CA1217339A - Method for perennially mass-propagating the useful annual plants by use of the shoot primordia - Google Patents
Method for perennially mass-propagating the useful annual plants by use of the shoot primordiaInfo
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- CA1217339A CA1217339A CA000443717A CA443717A CA1217339A CA 1217339 A CA1217339 A CA 1217339A CA 000443717 A CA000443717 A CA 000443717A CA 443717 A CA443717 A CA 443717A CA 1217339 A CA1217339 A CA 1217339A
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- primordium
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- Developmental Biology & Embryology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Abstract
Abstract of the Disclosure A method for perennially mass-propagating a useful annual plant by use of shoot primordium, comprises cutting off a shoot apex from the useful annual plant and transplanting the thus cut off shoot apex into an artificial culture medium containing an inorganic salt composition and a plant growth hormone; rotation-culturing the shoot apex under the conditions that a temperature is 15-30°C, an illumination intensity is 2,000-9,000 luxes and a number of revolution is 0.5-10 rpm to propagate the shoot primordium; and stationally culturing the shoot primordium to form young plants. The useful annual plant can be perennially mass-propagated in a short period of time while maintain-ing the genotype and chromosomal type for a number of years. This method is particularly effective with respect to the propagation of triploid water melon, hybrid vigor Indian corn, hybrid vigor rice plant, and hybrid morning glory.
Description
1;i~17339 The present invention relates to a method for mass-propagating the useful annual plan~s in a short period of time through perennation using the plant shoot primordia which is applicable to the fields of 05 the biology, agriculture, horticulture, pharmacology and the like.
The term "shoot primordium" used herein means ones consisting of primary shoot primordia of the cell conglomerate having a diameter of 50-1,000 ~m in which 0 the cells having plastids are not stratified and secondary shoot primordia of cell conglomerate having a diameter of 100-5,000 ~m in which the cells are doubly stratified, wherein these primordia circulate and vegetatively propagate to form hemispheric cell conglomerate.
Maintenance of the genotypes and the chromosomal types of the useful annual plants has been attained by the following two means of the conventional sexual reproduction and the recently developed vegetative propagation. But, the conventional methods have the following drawbacks:
(i) The sexual reproduction is a method in which the seeds are obtained through the meiosis and fertilization, whereby the progeny is reproduced. However, such a method needs a huge labor in screening among a large number of seeds in order to maintain the useful genotype and chromosomal type. Further, it is substantially impossible to maintain the genotype and the chromosomal lZ17339 type in the case of the useful annual plants of, for instance, triploid, hybrid vigor and the like.
(ii) The vegetative propagation relies upon the tissue and cell culture. According to this method, when a 05 part of a stem, shoot apex, leaf, root apex or the like is sterilized and then transplanted into an artificial culture to which a plant growth hormone has been added and cultured therein, dedifferentiation of the cells of the tissure takes place and callus (non-differentiated cell conglomerate) may be obtained. Thus obtained callus can be stored for a long period of time as vegetative cell and can be mass-reproduced, when this callus is subcultured. In case this callus is trans-planted into a culture medium for redifferentiation, somatic cell embryogenesis or nucellular embryony takes place to produce the juvenile young plants and to reproduce the juvenile plants. This method can be relatively easily applied to any annual plant until the stage of the dedifferentiation and the callus propaga-tion, but during the propagation, chromosomal mutationand gene mutation are often induced so that the same genotype and chromosomal type as the parent ones may not be obtained.
On the other hand, the redifferentiation is difficult in the case of some kinds of the annual plants. Even in the case of the annual plants in which the redifferen-tiation is possible, the long-term culture renders the ability of redifferentiation remarkably lower.
~217339 As mentioned above, in the case of the conven-tional vegetative reproduction, (a) it is difficult to maintain the genotypes and the chromosomal types of the annual plants for a long period of time as in perennial;
05 and (b) it is in most case impossible to redifferentiate the same types as the parent ones. Further, in the case of the callus cells, neither plastid, oil body, vacuole, assimilation substance nor storage substance is produed. Thus, the metabolites useful in the biochemical, agricultural and pharmacological fields can not be substantially produced.
The present invention aims at solving the above-mentioned problems of the prior art.
