Efficient method of protoplast culture

Details Efficient method of protoplast culture Efficient method of protoplast culture

2001-03-29

2003-05-27

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Plant cell or cell line, per se ; composition thereof;...

Culture, maintenance, or preservation techniques, per se

C435S420000, C435S430100

Reexamination Certificate

active

06569680

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to an efficient new method of protoplast culture.
BACKGROUND
All organisms, be it plants or animals, reproduce sexually by the fusion of male and female gametes, each containing a single set of chromosomes from either parent and this is possible only in the compatible and related species. Wide crosses are not common without human intervention. However, plant protoplast culture provides a unique system, wherein each protoplast has the potential to give rise to a whole plant and two or more protoplasts can be induced to produce a hybrid (fusion between nuclei) or a cybrid (fusion between cytoplasm of one and the nucleus of another). This technique is used to overcome sexual incompatibility barriers through the production of unique somatic hybrids involving vegetative cells. The lack of cell wall barriers offers significant advantages for the introduction of genetically engineered foreign DNA into the naked cells—that are protoplasts.
Hanstein for the first time used the term protoplast in 1880 (cf. Cocking E. C. 1972. Plant cell protoplasts, isolation and development. Ann. Rev. Plant Physiol. 23: 29-50). The isolation of protoplasts from plant cells was first achieved by Klercker by microsurgery on plasmolysed cells in 1892 (cf. Cocking E. C. 1972. Plant cell protoplasts, isolation and development. Ann. Rev. Plant Physiol. 23: 29-50).). With refinements in the technique, protoplasts were beginning to be isolated in large numbers by enzymatic removal of cell wall as pioneered by Cocking in 1960 (A method for isolation of plant protoplasts and vacuoles. Nature 187: 927). The plant species, condition under which plants are grown, plant age, method of protoplast isolation and protoplast culture are often critical for sustained division of protoplasts. Therefore, there are no standard methods for the isolation and culture of protoplasts. Considerable success has been achieved over the past two decades, when a number of techniques have been employed for the culture of protoplasts of numerous crop species.
In one such method (Binding H. 1974. Cell cluster formation by leaf protoplasts from axenic cultures of haploid
Petunia hybrida
L. Plant Sci. Lett. 2(3): 185-187), the protoplasts at a desired density are suspended in the optimum quantity of liquid medium in a petri dish and incubated at 25-28° C., generally in the dark or diffused light. Such a method provides opportunity to gradually change the osmolarity of the medium for better protoplast growth. But in this method, protoplasts generally tend to aggregate and some even degenerate which adversely affects the dividing protoplasts. This method also suffers from the fact that it requires relatively larger volume of protoplast suspension. Inadequate aeration also adversely affect protoplast growth.
Another method is the hanging/sitting drop culture method (Kao K. N., Keller W. A. and Miller R. A. 1970. Cell division in newly formed cells from protoplasts of soybean. Exp. Cell Res. 62: 338). In this method, small drops (40-100 l) of protoplast suspension are placed on the inner side of the lid of a petri dish so that the drops containing the protoplasts are hanging from the lid. The drops can also be placed at the bottom of the petri dish. Fresh medium can be added in small volumes whenever required. However, this method is generally employed where protoplast yield is low and also requires dexterous handling as a slight shaking disturbs the hanging drop.
Yet another technique used for protoplast culture is the micro-chamber culture method (Bawa S. B. and Torrey J. G. 1971. “Budding” and nuclear division in cultured protoplast of corn, Convolvulus and onion. Bot. Gaz. 132: 420) which is similar to drop culture method except that in this case a cavity microslide or a micro-chamber is used in order to follow the development of individual protoplasts. Here, either a conditioned medium is used or else a feeder layer is required for protoplast growth. However, this method suffers from the defect of quick drying and requires special efforts to ensure aeration.
In yet another method called Micro-drop Array (M.D.A.) technique (Potrykus I., Harms C. T. and Lorz H. 1979. Multiple-drop array (MDA) technique for the large-scale testing of culture media variations in hanging microdrop cultures of single cell systems. 1. The technique. Plant Sci. Lett. 14:231), which is usually employed to test the response of protoplasts to different culture media under varying combinations and permutations. The drop size is reduced to 40 l to accommodate many drops per petri dish. This method provides opportunity to use smaller number of petri dishes. However in this method it is not easy to handle small drops where the size of the drop is too small and it tends to dry quickly.
Another method used is the Micro-droplet Culture Technique (Gleba Y. Y. 1978. Microdroplet cultures: tobacco plants from single mesophyll protoplasts. Naturwissenschaften 65: 158), wherein the size of culture droplet is reduced to about 0.25 to 0.50 l so that each droplet containing only one protoplast is placed separately in numbered wells of special Cuprak petri dishes. However, since only one protoplast is being grown, improved media and pre-culturing of the protoplast is recommended before placing this into micro-culture.
The protoplasts/cells have also been known to be cultured in semisolid media using different gelling agents such as agar, agarose, K-carrageenan, alginate, gelatin and polyacrylamide for secondary metabolite production. In this technique, the protoplast suspension at double the required density is gently mixed with double strength molten agar at 40-45° C. in a petri dish so that protoplasts get embedded in the gel matrix upon solidification. However, a careful monitoring of agar gel temperature needs to be done to avoid damage to the protoplasts because of high temperature. The impurities present in agar also sometimes adversely affect the growth of the protoplasts.
Another method has been the use of semi-solid media using agarose (Lorz H., Larkin P. I., Thomson I. and Scowcroft W. R. 1983. Improved protoplast culture and agarose media. Plant Cell Tissue Org. Cult. 2: 217), wherein the protoplast suspension is made in agarose till micro-calli formation, which is then recovered by remelting the medium at 40° C. But, reheating adversely affects cell growth. An improvement over this method is the use of alginate as the gelling agent (Adaoha-Mbanaso E. N. and Roscoe D. H. 1981. Alginate: an alternative to agar to plant protoplast culture. Plant Sci. Lett. 25: 61), which can then be dissolved in osmotically adjusted sodium citrate solution and the protoplast derived micro-colonies could be easily recovered without affecting the growth. However, another improvement of protoplast regeneration efficiency has been the use of a combination of both semi-solidified blocks/thin layers and liquid media. One such technique is bead culture method, wherein the protoplasts are suspended in agar or agarose. The gel blocks containing embedded protoplasts are transferred to liquid medium and placed on a shaker. In this manner, an improvement in the protoplast growth could be accomplished but again, this method is quite cumbersome and involves many steps. Out of all gelling agents, use of agarose has improved protoplast culture efficiency and also the production of secondary metabolites, when protoplasts are embedded in thin layers of agarose placed on top of the solidified medium in a petri dish. However, the cost and the handling of agarose is a major constraint. Moreover, the recovery of micro-colonies is difficult in the embedding procedure using agar or agarose as it requires heating and remelting of agar/agarose which may adversely affect the growth of protoplasts.
An improvement over all the methods described above is the the Thin Alginate Layer (TAL) technique (Golds T. J., Babezinsky J., Rauscher G. and Koop H. U. 1992. Computer controlled tracking of single cell development in
Nicotiana tabaccum
L. and
Hordeum vulgare
L. P

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