CULTURE MEDIUM SUPPORTS FOR PLANT TISSUE CULTURE

Abstract: The culture medium support is prepared from ceramic fibers releasing substantially no components harmful to plant tissue culture, is used in combination with a liquid culture medium, promotes the propagation, regeneration, and root formation of cultured plant tissues, improves the rate of survival during acclimatization and the growth after acclimatization of the plantlets, is applicable to a variety of plants, can be used continuously from tissue culture to planting after acclimatization and accomplishes labor saving during acclimatization.

Description: DESCR I PT I ON CULTURE MEDIUM SUPPORTS FOR PLANT TISSUE CULTURE Techn i ca I Field This invention relates to a culture medium support used in combination with a l iquid culture medium for plant tissue culture, more particularly to a culture medium support made of ceramic fibers, .and further relates to a culture medium support for plant tissue culture usable also as a acclimatization culture medium for plantlets. Background Art Agar has been used most commonly as a culture medium support in plant tissue culture and a combined use of liquid culture media and special supports such as glass fibers and absorbent cotton has also been known. Agar, however, contains unidentified substances which hinder the propagation, regeneration, and/or growth of the root of plant tissue cells and even purified agar consi erably retards the propagation, regeneration, and/or root formation in some plants. Besides, a culture medium containing agar is a gel and the waste matters and the harmful substances produced by the cultured tissues may diffuse so slowly as to make culture extremely difficult for some plants. Moreover, in the plantlets obtained by tissue culture in a gelled culture medium such as agar, the roots regenerate from the cultured tissues and grow straight with no roothair in smal l numbers or they are the so-cal led underwater roots. These plantlets show extremely poor survival when directly transplanted to a accli atization culture medium and acclimatized. Any culture medium adhering to the roots, if brought into the acclimatization culture medium, may cause decay and death of the plant as a result of contamination by fungi. A better removal of the culture medium from the plantlets wil l damage the plant more and result in retarded growth of the plant after accli atization. Hence, with the conventional culture method using agar or the like, there was a need to use the acclimatization culture medium prepared from special ly sterilized perlite or vermiculite at the time of ace I i mat i zat i on. Glass fibers, when used as culture medium support, present a problem in that the alkaline components pass into the medium while in use and change the pH and chemical composition of the culture medium. Absorbent cotton is known to yield worse results than agar culture media. Therefore, an object of this invention is to provide a novel culture medium support to be used in combination with a l iquid culture medium for p I ant t i ssue cu I ture. Another object of this invention is to provide a culture medium support which promotes the propagation, regeneration, and root formation of plant tissues and improves the rate of survival during accl imatization and growth after acclimatization of plantlets. A further object of this invention is to provide a culture medium support which enables one to carry out culture of plant tissues that can be cultured with difficulty by a gel cult re medium such as agar. A stil l further object of this invention is to provide a culture medium support which can be used continuously from the culture of tissues to the planting of the plant after accl i atization, saves labor in the ace I i mat i zat i on work, and improves the rate of survival at the time of accl imatization and the production rate after ace I i mat i zat i on. Disclosure of Invent i on Thus, this invention provides a culture medium support to be used in combination with a liquid culture medium for plant tissue culture prepared from ceramic fibers which do not substantial ly release components harmful to plant t i ssue cu I ture. Ceramic fibers which do not release components harmful to the propagation, regeneration, and root formation of plant tissues in any step of tissue culture, for example, in the impregnation with a liquid culture medium and the subsequent sterilization, can be used in accordance with this invention. Moreover, it is important that the ceramic fibers do not substantially react with the liquid culture medium to be used in combination and do not affect the pH of the liquid culture medium. In particular, aluminum ions dissolving out of the support will react with phosphate ions in the liquid culture medium to form water- i nso I ub I e aluminum phosphate, resulting in shortage of phosphate ions. Furthermore, release of alkal i ions wil l change the pH of the liquid culture medium in use and hinder the propagation, regeneration, and root formation of plant tissues. The ceramic fibers to be used in accordance with this invention include fibers based on silica and