive. These false positives during the initial selection at the M2 level, can easily be eliminated during the re-evaluation of events in the next generation. Cross lines that showed significant ethylene insensitivity and were confirmed in the ethylene test were re-harvested and grown in a greenhouse under the conditions of regular lettuce production to produce mature heads that were evaluated by brown stippling and yellowing ( see also the examples). As negative controls, ethylene-sensitive plants were grown which originate from the population which is used to select events insensitive to ethylene. Surprisingly, the seeds of all the ethylene-insensitive mutants germinated normally, that is, comparable to the seeds of an almost isogenic ethylene-sensitive control plant when planted in the soil of the pot. This clearly contrasts the situation in other plant species such as Arabidopsis thaliana which shows a strong reduction in germination capacity when planted in the soil of the pot (Harpham, N.J. V. et al (1991) Annals of Botany 68, 55-61). Apparently for lettuce, it is possible to grow the ethylene-insensitive mutants according to a normal cultivation practice in which many cases include sowing in soil blocks of the pot or soil plugs in the pot. After cultivation, the mature heads were harvested and exposed to ethylene. One week after incubation after harvesting the heads at 8 ° C under an atmosphere containing ethylene resulted in a strong induction of brown stippling of the heads of the ethylene-sensitive control plants. However, 29 out of 37 of the ethylene-insensitive events that were evaluated show no signs of brown stippling at all, which surprisingly demonstrates that ethylene resistance which is selected for the seedling level can reduce physiological disturbances in the level of the mature plant, even in the stage after the harvest. The resistance to yellowing could be demonstrated by a number of events insensitive to ethylene under ethylene-free storage conditions at sub-optimal temperature. This is surprising, because until now yellowing resistance has only been reported in the presence of ethylene (Saltveit et al., Postharvest Biology and Technology 27: 277-283 (2003)). The present invention will be illustrated in the examples that follow and are not intended to limit the invention in any way. More particularly, the experiments in the examples are carried out with the lettuce but the invention is more widely applicable to other plant species that encounter similar difficulties after harvesting when connected with ethylene.

EXAMPLES EXAMPLE 1 Genetic modification of lettuce by ethyl methane sulfonate (ems) The seeds of the Troubadour, Apache and Yorvik lettuce varieties (all three from Rijk Zwaan, De Lier, the Netherlands) were treated with ems by immersion of approximately 2000 seeds per variety in an aerated solution of either 0.05% (w / v) or 0.07% (w / v) ems for 24 hours at room temperature. Approximately 1500 seeds treated by one variety per dose ems are germinated and the resulting plants were grown in a greenhouse in the Netherlands from May to September to produce the seeds. After maturation, the M2 seeds were harvested and increased in size in one group by one variety per treatment. The 6 groups resulting from the M2 seeds were used as starting material to identify the individual M2 plants containing the susceptibly reduced alleles. The effectiveness of the genetic modification procedure was evaluated by determining the presence of discolored plants, which are indicative for the loss of chlorophyll due to modifications in genes directly or indirectly involved in the formation or accumulation of chlorophyll. In total, 6 groups of the individual plants of the M2 seeds, which are bleached, are observed which demonstrate that the treatments applied result in genetic modifications.