An object of the invention is therefore to provide a method for mass-propagating the useful annual plants by use of somatic cells, while maintaining the genotype and chromosomal type of the annual plants for a number of years, whereby the useful annual plants can be reproduced and propagated.
Another object of the invention is to provide a method for maintaining and propagating the triploid, heteroploid, chromosome mutation type, chromosomal rearrangement type, mutation genotype, hybrid vigor, hybridous genotype of the useful annual plants for a long period of time.
According to the present invention, the shoot apex is cut off from a useful annual plant and trans-planted in an artificial culture. Thereafter, the 12~339 culture is carried out under rotation while the circum-stance is held in the conditions that the temperature is 15-30C, an illumination intensity is 2,000-9,000 luxes, and the number of revolution is 0.5~10 rpm to obtain 05 the shoot primordia. Then, the thus obtained shoot primordia are stationally cultured to form young plants.
In this method, since the substances such as the plastid, Gil body, vacuole, assimilation substance, and storage substance which are usefui in the pharma-cological and medical fields are produced in a body ofthe shoot primordium as the origin for the propagation and reproduction. Thus, the method according to the present invention can be applied to the production of the substances which are useful in the pharmacological and medical fields. Further, it is also applicable to the propagation and reproduction of edible plants and decorative plants.
These and other objects and features of the invention will be well appreciated upon reading of the following description of the invention in conjunction with the attached drawings with the understanding that any modification, change and/or variation can be easily made by the skilled in the art to which the invention pertains without departing from the spirit of the invention and the scope of the appended claims.
The invention will now be described in greater detail with reference to the accompanying drawings~
wherein:
~2~'7339 Fig. 1 is a view showing the shoot primordium conglomerate of triploid water melon as magnified 5 times;
Fig. 2 is a view showing the shoot primordium 05 conglomerate of the hybrid vigor corn as magnified
The term "shoot primordium" used herein means ones consisting of primary shoot primordia of the cell conglomerate having a diameter of 50-1,000 ~m in which 0 the cells having plastids are not stratified and secondary shoot primordia of cell conglomerate having a diameter of 100-5,000 ~m in which the cells are doubly stratified, wherein these primordia circulate and vegetatively propagate to form hemispheric cell conglomerate.
Maintenance of the genotypes and the chromosomal types of the useful annual plants has been attained by the following two means of the conventional sexual reproduction and the recently developed vegetative propagation. But, the conventional methods have the following drawbacks:
(i) The sexual reproduction is a method in which the seeds are obtained through the meiosis and fertilization, whereby the progeny is reproduced. However, such a method needs a huge labor in screening among a large number of seeds in order to maintain the useful genotype and chromosomal type. Further, it is substantially impossible to maintain the genotype and the chromosomal lZ17339 type in the case of the useful annual plants of, for instance, triploid, hybrid vigor and the like.
(ii) The vegetative propagation relies upon the tissue and cell culture. According to this method, when a 05 part of a stem, shoot apex, leaf, root apex or the like is sterilized and then transplanted into an artificial culture to which a plant growth hormone has been added and cultured therein, dedifferentiation of the cells of the tissure takes place and callus (non-differentiated cell conglomerate) may be obtained. Thus obtained callus can be stored for a long period of time as vegetative cell and can be mass-reproduced, when this callus is subcultured. In case this callus is trans-planted into a culture medium for redifferentiation, somatic cell embryogenesis or nucellular embryony takes place to produce the juvenile young plants and to reproduce the juvenile plants. This method can be relatively easily applied to any annual plant until the stage of the dedifferentiation and the callus propaga-tion, but during the propagation, chromosomal mutationand gene mutation are often induced so that the same genotype and chromosomal type as the parent ones may not be obtained.
On the other hand, the redifferentiation is difficult in the case of some kinds of the annual plants. Even in the case of the annual plants in which the redifferen-tiation is possible, the long-term culture renders the ability of redifferentiation remarkably lower.
~217339 As mentioned above, in the case of the conven-tional vegetative reproduction, (a) it is difficult to maintain the genotypes and the chromosomal types of the annual plants for a long period of time as in perennial;
05 and (b) it is in most case impossible to redifferentiate the same types as the parent ones. Further, in the case of the callus cells, neither plastid, oil body, vacuole, assimilation substance nor storage substance is produed. Thus, the metabolites useful in the biochemical, agricultural and pharmacological fields can not be substantially produced.
