Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-07-21
2001-12-25
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S295000, C800S298000
Reexamination Certificate
active
06333450
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to plants and plant parts exhibiting inhibited or delayed fruit ripening or tissue senescence. In particular, the invention relates to edible Brassica plants which display a delayed senescence, at least at room temperature.
2. Description of the Related Art
A problem faced by growers of plants destined for consumption is that harvesting of plants of a marketable age generally has to be performed over a time course of from about one to four weeks. If the time course for harvesting is too long, the edible portions of plants start visibly to deteriorate on the plant and become of limited commercial interest to the grower and/or retail outlet supplier. Retail outlets such as super-markets and the like try to extend the shelf life of plant parts by utilizing measures such as cooled shelving, water sprays, the use of plastic foil wrappings and gas manipulation. Although these work to a degree, there is still wastage of plant material, and hence loss of sales. Edible portions include those portions which are typically sold in the fresh produce shelves of super markets and the like or are sold to the canning and/or pickling industries.
In climacteric plants, including many members of the Brassica family, there is a rise in the rate of respiration and an associated production of ethylene at the onset of fruit ripening and the senescence of green tissues. In non-climacteric plants these changes are not observed. Ethylene is believed to trigger the ripening of fruits and senescence in leaves of climacteric plants and it has previously been shown that the exogenous addition of ethylene to such plants promotes these responses.
Cytokinins have a pronounced effect on the delay of senescence in fruits and other plant parts of climacteric plants. Cytokinins also play a role in cell division, the formation of lateral shoots, sprouts, flowers and fruits. Cytokinins are believed to be actively involved in the senescence process of plants when in the active free base and riboside configurations. Conversion of the active configuration of cytokinin into an inactive configuration in Brassica plants involves the addition of a sugar or amino acid moiety to the N
7.9
-of the adenine ring of the cytokinin.
SUMMARY
The present invention provides inter alia, edible Brassica plants or edible parts thereof, exhibiting a modified ripening or senescence metabolism, the edible parts displaying substantially non-climacteric behavior.
According to the present invention there is provided a method of obtaining plants which exhibit delayed or inhibited fruit ripening and/or vegetable tissue senescence comprising:
(i) growing seedlings in the presence of an auxin polar transport inhibitor and selecting those seedlings the hypocotyledon of which grows at an acute angle of less than about 70° to the horizontal; or—
(ii) contacting the seeds of plants which have a low or reduced auxin polar transport with a conjugation inhibitory amount of an inhibitor of cytokinin-N
7.9
glycosyl conjugation and selecting from the thus contacted seeds those which germinate.
A preferred embodiment of the method comprises performing steps (i) and/or (ii) indicated above to obtain selected plant products, selfing the thus obtained products and selecting by use of the said steps those that exhibit the traits of delayed or inhibited fruit ripening and/or vegetable tissue senescence, thereby to obtain a substantially homogeneous line with respect to these traits, and optionally crossing individuals of the said line, or the progeny thereof, with plants optionally exhibiting the said traits. It is particularly preferred that the said selfing and selection steps are repeated at least five times in order to obtain the homogeneous line.
It is preferred that the angle which the growing hypocotyledon makes with the horizontal is less than about 50°, still more preferred that the angle is less than about 25° and still more preferred that the angle is less than about 10°. Those plants obtained by the method of the invention which have the longest shelf life or delayed or inhibited senescence characteristics have hypocotyledons which grow substantially horizontally when seedlings thereof are subjected to inhibitors of polar auxin transport, even at very low concentrations, such as less than or equal to about 20 &mgr;M. Indeed a typical concentration of transport inhibitor used to select plants which have these desirable delayed senescence characteristics is about 1 to 10 &mgr;M, with 5 &mgr;M being substantially ideal for the selection of Brassicas, for example. It is particularly preferred that the transport inhibitor is chosen so that its effect on the seedling is substantially similar to that provided by about 5 to 10 &mgr;M HFCA (see below).
By “plants which have a low or reduced auxin polar transport” is meant plants in which the transport of auxin from its site of synthesis in the apical regions of the meristem to its site of utility in the known auxin sinks (such as leaves, for example) is at least 30% lower, more preferably at least 50% lower, still more preferably at least 80% lower, and still more preferably at least 90% lower than that in like plants which do not have a low or reduced auxin polar transport. One skilled in the art is aware of how auxin polar transport may be measured. One obvious way (as exemplified in Okada et al (1991) The Plant Cell Vol 3, pp. 677-694) to assess the auxin polar transport capacity of a plant is to add radiolabelled indole 3 acetic acid to one end of a cut inflorescence axis of plants suspected of having a low auxin transport and control plants known not to be deficient in this respect. Cut axes from the control plants are able to transport the radiolabelled auxin from one end to the other through the infloresence tissues in an inverted orientation, i.e. in a polar manner against gravity from the apical side to the basal side of the cut inflorescence. When incubated in a non-inverted orientation the cut inflorescence axis accumulates the radioactive auxin at the basal end of the tissues. Plants which have a low or reduced auxin polar transport are thus those which are substantially incapable of transporting auxin against gravity from the apical side to the basal side of, for example, a cut infloresecence.
“Delayed or inhibited fruit ripening and/or vegetable tissue senescence” provides for long shelf life, and these terms are more or less interchangeably used within the context of the present application. Delayed or inhibited fruit ripening or senescence (viz extended shelf life) can be determined in a manner known per se, for example, by visual comparison of plant parts after storage under standard conditions (time, temperature, humidity, sun-light etc.) with corresponding parts of like plants which do not constitute part of this invention (see below), or by determination of metabolic parameters of test plant parts with like parameters obtained from analysis of known plants of the same species. Examples of metabolic parameters suitable for indicating extended shelf life, include insensitivity to ethylene by plant parts which ripen or senescence.
By “polar transport inhibitor” is meant any compound or chemical which reduces or substantially prevents the transport of auxins from their site of synthesis in the apical meristem to their site of utility in the sink regions of the plant. The term thus includes compounds which bind auxins and as a consequence thereof prevent or otherwise inhibit auxin transport, as well as compounds which interfere with components of the auxin transport pathway. Known transport inhibitors include, amongst others, 9-hydroxyfluorene-9-carboxylic acid (HFCA), N-(1-naphthyl)phthalamic acid (NPA), silver thiosulfate (STS) and 2,3,5-tri-iodobenzoic acid (TIBA).
By “inhibitor of cytokinin-N
7.9
-glycosyl conjugation” is meant any compound or other agent which prevents or reduces conjugation at the N
−7
or N
−9
position of cytokinins by amino acids or sugars, in particular glucose, xylulose o
Broer Jacobus
De Nijs Johannes Jacobus Maria
Van Doorn Johannes Elizabert
Kruse David H
Nelson Amy J.
Syngenta Participations (AG)
Vrana Bruce
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