CA1231071A - Medium for plant protoplast culture - Google Patents

Medium for plant protoplast culture

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Publication number
CA1231071A
CA1231071A CA000461772A CA461772A CA1231071A CA 1231071 A CA1231071 A CA 1231071A CA 000461772 A CA000461772 A CA 000461772A CA 461772 A CA461772 A CA 461772A CA 1231071 A CA1231071 A CA 1231071A
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Prior art keywords
serum
medium
plant
protoplasts
improvement
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CA000461772A
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French (fr)
Inventor
Ning-Sun Yang
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Agracetus Inc
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Agracetus Inc
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Priority claimed from US06/526,496 external-priority patent/US4533636A/en
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

MEDIUM FOR PLANT PROTOPLAST CULTURE

Abstract of the Disclosure A culture medium and method for the isolation and culture of plant protoplasts is disclosed which includes in it a quantity of neutral mammalian blood serum. An addition of 1 to 25% serum to protoplast media results in a greater yield of viable protoplast cells, helps to protect the protoplasts in culture, and makes it possible to obtain and maintain corn protoplasts, a heretofore difficult task.

Description

:
1~3~7~L

MEDIUM FOR PLANT PROTOPLASM CULTURE

Field of the Invention The present invention relates to the husbandry and maintenance of plant cell protoplasts in general, and no-fates, in particular, to a medium particularly adapted forth isolation, maintenance, and sustenance of plant cell protopasts in vitro and a method of using the same.

Description of the Prior Art The techniques of isolating, maintaining, and culturing lo protoplasts of plants are becoming more widely used as a key mechanism for use in the genetic engineering of plants and for the asexual prorogation of plants from protoplasm cell cultures into adult normal plants. Plant cell protoplasts are viable, intact, plant cells that have been removed from their cell walls. Much effort is currently being made to develop suitable techniques for the handling and care of protoplasts through the various steps of regenerating whole plants. To begin with it is necessary to isolate viable _ protoplasts, i.e. by removing plant cells from the structure of the plant and by stripping from the cells their own cell walls, ideally without damaging the cytoplasmic membranes and the remainder of the cells themselves. Once isolated, _ the problem becomes the maintenance of active protoplasts during in vitro manipulation, especially during insertion ! 25 and removal of genetic materials, if that is a part of the procedure. Once the protoplasts are obtained and manipulated ~L23~0~1 or engineered as desired, the task is then to regenerate the protoplasts back into whole plant cells in culture media as a normal cell wall containing plant cells, and later as plant callus cultures and plant cell aggregates, in an attempt to start the process of reproducing whole intact and structurally normal plants.
Most of the current procedures used for -the maintenance of protoplasts have been found deficient in one or more regards. In particular, no method or product has yet been developed which is reliably usable for the isolation and maintenance of protoplasm cells in most cereal plants of wide economic interest such as corn and wheat. The print supply difficulty in the formulation of such methods and media is the lack of knowledge about the character of any constituents or reagents which will protect plant proton plats in general from louses under various in vitro con-dictions. Typically in attempted in vitro maintenance and regeneration of plants from plant protoplasm cultures, most if not all of the protoplasm cells are lucid inadvertently at one or more steps during the procedure of their isolation, maintenance or handling. oven when a protoplasm culture can be maintained so that some yield of protoplasm cells can be achieved, the yield of viable protoplasm cells is often very low. Therefore it is necessary to develop additional methods and media which are suitable to generating a larger yield of viable protoplasts during the necessary in vitro manipulations of protoplasts if genetic engineering technic quest are to be successfully or commercially applied to plant protoplasts in general and to cereal grain plant protoplasts in particular.
' There are broadly speaking two main types of threats to Jo protoplasm viability using in vitro techniques, chemical damage and physical damage.
helical damage to plant protoplasts typically occurs through enzymatic action from crude or only partially purified cell wall degrading enzymes and also from various endogenous enzymes released from tangent plant tissues and cells. Currently commercially available cell wall degrading enzymes, such as cellulases and pectinases, necessary to the ~231~

