GB1584854A - Plant propagation method - Google Patents

Description

(54) PLANT PROPAGATION METHOD (71) We, AGENCE NATIONALE DE VALORISATION DE LA RECHERCHE ANVAR a body corporate organised under the laws of France of 13 Rue Madeleine Michelis, 92522 Neuilly S/Seine, France, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention, which was made at the Office de la Recherche Scientifique et Technique Outre mer (Overseas, Scientific and Technical Research Office), 24 rue Bayard, 75008 Paris, relates to a process for the vegetative propagation of monocotyledones which are palms as herein defined. By "palms" we mean herein plants of the family palmae, including in particular the oil palm.The invention also includes the sets obtained by this process.

Fats have a most important place among substances useful to the human race, either for use as foodstuffs or for industry. Their alimentary energy (about 8.45 calories per gram) is about twice that of the glucides (about 3.88 calories per gram) or of proteins (about 3.68 calories per gram); this comparison applies only to the small molecules of fatty substances. The difference is greater where long-chain lipides are concerned; a molecule of glucose liberates in fact 673 calories, whereas a molecule of stearic acid liberates 2710, that is to say over four times as much.

In industry the lipides find numerous applications: lubricants, paints, thermal and electrical insulating materials, detergents, plastics materials, fuels, etc. The economic importance of lipides is therefore considerable.

As far as we are aware, the oil palm is the plant which gives the highest yield of oil per hectare.

By means of selection it has been found possible to obtain plantations in which the average yield is about 4000 kg of oil per hectare, whereas oleaginous plants, such as for example the peanut, soya, colza, and the castor-oil plant give yields no higher than 200 to 400 kg of oil per hectare.

Since the oil palm is reproduced solely by means of seeds, a certain heterogeneity of plantations is inevitable, and it would appear that the yields achieved cannot be further substantially improved.

Research into vegetative propagation of palm trees is at present being undertaken.

With regard to the date palm, it would appear that the problem concerning the obtaining of sets by vegetative reproduction is far from being solved (Oppenheimer Ch., O. REUVENI Final report of research, Agric. res. organ.; Volcani Center, Div.

of subtrop. hortic., Bet-Dagan, Israel, 91 p. (1972); REUVENI O. et al 49th annual Date Grower's Institute: 17-24 (1972). As regards the coconut palm, research is also encountering serious difficulties (EEUWENS C. J. - Physiol. Plant 36: 23-28 (1976); FULFORD R. M., S. H. F. W. JUSTIN East Malling Res. Stat. Report 1971; p. 65, Report 1972; pp. 99-100; NOERHADI E., N. L. TORUAN (FAO Consultation -- Problems in palm tree breeding, Rome 29th September-lst October 1975)).

On the other hand, where the oil palm is concerned, study has shown that in vitro culture of cells and certain tissues of the oil palm could make it possible to solve the problem of the vegetative reproduction of this tree. For this purpose, reference may be made to the following articles: - RABECHAULT H. et al. C. R. Acad. Sci., Paris 270: 3067-3070 (1970); this article describes the production of embryoids from tissue cells of seed embryos; - STARITSKY, G., Euphytica, 19: 288-292 (1970); this article shows that it has been found possible to obtain in vitro a certain apex development of young palms.

Furthermore, rhizogenesis of root calluses has been achieved with various culture media (MARTIN J. P. et al. Oleaginous 27th year; No. 6, 303-305 June 1972); however, these calluses have not given aerial portions.

In addition, SMITH W. K. and J. A. THOMAS (Oleagineux 28: 123-127 (1973)) have obtained cultures of root tissues giving rapid growth and capable of abundant rhizogenesis; it would however appear that the capacity of these tissue cultures to form chlorophyllian tissues has remained in the latent state despite considerable proliferation. Other research workers, including JONES L. H. - Oil Palm News, 17: 1-8 (1974), working with segments of aseptic plants have succeeded in effecting the dedifferentiation of regions near the cotyledonary node and the formation of embryoids evolving into planflet's.

Finally, from fragments of the apex and heart of the palm, complete organogenesis has been obtained under particular conditions (RABECHAULT H. et al.

Oleagineux, 27th year No. 11, 531-534, November 1972). In this last article it is indicated that different nutrient media have been tested and that the best combination found for achieving complete organogenesis consisted of a process comprising the following stages: 1) cultivation in darkness of fragments of the apex and heart of the palm on a nutrient medium containing mineral salts (1800 mg/l), trace elements, saccharose (20%o) and, as growth substance, 2,4 - dichloro - phenoxy - acetic acid (2,4 D) and kinetin or benzylaminopurin; 2) planting out the culture on a medium which is identical to that previously used except in that the amount of mineral salts is increased up to 3000 mg/l, while the 2,4D is replaced by alpha-naphthalene-acetic acid (ANA), this cultivation taking place in darkness once again;; 3) planting out again on a liquid or semi-liquid medium of composition identical to that previously used except in that the concentration of saccharose has been increased from 20 to 400/or; and 4) finally, planting out on media exposed to the light and identical to the medium previously used except in that the concentrations of mineral salts and saccharose have been reduced to their original values of 1800 mg/l and 20%o respectively, while alpha-naphthalene-acetic acid is replaced by beta-indole-acetic acid (AIA).

