Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
1999-08-20
2003-02-18
Campell, Bruce R. (Department: 1661)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C435S430100, C435S431000, C435S410000, C800S320000, C800S295000, C047S057600, C047S05810R
Reexamination Certificate
active
06521452
ABSTRACT:
This invention relates to sugar cane plants and more particularly to methods of producing such plants.
Sugar cane is well known as an extremely important crop plant because of its use in the production of foodstuffs and the use of its by-products such as molasses, bagasse, filter mud and ethanol, all of which are of great value to both developing and developed countries.
Generally, sugar cane is cultivated as a perennial crop. The plant is allowed to grow for one year and is known as “plant cane”. The plant cane is cut at this stage and processed to produce sugar. The plant cane root and a small amount of cane stem is left in the ground and this grows over the next year to produce the “first ratoon”. This is then harvested to produce sugar and the residue of the plant is left to grow for a further year to produce the “second ratoon”. In some countries this growing cycle is repeated up to a “fifth ratoon” before the plant is finally uprooted.
Sugar cane is wind-pollinated and this leads to great variability in the plants obtained from seeds. This is useful for breeding programmes because it produces many different plant variations which can be used to select new useful clones. However, for normal commercial propagation the natural reproductive method does not give true-to-type plants. As a result, up until now, vegetative propagation has been the only practical means of propagating sugar cane. The existing method of propagating sugar cane relies on using the stems of mature sugar cane. This method is very wasteful in terms of reducing the overall productivity of the sugar cane crop, is expensive in terms of labour costs and can also produce problems in terms of spreading virus diseases such as Fiji disease, bacterial diseases such as red rot and the main fungal disease of smut. Disease problems in sugar cane production can be extremely serious since they are spread by many different vectors and can very rapidly spread throughout sugar cane plantations.
Unfortunately, vegetative propagation is very expensive because the plant has to grow for 9-12 months before the stem can be cut down into segments of about 30 cm. The main problem is that the cut ends of the cane are exposed to contamination, especially due to the presence of the nutrient sucrose in the cane sap. As a result, it is easy to spread viral, bacterial and fungal diseases which can have serious effects on sugar cane production.
Because of all these problems with the production of new sugar cane plants, tissue culture methods have been developed for breeding and propagation programmes and for the elimination of viruses. For example, Barredo R., Luzaman R and Dequinto B. (1994) used the spindle leaf (the innermost leaf of the tiller) to produce 13,500 plantlets from variety Phil 74-64. However, they found that it took 8 months from the beginning of the laboratory work to produce seedlings capable of being planted in the field. Barba R. C., Zamora A. B., Linga C. K. and Thai Van N. (1975) used a callusing method which allowed them to produce 4,000 plantlets from a 3 cm piece of sugar cane shoot but disadvantageously this method can lead to genetic variations.
Recent advances in biotechnology have offered new opportunities for crop improvement and propagation through plant tissue culture techniques.
Somatic embryos are bipolar structures which contain both shoot and root meristems. They may be formed in both callus and cell suspension culture and lead directly to the formation of mature plants. Somatic embryogenesis offers a potential system for large scale production of plants. However, for somatic embryogenesis to be practical for the large scale production of plants, numerous problems have to be overcome which, up until the present invention, have remained obstacles to the use of the method for the production of sugar cane plants. Up until the present invention, mature somatic embryos have only been produced from callus grown on solid medium. Cells grown in callus are known to be genetically unstable and so the embryos grown from callus are likely to be genetically distinct. Moreover, callus-derived embryos do not grow in synchrony and manipulation is labour-intensive. Such problems render callus-derived somatic embryogenesis unsuitable for large-scale commercial propagation. By contrast, cell suspensions in liquid culture show greater genetic stability, can be synchronised by modifying growth conditions and can be subjected to automated manipulation. Up until the present invention, attempts at liquid culture only succeeded in producing immature globular embryos.
The present invention has been provided through a consideration of the above mentioned problems. The present invention addresses those problems and provides an efficient and reliable method of producing sugar cane plants which is particularly suitable for use on a large scale for commercial purposes.
According to the present invention there is provided a method of producing sugar cane somatic embryos from sugar cane explants comprising the steps of:
(1) culturing immature embryos from explants
(2) culturing mature sugar cane somatic embryos from those immature embryos
The present invention provides a system involving somatic embryogenesis for the production of somatic embryos for use in the production of sugar cane plant as mature somatic embryos may be produced quickly and in large quantities and the original plant gene characteristics are maintained in the somatic embryos.
The sugar cane somatic embryos produced by the method of the present invention may subsequently be germinated to produce sugar cane plants or may be encapsulated in an encapsulating agent to produce “artificial seeds” for direct delivery to the field for subsequent germination of the embryos in situ.
Most preferably at least step 2 occurs in liquid suspension culture as this is the most practical media for use of the method on a large scale.
The production of sugar cane somatic embryos by the method of the present invention involves somatic embryogenesis. This method is extremely valuable because it is possible to produce vast numbers of embryos in small volumes of culture media in an approximately synchronous manner. A high multiplication rate, however, is only the first of several potential advantages offered by somatic embryogenesis for the subsequent production of sugar cane plants in comparison with other methods of vegetative propagation of sugar cane plants. For example, both the growth of embryogenic tissue in step (1) and the subsequent development of the embryos to maturity in step (2) can be accomplished in liquid medium, making possible the manipulation of very large numbers of propagules with minimal handling. In addition, the product of somatic embryogenesis is an embryo that is capable of developing into a regenerated sugar cane plant with very little further input of labour. The advantage of somatic embryogenesis for the production of sugar cane plants over the prior art systems of vegetative propagation and micro-propagation is the presence of both root and shoot meristems in the same unit and therefore there is no requirement for the laborious transfer operations, thus reducing operating costs very significantly.
In addition, this absence of repetitive transfer operations advantageously decreases contamination which is spread by contact. A further advantage is that the embryogenic systems are capable of producing separated individual embryos, unattached to either mother tissue or other embryos. Thus, embryogenic cultures produce propagules that are not only complete but also discrete. The combination of those-two properties gives somatic embryos potential for direct delivery to the greenhouse or field for example, as components of artificial seeds or in a fluid drilling system.
The method of the present invention advantageously provides an in vitro multiplication tool providing the new possibility of quickly obtaining large quantities of sugar cane embryos in a liquid medium. This permits much greater and faster propagation of sugar cane plants than can presently be obta
Abdelrahman Layla Zakaria
Campell Bruce R.
Grunberg Anne Marie
Tolpin Thomas W.
Welsh & Katz Ltd.
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