creation of plant cell protoplasts, since these enzymes are used to remove the plant cell walls from the cells, are not greatly purified and are generally contaminated with proteolytic enzymes. When the currently available cell wall degrading enzymes are used to degrade plant structural tissues, the contaminating proteolytic and Lopez enzyme activities often damage or at least degenerate severely the newly exposed protoplasm cell membranes. Thus many cells are lucid by the exact mechanism needed to create the protoplasts. Also, lo even if highly purified cellulases or pectinases were used in cell wall digestion, it is likely that cellular endogenous pro teases and other hydrolytic enzymes, such as lapses, would be released into the host medium from the wounded cells or tissues used as starting materials. These endogenous enzymes would act on protoplasts as readily as introduced exogenous enzymes. Furthermore, when protoplasts are exposed to elevated concentrations of osmoticum, which is routinely used in the isolation of protoplasm procedure to prevent cell collapse, they often are found to generate high levels of protozoa and RNase activities. Thus the cycle of proton plats degeneration feeds upon itself as the protoplasts lucid by the contaminated exogenous pro teases or by initial _ wounding induced endogenous pro teases or lapses will in ; turn release large amounts of endogenous cellular pro teases and lapses into the surrounding cell tissue medium which then catalyze further damage in the membranes of the newly generated population of protoplasts.
ilk regard to physical damage, protoplasts are very vulnerable organisms and can be very sensitive and fragile to shearing, shaking or any kind of severe packing or sup-pension. Cell protoplasts are also very vulnerable to -changes in the osmotic pressure of the cell medium which can rapidly cause expansion, and bursting, of the protoplasts, if not carefully monitored.
_ 35 Both the chemical and physical damage to protoplasts is directly related to the basic biochemical nature of plant protoplasm surfaces, lye. from the fact that the cell membrane instead of the cell wall acts as the cellular envelope in a protoplasm. This problem is very analogous ~2;~)7~

to similar problems which exist in the culture of mammalian tissues as single cells and the transfer of mammalian cells into sub-cultures. In the past, it has been believed that particularly specialized media were necessary for the main-S tenancy of plant cell protoplasts and that the media con-j stituents should be primarily derived from plant sources.
An example of use of a plant cell culture media derived from plant sources, in this case micro algae, can be found in European Patent application 00~9632. An example of the use of serum in a culture media used for animal cell lines is shown in POT published application IT 82/02900.

Summary of the Invention The present invention is summarized in that a medium for the maintenance and propagation of plant protoplasts in vitro includes therein an effective amount of neutral mammalian blood serum.
The present invention is also summarized in that a method is provided for the in vitro maintenance and pro-pagation of plant protoplasts including the steps of Foss toning the growth of plant protoplasts in a media supple-minted with tile biologically active amount of neutral mammalian blood serum.
It is an object of the present invention to provide a medium which fosters the growth of plant cell protoplasts and which yield a far greater yield of viable protoplasts than was possible using other media.
It is another object of the present invention to pro-vise a method for the isolation and propagation of plant cell protoplas-ts which provides a larger yield and better viable result -than previous techniques.
It is yet another object of the present invention to enable the protoplasm cultivation of cells from common cereal grain plants such as corn.
It is another object of the present invention to pro-vise such a media and enable such a method using widely and commonly available materials.
Other objects, advantages, and features of the present I I

invention will become apparent from the following specific cation and accompanying example.

Brief Description of the Drawing The single drawing figure is a graphic representation of the experimental results from a suspension cell line maintenance experiment as described in Example 5 below.

Detailed Description of the Invention . _ In summary, the present invention makes it possible to viably propagate plant protoplasts of many species with good yield by adding to the medium in which the plant protoplasts are cultured a biologically effective amount of whole or fractionated neutral mammalian blood serum. The fact that this medium helps to foster and protect plant protoplasm growth is a surprising one. It had previously been believed that plant cell cultures were more properly conducted using media generated from plant derivatives, but it has been found instead that mammalian serum is a very advantageous constituent of the protoplasm medium.
It has been found that a media containing between 1 and 20 25 percent fetal calf serum, and preferably between 5 and 15 percent, is an extremely effective and positive addition -to plant cell protoplasm maintenance and generation. This additive serves useful purposes in all the steps of plant -1 protoplasm separation, isolation and initial propagation and provides a measure of effective control of several of the previously experienced problems in plant protoplasm main-tenancy.
In particular, it has been found that the provision in the plant protoplasm medium of a supplement of between S and _ 30 lo percent neutral fetal calf serum is very generally effective in helping to protect and stabilize plant proton plats against cellular louses and deactivation in vitro. It has been found experimentally that the use of a heretofore - typical plant protoplasm media with mammalian serum added thereto is of benefit in reducing destruction of plant - protoplasm cells in a variety of stages in the procedure of 123~