This process therefore consists in cultivating, first in darkness and then in the light, tissues originating from a clearly determined part of the palm, using nutrient media whose concentration of saccharose and mineral salts and also the nature of the growth substances are varied.

The results reported to date are of great importance, but as regards those obtained with embryos or aseptic plantlets the genetic future of the plants produced cannot be predicted.

Moreover, it must be noted that the removal of the terminal bud, or of fragments of the terminal bud, will entail the death of the tree (see the articles by STARITSKY and RABECHAULT cited previously). This is not the case with other organs; the roots are easy to take off, but as they harbour numerous microorganisms, their disinfection is difficult; the young inflorescences protected by spathes offer naturally sterile tissues, but up to the present time the formation of calluses and the culture of tissues have been slow and difficult.

It is an object of the present invention to provide a process for obtaining, by vegetative propagation, palm sets from leaves of plantation palm trees, this process consisting of utilising cultures of tissues of palm leaves.

In its more general aspect the present invention relates to a process for the vegetative propagation of monocotyledones which are palms as herein defined, which process comprises: 1) placing in isolation previously disinfected fragments of leaves on a basic nutrient medium; 2) effecting the dedifferentiation of the leaf tissues resulting from stage 1) by bringing them into contact with a nutrient medium which is similar to that of stage 1) but which contains additionally one or more growth substances in an amount sufficient to achieve the formation of calluses and the proliferation of the tissue cultures;; 3) then effecting the differentiation of the tissues resulting from the dedifferen tiation stage, on a nutrient medium similar to the medium used in stage 1) but add tionally containing cytokinin in an amount sufficient to effect the formation of embryoids; and 4) transferring the resulting embryoids to a nutrient medium similar to that of stage 1), this medium being substantially free from growth substance, and keeping the said embryoids on this medium until plantlets are obtained, the said plantlets then being transplanted in a greenhouse.

The present invention also relates to a process for the vegetative propagation of palms, which process comprises: I) placing in isolation previously disinfected fragments of leaves on a basic nutrient medium containing mineral salts rich in phosphorus, trace elements, iron, a sugar, vitamins, and other nutritive additives, the said medium being substantially free from growth substances and having a pH between 4.0 and 6.0; 2) effecting the dedifferentiation of the leaf tissues resulting from stage 1) by bringing them into contact with a nutrient medium which is identical to that used in stage 1) except in that the sugar content has been modified and in that it contains one or more growth substances in sufficient quantity to effect firstly the formation of calluses and then the proliferation of the tissue cultures thus obtained, the pH of the medium being between 4.0 and 6.0;; 3) then effecting the differentiation of the tissues resulting from the dedifferentiation stage, on a nutrient medium which is identical to the medium used in stage 1) except in that its sugar content is lower and in that it additionally contains a cytokinin, either alone or in the presence of a small amount of auxin, in an amount sufficient to effect the formation of embryoids; and 4) transferring the resulting embryoids to a nutrient medium which is identical to that of stage 1) except in that its sugar content is lower, this medium being substantially free from growth substance, and keeping the said embryoids on the said medium until plantlets are obtained, these plantlets then being transplanted in a greenhouse.

In a process according to the invention, it is advantageous in addition to make use of a "sequential" effect as hereinafter defined, to stimulate cellular propagation and the growth of the tissue cultures, particularly during the dedifferentiation stage. The ease with which true tissue cultures can be obtained by dedifferentiation of vegetable tissues is in fact very variable from one family to another, from one species to another, and even from one individual to another. This ease is connected with the sensitivity of the tissues to the physical and chemical factors of the medium and to their variations.

In nature, in fact, the growth of organisms and their tissues is never continuous but proceeds by stages and successive steps, even when the climate is substantially constant, as it is in equatorial countries.

Sensitivity to the factors of the medium and sensitivity to the variations of these factors thus in fact exist.

As regards palms, particularly the oil palm, tissues cultivated on a medium which is favourable to dedifferentiation and constant may remain for several years without developing, unless this occurs in an exceptional manner; cellular multiplications are certainly caused by the chemical constituents of the culture medium, but they soon stop for one reason or another. This stopping may be due to: 1) the lack in the medium of an essential element which has been exhausted, so that the medium must be renewed and this substance added; or 2) the fact that the process is blocked by the excess of a substance (for example an excessive dose of auxin, in which case it is advantageous to transfer the cultures to a medium in which the amount óf auxin is smaller); or 3) the fact that the process is blocked through the absence of variations of the factors of the medium.