The present invention aims at solving the above-mentioned problems of the prior art.
An object of the invention is therefore to provide a method for mass-propagating the useful annual plants by use of somatic cells, while maintaining the genotype and chromosomal type of the annual plants for a number of years, whereby the useful annual plants can be reproduced and propagated.
Another object of the invention is to provide a method for maintaining and propagating the triploid, heteroploid, chromosome mutation type, chromosomal rearrangement type, mutation genotype, hybrid vigor, hybridous genotype of the useful annual plants for a long period of time.
According to the present invention, the shoot apex is cut off from a useful annual plant and trans-planted in an artificial culture. Thereafter, the 12~339 culture is carried out under rotation while the circum-stance is held in the conditions that the temperature is 15-30C, an illumination intensity is 2,000-9,000 luxes, and the number of revolution is 0.5~10 rpm to obtain 05 the shoot primordia. Then, the thus obtained shoot primordia are stationally cultured to form young plants.
In this method, since the substances such as the plastid, Gil body, vacuole, assimilation substance, and storage substance which are usefui in the pharma-cological and medical fields are produced in a body ofthe shoot primordium as the origin for the propagation and reproduction. Thus, the method according to the present invention can be applied to the production of the substances which are useful in the pharmacological and medical fields. Further, it is also applicable to the propagation and reproduction of edible plants and decorative plants.
These and other objects and features of the invention will be well appreciated upon reading of the following description of the invention in conjunction with the attached drawings with the understanding that any modification, change and/or variation can be easily made by the skilled in the art to which the invention pertains without departing from the spirit of the invention and the scope of the appended claims.
The invention will now be described in greater detail with reference to the accompanying drawings~
wherein:
~2~'7339 Fig. 1 is a view showing the shoot primordium conglomerate of triploid water melon as magnified 5 times;
Fig. 2 is a view showing the shoot primordium 05 conglomerate of the hybrid vigor corn as magnified
2.5 times;
Fig. 3 is a view showing the shoot primordium conglomerate o~ the hybrid vigor rice plant as magnified 6 times;
Fig. 4 is a view showing the shoot primordium conglomerate of the hybrid morning glory as magnified 8 times;
Fig. 5 is a vertically sectional view showing the primary shoot primordium of the triploid water melon as magnified 30 times at the central project portion thereof;
Fig. 6 is a vertically sectional view showing the secondary primordium of the triploid water melon as magnified 84 times; and Fig. 7 is a view showing the chromosomes 2n=33 in the shoot primordium of the triploid water melon during the meiosis middle stage in which the right and left side views are photographis of the same image taken with different focuses.
The invention is widely applicable to the useful annual plants including the medical annual plants and the agricultural and horticultural plants, parti-cularly to the triploid water melon (Citrullus battich), 12~7339 ~ 7 -the hybrid vigor Indian corn (Zea mas) and the hybrid vigor rice plant (Oryza sativa) and the hybrid morning glory (Ipomoea _il Roth) for the purpose of the mass-production while maintaining the ~riploid, hybrid vigor 05 and hybrid thereof. In addition to the above water melon, Indian corn, rice plant and morning glory, the invention is also applicable to the medicinal plant such as Japanese green gentian (Swertia japonica), poppy (Paraver somniferum), the useful edible plants such as wheat (Triticum Salivum), soy bean (Glycine max), the useful industrial plant such as rape (Brassica campestris) and safflower (Carthamus tinctorius), the useful horticultural plant such as petunia (Petunia hybrida) and the like.
The method according to the present invention for vegetatively and perennially mass~propagating the useful annual plants over a long period of time by use of the shoot primordia will be explained more in detail with reference to the specific features thereof.
First, after the shoot apex of the useful annual plant is sterilized with a sterilizing solution and washed with sterilized water, the shoot apex portion is cut off under observation using a stereomicroscope.
Then, the thus obtained shoot apex is transplanted into an artificial culture medium containing an inorganic salt composition and a plant growth hormone and subjected to the culture under rotation (hereinafter referred to as "rotation culture") under the conditions that the ~217339 temperature is 15-30C, the illumination intensity is 2,000-9,000 luxes and the number of revolution is 0.5-10 rpm to obtain shoot primordia.
