as discussed above , one of the purposes of the invention is to provide a solution to vitrification disturbances without loss of the rate of plant multiplication . one characteristic of this invention therefore provides a method to fight against growth disturbances , referred to as vitrification , vitrescence or hyperhydria , in the micropropagation of plants that are multiplied in vitro . in this method one incorporates an efficient amount of agar hydrolysate in the culture medium used to grow the plants . in a preferred embodiment of the invention , the amount of agar hydrolysate used in the culture medium equals at least 0 . 1 g / l ( calculated on the basis of agar before hydrolysis ) of nutritional medium . the amount of agar hydrolysate which can be used ranges from 0 . 1 g / l to 100 g / l . and even higher amounts of agar hydrolysate can be used . in another embodiment the invention provides a composition for fighting the vitrification of plants that are multiplied by way of in vitro micropropagation . this composition contains an agar hydrolysate acting as an active principle . the agar hydrolysate can be used in a range of from 0 . 1 g / l to 500 g / l , preferably 0 . 1 g / l to 100 g / l , of culture medium . another embodiment of this invention provides a process for preparing the composition discussed above . in this process one hydrolyzes agar in an acid medium , at a temperature of between 20 and 200 ° c ., for a period of time of from 10 minutes to 7 days . preferably , the hydrolysis is performed at a temperature between 100 and 120 ° c ., for 30 to 60 minutes , at ph = 3 to 4 . the acid medium used for the hydrolysis can be acifified water or an acidified culture medium . the choice between these two materials as acid medium depends on whether one wishes to obtain a pure agar hydrolysate which can be added to a culture medium , or to prepare directly the culture medium which contains agar hydrolysate therein in a desired amount . in a preferred embodiment of the invention one introduces into the nutritional medium an effective amount of agar hydrolysate . according to tests that were conducted by the inventors , the amount of agar hydrolysate is not critical , as long as it is greater than 0 . 1 g / l ( calculated on the basis of agar before hydrolysis ) of nutritional medium , a threshold under which no activity has been observed . the invention applies to all the plant species . it especially applies to the ligneous species , including in particular prunus and malus from among the fruit trees , for which the inventors have a vitrification induction method , and especially for which exemplary embodiments are provided . the present invention is also advantageously applied to nonligneous species however . agar hydrolysate is liquid . therefore it does not solidify the culture medium . multiplication in the present invention occurs in a liquid medium , in other words , under the most favorable conditions from the standpoint of the rate of multiplication , but with a greater risk that plant vitrification will appear . the agar is hydrolyzed , either alone in water , or with the liquid culture medium . any culture medium well known in the art can be used with this invention . for example , medium recommended by druart ( international colloquium on in vitro culture of wood essences , pp . 101 - 108 , 1981 , iufro fontainebleau , france ) can be advantageously used . the vitrification induction model recommended by paques ( arch . int . phys . et bioch . 1984 , 92 , 20 ), which uses a liquid medium in which the bases of stems from microplants are submerged , can act as an activity efficiency control for the agar hydrolysate . one implementation of the invention involves the introduction of agar in the selected culture medium , as if one were preparing a solid conventional culture medium , in the amount of 0 . 1 to 100 g / l . then , the unit is hydrolyzed in an acid medium , ph = 0 to 7 , preferably ph = 0 to 4 , more preferably ph = 3 to 4 , at a temperature in the 20 - 200 ° c . range , preferably 50 - 150 ° c , more preferably 75 - 120 ° c ., for periods of time that vary as a function of the temperature and the ph used and which are typically from 10 minutes to 7 days . the acid used for acidification is not a parameter of the invention , and any ordinary acid can be used as long as it does not interfere with the multiplication process . for instance hydrochloric acid , sulfuric acid , phosphoric acids , carboxylic acids and analogues can be used . a preferred hydrolysis according to the invention involves hydrolysis at ph 3 - 4 , at a temperature of 75 to 120 ° c . hydrolysis lasts between 30 and 40 minutes . the hydrolysate that is obtained , or the floating remainder stemming from centrifuging , is used then as a culture medium . indeed , tests have shown that the centrifuging base was inert , and did not play a role in the process of fighting against vitrification according to the invention . therefore , if so desired , it can be eliminated for the sake of convenience . hydrolysis of the culture medium is not useful however according to the invention and one can also prepare a &# 34 ; pure &# 34 ; agar hydrolysate , in other words free of culture medium . thus , one can prepare a concentrated hydrolysate , for instance by adding to the agar the amount of water that is strictly needed to achieve hydrolysis . this concentrated hydrolysate can be added then as such to the selected culture medium , in order to protect the plants from the risk of vitrification . other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof . in this description , the amounts of agar hydrolysate are given in grams / liter of culture medium , and they refer to the weight of agar before hydrolysis . as discussed above , the inventors have found that the amount of agar hydrolysate was not a significant parameter of the invention , so long as it is equal to or greater than the efficient amount , or about 0 . 1 g / l . preferred amounts are 1 to 100 g / l , better 7 to 12 g / l , or amounts equal to those used presently for solidifying the culture media . moreover , the origin of agar does not influence the anti - vitrification properties . the agar used by the inventors is &# 34 ; bacto &# 34 ; quality agar from difco . microcuttings of apple - tree m 26 stock are grown either on a liquid proliferation medium which contains an agar hydrolysate , or on the same medium free of that hydrolysate . medium with agar hydrolysate : the solution is comprised of macroelements , microelements , vitamins , growth and sucrose substances in concentrations recommended by druart ( 1981 ). &# 34 ; bacto &# 34 ; agar from difco is added to a 7 g / l concentration . medium free of agar hydrolysate : this medium contains all of the previously mentioned elements except for agar . the ph of the created media is adjusted to 3 . 5 with hydrochloric acid , at ambient laboratory temperature . then , the solutions are either brought to boiling , distributed in the culture flasks then sterilized for 40 minutes at 110 ° c ., or directly sterilized under the previously mentioned conditions then distributed sterilely in the culture flasks . the media thus obtained are liquid , however , the medium which contains hydrolyzed agar flocculates slightly . the agar - free medium ( medium a ) is used as such ; the medium which contains the agar hydrolysate being used either as such ( medium b 1 ), or centrifuged at 300g for 10 minutes . the floating remainder which is obtained comprises the culture medium b 2 . the base which is suspended again in the agar - free medium is medium b 3 . 20 microcuttings of m 26 , 2 cm long , are planted on the four nutritional media described above . the recorded observations , after thirty days of culture are listed in the following table : ______________________________________ mediaobservations a b . sub . 1 b . sub . 2 b . sub . 3______________________________________vitrification 20 / 20 0 / 20 0 / 20 19 / 20ratenumber of 4 - 7 5 - 7 6 - 7 5 - 6buds / plant______________________________________ obviously , numerous modifications and variations of the present invention are possible in light of the above teachings . it is therefore to be understood that within the scope of the appended claims , the invention may be practiced otherwise than as specifically described herein .
the invention relates to a culture medium containing an effective amount of agar hydrolysate . this material is advantageously used to fight against the appearance of the plant vitrification phenomenon during in vitro micropropagation of plants .