In the oil palm a stimulation was for example observed when the tissues were transferred from a liquid medium to a solid medium, or when the nature and proportion of the growth regulators, sugars, etc. were changed. This effect, referred to not only in the prior art but also in the present specification under the name of "sequential effect", was discovered by Kent A. E. and F. C. Steward (Amer. J. Bot., 52: 619 (1965) and Ann. N. 4 Acad. Sci., 144: 326334(1967)) for the culture of cells. It is here applicable to the culture of tissues.

Those skilled in the art can determine the nature and duration of the sequences in dependence on the sensitivity of the vegetable material used to the variations of the factors of the medium.

For the sake of greater convenience the invention will now be described with reference more particularly to the oil palm.

The starting material used according to the present invention consists of leaves of open-air palms; the young leaves, still white, situated slightly above the apex become brown less quickly through the effect of the dedifferentiation treatment than adult leaves, and therefore are preferred according to the invention provided that their removal does not entail the risk of endangering the life of the tree.

The palm leaves or fragments of leaves used in the process of the invention are first disinfected, for example by immersing the fragments of leaves for about 20 minutes in a 1% aqueous solution of mercuric chloride. In order to increase the effectiveness of the mercuric chloride, it is advantageous to add to the aqueous solution a wetting agent, such as e.g. the product known under the registered Trade Mark "TWEEN 80", and in order to limit osmotic shocks it is desirable to add glucose. When glucose is used, the tissues must also be rinsed with a glucose solution.

Contamination of fragments of leaves or explants is due to the action of fungi and above all of bacteria, and may vary depending on the time when the leaf or explant is taken from the plant. The isolation stage makes it possible to eliminate explants which are still contaminated despite disinfection.

Tests have shown that in the case of the palm it was not necessary to add anti biotic such as dihydrostreptomycin and griseofulvin, to the nutrient medium used in the isolation stage.

As has been stated previously, the nutrient medium (hereinafter referred to as base or basic medium) used in the different stages of the process of the invention contains: mineral salts rich in phosphorus, trace elements, iron, a sugar, vitamins, and other nutritive additives.

The mineral salts contained in the nutrient medium used in the process of the invention are for example the following compounds: potassium and ammonium nitrates, magnesium sulphate (MgSO4, 7H2O), calcium chloride (CaCl2, 2H20), and potassium monophosphate. This combination of mineral salts constitutes what those skilled in the art normally call the macroelements of MURASHIGE and SKOOG (Physiol. Plant., 15: 473497 (1962)); according to the invention the mineral salts are preferably the macroelements of MURASHIGE and SKOOG in a concentration of about 2/5, although the concentration of phosphorus, expressed in mg, is desirably higher than in the MURASHIGE and SKOOG formula, for example multiplied by about 5; the complete disappearance of this element after 21 days of culture has in fact been noted.

It is therefore advantageous to use the following concentrations of mineral salts: KNO3 600 mg/l NH4NOs 650 mg/l KH2PO4 350 mg/l CaCI2, 2H2O 180 mg/l MgSO4, 7H2O 150 mg/l The trace elements used in the nutrient medium may consist for example of manganese, zinc, and boron; it is in particular possible to use the trace elements of MURASHIGE and SKOOG modified by NITSCH (NITSCH et al. C. R. Acad. Sci., Paris 269: 1275-1277(1969)).

Iron is desirably added to the nutrient medium in the proportion of about 6 mg/l in the form of iron ethylene-diamine-tetraacetate (EDTAte of Fe). Iron may advan tageously be used in accordance with the indications of MURASHIGE and SKOOG (Physiol. Plant. 15: 473-497 (1962)).

The vitamins and other nutritive additives desirably comprise thiamine, pyridoxine, Ca pantothenate, nicotinic acid, ascorbic acid, inositol, casein hydrolysate, and yeast extracts.

The sugar contained in the nutrient medium used in the process of the invention may be a non-reducing sugar, such as for example saccharose, or a reducing sugar, or a mixture of reducing sugars, such as for example glucose, or a mixture of glucose and levulose (or fructose). Saccharose is very particularly preferred. The sugar content of the nutrient medium varies from one stage to another. As will be indicated later on, it may be advantageous to modify the nature of the sugar in the course of the stage of the process, particularly the dedifferentiation stage.

The pH of the nutrient medium of the isolation stage is adjusted to a value between about 4.0 and 6.0, for example 4.5, with the aid of a solution of sodium hydroxide or sulphuric acid, for instance.