alumina such as sil ica-alumina (S i 02 - A 12 0 3 ) fibers, sil ica-alumina-zirconia (S i 0 2 - Al 2 O3 -Zr0 2 ) fibers, and si lica- alum i na-chrom i a (S i 02 -A I 2 °3 ' Cr 2 °3 ) fibers; fibers based on si l ica; fibers based on alumina; and carbon fibers. Sil ica-alu ina fibers are most desirable from the standpoint of release of undesirable components, reactivity with l iquid culture medja, stabi lity, and price. Silica-alumina fibers are commercially manufactured in large quantities as refractory and insulating materials in building furnaces and as raw materials for inorganic papers. As these fibers often contain l bricants or other additives for improved processab i I i ty, those which have been heated at or above temperatures where such additives can be el iminated should preferably be used. The bulk density of the ceramic fibers of this invention ranges from 0.005 to 0.3 gp / cm , preferably from 0.01 to 0.2 9 / cm , depending upon the intended use. Fibers with a bulk density of less than 0.005? / as 3 are difficult to handle and those above 0.3 g? / cm hinder the growth of roots. The ceramic fiber culture medium supports of this invention are hydrophil ic by themselves, but they can be improved in the hydrophi lic property by addition of substances harmless to the growth of plants such as nonionic surfactants and higher a I coho I s. - The ceramic fiber culture medium support of this invention (hereinafter referred to as the support of this invention) may be in the form of bulk, but it should preferably be in the form of a needled or unneedled blanket or loose wool in which the fibers are oriented in one direction. When the main objective is the propagation and regeneration in a culture vessel, the support of this invention can be used in the form of paper, sheet, blanket, or loose wool matching in shape to that of the culture vessel. The fibers in the support of this invention may be oriented ' vertical ly, " horizontal ly, or at random, but the vertical orientation is desirable in consideration of the rise of liquid culture medium, the division of plantlets, and the growth of roots. The support of this invention is used in combination with the known l iquid culture media and their modifications such as Murash i e-Skoog, White, Knudson, . L i nsma i er-Skoog, Hel ler, Gautheret, N i tsch- i tsch, Ericksson, Ga borg- M i I I er-Oj i a, and Tsuka oto-Kano. Any of these l iquid culture media can be combined with the support of this i vention preferably by impregnation, but also by dripping or spraying. For culture of plant tissues with the use of the support of this invention, the support of a suitable amount is placed in a culture vessel, impregnated with a required amount of l iquid culture medium, steril ized in an autoclave or the like, al lowed to cool, and the previously steri l ized plan tissues are explanted to the culture medium thus prepared. The same care as in the case of culture with an agar c lture medium is taken here, but with the use of the support of this invention, further addition of the l iquid culture medium, addition of growth hormones, and cleaning and exchange of the l iquid culture medium can be carried out without rans lanting of the cultured tissues in an operation under sterile cond i t i ons. The support of this invention can be used as accl imatization culture medium for plantlets, if necessary in combination with commercial liquid ferti l izers. In the combined use, a l iquid fertil izer prepared at a required concentration is suppl ied to the support of this invention by dripping, spraying, or sprinkl ing. For accli atization of plantlets with the accli atization culture medium thus prepared, when the plantlets are formed, the culture medium is washed from the root system with running tap water, and after that the plantlets are placed in a suitable amount of the accl imatization culture medium in a culture vessel with the roots of the plantlets enclosed in the medium, being kept in a greenhouse for one week to one month under suitable shielding of l ight and humidity. The care to be exercised here is the same as with the use of perlite or ver iculite. However, as the support of this invention is capable of hol- ing water, sprinkling of water is usually unnecessary and the plantlets after accl imatization can be transplanted to ordinary soi l culture or soi l less culture without removal of the culture medium. Thus, the support of this invention provides the roots of the plantlets with a suitable amount of water and aeration and, because of high contents of voids in the support, the culture medium appears to maintain a large volume of air inside even after sprinkl ing and prevent a shortage of oxygen needed for the roots. As described above, the support of this invention can be used not only as supports for culture media for the propagation, regeneration, and root formation in tissue culture but also as supports for acclimatization culture media for the acclimatization of the plantlets from the tissue culture. It is therefore possible to carry out the propagation, regeneration, and root formation of plant tissue and the accl