In preparing the shoot primordia, the composi-05 tion and concentration t.hereof in the artificial culturemedium should be slightly varied depending upon the intended plants. As the inorganic salt composition in the artificial culture medium, the composition contained in the known culture media such as Murashige-Skoog (hereinafter referred to briefly as "MS") and Gamborg B5 (hereinafter referred to briefly as "B5") and the like may be used with a slight change in the ingredients thereof. As the plant growth hormone, auxins such as indolylacetic acid, naphthaleneacetic acid, 2,4-dichloro-phenoxyacetic acid, cytokinins such as kinetin,benzylaminopurine may be employed. The culture temper-ature is preferred to be a constant temperature of 15-30C. If it is lower than 15C, the rate of the propagation is low, while if the temperature is too high, the growth is not good and is unstable.
In order to culture the shoot primordium, strong light beams are necessary. The illumination intensity of continuous light beams of 2,000-9,000 luxes is preferable. In the illumination intensity beyond this range, the growth of the shoot primordi~lm is poor.
With respect to the culture, the rotation culture is suitable for the present invention rather than the :~Z17339 g stational culture in which the culture medium is not rotated but kept still. According to the present invention, the good results can be obtained at the low number of revolution from 0.5-10 rpm. In case the 05 stational culture is employed, the growth is slow and it is difficult to form the tissue of the shoot primordium, while if the number of the revolution is too large, much more callus portions are produced and therefore, no excellent results can be obtained.
When the propagation method according to the invention is applied to, particularly, the triploid water melon, the hybrid vigor Indian corn, hybrid vigor rice plant and hybrid morning glory, the shoot primordia which propagate actively can be obtained. Tables 1-4 show examples of the optimum culture media prepared by varying the composition and the concentrations thereof where the shoot primordia of these plants are produced.
The artificial culture medium for obtaining the most rapid and stable propagation of the shoot primordium is one consisting of B5 and benzylaminopurine (BAP 20 ppm) for the triploid water melon (Table 1); one consisting of B5 and naphthaleneacetic acid (NAA, 0.25 ppm) and benzylaminopurine (BAP, 0.125 ppm) for the hybrid vigor Indian corn (Table 2); one consisting of B5 and naphthaleneacetic acid (NAA 0.25 ppm) and benzylamino-purine (BAP 0.125 ppm) for the hybrid vigor rice plant (Table 3); and one consisting of B5 and 2,4-dichloro-phenoxyacetic acid (2,4-D, 0.25 ppm) for the hybrid ~2~7339 morning glory (Table 4).
In the following Tables, NAA, 2,4-D, BAP and K
denote naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, benzylaminopurine and kinetin, respectively.
Table 1 mg/Q)¦ B P K
auxin ~ 0.125 2.0 0.125 2.0 juvenile shoot ~; juvenile juvenile O withered plant primordium primordium plant shoot shoot shoot 0.25 withered . . . . . . withered NAA prlmordlum prlmordlum prlmordlum 1.0 withered withered withered withered withered _ 2,4-D 0.25 withered withered withered withered withered 1 0 w thered withered withered withered withered Note: * the optimum culture medium for the formation of the shoot primordium of trifloid water melon ~Z1733~
Table 2 ~ , \ ytokinin _ BAP K
~ mg/Q) O
(mg/Q) \ 0.125 2.0 O.l25 2.0 ~ . _ O juvanile juvanile withered juvanile withered plant plant plant 0 25 juvanile shoot ~* juvanile juvanile withered . plant primordium~plant plant NAA _ l O juvanile shoot juvanile¦juvanile withered plant primordium plant Iplant 0 25 juvanile juvanile juvanile juvanile withered . plant plant plant plant 2,4-~
1 0 juvanile juvanile juvanile juvanile withered . plant plant plant plant I . I . I
Note~ the optimum culture medium for the formation of the shoot primordium of hybrid vigor Indian corn Table 3 ~/ i O Bn r = K
auxine \ 0.125 2.0 0.125 2.0 O Juvanile withered withered withered withered 0 25 callus shoot '~ shoot _ . primordium primordium NAA _ 1.0 callus shoot withered _ __ ~ prim~rdium 0 25 withered shoot shoot callus callus . primordium primordium 2,4-D _ _ 1.0 withered callus withered jwithered callus Note~ the optimum culture medium for the formation of the shoot primordium of hybrid vigor rice plant ~Z~73;~9 Table 4 ( ~ j --T 'P K
aux ne ~ 0.125 2.0 O.l~ ; O
O Withered liUvanile juvanile juvanile¦juvanile r---- plant plant plant Iplant 0.25 callus ~ callus callus callus ~ callus NAA . !