EXAMPLE 2 Identification of lettuce plants which have reduced susceptibility obtained from alleles for ethylene M2 lettuce seeds were germinated on paper in a small plastic container with an ethylene concentration of 10-20 vpm (parts by volume) per million) at 16 ° C in the dark. 1 vpm contains 0.41 μmol / liter or 1.14 μg / liter. Ethylene-insensitive mutants were compared to ethylene-sensitive controls, and selected at the base of hypocotyl and / or elongate root (ie triple response test). These ethylene insensitive mutants were cultured to produce M3 lines by self-fertilization. These M3 lines were retested with the triple response test to confirm insensitivity 2 of ethylene. When a line was segregated during ethylene insensitivity, plants were selected followed by one or two additional inbreeding cycles, and a final triple response test to select homozygous ethylene-insensitive lines, if possible. In one case an M3 plant growing from the M3 seed was selected for reduced sensitivity of ethylene in a triple response test, and crossed with a sensitive precursor of brown stippling and sensitive to ethylene, Troubadour to obtain seed Fl. A plant Fl that grows from the seed Fl was purified to produce F2 seeds. An F2 plant growing from these F2 seeds was selected for reduced sensitivity to ethylene in a triple response test, and purified to produce F3 seeds. The resulting line F3 was added to the set of inbred lines 53 M3, M, and M5. In this case, the F3 line was only the representative of the original M2 mutant plant, because it had not left its own seeds. This set of lines 54 M3 and M4 of Example 1 were germinated in peat blocks in a closed vessel under ethylene concentration between 4 and 4.5 vpm in the dark. Mutants insensitive to ethylene were identified by their larger hypocotyls compared to sensitive control varieties (Troubadour, Apache, Yorvik, Sensa?). The results are presented in Table 1. 42 of 54 lines that were identified by the triple response test appear to be at least partially insensitive to ethylene in the ethylene test. These lines represent 40 M2 plants, because 2 M2 plants were represented twice.

Table 2 shows the results. Table 2 The ethylene response of mutant lines in a closed vessel test and a triple response test (trt). Observations in hypocotyl and root are separately indicated, when the non-consistent response is shown. The broker response is indicated as partial. R = insensitive to ethylene; S = sensitive to ethylene; R / S = segregation; T = Troubadour; A = Apache; Y = Yorvik; hypo = hypocotyl seed plot no. original etilepo 1 YORVIK S 2 TROUBADOUR S 3 APACHE S 4 00D 88531 Y S 5 00D 88539 Y S 6 01 D 85717 Y S 7 01 D 85720 Y S 8 01D.85732 Y S 9 00D 88533 Y S 10 00D 88538 Y S 11 03D 7400B Y R 12 02O 90749 Y S EXAMPLE 3 Identification of lettuce plants having reduced susceptibility obtained from alleles for brown dotting The thirty-seven ethylene-insensitive lines of the forty-two lines found in Example 2 were planted in a greenhouse to produce mature heads under production conditions. regular lettuce (location: Maasdijk, the Netherlands, seeded: January 10, transplanted: February 17, harvested: April 18). Harvested mature heads were stored in a closed container at a temperature of 8 ° C in the dark. These were exposed to ethylene gas at a concentration of between 6 and 7 vpm (parts by volume per million). 1 vpm contains 0.41 μmol / liter or 1.14 μg / liter. After 9 days, the plants were identified showing brown stippling. All the control plants except Yorvik showed brown dotting. The 29 of 37 test lines show absence of brown stippling or less brown stippling compared to the original variety (Table 3). Six lines were chosen for multiplication and deposition at NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom. The first line is numbered 02D.91445. It is an M4 line descending from the ethylene insensitive M2 plant Troubadour-M2 OOD.7856. An M4 plant insensitive to ethylene was selected from 02D.91445 in the closed test vessel in Example 2 and purified to produce M5 seeds. Sixteen M5 plants were grown from these seeds to produce a batch of M6 seed by purification. This seed lot is numbered 07D.826509 and is deposited as NCIMB- number 41449 (first line). The second line is number 02D.90047. It is an M4 line descended from the M2 Troubadour plant insensitive to ethylene OOD.6876. An M4 plant insensitive to ethylene was selected from 02D.90047 in the closed test vessel in Example 2 and purified to produce M5 seeds. Sixteen M5 plants were grown from these seeds to produce a batch of M6 seed by purification. This seed lot is numbered 07D.826514 and is deposited as NCIMB number 41450 (second line). The absence of brown dotting for this origin was established by testing line M5 03D.90323, descending from an M4 plant of 02D.90047. The third line is numbered 00D.88578. It is an M3 line descending from the M2 plant of Troubadour insensitive to ethylene 00D.7871. Sixteen M3 plants were grown from OOD.88578 to produce a batch of M4 seed by purification. This seed lot is numbered 07D.826502 and is deposited as NCIMB number 41448 (third line). The fourth line is numbered 03D.90452. It is an M5 line descending from the M2 plant Apache insensitive to ethylene OOD.6883. An M5 plant insensitive to ethylene was selected from 03D.90542 in the closed test vessel in Example 2 and purified to produce M6 seeds. Sixteen M6 plants were grown from these seeds to produce a batch of M7 seed by purification. This seed lot is numbered 07D.826522 and is deposited as NCIMB number 41451 (fourth line). The fifth line is numbered OID.85780. It is an M3 line descended from the M2 Apache plant insensitive to ethylene OOD.6896. An M3 plant insensitive to ethylene was selected from OID.85780 in the closed test vessel in Example 2 and purified to produce M4 seeds. 16 M4 plants were grown from these seeds to produce a batch of M5 seed by purification. This seed lot is numbered 07D.826540 and is deposited as NCIMB number 41452 (fifth line). The sixth line is numbered 04D.801660. It is an F3 line descended from a cross between the M3 Yorvik plant insensitive to ethylene 02D.8484 and a plant of the Troubadour variety sensitive to ethylene. The plant M3 02D.8484 descending from the M2 Yorvik plant insensitive to ethylene OOD.7845. An ethylene insensitive F3 plant was selected from 04D.801660 in the closed test vessel in Example 2 and purified to produce F4 seeds. 16 F4 plants were grown from these seeds to produce a batch of F5 seeds by purification. This seed lot is numbered 07D.826542 and deposited as NCIMB number 41453 (sixth line). Figures 1A-1B show resistance and control of plants.