All the elements used in the basic nutrient medium are first sterilised by filtration or autoclaving, filtration obviously being preferable in the case of thermolabile substances. The liquid nutrient medium is desirably aerated by agitation and kept at a temperature of about 25 to 300C.

After disinfection the explants are sown under sterile conditions in the medium defined above at the rate of about 1/2 cc of explants to 15 ml of medium.

When saccharose is used in the basic nutrient medium utilised in the isolation stage, it should desirably be present in a concentration of about 20,000 mg per litre.

Isolation treatment is preferably effected in darkness in order to slow down the biosynthesis of polyphenols.

Residual contaminations start to develop after culture for one week, and the contaminated explants are eliminated. In the case of young leaves the laminae unfold; it is expedient to utilise or plant them out before the nutrient elements of the medium are exhausted. After isolation for from 2 to 4 weeks the leaf tissues are ready for de differentiation.

The dedifferentiation stage consists in first inducing the formation of calluses by a heavy dose of auxin or a mixture of growth regulators, then separating the calluses from the foliaceous explants, in order to effect their proliferation into true tissue cultures.

The grouping of the calluses on the surface of the medium causes what is called "a mass effect" which is favourable to growth; this effect stimulates the proliferation of the calluses.

In this stage fragments of about 1 cm2 of leaf laminae are taken from the isolation explants. Induction is preferably effected in darkness, on a gelose-containing medium, the upper face of the laminae being placed against the medium. It should however be noted that light tends to accelerate the phenomenon, although it increases browning.

The important factors, determined after numerous tests, are: the concentration of sugars and of mineral salts, the pH, and the nature and concentration of growth regulators.

The nutrient medium used in the dedifferentiation stage consists of the base nutrient medium whose sugar concentration has been increased. When the sugar is saccharose, this concentration is from about 30 to 60%, preferably 40%. The sugar used may instead be glucose, for example in a concentration of about 20%o, or a mixture of glucose (10%) and fructose (10%o). Gelose is preferably added to this medium; it may for example contain 8% of gelose.

The pH of the nutrient medium of the dedifferentiation stage is an important factor; it should be between about 4.0 and 6.0, preferably about 4.5.

Throughout the dedifferentiation stage the pH of the nutrient medium may vary between the limits indicated above; however, it should be noted that the transfer of the cultures from a medium having a given pH to a medium having a higher pH is generally unfavourable, whereas a transfer in the opposite direction is beneficial, particularly with a high dose of saccharose. The pH of the medium is adjusted as 'indicated previously.

The thermolabile elements added to the base nutrient medium in order to form the dedifferentiation nutrient medium must be sterilised, preferably by filtration. These elements are added at the last moment before the medium solidifies.

The growth substances which can be used in this stage comprise auxins, such as: 2,4-dichlorophenoxyacetic acid (2,4D), 2,4,5-trichlorophenoxypropionic acid (2,4,5 TCPP), alpha-naphthalene acetic acid (ANA); these auxins may be used in combination with cytokinins, such as kinetin or benzyl aminopurine (BAP) and its derivatives.

The auxins initially used in the dedifferentiation nutrient medium are generally 2,4 - dichlorophenoxy - acetic acid (2,4D) or 2,4,5 - trichlorophenoxypropionic acid (2,4,5-TCPP); their initial concentration generally varies between 2 and 10 mg/l.

It has been found that certain explants of the oil palm Elaeis quineensis Jacq can be dedifferentiated only in the presence of heavy doses of 2,4D or when the concentration of mineral salts is for example twice that of the base nutrient medium.

The variation of the composition of the medium during the culture, both as regards the nature and as regards the concentration of the auxins, salts, and sugars, has a beneficial effect which is a "sequential" effect as herein defined. Thus, passing from a nutrient medium containing 2,4D to a nutrient medium containing ANA, or vice versa, changing from a medium containing 2,4D to a medium containing a mixture of auxins (for example 2,4-D 2 mg + ANA 2 mg/l) or a mixture of an auxin and of a cytokinin (for example 2,4,5-TCPP + BAP, or 2,4,5-TCPP + kinetin 0.5 mg/l), and changing from a medium containing a reducing sugar to a medium containing a non-reducing sugar have given positive results.

In certain cases the prolonged use of heaxy doses of auxin, while giving rise to localised dedifferentiation, can then prevent cellular proliferation, and it has been found that the formation of calluses can then be obtained by considerably reducing the amount of auxin used for the induction, for example by transferring the cultures from a medium containing from 2 to 5 mg/l of 2,4D to a medium containing 0.2 to 0.5 mg/l of 2,4D, or replacing the 2,4D by a small amount of alpha-naphthalene acetic acid (ANA) of between 0.5 and 2 mg/l.