solacing and propagating protoplasts, including enzymatic digestion of the plant cell wall tissues, the rinsing, isolation and purification of protoplasts from plant cell wall tissue and other cell debris, and in the manipulation, 5 plating and growth of protoplasts and the plant cells in culture. In all stages of these kinds of procedures, widely used in plant cell culture and in gelletic manipulation techniques of plants, a far greater yield of viable proton plats is achievable using a serum supplement to the medium 10 than is achievable without it. Furthermore these ad van-tageous results have been achieved in a variety of useful plant species, including corn, carrot, cotton and tomato.
For use in the present invention i-t is preferred, but not essential, that the mammalian blood serum be neutral, 15 meaning, in this context, that the serum is partially de-natured. To neutralize the serum a two-stage technique is preferably utilized to remove therefrom potentially undesir-ably biologically active compounds in the untreated serum which could potentially exhibit detrimental effects on plant 20 protoE~lasts in culture. The first step in the neutralizing process is to use activated charcoal to filter the serum to remove from it the steroids and other biologically active small molecular weight compounds. This step is preferably performed overnight. The second step in the procedure is 25 preferably to use heat treatment (approximately 57~ C for 7 1/2 to 4 hours) to deactivate and denature the peptize hormones and growth factors which may be objectionable in the serum. Other serum constituents apparently not dotter-mental to plant protoplasm isolation, maintenance, and growth, 30 such as the serum trypsin-like protozoa inhibitors, are heat resistant to a limited heat treatment of this type and are not affected by this treatment. Cell biologists working with mammalian cells have also generated several types of neutral blood sofa, specifically adapted for use with special 35 mammalian cell culture systems, and most of these sofa will be usable in the technique of the present invention on plant protoplasts cultures also. The serum as used in the present invention can be whole or fractionated serum. Whole serum will generally be preferred, for reasons of cost, but appear-~Z3~0~

private serum fractions still containing protozoa inhibitors and proper osmotic and viscosity characteristics would also be usable.
While it is not possible to state with certainty the exact chemical and biochemical qualities of the mammalian blood serum which are beneficial to the plant cell proton plats, there are some general biochemical and physiological characteristics of such a serum which can be fairly char-acterized as the agents which are most likely to be the most helpful to the maintenance and propagation of plant cell protoplasts. Mammalian blood serum contains high levels of potent trypsin-like protozoa inhibitors which provide potent anti-proteolytic activity and which act as an agent to inhibit cell louses in plant as well as animal cells. These inhibitors are of particular value since general protozoa activity is a strong threat to high proton plats yields, as mentioned above. Mammalian blood serum also includes various large and small molecules, such as albumin, which greatly raise the viscosity of solutions into which they are introduced, and which thus, when introduced into a culture medium, reduce the likelihood of physical damage to cells in the medium which might be caused by shearing, shaking, or packing of the cells. Such an in-creased viscosity medium thereby reduces the likelihood of cell louses because of physical damage during experimental manipulations of the protoplasm culture. rlammalian blood serum also contains high concentration of soluble salts and other components which are present both intracellularly and intercellularly in mammalian organisms. In viva in animals there is an exchange of osmotic fluids between the cytosol and the extra cellular voids and the maintenance of proper osmotic control is necessary to allow transfers of sugars, amino acids, intermediate metabolizes and soluble proteins into and between cells. Since the mammalian serum is the medium which is responsible for the maintenance of proper osmotic and nutritional conditions to allow such transfers, it can also naturally serve as an effective naturally occurring osmoticum and analog for cytosol for cultured mammalian cells in vitro. In accordance with the present invention it ~Z3~07~

has been determined that it can also serve the same purpose for plant cells, once they are removed from their protective plant cell walls.
In general the functioning of the present invention is another indication that the basic cellular physiology observed for plant and animal cells grown in culture may be surprisingly similar once the cells walls are removed from plant cells. Chile this result is surprising because it was previously believed that vastly different media were neck essay for plants as opposed to animal cells, the function-in of the present invention is an indication that the fundalllental biochemical differences between plant and animal cells may be somewhat less Tony previously perceived.
It should be understood within the scope of the present invention in that the particular species of mammalian serum provided is no-t of critical importance. It has already been generally recognized that mammalian sofa from a diverse group of species exhibits similar general biological and biochemical characteristics, independent on whether the source ox the serum is of any specific species or any specific maturity level within a species.
One particularly strong advantage of the present in-mention is that it helps enable protoplasm cultivation of cells of cereal grain plants, such as corn. The maintenance of corn propolasts is an important step in the development of techniques to cell culture and to genetically engineer corn and other important cereal grain plants. Ire isolation and maintenance of corn protoplasts has heretofore been relatively difficult and it has been found that the addition of serum to the protoplasm host media significantly lessens ' that difficulty.
- The following examples are illustrative of the scope of the proselyte invention but are not meant or intended to define the same.

