CA2064603A1 - Process for adaptation to the direct open-field pricking-out of plants propagated by tissue culture - Google Patents

Abstract

PROCESS FOR ADAPTATION TO THE DIRECT OPEN-FIELD PRICKING-OUT
OF PLANTS PROPAGATED BY TISSUE CULTURE

A b s t r a c t The invention relates to a process for the adaptation to the direct open-field pricking-out of plants propagated by tissue culture characterized thereby that the plants propagated in a known manner a) are further cultivated in one or more step(s) on a nutrient medium having an osmotic pressure corresponding to 0.2 to 0.4 M sucrose concentration and, in the case of a multi-step cultivation, the osmotic pressure of the nutrient medium is adjusted to a value increasing step by step within the said range and/or b) they are stored at 2 to 10 °C for at least 30 days and the plant thus obtained is directly pricked out to open-field or marketed to such purpose.

Description

206~603 PROCESS FOR ADAPTATION TO THE DIRECT OPEN-FIELD PRICKING--OUT OF PLANTS PROPAGATED BY TISSUE CULTURE

This invention relates to a process for the adaptation, acclimatization to direct open-field pricking-out of plants propaqated by tissue culture.
The micropropagation of plants, the tissue-cultivating propagation techniques are worldwide used. The principle of these processes consists therein that complete plants are grown under sterile conditions from a relatively little-size plant part on a nutrient medium containing hormones and vi-tamines. By using a nutrient medium of suitable composition, the plant is prompted to form many shoots and the shoots de~eloped are urged to root.
However, due to their loose tissue structure and sen-sitivity to W irradiation, withering and temperature changes, the plants rooted in this manner are unsuitable for pricking out to the garden or open field. They are burnt by sunshine, loose their water content and finally die. Thus, the tissue-culturing laboratories usually have a green-house, foil tent or an other establishment with a closed air space or at least, they lease such a so-called training sur~ace [E.F. George and P.D. Sherrington: Plant Propagation by Tissue Culture, Exegetics Ltd., England, page 42, (1984)].
In the green-house or foil tent the plants are gradually adapted to the unfavourable conditions, lower moisture con-tent, wider fluctuations of temperature and intense light.
This procedure takes about 6 to 8 weeks and 10 to 80 % of -2- 2~6~603 the plants prepared in the laboratory die during this habituation. Thus, not only much efforts and materials are lost but it has also to be considered that tne training and habituation in an isolated air space are rather expensive cultivation of plant~ for 6 to 8 weeks costs 8 to 10 forints/plant in a green-house and 5 to 7 forints in a foil tent. However, by usinq foils, the operation of the equipment is very uncertain in the early spring and winter.
Thus, a demand exists on a process of tissue cultivation resulting in plants being suitable to be pricked out directly in the open air. Such a process has not been available up to the present: the propagating material obtained by tissue cultivation is everywhere pricked out to the open field after an appropriate adaptation.
The invention starts from the fact that the factors influencing the plants under open-air conditions such as temperature fluctuation, occasional water deficiency, rain-fall, wind and the like are responsible for the development of a "tr~ined" plant, i.e. for the change by which an individual having a habit corresponding to its own species is developed from the sensitive tissue-cultivated plant consisting of loose tissue. If it were possible to assert the factors mentioned above in the tissue culture period, the plant rooted on the sterile soil would not need a lengthy habituation process, i.e. the adaptation to the open-fie~d conditions.
Obviously, it is hardly possible to produce the "training" factors in a tissue culture. It has emerqed, 206~6~3 however, that it would perhaps be worth trying to model one or other factor separately.
The water uptake of plants is based on the osmotic pressure, more precisely on the difference between the osmotic pressure of the soil and that of existing in the root of the plant. Under native conditions the iso-osmotic pressure of plants is substantially equivalent of the osmotic pressure of a 0.3 M sugar solution; under in vitro conditions it is somewhat lower, about 0.25 M. Under arid conditions, the osmotic pressure of soil exceeds that of the root cells, the soil withdraws water from the plants and therefore, the plants are thirsting. When the osmotic pressure is higher than iso-osmotic, the withdrawal of water may be of such extent that, due to the intracellular proto-plast formation, the interconnection of the cells will be abolished.
The invention is based on the recognition that water deficiency can be induced for the plants in culture bottles with a relative moisture content of otherwise 100% by adding to the nutrient medium one or more non-toxic additive(s) being suitable to adjust the osmotic pressure in a concentration resulting in the osmotic pressure of an 0.2 to 0.4 M sugar solution and/or permanently decreasing the temperature below from 2 to 10 C. Decrease of temperature also induces water deficiency since the membrane processes of water uptake depend on the temperature. By altering the above-said factors, i.e. osmotic pressure and temperature, the "training" effect of the native conditions can yet be 2~64603 simulated during the tissue cultivation whereby the plant becomes capable of directly pricking out. It is the same for the plant whether water is present or not when it cannot resorb water; thus, the plant surprisingly responses to the increased osmotic pressure of the culture medium or the decreased temperature by intense development of roots and transformation of its habit.