A beneficial effect consisting of the appearance of rapidly growing tissue cultures has also been found when the change of auxin in respect of quality and quantity was in certain cases associated, or in certain other cases was not associated, with a change of the texture of the medium (changing from a liquid medium to a solid gelose medium) or from a medium containing one or more reducing sugars (for example glucose or glucose + levulose) to a medium containing a non-reducing sugar (for example saccharose).

It is also possible to utilise sequential effects, as herein defined, due to variations of the physical factors of the environment (temperature, light, osmotic pressure).

The determination of the nature and duration of the sequences is within the scope of those skilled in the art, who should adapt them to the sensitivity of the plant material used.

The tests carried out on the oil palm Elaeis quineensis Jacq have shown that the addition of ferulic acid (10 mg/l), of adenine sulphate (60 mg/l), or of a mixture of amino acids according to the formula of REINERT and WHITE (Physiol. Plantar., 9: 177-189 (1956)), had no appreciable effects.

The duration of the dedifferentiation stage varies between 3 and 24 months depending on the sensitivity of the tissues to auxins.

Until their active proliferation occurs, the calluses are advantageously transferred so as to be grouped together on gelose media in order to take advantage of the mass effect. Once proliferation is established, it is advantageous to reduce the amount of auxin; an amount of alpha-naphthalene acetic acid of the order of 2 mg/l is particularly appropriate.

The presence of a cytokinin, even in a small proportion, and particularly when zeatin is used, does not improve proliferation, and tends to cause browning of the tissues.

Established cultures must be replanted every 1 to 2 months. It has in fact been found that, after 80 days on the same medium without replanting, a third of the cultures form embryoids, with a slight advantage in the case of those developing in light (43%.

in the case of cultures in light as against 30% for those in darkness). Similarly, cultures effected on filtered media, despite greater browning, form a higher proportion of embryoids (47%) than those on autoclaved media (23%?. The number of embryoids neoformed under these conditions varies considerably from one culture to another. It seems that this early differentiation is due to a relative exhaustion of the medium and not to a reduction of the auxin content, because the complete suppression of this does not improve the formation of embryoids.

The fresh weight of the organogenic tissues obtained in the dedifferentiation stage increases by 10 to 30 times every two months approximately. These tissues contain up to 9596% of water and are more or less granular and of a yellowish white colour.

Certain strains are heterogeneous, as can be confirmed by observing the variability of their coefficients of growth and the morphological appearance of their descendants. It is thought that this phenomenon can be explained by the different tissual origin of their constituents.

Then follows the differentiation of the tissues resulting from the dedifferentiation stage, on gelose-containing nutrient media without auxin, or with a very low concentration of auxin, and in the light.

The differentiation medium is the basic nutrient medium mentioned above to which however, a cytokinin, a substance necessary for differentiation, has been added.

As cytokinins which are suitable for the purposes of the invention, mention may be made of benzylaminopurin (BAP), kinetin, and zeatine, benzylaminopurin and its derivatives being preferred. Tests made with tissues of the oil palm Elaesis quineensis Jacq show that the effect of benzylaminopurin increases with the quantity used when this quantity passes from 0.1 to 2.5 mg/l. At doses between 0.05 and 0.25 mg/l, zeatine gives rise to the formation of a few embryoids, and at a dose of about 0.1 mg/l the formation of only a few roots was observed.

The differentiation stage takes about 45 days.

When the embryoids are well formed, they are transferred to a nutrient medium capable of effecting the formation of plantlets; this plantlet formation stage takes place in the light, and preferably still on gelose-containing media.

The normal process of organogenesis of the embryoids must start by the growth of the caulinary part. When the root develops first, the growth of the bud is inhibited.

The nutrient medium suitable for the formation of plantlets is the previously described basic nutrient medium, but with a progressively reduced content of sugars.

In general no auxin is added to the nutrient medium for the formation of plantlets; nevertheless, when rhizogenesis is difficult it is advantageous to add a small amount of ANA.

When the roots have a certain development (length about 1 cm) the plantlets are transferred to a non-gelose medium in tubes aerated by agitation in a roller tube in the light, in order to fortify the root system.

Transplanting in a mixture of peat and vermiculite in greenhouses is effected under optimum conditions of hygrometry.

The process of the invention makes it possible to obtain viable palm sets.

Multiplication potential is very high. On the moderate basis of a proliferation coefficient of 10 every two months, and obtaining some fifty viable plantlets per gram of tissue subjected to differentiation, it will be seen that starting with 1 g of culture there- should be 1 tonne at the end of a year, that is to say 50 million young palm trees.

