Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters ethylene production in the plant
Reexamination Certificate
2002-08-06
2004-04-27
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters ethylene production in the plant
C800S278000, C800S287000, C800S286000, C800S298000, C800S295000, C435S419000, C435S468000, C435S320100, C435S183000, C435S189000, C536S023200, C536S023600, C536S024100, C536S024500
Reexamination Certificate
active
06727406
ABSTRACT:
BACKGROUND OF THE INVENTION
Coffee is prepared from the roasted beans of the plants of the genus Coffea, generally from the species
C. arabica
(Caturra coffee) and
C. canephora
(Robusta coffee), and hybrids of these. Beans are the seeds of the coffee plant and are obtained by processing the coffee fruit, ideally the mature coffee fruit which commands the best price due to its superior quality. In order to obtain high quality “gourmet” coffee, it was considered necessary in the past to pick the coffee tree fruit by hand because the fruits of a coffee tree do not ripen uniformly and, thus, there are both mature and immature fruit on the same tree. This did not previously present a serious problem, as most coffee is grown in areas of the world where labor is plentiful and not expensive. However, recently, a lack of abundant and inexpensive labor has bercome a major contributor to decreased coffee production. In order to increase productivity, countries in some regions of the world, such as the largest coffee producing country, Brazil, have resorted to strip harvesting where workers rapidly remove all fruit from a branch whether ripe or unripe. The speed of harvesting is thus increased, but the yield of the highest quality beans is decreased because much of the harvested fruit is immature (green).
The lack of uniform ripening of coffee fruit on the tree has also seriously limited the effectiveness of mechanical harvesting. The force required to remove mature fruit (cherry) from the tree is similar to the force required to remove green fruit. Thus, mechanical harvesters do not distinguish well between green fruit and cherry and a large amount of immature fruit is harvested along with mature fruit. If coffee fruit ripening could be controlled so that all fruit ripened at one time, both the strip method of hand harvesting and mechanical harvesting would be much more efficient and a higher percentage of the harvested fruit would be in the higher quality grades, resulting in increased profitability of coffee production.
Ripening of fruit involves a number of changes in the fruit. In fleshy fruits, chlorophyll is degraded and other pigments often form, changing the color of the fruit. Simultaneously, the fleshy part softens as a result of the enzymatic digestion of pectin, the principal component of the middle lamella of the cell wall, and starches and organic acids are metabolized into sugars. Fruits are divided into two major groups, based on the respiratory behavior observed during the ripening process. In the climacteric fruits, such as tomatoes, avocados, bananas, apples and pears (i.e., pome fruits), and papaya, there is a large increase in respiration (i.e., a large increase in oxygen uptake termed the “climacteric rise”) concomitant with a burst of ethylene synthesis, producing marked changes in fruit composition and texture. In these fruits, the “climacteric” is required for the final stages of ripening when softening and development of color and flavor occurs. Other plants do not have a climacteric and ethylene does not seem to be important in their fruit ripening. Such fruits that show a steady decline or gradual ripening are called “non-climacteric fruits” (e.g., citrus, grapes, watermelon, cherries, pineapples, strawberries, and most vegetable crops such as carrots, onions, celery, spinach, crucifers, peas and beans).
Once climacteric fruit reach a certain stage of maturity, it is known that they can be induced to ripen by the exogenous application of ethylene, such as during storage and/or transport. Techniques to avoid exposure of climacteric fruits to ethylene until just before marketing have been used to control and regulate the timing of the ripening process, and have had a major impact on the quality of fruit sold. For example, tomatoes are often picked when they are green, and then stored in the absence of ethylene until just before marketing, at which time they are exposed to exogenous ethylene to induce simultaneous ripening. Exogenous ethylene has also been used commercially to promote loosening of fruit such as cherries, blackberries, grapes, and blueberries, thereby facilitating mechanical harvesting of these fruit crops.
In view of the foregoing, it would be very advantageous to be able to characterize coffee plants as to whether or not they are climacteric and, if shown to be climacteric, to control the ripening of coffee fruit by exogenously applied ethylene. Until the investigations described herein and in our co-owned U.S. Pat. No. 5,874,269, the disclosure of which is hereby incorporated by reference in its entirety, it was not known whether coffee fruit is climacteric or non-climacteric. Although it was observed that coffee fruit ripened in response to ethylene after reaching a certain stage of development [Crisosto, C. H., et al.,
J. Haw. Pac. Agri
. 3:13-17 (1991)], it was not possible to measure ethylene evolution or a respiration increase in ripening fruit. This may be because of the small size of the fruit and the lack of uniformity of ripening.
The biosynthesis of ethylene begins with the reaction of methionine and ATP to form S-adenosylmethionine (SAM). The enzyme ACC synthase catalyzes the conversion of SAM to 1-aminocyclopropane-1-carboxylic acid (ACC). In most plants this is the rate limiting step. The ACC is then converted to ethylene, in a reaction that is catalyzed by ACC oxidase [Yang and Hoffman,
Ann. Rev. Physiol
. 35, 155 (1984)].
It is well known that ethylene is related to various events in plant growth and development, including fruit ripening, seed germination, leaf and flower senescence and abscission, and root and leaf growth. Ethylene production is strictly regulated by the plant and can be induced by a variety of external stress factors, including the application of auxins, wounding, anaerobic conditions, viral infection, chilling, drought, ions such as cadmium and lithium ions, and the like.
Recombinant DNA technology has been used to isolate a number of ACC synthase genes from, for example, rice, petunia, winter squash, zucchini, tomato, tobacco, mung bean, soybean, and apple. Examples of these ACC synthase genes are described in our co-owned U.S. Pat. No. 5,767,376, the disclosure of which related to these examples is hereby incorporated by reference. However, with the exception of the apple and a subset of the tomato ACC synthase gene sequences, none of the described ACC synthase genes are involved with the ripening of fruit. Therefore, ethylene production in plants is apparently governed by a family of ACC synthase genes, at least in the above examples, not all of which are expressed during fruit ripening, e.g., some would be active in wound response, and the like. Similarly, it is considered likely that there is a family of ACC oxidase genes in plants that are variously active at different stages of plant growth and fruit ripening. The DNA sequences of the members of the ACC synthase gene family or the members of the ACC oxidase gene family in a plant such as coffee are therefore thought to be different from each other, although they would be related. For example, ACC synthase is encoded by at least six divergent genes in tomato. J. E. Lincoln et al. [
J. Biol. Chem
. 268 (no. 26), pp. 19422+, September 1993] compared the gene sequences of two ACC synthases thought to be involved in fruit ripening in tomatoes and found a sequence homology of only 71%. Two other ACC synthase genes from tomato had a sequence homology of 96% with each other. However, the sequence homology between the two sets of ACC synthase genes was only 68% and they had only a 51% sequence homology with an ACC synthase gene from rice. It is similarly expected that the genes coding for ACC synthases involved in fruit ripening from different varieties of coffee, such as
C. arabica, C. canephora
, and blends of these, such as the Timor hybrid and the like, would show a high sequence homology, but would not be identical. Moreover, the findings in the tomato demonstrate the importance of using ripening coffee fruit tissu
Moisyadi Istefo
Neupane Kabi Raj
Stiles John I.
Fox David T.
Ibrahim Medina A.
Jones Day
University of Hawai'i
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