imatization of the plantlets in succession simply by washing out the l iquid culture medium previously used with water and adding the next l iquid culture medium without transplan ing. This consecutive procedure without transplanting markedly reduces damages to the plantlets, improves the yield of plantlets by the tissue culture method, and enhances the rate of survival of plants after accl imatization. Moreover, the plants after accl imatization can be transplanted to normal soi l culture, hydroponic culture, sand culture, gravel culture, and rock wool culture, and especial ly suited to rock wool culture which, simi larly to the support of this invention, u es inorganic fibers. With the use of the support of this invention, the chemical stability and physical properties of ceramic fibers make it possible for a particular liquid culture medium in combined use to manifest its full capabil ity. Furthermore, the support of this invention has a high content of voids and most of such voids remain after the support is combined with a l iquid culture medium. The voids are likely to promote the propagation, regeneration, and root formation of plant tissues or cells and the growth of cultured tissues during acclimatization. It is also likely that the l iquid culture medium in combined use is present among the ceramic fibers as a result of the capillary action or adhesion and this helps the cultured tissues to absorb the liquid culture medium or facilitates removal of the liquid culture medium at the time of acclim-atization. Best Mode for Carrying Out the Invention This invention wil l be i l lustrated with reference to the examples. Exa le 1 : The silica-alu ina fibers (S i0 2 , about 53 wt %; Al 2 0 3 , about 47 wt % ) used as ceramic Fibers had the following properties: 1? of the fibers released 229 ppm Al when heated with 150 ml of 0.5 N hydrochloric acid at 30 * C for 1 hour and showed a pH of 6.5 when immersed in an 8-fold quantity of test water of pH 7 in a closed container at room temperature for 6 days. The fibers were oriented un i d i rect i ona I I y in layers, an organic lubricant was added, and the lubricated fibers were needled and then heated at about 700 °C for about 20 minutes to decompose the lubricant. The resulting sil ica-alumina fiber culture medium support was in the form of a blanket having a bulk density of 0.13 % / as 3 . The support was cut into 2 x 2 x l cm cubes, 1.0 §f of such cubes was placed at the bottom of a tube, 5 en in diameter and 12 cm in height, with the fibers running vertically, 8 ml of the Murash i ge-Skoog culture medium (containing 0.1 m I b of naphthaleneacetic acid, 0.1 w% I b of benzyladenine, and 30 - I „ of sucrose) was added in portions, and the tube was capped with aluminum foil, steril ized at 120 °G for 15 minutes in an autoclave, and allowed to cool at room temperature. The stem base of Cyperus pulchella (cv:nanas) soaked in a (7%) solution of sodium hypochiorite (for 10 rain. ) in advance was cut into 2 mm cubes, explanted in the tube inside a laminar air flow cabinet, . cultured at 25°C in a light and dark cycle of 12/12 hr. , l ight intensity of 3000 lux for 3 weeks, and the rate of survival, length of shoots, total growth of shoots, number of shoots , and root formation were examined. The resu I ts are shown i n Tab I e 1. ExampI e 2 : Silica-alumina-zirconia fibers ( S i 0 2 . about 50 wt % ; Al 2 0 3 , about 35 wt % Zr0 2 , about 15 wt % ) which released 77.7 ppm Al and showed a pH of 6.5 when tested as in Example 1 were converted into a .s i I i ca-a I um i na-z i rcon i a culture medium support in the .form of a blanket (bulk density 0.13 gt / ^ ) in the same manner as i n Examp I e 1. With the use of this support, the same plant tissues as in Example 1 were cultured and examined under the same conditions as in fxample 1. The resu Its are shown i n Tab I e 1. Examp I e 3 : Silica-alumina-chromia fibers (S i 0 2 > about 55 wt %; Al 2 0 3 , about 42 wt %; Cr 2 0 3 , about 3 wt X) which showed a pH of 6.5 when tested as in Example 1 were converted into a silica- alumina-chromia culture medium support in the form of a blanket (bulk density 0.13 g / ακ 3 ). With the use of this support, the same plant tissues as in Example 1 were cultured and examined as in Example 1. The results are shown in Table 1. Comparat ive Examp I e 1 : Commercial purified agar for culture use (Difco Bacto agar) was added at a ' rate o f & 9 / i to the same Murash i ge-Skoog culture medium as used in Examples 1-3 and the resulting agar culture medium, 8 ml, was poured into a tube of the same type as used in Examples 1-3, and the same plant tissues as in Example 1 were cultured and examined as in Example 1. The results are shown in Table 1. Comparat i ve Examp I e 2 : Using commercial rock wool (loose wool, hydrophilic type) for plant culture use (Sio 2 , about 46 wt %; -Al 2 0 3 , about 13 wt % ; CaO, about 18 wt % , MgO, about 12 wt %; Fe 2 0 3 , about 8 wt %), the same plant tissues as in Example 1 were cultured and xa ined as in Example 1. The results are shown in Table 1. Comparat i ve Examp I e 3 : Using commercial glass fibers in bulk (S