EXAMPLE 4 Identification of lettuce plants showing less yellowing after harvest The growth of ethylene-insensitive lines under greenhouse conditions as described during Example 3 were harvested and the mature cut heads were stored in an ethylene-free storage chamber. at 8 ° C. After two weeks, the base leaves of the green control variety Yorvik and Troubadour begin to turn yellow. At that time and even one week 1 after three lines it appears to have fewer yellow base leaves than its home varieties, in which controls were used in this trial. These lines were numbered 04D.800900, 03D.90323, 04D.801660. Although a relationship is reported between leaf yellowing and the presence of ethylene (Saltveit et al (2003) Postharvest Biology and Technology 27: 277 283), it is surprising that even under conditions without ethylene, some of the lines insensitive to ethylene express a lower yellowing phenotype.

Table 3 Observations of brown stippling on mature heads after 9 days of storage under ethylene conditions 0 = absence of symptoms; l = weak symptoms; 2 = strong symptoms; NA = not available seed no. Ethylene trt dotted brown seed no. Ethylene trt dotted p 01D.85762 R partial hypo R; 2 OOD 88565 R R (root S 03D.90452 R R 0 02D.91445 R R (0.5 01D.85764 R / S hypo R; 1 00D.88577 R / S hypo R; (0 Partial root S ralz S 02D 90070 R R 0 02D.91446 R R (0 03O 90457 R R 0 02D 91447 R R (0 01D.85768 R R 0 01D.85754 S? R / S 0 02D.90128 R R 2 01D.85756 R R I 0 02D.90129 R R 2 02D.90047 R R I NA 02D 90130 R R 2 01 D 85758 R R (0 03D 90462 R R 2 02D.91442 R R I 0 Apache S S 2 Troubadour S S 1 03D 90464 R R 0 OOD 88569 R R 0 02D 90133 R R 2 O0D.88573 R R I 0 02D.90134 R R 2 O0D.88578 R R OOD.88600 R / S R NA 00D.88582 R R 02D 91479 R R 0 01D.85748 R / S R / S 0 1 01 D 85772 R hiccup S; 0 Rlz R 04D 800957 R hiccup S; 0 02D.90036 R partial hypo S; NA root R root R 02D 90091 R R 0 01D.85755 R / S R / S NA 04 D 800960 R hipo S; 0 04D.800900 R hipo S; 0.5 root R root R 04D 800963 R R 2 03D.90323 R R 0 01 D 85780 RR 0 4D 801660 R partial hypo R / S 02 ralz S Apache Yorvtt It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (23)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for systematically identifying a population of plants for the presence therein of induals showing reduced susceptibility to ethylene and physiological disorders, in particular brown stippling and yellowing, compared to a control plant, characterized in that it comprises: a) providing a population of seeds; b) germinate seeds in the dark and in the presence of ethylene to obtain seedlings; c) select seedlings that show hypocotyls longer than the hypocotyls of an ethylene-sensitive control; d) subject the selected seedlings to pollination to produce seeds; e) germinate a part of the seeds produced from each selected seedling in the dark and in the presence of ethylene and another part of the seeds of each selected seedling in the dark in air; and f) measure the relative growth of hypocotyls of the seedlings germinated under ethylene against the growth of the hypocotyls of germinated seedlings in air to distinguish plants that have a longer hypocotyl compared to the control sensitive to the original ethylene both in ethylene and in air of plants having a longer hypocotyl compared to the original ethylene sensitive control only under ethylene, wherein the plants having a longer hypocotyl compared to the original ethylene sensitive control only under ethylene are identified as plants showing a reduced susceptibility to ethylene and physiological disorders, in particular brown stippling or yellowing.