Example I - Protoplasm Isolation protoplasm isolation technique was practiced on a stable in vitro cell line of carrot cells known as whelk.

~2;~t)7~
g The cells in this cell line are in suspension and are viable Jo as normal plant cells in culture and contain cell walls. To create protoplasts, equal volumes of cell suspensions were digested for 16 hours at room temperature ~25C) in an enzyme digestion mixture including cellulose, hemicellulase, pectins and buffering salts. To one sample an amount of neutral, whole fetal calf serum equal to 5% of the sample by volume was added. After the digestion, the yield of newly created protoplasts was collected by differential centric fugation and then counted using a sampling technique on the basis of the number of remaining viable protoplasm cells per millimeter of packed volume of starting cell material con-cent rated. In the sample without the serum, 1.2 x 106 protoplasts per milliliter were obtained while in the sample with the serum, the corresponding count was 10.1 x 106 protoplasts per ml, an eightfold better yield.

Example 2 - Protoplasm Isolation . _ _ The same protoplasm isolation technique was followed as in Example 1 above on a stable BMS-Y2 line of corn cells which were again propagated in a cell suspension and which again normally have cell walls. Again I by volume of fetal calf serum was added to the experimental sample and not to the control and both samples were digested, concentrated and counted. The yield for the control was 0.3 x 10 proton 1 25 plats per ml while the yield for the experimental sample was 3.4 x 106 protoplasts per ml.

Example 3 - Protoplasm Isolation In this example protoplasts were isolated directly from plant tissue. A sample of leaf misfile tissue was removed from the young leaves of a young corn plant. The sample pieces of tissue, about 1/2 cm in size, were first con-ditioned in a nutrient medium, cut into smaller pieces on the order of .1 cm in size, and digested with the enzyme digestion media as also used in example 1 above in identical experimental and control samples with and without the addition of fetal calf serum. The protoplasts were enriched ~23~L07~

by differential centrifugation in a rinse medium and again -_ counted. Two separate test procedures were run, and the results were as tabulated below.
Treatment Number of Protoplasts per g.
of fresh tissue (x105) _ _ - Viable Dead Procedure 1 Control 7.9 7.3 Experimental (5% serum) 35.9 6.8 Procedure 2 Control 0.3 ---Experimental 1 (5% serum) 16.6 ---Experimental 2 (12.5% serum) 24.5 ___ Example 4 - Protoplasm Maintenance Regeneration Previously isolated tomato and corn protoplasts were platted onto a preparation of Gamborg's protoplasm medium to which lmg/ml of 2,4-D had been added. The controls had no serum added while the experimental plates had neutral, whole, fetal calf serum added to make a total of 5% by volume of the serum. The results were as follows, represented as a percentage of total protoplasts plated.
Treatment Cell Wall Cell Budding Survival _ Formation or Division Tomato:
After 5 days:
Control 5% 2% 95 Experimental (5% serum) 30% 25% 95%
After 10 days:
Control 5% 2% 55%
Experirmen tat I swarm% 40% 85%
Corn:
After 5 days:
Control 30% 0% 90%
Experimental (5% swarm% 5% >90%

I

Example 5 - Cell Suspension Maintenance _ Identical cultures of rums cell line corn cells were grown in a modified MS medium inoculated with lmg/ml of 2,4-D as described in "Plant Regeneration from Tissue Culture of Maize", Green & Phillips, Crop Science, Vol. 15, p. 41 (1975). The number of cells was sampled and counted periodic gaily and the results are shown in the graph of the drawing figure. The suspension labeled "Experimental" contained again 5% neutral fetal calf serum by volume, while the control did not.

Example 6 -callus Maintenance Samples of AYE corn cells of muscatel tissue from young seedlings were inoculated onto ajar plates. The resulting callus cultures were maintained for 45 days, both with and without the addition of 5% fetal calf serum. The serum inoculated samples grew to be approximately 10~-15%
greater in size than the controls. After 45 days all cultures were still healthy and viable.