i 0 2 . about 59 wt % ; Al 2 0 3 , about 4 wt % CaO, about 16 wt % ; MgO, about 5 wt % B 2 0 3 , about 3 wt % ; Na 2 0, about 11 wt % ) , the same plant tissues as in Example 1 were cultured and examined as in Example 1. The results are shown in Table 1. The numerical values in Table 1 are relative to the average values of Comparative Example 1 which are taken as 100, except for the root formation which is shown in actual ly determined va I ues. As is apparent from Table 1, the use of ceramic fiber culture medium supports in Examples 1-3 has considerably improved the survival rate, growth of shoots, and root formation compared with Comparative Examples 1-3. Table 1 Examp I e 4: The same silica-alumina fibers as in Example 1 were converted into a support in the form of a blanket (bulk density 0.1 g / cm 3° ) The support was cut into 2 x 2 x 2 cm cubes, 0.8 g of such cubes were placed at the bottom of a tube, 5 cm in diameter and 12 cm in height, with the fibers running vertical ly, and using 8 ml of the same Murash i ge-Skoog culture medium as in Example 1, the 2 mm cubes of the stem base of Cyperus pulchel la (cv:nanas) were cultured for 3 weeks under the same conditions as in Example 1, and the survival rate, length of shoots, number of shoots, and root formation were examined. The resu Its are shown i n Tab I e 2. As in Examples 1-3, the control culure was carried out here as in comparative Example 1 and the results were expressed relative to the average values, taken as 100, of the control. Table 2 As is apparent from Table 2, the use of the si l ica-alumina culture medium support here has considerably promoted the growth of shoots. The plantlets of Cyperus obtained in this Example were taken out of the tube together with the silica-alumina fiber culture medium support, washed with running water, placed with the support in a plastic container, subjected to acclimatization in a greenhouse with lb % shielding of light for 10 days, and the rate of survival was exam i ned. Separately, a culture soil for acclimatization use consisting of a mixture of 2 weight parts peat moss, 1 weight part perlite, and 0.5 weight part vermicul ite was placed in'a plastic container of the same type as in this Example to a thickness of 2 cm . The plantlets of Cyperus obtained in the control culture in this Example (the plantlets were removed from the tubes and the agar was washed from the root system with running tap water) were planted, accl imatized, and examined under the same conditions. With the rate of survival of the control taken as 100, that of this example is 143. Thus, the use of the si l ica-alumina fiber culture medium support improved the rate of survival at the time of ace I i mat i zat i on. Examp I e 5 : Using the same si lica-alumina fiber culture medium support as in Example 4, 5 mm sections of the tip of the runner of Nephrolepis exaltata var. marshal I i i were explanted on the culture medium as in Example 4, cultured for 8 weeks under the same conditions as in Example 1, and survival rate, length of shoots, number of shoots, and root formation were examined. The results are shown i n Tab I e 3. Example 6 : The same s i I i ca-a Iumi na-zi rcon ia fibers as in Example 2 were converted into a sil ica-alumina- zirconia fiber culture medium support in the form - * > of a blanket (bulk density 0.1 gp / c ). Using this support, the same plant tissues as in Example 5 were cultured and examined as in Example 5. The results are shown in Table 3. As in Examples 1-3, the results of Examples 5 and 6 were expressed relative to the control, taken as 100, cultured as in Comparative Example 1 . As is apparent from Table 3, the culture medium supports prepared from two different ceramic fibers were found to promote the growth of shoots and root formation considerably compared with the contro I . Table 3 Examp I e 7 : The same silica-alumina fiber culture medium support (bulk density 0.1 g / .OH 3 ) as in Example 4 was cut into 1.8cw cubes, about 0.6 g of such cubes was placed in a bottle, and 6 ml of the same Murashi ge-Skoog culture medium as in Example 1 was poured into the tube. Sections of a leaf of Saintpaul ia ionantha Hybrida A, in 2 mm cubes, were explanted on the culture medium as in Example 1, cultured for 40 days under the same conditions as in Example 1, and the survival rate, diameter of callus, length of shoots, number of shoots, and root formation were examined. Expressed relative to the averages, taken as 100, of the control cultured in an agar culture medium as in Comparative Example 1, the results were: survival rate 144, iameter of callus 269, length of shoots 288, number of shoots 260 , and root formation 135 . As is apparent from the results, the use of the si l ica-alumina fiber support in this Example considerably promoted the growth of callus and regeneration and growth of shoots. Exampl e 8: The same silica-alumina fibers as in Example 1 were converted into a sil ica-alumina fiber culture medium support in the form of a blanket (bulk densi ty 0.07g / cm 3 ). The support was cut into 2 x 2 x 2 cm cubes, 0.56g of such cubes was placed on the bottom of a tube, 