2. The method according to claim 1, characterized in that it further comprises the step: g) testing the plants identified as showing reduced susceptibility to ethylene for their resistance to brown stippling and / or yellowing.

3. The method according to claim 1 or 2, characterized in that the plants are leafy vegetable plants.

4. The method according to claim 3, characterized in that the plants belong to the genus Lactuca and in particular to the species Lactuca sa tiva.

5. The method according to any of claims 1-4, characterized in that the population of plants is a population of mutant plants, a collection of germ plasm or a population of transgenic plants.

6. The method according to claim 5, characterized in that the population of mutant plants is obtained by a mutagenesis treatment using chemicals and / or radiation.

The method according to any of claims 1-6, characterized in that the concentration of ethylene in step b) is at least 10 μg / liter, preferably between 11 and 25 μg / liter.

8. The method according to any of claims 1-6, characterized in that the concentration of ethylene in step d) is about 4 to 5 μg / liter.

9. A plant showing a reduced susceptibility to ethylene and physiological disorders, in particular brown stippling and yellowing, compared to a control plant, characterized in that the plant is obtained by subjecting a population of plant seeds to a method of systematic identification of conformity with any of claims 1-8 and plants selected from the population showing longer hypocotyls compared to an ethylene sensitive control such as plants showing reduced susceptibility to ethylene and physiological disorders, in particular brown stippling or yellowing.

10. The plant according to claim 9, characterized in that it is a plant of vegetables with leaves.

11. The plant according to claim 10, characterized in that it belongs to the genus Lactuca and in particular to the species Lactuca sativa.

12. The plant according to any of claims 8-10, characterized in that the seed of the plant is deposited with the NCIMB on January 3, 2007 and has been given the access number as listed in Table 1.

13. The plant according to any of claims 9-11 , characterized in that the plant is obtained by crossing a plant according to claim 12 with another plant of the same species.

14. The progeny of a precursor plant according to any of claims 9-13, characterized in that it has reduced susceptibility to ethylene and physiological disorders, in particular brown spotting or yellowing.

15. Part of the plant according to any of claims 9-14.

16. Part according to claim 15, characterized in that it is selected from leaves, heads, cut leaves, processed heads, tender leaves.

17. The regenerated plant of a part of the plant according to claim 15 or 16, characterized in that it has reduced susceptibility to ethylene and physiological disorders, in particular brown stippling or yellowing as found in the precursor.

18. A seed of the plant according to any of claims 9-14 and 17.

19. The progeny of the seed according to claim 18, characterized in that it shows reduced susceptibility to ethylene and physiological disorders, in particular brown stippling or yellowing as it is in the forerunner.

20. The plant product, characterized in that it comprises the plant or a part thereof according to any of claims 9-19.

21. The vegetable product according to claim 20, characterized in that the vegetable is a leafy vegetable.

22. The vegetable product according to claim 21, characterized in that the vegetable is lettuce.

23. The plant product according to any of claims 19-22, characterized in that the plant product when systematically identified in the method according to any of claims 1-7 shows reduced susceptibility to ethylene and physiological disorders, in particular dotted brown or yellowing.