As previously indicated, according to the invention it is important that the base nutrient medium should be rich in phosphorus. It should also be noted that the isolation stage is necessary according to the invention; it makes it possible in fact to prevent operation shocks and to limit contamination. It is also advantageous to utilise the mass effect obtained by grouping the calluses together at the same point in the medium, and to subject the cultures to sequential effects, as herein defined, by varying environmental factors (light, temperature, pH, and transfers to media of different compositions, e.g.

changing from a medium containing reducing sugars to a medium containing non reducing sugars, or changing the growth substance).

The invention will be illustrated in greater detail by the non-limitative examples given below.

EXAMPLE 1.

Isolation stage Fragments of leaves of the oil palm Elaeis quineei2sis Jacq were used.

These fragments were disinfected by immersion for 20 minutes in a 1%ill aqueous solution of mercuric chloride containing 2 drops of the wetting agent "TWEEN 80" (the word "TWEEN" is a registered Trade Mark), and 20% glucose.

The leaf fragments were then isolated on the basic liquid nutrient medium whose composition is as follows: Composition of basic liquid nutrient medium 1) Mineral salts Content in mg/l KNO2 600 NH4NO13 650 KN2PO4 350 CaCI,, 2H2O 180 MgSO4, 7H2O 150 2) Trace elements MnSO4, 4H2O 25 ZnSO4, 7H,O 10 HOBOS 10 3) Vitamins and additives Thiamine HCI 1 Pyridoxine HCI 1 Ca pantothenate 1 Nicotinic acid 1 Ascorbic acid 100 Myo-inositol 100 Casein hydrolysate 250 Yeast extracts 250 4) Iron 6 mg/l of Fe In the form of Fe EDTAte 5) Saccharose 20,000 mg per litre pH 4.5 The basic liquid nutrient medium was aerated by agitation.

After disinfection the explants were sown under sterile conditions on this medium at the rate of about 0.5 cc per 15 ml of medium.

Isolation took place in darkness. After about 3 weeks the leaf tissues were ready for dedifferentiation.

EXAMPLE 2.

Influence of the saccharose content and pH in the dedifferentiation stage.

The dedifferentiation medium used consisted of the basic liquid nutrient medium defined in Example 1, in which however the saccharose content was modified.

The growth substance used was 2,4 - dichlorophenoxyacetic acid (2,4D) at the rate of 2 mg/l; in addition, the nutrient medium contained gelose (8%o of gelose).

In order to study the influence of the saccharose content, the increases of fresh weight of the cultures (as percentages)were compared for two saccharose contents, and the pH value was also taken into account.

The results obtained are shown in the graph in Figure 1; on the vertical line A are plotted the percentages of increase of fresh weight of the cultures obtained with saccharose contents of 20% and 40%O respectively, the pH of the nutrient media not being modified between the two planting operations, which took place at an interval of 60 days; for each point the pH value is indicated. On line B are plotted the percentages of increase of fresh weight obtained by transposing the pH values on the second planting-out. These results show that when the saccharose content is high a reduction of the pH between two planting-out operations is favourable.

EXAMPLE 3.

Influence of sterilisation.

The dedifferentiation stage was carried out on the nutrient medium defined in Example 2, in which the saccharose concentration was 40%.

The additives added to the basic nutrient medium were sterilised by filtration or by autoclaving.

After 46 days the increase of the fresh weight of the explants was measured.

The results obtained are given in Table I below, in which an estimate of the browning of the tissues is also given; the results obtained with nutrient media in which the additives were filtered, and to which extracts of palm buds (which may contain specific stimulating substances) were added, are likewise shown in this Table.

TABLE I

Culture media Filtered additives Filtered Autoclaved + 15% of Observations additives additives bud extract Increase of fresh weight of explants at the end of 46 days 224% 180% 154% Estimate of browning of tissues (1 = very light) 1.10 1.34 1.45 EXAMPLE 4.

Dedifferentiation stage.

The leaf tissues resulting from Example 1 were subjected to dedifferentiation on a gelose-containing nutrient medium consisting of the basic nutrient medium in which the saccharose content was brought to 40%; the additives introduced into the medium were sterilised by filtration and the pH was fixed at 4.5; the growth substance was 2,4D (2 mg/l).

When proliferation had been established the callulses were transferred to a nutrient medium which was identical to that previously used except in that it contained ANA (2 mg/i) as growth substance. The calluses were replanted every 45 days.

After about 20 months the dedifferentiated tissue cultures were ready for differentiation.

EXAMPLE 5.

Differentiation and formation of plantlets.

The tissue cultures obtained in Example 4 waere transferred to the differentiation medium. This medium was the basic nutrient medium defined in Example 1, to which ANA (0.1 mg/l) and benzylaminopurin (2.5 mg/l) were added. This differentiation lasted about 45 days. The embryoids formed were then transferred to a medium capable of forming plantlets.