1 o shoot callus callus callus callus primordium _ _. _ 0.25 shoot '~ '; callus eallus callus callus 2,4-D primordium _ 1.0 shoot callus callus callus calus J primordium , L i l Note: lJ,~.t~J,; the optimum culture medium for the formation of the shoot primordium of hybrid morning glory The resulting shoot primordia are in a form of hemispherical conglomerate. The conglomerates of the triploid water melon (see Fig. 1) and the hybrid morning glory (Fig. 4) indicate light green color or dark green color and have the callus at the base portions thereof. Meanwhile, the conglomerates of the hybrid vigor Indian corn (Fig. 2) and the hybrid vigor rice plant ~Fig. 3) indicate light green color. These shoot primordia prepared in the invention have been actively propagating now for 6-7 months.
Next, when these shoot primordia are trans-planted to solid culture media for forming young plant and the culture media are subjected to stationary :lZ~7339 culture at 15-30C and at the illumination intensity of 1,000-4,000 luxes, a large number of fine cormuses are firstly ormed and roots are formed from the base portions thereof. Two to four months after the culture, 05 the plant having the same genotype, chromosomal type and phenotype as the parent plant can be obtained.
The shoot primordium is a projection having a diameter of 50-80 ~m, of which the surface is smooth at the initial stage (Fig. 5). The constituting cells are uniform samll polygonal cells which effect polyaxial division in which the axis of the division is vertical, parallel, oblique or the like. When the shoot primordium (primary shoot primordium) gradually enlarged and the diameter becomes 100-1,000 ~m (see Fig. 6), two strata of an epidermal system and a cortexal system are formed.
The outermost stratum consists of one or two cell strata in which the axis of the cell division is only parallel. While the cortexal system in -the inner side from the outermost stratum consists of a number of more or less larger cell conglomerate, and at inside of this inner side cortexal system are observed a number of well-developed chloroplast, vacuole, and storage substance grains. The latter shoot premordium (secondary shoot primordium) grows up in a form of the trapizoidal projection having a diameter of not less than 500 ~m, and in the cells of the epidermal system of the outermost stratum at this stage, these are large oil bodies and in the cells of the endodermal system of the inner 1;~17339 stratum, the number of chloroplast is increased and the vacuole is highly grown. ~t this stage, several primary shoot primordia are newly produced around this trapizoidal projection. Through this route, the shoot primordium 05 continously propagates and one cycle of this route terminates in about 7-14 days. Accordingly, one shoot primordium propagates about four times within about 7-14 days.
The observation of the chromosomes in the triploid water melon (2n=33) was made for the purpose of the inspection of the stability in inherency of the shoot primordia thereof, and it was confirmed that all the shoot primordia examined had the same chromosome number of 2n=33 as the parent plant (Fig. 7). It is now therefore possible to vegitatively propagate the triploid water melon while perennially maintaining the triploid. Thus, it is possible to mass-propagate the useful annual plants with the same genotype and chromosomal type over a number of years by using the shoot primordia.
On the other hand, the plastids, vacuoles, oil body, storage substance grain (starch, protein, etc.) are actively produced in the shoot primordia.
To the contrary, in the conventional callus cell method, substantially no visual secondary metabolites are produced. Meanwhile, according to the present invention, there is likelihood that the useful substances can be industrially mass-produced from the living cells by ~Z~7339 using the shoot primordia.
It is also possible to vegitatively propagate the shoot primordia in an inherently and chromosomaly stable state through the subculture at l time per 05 0.5-2 months.
The effects obtained by the invention are that it is possible to vegitatively and perennially propagate the useful annual plants by using the shoot primordia and it is also possible to obtain a large quantity of the population thereof while perennially maintaining the genotype and chromosomal type in the useful annual plants. Moreover, it is possible to manufacture the medical, edible and industrial useful substances using those produced by the living cultured cells.