Accordingly, the present invention relates to a process for the adaptation to the direct open-field pricking-out of plants propogated by tissue culture. According to the process of the invention, the plants propagated by tissue cultivation in a manner known ~E se a) are further cultivated in one or more step(s) on a nutrient medium having an osmotic pressure corresponding to 0.2 to 0.4 M sucrose concentration and, in the case of a multi-step cultivation, the osmotic pressure of the nutrient medium is adjusted to a value increasing step by step within the said range and/or b) they are stored at 2 to 10 C for at least 30 days.
Surprisingly, the plants grown in this way can directly be pricked out to the open field without any process of adaptation or they can be stored at a low temperature (5 to 10 C) for 3-4 months.
The substance used for adjusting the osmotic pressure has to comply only with two demands. One of these is to have influence on the osmotic pressure. A number of native oligo-and polymers, e.g. agar or gelatine have only an insignificant osmotic pressure ~d'Ans, Lax: "Taschenbuch f~r 2û64603 Chemiker und Physiker", 2nd edition, Springer-Verlag Berlin--Gottingen-Heidelberg, page sos)~
It is known that the osmotic pressure depends only on the number of particles being present in the medium but not on the nature of the substance used. Thus, e.g. 34.2 g tO l mol) of sucrose in 1 litre of medium results in the same osmotic pressure as 3.73 g/litre of potassium chloride does since, in addition to the lower molecular weight of the latter one, the dissociation of the salt has also to be considered, i.e. both chloride and potassium ions contribute to the osmotic pressure as separate particles. However, these facts are known to a person skilled in the art and the amount needed of the substance used is easy to calculate.
The other requirement is the absence of any phytotoxicity of the substance in question. Therefore, sodium salts are excluded.
Of the substances being suitable in principle, those otherwise utilized in the tissue cultivation such as sucrose, other sugars, sugar alcohols, nutritive salts generally used in a nutrient medium and the li~e are preferred.
Regarding that the water uptake of plants by osmotic pressure difference is a quite universal principle, the proces~ according to the invention can be used safely also for the propagation of all arborescent and soft-stalk annual and perennial plants, supposing that the plant is suita~le for propagation by tissue culture at all. ~ereinafter, some plants are enumerated by way of illustration which were -6- 206~603 successfully propagated by using the process according to the invention.
Garden-trees and ornamental shrubberies, e.g. willow-wood and rhododendron;
greens such as asparagus, tomato, broccoli, chicore, pumpkin, melon, kohlrabi, cucumber, potato, beet;
potted ornamental plants such as Phoenicopterus ruber roseus, rubber-plant, violet of Cape Colony, philodendron, bromelia, Dieffenbachia, begonia, cyclamen, dragon lily, pelargonlum;
bulbous ornamental plants such as gladiolus, freesia, hyacinth, iris, lilies;
cut flowers such as Michaelmas daisy, rose, chrysanthemum, orchidea, bell-flower, pink, gerbera.
The plants grown according to the in~ention can directly be pricked out to the site or nursery-garden. Their survival ratio exceeds that of the plants subjected to the traditional adaptation process. Considering that the pricking-out directly follows the period of tissue culture, the proce6s is named Tissue Culture Direct process and the plant obtained is called TCD plant.
The process according to the invention is illustrated in detail by the following non-limiting Examples.
Ex~mpl~ 1 Limonium latifolium Two-step process Limonium latifolium ~a perennial ornamental plant) was propagated on the following traditional tissue culture 20646~3 nutrient medium:
NH4N3 1600 mg/l CCl2 62 0.025 mg/l KN03 1900 mg/l Mesoinositol lO0 mg/l CaCl2.2H20 440 mg/l Vitamin B1 0.1 mg/l MgS04.7H20 370 mg/l Vitamin B6 0 5 mg/l Na2EDTA+FeSo4-7H2o 25 mg/l Nicotinic acid 0.5 mg/l H3B03 6.1 mg/l Glycine 2.0 mg/l MnS04.4H20 22.3 mg/l Kinetin 2.0 mg/l ZnS04 4H2 8~6 mg/l ~-Na~thylacetic acid 0.02 mg/l KI 0.83 mg/l Sucrose 30000 mg/l Na2M004-2H20 0.25 mg/l Agar 11000 mg/l CuS04-5H20 0.025 mg/l pH 5.7 The above components were present in 1 litre of nutrient medium therefore, the medium contained 3 % of sucrose and 1.1 % of agar. It should be noted that the sucrose content of this traditional tissue-cultivating nutrient medium remained far below the amount needed to adjust the osmotic pressure to the desired value; it served in the nutrient medium as an energy source of plants scarcely assimilating under the tissue culture conditions.
After achieving a satisfactory growth (about 30000 pieces), the plants were transplanted for rooting to a nut-rient medium having the following composition. Ten pieces were transplanted in each glass-bottle of 400 ml volume:
NH4N3 1600 mg/l CoCl2.6H20 0.025 mg/l KN03 1900 mg/l Mesoinositol 100 mg/l CaC12.2H20 440 mg/l Vitamin B1 O.S mgJl MgS04.7H20 370 mg/l Vitamin B6 l.0 mg/l Na2EDTA+FeS04.7H20 25 mg/l Nicotinic acid 5.0 mg/l H3B03 6.2 mg/l Pantothenic acid 2.5 mg/l MnS04-4H20 22.3 mg/l Naphthylacetic acid 0.5 mg/l ZnS04.4H20 8.6 mg/l Sucrose 70000 mg/l KI 0.83 mg/l Agar 11000 mg/l Na2MoO~ 0.25 mg/l pH 5.7 The sugar content and the osmotic pressure of the root-ing nutrient medium was more than two-fold of the propagat-ing nutrient medium therefore, its osmotic pressure equalled that of a 0.21 M sucrose solution. The transformation of the plants started in this nutrient medium. After achieving a height of 5 to 6 cm of the plants and beginning of rooting, the plants were transferred onto a third nutrient medium which was different from the second (inductive) nutrient medium only therein that it contained 110000 mg/litre (0.32 M) of sucrose and additionally 250 mg/litre of activated carbon. The latter one was necessary for protecting the roots developed from the light. 25 ml of the above nutrient medium each were filled into transparent plastic bottles equipped with a sterile cover. One single plant was put into each small-angle truncated pyramid-form bottle of 6 cm in height. The bottles were maintained at 22 to 25 C
temperature under illumination until the nutrient medium was interspersed ky the roots. At this point the plant was a complete TCD plant which could be pricked out without adaptation or be stored in a refrigerator at S to 10 C
temperature for 3 to 4 months until the use.
Example 2 g Preparation of a Spathyphylum TCD plant So-called microshoots, i.e. unrooted shoots were prepared from Spathyphylum plants (a potted ornamental plant) which were then transplanted into plastic bottles filled up to a height of 2 to 3 cm with the inductive nutrient medium of Example 1. Either three microshoots or one having three shoots branching at the stock were planted into each bottle. The bottles were kept at 22 C
under daylight illumination. Thus, they achieved the TCD
quality after about 6 weeks. ~he plants could i~mediately be potted without any green-house adaptation therefore, they could be grown to quickly marketable ornamental plants.
Example 3 Preparation of a Gerbera jamesoni TCD plant ~ erbera jamesoni plants were propagated by tissue culture and then rooted in a known manner. The plants having roots of 1 to 1.5 cm in height were placed into plastic bottles filled with the nutrient medium up to a height of 2 to 3 cm. Each plant was put in a separate bottle. The inductive nutrient medium described in detail in Example 1 was used, except that it contained 130 g/litre of sucrose and 500 mg/litre of activated carbon. The seedlings became useful for planting after growing for 6 weeks.
Example ~
Preparation of cucumber TCD plants in two steps Clones of cucumber F1 hybrids propagated by tissue culture in a known manner were placed onto a rooting nutrient medium with a composition corresponding to that of 2~6~603 the rooting (inductive) nutrient medium of Example 1, except that it contained 50 g/litre of sucrose and 0.01 mg/litre of naphthylacetic acid. The cucumber plants were grown on this nutrient medium up to the 4 to 5-bud (leaf) stage and subsequently, the rooted plants were transferred to a nutrient medium containing 50 g/litre of glucose (i.e.
having an osmotic pressure corresponding to that of 100 q/litre of sucrose~ and 0.01 mg/litre of naphthylacetic acid. After growing on this nutrient medium for at least 2 weeks, the plants could be used for plantation or stored at 5 to 10 C.
Example 5 Preparation of asparagus TCD plants a) Preparation of the propagating material The plants rooted in a known manner were put under sterile conditions onto the surface of the solidified nutrient medium. The composition of the medium corresponded to the first medium of Example 1, except that it did not contain plant hormones. The flask planted with 15 to 20 plants were incubated at 17 C under short daylight ~llumination for 21 days. The incubation was considered to be finished when a bud of 1 to 2 mm size appeared beside the fresh shoots. The resting buds were separated with the root part or the root stem developed was divided and transplanted into fresh flasks until 10000 starting plants were obtained.
b) Preparation of a TCD plant by cooling A part of the plants prepared were transferred to a cooling chamber and stored under cooling at 5 C. A few flasks were taken out in every 10 days and the plants were pricked out into a soil of 25 C which is the optimum temperature for asparagus. Before pricking the old shoots were cut back in a height of 0.5 cm. Care should be taken to make the soil free from weeds for a long period (e.g. by treatment with methyl bromide). Before pricking out, the soil was sprinkled and a furrow of 12 cm in depth was opened on pricking. Directly before transferring the plants, a water-retaining material, e.g. a swellable acryl polymer, turf or the like was introduced to the furrow. The plants were placed onto this layer, then covered by a soil layer of 8 to 10 cm in thickness and the soil was compacted by using light roller. The shooting of the plants pricked out in intervals of 10 days each was observed. The results are summarized in the following Table:

Duration of storage at 5C Shooted plants Rate of shooting day % day It is shown that the plants treated according to the invention shooted safely at a much more rate than the untreated plants from which only a few shooted after 40 days c) Preparation of TCD plants by changing the osmotic pressure 2~64603 The other part of plants prepared according to a) above were inoculated onto a nutrient medium corresponding to the inductive nutrient medium described in Example 1, except that it contained 0.3 mol/litre of sucrose. The plants were kept on this nutrient medium at 25 C under continuous illumination for 30 days, then taken out from the flasks and the old shoots being present were cut back as described under b) above. The plants were pricked out into a soil of 25 C temperature as described under b) above. The behaviour of the transferred plants was similar to that of the asparagus seedling.

Claims (4)

1. Process for the adaptation to the direct open-field pricking-out of plants propagated by tissue culture, which comprises that the plants propagated in a manner known per se a) are further cultivated in one or more step(s) on a nutrient medium having an osmotic pressure corresponding to 0.2 to 0.4 M sucrose concentration and, in the case of a multi-step cultivation, the osmotic pressure of the nutrient medium is adjusted to a value increasing step by step within the said range and/or b) they are stored at 2 to 10 °C for at least 30 days and the plant thus obtained is directly pricked out to open-field or marketed to such purpose.

2. A process as claimed in claim 1 under a), which comprises using sucrose, glucose, a vegetable nutritive salt or a mixture thereof to adjust the osmotic pressure.

3. A process as claimed in claim 1 under a), which comprises individually putting the plants together with the nutrient medium of the last step under sterile conditions into transparent plastic bottles and further growing them therein.

4. A process as claimed in claim 1 under b), which comprises storing the plants at a temperature of 5°C for 30-50 days.