This medium consisted of the basic nutrient medium defined in Example 1, in which the concentration of saccharose was only 10%o. It contained no growth substance; however, in cases where the rhizogenesis of the embryoids appeared difficult, alpha-naphthalene acetic acid was added with a dosage between 0.05 and 0.1 mg/l.

When the roots had a certain development (length about 1 cm) the plantlets were transferred to tubes aerated by agitation in a roller tube in light (1000 lux) in order to fortify the root system. The plantlets were then transplanted in a mixture of peat and vermiculite in greenhouses, and viable palm sets were thus obtained.

EXAMPLE 6.

Influence of rhe nature and dosage of cytokinin used in the differentiation stage.

The differentiation of the tissue cultures obtained in Example 4 was then effected on nutrient media, each containing different cytokinins (BAP, kinetin or zeatine, in doses changing from 0 to 2.5 mg/l).

The number of embryoids obtained on each medium was measured for each concentration.

The results obtained for the two best substances are shown in the graph in Figure 2; in this graph the number of embryoids formed in 6 weeks per gram of tissue subjected to differentiation is plotted on the ordinate, and the concentration of growth substance in mg/l is plotted on the abscissa. The curve (BAP) relates to benzylaminopurin and the curve Kin relates to kinetin. These results show that benzyl aminopurin is preferable to kinetin.

It has been found that with 2.5 mg/l of BAP and 0.1 mg/l of ANA an average yield of 62 embryoids per gram of fresh weight of tissue is obtained.

Figure 3 groups together representations illustrating the different stages of the process of the invention; the legends to this Figure are to be interpreted as follows:

Representation Stage Observations A Dedifferentiation, induction Piece of leaf (f) with calluses of calluses (c) formed along the veins B Dedifferentiation, culture Rapid growth tissue culture of tissues C Early organogenesis Culture of tissues carrying an embryoid e D Differentiation Culture of tissues in differentiation showing numerous embryoids e E Formation of plantlets Embryoids placed on a medium favourable to rhizogenesis F Bud b; root r G Formation of plantlets Commencement of formation of a complete plantlet r: roots pn: pneumatode b: bud f: first leaf H Formation of plantlets Complete plantlet f: first two leaves r: root with root cap co t: residues of tissue cultures I Formation of plantlets Another plantlet f: leaves r: roots pn: pneumatodes J Young palm tree in greenhouse WHAT WE CLAIM IS: 1. A process for rhe vegetative propagation of monocotyledones which are palms as hereindefined, which process comprises: 1) placing in isolation previously disinfected fragments of leaves on a basic nutrient medium; 2) effecting the dedifferentiation of the leaf tissues resulting from stage 1) by

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (18)

**WARNING** start of CLMS field may overlap end of DESC **. Figure 3 groups together representations illustrating the different stages of the process of the invention; the legends to this Figure are to be interpreted as follows: Representation Stage Observations A Dedifferentiation, induction Piece of leaf (f) with calluses of calluses (c) formed along the veins B Dedifferentiation, culture Rapid growth tissue culture of tissues C Early organogenesis Culture of tissues carrying an embryoid e D Differentiation Culture of tissues in differentiation showing numerous embryoids e E Formation of plantlets Embryoids placed on a medium favourable to rhizogenesis F Bud b; root r G Formation of plantlets Commencement of formation of a complete plantlet r: roots pn: pneumatode b: bud f: first leaf H Formation of plantlets Complete plantlet f: first two leaves r: root with root cap co t: residues of tissue cultures I Formation of plantlets Another plantlet f: leaves r: roots pn: pneumatodes J Young palm tree in greenhouse WHAT WE CLAIM IS: 1.A process for rhe vegetative propagation of monocotyledones which are palms as hereindefined, which process comprises: 1) placing in isolation previously disinfected fragments of leaves on a basic nutrient medium; 2) effecting the dedifferentiation of the leaf tissues resulting from stage 1) by bringing them into contact with a nutrient medium which is similar to that of stage 1) but which contains additionally one or more growth substances in an amount sufficient to achieve the formation of calluses and the proliferation of the tissue cultures; 3) then effecting the differentiation of the tissues resulting from the dedifferentiation stage, on a nutrient medium similar to the medium used in stage 1) but additionally containing a cytokinin in an amount sufficient to effect the formation of embryoids; and 4) transferring the resulting embryoids to a nutrient medium similar to that of stage 1), this medium being substantially free from growth substance, and keeping the said embryoids on this medium until plantlets are obtained, the said plantlets then being transplanted in a greenhouse. 2. A process for the vegetative propagation of palms, which process comprises:

1) placing in isolation previously disinfected fragments of leaves on a basic nutrient medium containing mineral salts rich in phosphorus, trace elements, iron, a sugar, vitamins, and other nutritive additives, the said medium being substantially free from growth substances and having a pH between 4.0 and 6.0;

2) effecting the dedifferentiation of the leaf tissues resulting from stage 1) by bringing them into contact with a nutrient medium which is ' identical to that used in stage 1) except in that the sugar content has been modified and in that it contains one or more growth substances in sufficient quantity to effect firstly the formation of calluses and then the proliferation of the tissue cultures thus obtained, the pH of the medium being between 4.0 and 6.0;;

3) then effecting the differentiation of the tissues resulting from the dedifferentiation stage, on a nutrient medium which is identical to the medium used in stage 1) except in that its sugar content is lower and in that it additionally contains a cytokinin, either alone or in the presence of a small amount of auxin, in an amount sufficient to effect the formation of embryoids; and

4) transferring the resulting embryoids to a nutrient medium which is identical to that of stage 1) except in that its sugar content is lower, this medium being substantially free from growth substance, and keeping the said embryoids on the said medium until plantlets are obtained, these plantlets then being transplanted in a greenhouse.

3. A process according to Claim 1 or 2, in which process the leaves or fragments of leaves used are previously disinfected, with the aid of an aqueous solution of mercuric chloride or another effective disinfecting agent.

4. A process according to Claim 2, or Claim 3 when read with Claim 2, in which process the sugar is a reducing sugar or a non-reducing sugar.

5. A process according to Claim 4, in which process the sugar is selected from saccharose, glucose, or mixtures of glucose and levulose (or fructose).

6. A process according to Claim 2 or 3, in which process the concentration of saccharose in the nutrient medium of the isolation stage is about 20,000 mg per litre.

7. A process according to any of Claims 1 to 6, in which process the one or more growth substances used comprise one or more of the auxins: 2,4-dichlorophenoxyacetic acid (2,4D) 2,4,5-trichlorophenoxypropionic acid (2,4,5 TCPP) alpha-naphthalene acetic acid (ANA), and of the cytokinins: benzylaminopurin (BAP) and its derivatives, kinetin.

8. A process according to any of Claims 1 to 7, in which process the isolation and dedifferentiation stages are carried out in darkness and the differentiation and plantlet formation stages are carried out in the light.

9. A process according to Claim 2, or any of Claims 3 to 8 when read with Claim 2, in which process fragments of leaves of the oil plant Elaeis quineensis Jacq are used, and the sugar is saccharose, the saccharose content of the isolation medium being about 20%o, that of the dedifferentiation medium being about 40%o, and that of the plantlet formation medium being about 10%o.

10. A process according to Claim 9, in which process the growth substance used in the dedifferentiation medium is an auxin, the differentiation medium containing a growth substance which is a cytokinin, alone or in the presence of a small amount of auxin.

11. A process according to Claim 9 or 10, in which process the intial concentration of growth substance in the dedifferentiation medium is between 2 and 10 mg/l.

12. A process according to any of Claims 2 to 11, in which process the mineral salts of the basic medium are MURASHIGE and SKOOG salts at a concentration of 2/5, the concentration of phosphorus being multiplied by 5; the trace elements are MUASHIGE and SKOOG trace elements modified by NITSCH; and the vitamins, and additives are: thiamine HC1 1 mg/l pyridoxine HCI 1 mg/l Ca pantothenate 1 mg/l nicotinic acid 1 mg/l ascorbic acid 100 mg/l myo-inositol 100 mg/l casein hydrolysate 250 mg/l yeast extracts 250 mg/l; iron being present in the proportion of 6 mg/l in the form of iron ethylenediaminetetraacetate.

13. A process according to any of Claims 1 to 12, in which process the proliferation of the calluses is stimulated by placing them in groups on the surface of the culture medium.

14. A process according to any of Claims 1 to 10, in which process, in order to stimulate the formation and proliferation of calluses, use is made of a sequential effect, as herein defined, produced by modifying the culture medium in the course of the dedifferentiation phase.

15. A process according to Claim 14, in which process the modifying of the dedifferentiation culture medium comprises varying its sugar content, varying the physical factors cf the environment, varying the texture of the medium, or varying the content of auxins.

16. A process according to Claim 14 or 13, in which process, in order to modify the culture medium in the course of the dedifferentiation stage, the concentration of auxins is varied, particularly from 2--1C mg/l to 0.2-0.5 mg/l.

17. Palm plantlets and sets obtained by a process according to any of Claims 1 to 10, and also vegetable organs utilised in a process according to any of Claims 1 to 10.

18. A process according to Claim 1, substantially as described hereinbefore with particular reference to the Examples and the Figures of the accompanying drawings.