It has also been found that the optimal and the toter-able levels of blood serum used in a culture both vary considerably depending on the species of plant and the stage of cell growth. For example, tentative results seem to indicate that 12.5% by volume of neutral fetal calf serum is very effective in a medium for enzymatic digestion of corn tissue to isolate corn protoplasts, while a 5% level of serum has been found to better foster corn protoplasm pro-pogation and growth both in ajar plate culture and in sup-pension. For another example, while the addition of 5% by vilely of serum significantly increased cell growth in corn suspension cell lines over a 45 day period, that same level seemed to inhibit growth of carrot cells under similar conditions in a long term culture, although in a short term culture a serum level of 10~ by volume seems to improve protoplasm yield in carrot during enzymatic protoplasm - 35 isolation. Thus some empirical experimentation is necessary to establish the precise optimal serum level for a given 37~
species, although it is to be understood that the optimal level will be within the range of no less thin% nor more than 25% serum by volume in the medium.
It is to be understood that the present invention is not limited to the particular embodiments and examples disclosed herein, but embraces all such modified forms thereof as come within the scope of the following claims.

Claims (26)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A medium for the in vitro isolation and culture of plant protoplasts characterized by containing a biologically effective amount of mammalian blood serum.
2. A medium for the in vitro isolation and maintenance of plant protoplast cultures including buffer salts, plant nutrients, sugar and plant hormones in aqueous solution and containing a growth promoting amount of mammalian blood serum which has been treated to render said serum essentially free of potentially phytotoxic biological substances therefrom.
3. A medium as claimed in claim 1 or 2 wherein the medium contains at least 1% neutral mammalian blood serum.
4. A medium as claimed in claim 1 or 2 wherein the medium contains no more than 25% neutral mammalian blood serum.
5. A medium as claimed in claim 1 or 2 wherein the serum is neutralized by filtration through activated charcoal and by heat treatment at approximately 57°C.
6. A medium as claimed in claim 1 or 2 wherein the serum is neutral whole serum.
7. A medium as claimed in claim 1 or 2 wherein the serum is fractionated serum.
8. A medium as claimed in claim 1 or 2 wherein the plant is selected from the group consisting of corn, tomato, carrot and cotton.
9. An improvement to a culture medium adopted for the in vitro isolation and maintenance of plant protoplast cultures comprising an addition to the medium of a biologically effective amount of neutral mammalian blood serum.
10. In a medium for the in vitro isolation and maintenace of plant protoplast cultures including buffer salts, plant nutrients, sugar and plant hormones in aqueous solution wherein the improvement comprises an addition to the medium of a growth promoting amount of mammalian blood serum which has been heat treated to render said serum essentially free of potentially phytotoxic biological substances therefrom.
11. An improvement as claimed in claim 9 or 10 wherein the addition of serum is at least 1% of the medium.
12. An improvement as claimed in claim 9 or 10 wherein the addition of serum is at least 1% and no more than 25% of the medium.
13. An improvement as claimed in claim 9 or 10 wherein the plant is selected from the group consisting of corn, tomato, carrot and cotton.
14. An improvement as claimed in claim 9 or 10, wherein the serum is neutralized by filtration through activated charcoal and by heat treatment at approximately 57°C.
15. A method for isolating and culturing plant protoplastics in vitro comprising the step of adding to the protoplast medium a biologically effective amount of mammalian blood serum.
16. In a method where protoplasts are cultivated in a media and are isolated wherein the improvement comprises adding to the protoplast medium a growth promoting amount of mammalian blood serum which has been heat treated to render said serum essentially free of potentially phytotoxic biological substances therefrom.
17. A method as claimed in claim 15 or 16 wherein the adding step includes adding no less than 1% and no more than 25% serum to the medium.
18. A method as claimed in claim 15 or 16 further comprising the step of neutralizing the serum by filtering with activated charcoal and by heat treatment at approximately 57°C.
19. A method as claimed in claim 15 or 16 wherein the plant is a cereal plant.
20. A method as claimed in claim 15 or 16 wherein the plant is selected from the group consisting of corn, tomato, cotton and carrot.
21. A method as claimed in claim 15 ot 16 wherein the serum is neutral whole serum.
22. An improvement to methods for isolating and culturing plant protoplasts in vitro comprising including the protoplast host medium a biologically effective amount of neutral mammalian blood serum.
23. An improvement as claimed in claim 22 wherein the quantity of serum is at least 1%.
24. An improvement as claimed in claim 23 wherein the quantity of serum is no more than 25%.
25. An improvement as claimed in claim 22 wherein the serum is whole serum.
26. An improvement as claimed in claim 22 wherein the plant protoplasts are selected from the group of corn, tomato, and carrot protoplasts.
CA000461772A 1983-08-25 1984-08-24 Medium for plant protoplast culture Expired CA1231071A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US06/526,496 US4533636A (en) 1983-08-25 1983-08-25 Medium for plant protoplast culture
US526,496 1983-08-25

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CA1231071A true CA1231071A (en) 1988-01-05

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