5 cm in diameter and 12 cm in height, with the fibers running vertically, sections of a leaf of Saintpaul ia ionantha Hybrida B, in 2 mm cubes, were explanted on the culture medium as in Example 1, cultured for 13 weeks under the same 'conditions as in Example 1, and the survival rate, cal lus formation, length of shoots, number of shoots, and root formation were examined. The results expressed relative to the averages, taken as 100, of the control cultured in an agar culture medium as in Comparative Example 1 were: survival rate 100, callus formation 100, length of shoots 276, number of shoots 105, and root formation 100. As is apparent from these results, the use of the silica-alumina fiber culture medium support of this Example considerably promoted the growth of Shoots. Examp I e 9 : The silica-alumina fiber culture medium support prepared in Example 1 was cut into 2 cm cubes, a cut was made in the center of such cubes, and the accl imatization culture medium was prepared from such cut cubes. The plantlets of Cyperus pulchella (cv : nanas) -obta i ned by tissue culture (in the Murash i ge-Skoog culture medium for 20 days) were removed from the tubes and the agar was washed from the root system with running tap wate . Then the plantlets were inserted into the cut in the aforesaid accl imatization culture medium. The plantlets thus planted were placed in a plastic culture vessel, acclimatized in a greenhouse with 75% shielding of light for 10 days, and the rate of survival was examined. Water was sprinkled once a day until the culture medium was saturated with water and any surplus water was discharged from the culture vessel. As a control in this Example, a culture soi l composed of 2 weight parts peat moss, 1 weight part perlite, and 0.5 weight part vermiculite was introduced as acclimatization culture medium into a plastic culture vessel of the same type as in Example 4 to a thickness of 2 cm , the same plantlets as used in Example 4 were placed in the accl imatization culture medium and accl imatized under the same conditions as in this Example above. The rate of survival was 143 with reference to that of the control as 100. Examp I e 10 : The plantlets of Alocasia macrorhiza cultured in the Mu ash i ge-Skoog culture medium in a tube for 60 days were planted in 3 x 3 x 3 c cubes of accl i atization culture medium and accl imatized under the same conditions as in Example 9, and the rate of survival was examined, The control in this Example was prepared and accl imatized under the same conditions as in Example 9 except for the thickness of the soil culture being 3 cm . The rate of survival of this Example was 125 relative to that of the control as 100. The plants accl imatized in this Example and its control were planted and their growth was examined. The test plant and the acclimatization culture medium were inserted into a hole, 3 cm in diameter and Zcm in depth, made at the center of a 7 x 7 x 6 cm commercial rock wool block for plant culture use, the block was placed in a greenhouse with 45% shielding of light, and cultured for 30 days while sprinkled once a day with a 2000-fold dilution of a Hyponex stock solution (liquid fertilizer manufactured by Murakami Bussan K.K..5 (N)-10 (P)-5 (K) and m i cronutr i ents) . As a control, the plant acclimatized as the control in this Example was taken out of the culture vessel together with as much of the culture soil a ' s possible, and planted in the center of a rock wool block for plant culture using the soil culture used in the acclimatization and cultivated in a greenhouse as above. When the growth of the plants was compared in terms of the number of leaves, the test plant in this Example produced growth of 150 relative to 100 for the contro I . Industrial Appl icabi lity As described above, a culture medium support of this invention is used in combination with a liquid culture medium for plant tissue culture, and if necessary in combination with commercial liquid fertilizers as supports for acclimatization culture medium for plantlets.

Claims: C LA I MS (1) A culture medium support for plant tissue culture comprising ceramic fibers releasing substantially no components harmful to plant tissue culture and used in combination with a ■ liquid culture medium. (2) A culture medium support for plant tissue culture according to Claim 1 in which the ceramic fibers do not substantially react with the liquid cuI ture medium. (3) A culture medium support for plant tissue culture according to Claim 1 in which the ceramic fibers are-hydrophilic. (4) A culture medium support for plant tissue culture according to Claim 1 in which the ceramic fibers are silica-alumina fibers. (5) A culture medium support for plant tissue culture according to Claim 1 in which the ceramic fibers are oriented vertically. (6) A culture medium support for plant tissue culture according to Claim 1 in which the bulk density of the support ranges from 0.005 to 0.3 g / cm 3 .

Inventors: OISHI TORU, OISHI, TORU
Applicants: NIPPON STEEL CHEMICAL CO, NIPPON STEEL CHEMICAL CO., LTD.
Application year: 1986
Main classification: A01G7
IPC codes: A01G31, A01H4