The propagation rate of the shoot primordia is extremely fast to such an extent that only one shoot apex can produce annually 412_452 shoot primordia, thus enabling the mass-propagation in a large scale.
The invention will be explained more in detail with reference to the specific examples which are never intended to restrict the scope of the invention.
Example l: Triploid water melon (Cirrullus battich) Propagation method:
A modified Gamborg ~5 was employed as the fundamental culture medium for culturing water melon, the composition of which is shown in Table 5.
~Z~7339 Table 5 Gamborg B5 modified culture medium for -triploid water melon mg/Q
NaH2PO4 2H2O 170.0 KNO3 '~L 2,500.0 (NH4) 2 S04 134.0 MgSo4-7H2o '` 250.0 CaCl2 113.0 Fe-EDTA 40,0 MnSO4 4H2O 13.0 H3BO3 3.0 ZnSO~ 7H2O 2.0 Na2MoO4 2H2O 0.25 CuS04 5H2O 0.025 CoCl2 6H2O 0.025 nicotinic acid 1.0 thiami~e, HCl 10.0 pyridoxine, HCl 1.0 myoinositol 100.0 sucrose 20,000.0 naphthaleneacetic acid ~L0-0 . 25 6-benzylaminopurine ~ 0.125-2.0 pH: 5.7-5.8 In Table 5, "~" markecl compounds are used for the modification.
~217339 First, the shoot apex portion having a length of about 15 mm was out off from a juvenile young plant of an actively growing water melon. After washing with sterilizing solution, the leaves were consecutively 05 peeled off from the outer side to the inner side under observation using a stereomicroscope with a pair of tweezers and a knife to pick up a shoot apex of about 1-1.5 mm with two or three fine leaf primordia at the innermost side. This picked-up shoot apex was cultured in the above foundamental culture medium. The rotation culture was effected in a test tube of 27 mm in diameter x 200 mm in length into which 25 mQ of the foundametal culture was poured, under the conditions that the temperature was 15-30C, the illumination intensity was 2,000-9.000 luxes, and the number of revolution was 0.5-10 rpm. The green shoot primordium conglomerate of about 10 mm in diameter was obtained one month after the outset of the culture. Thereafter, this conglomerate was cut every one month to obtain a subdivision of about 5-10 mm in diameter, which was then transplanted into a fresh culture medium as described above and propagated.
Young plant forming method:
As the fundamental culture medium for forming young plant, there was employed a solid culture medium prepared in such a manner that the above fundamental culture medium excluding sucrose, naphthaleneacetic acid and 6-benzylaminopurine was diluted 5 times, and lZ17339 20 g/Q of sucrose, 0.05-0.5 ppm of 6-benzylaminopurine, and 8 g/Q of gelatine were added thereto to obtain the culture medium as adjusted at pH 5.7-5.8. About 80 mQ
of this culture medium was poured into a 300 mQ conical 05 flask, on which was placed stationally a subdivided shoot primordium conglomerate of 3-5 mm in diameter.
The culture was effected under stationary conditions that the temperature was 15-30C, and the illumination intensity was 4,000 luxes (illuminated for 16 hours and non-illuminated for 8 hours). Two to three weeks after the stationary culture, 4-6 pieces of the dark green cormuses each having a length of 3-4 mm were formed per one shoot primordium conglomerate.
According to the invention, it is possible to perennially propagate the annual plants in a large amount. In the case of the triploid water melon, the proparation rate thereof is about three times per month, reaching the propagation rate of 3l2~-5xlo5 per year. That is, about 500,000 juvenile young plants can be produced in year from one parent plant. Thus, according to the present invention, the annual plants can be produced satisfactorily industrially. It was confirmed that the shoots thus obtained had the same triploid as the parent plant. Accordingly, the popula-tion having the same chromosomal type and genotype asthe parent ones can be industrially mass-produced.
Example 2: Hybrid vigor Indian corn (Zea mays) ~Z1~339 Propagation method:
As the fundamental culture medium for the hybrid vigor Indian corn was employed the same solid culture medium as in Example 1 except that 0.25-1 mg/Q
05 of naphthaleneacetic acid and 0.125 mg/Q of 6-benzyl-aminopurine were employed. Pursuent to the method in Example 1, about 1 mm of the shoot apex was cut off and subjected to the culture in the above fundamental culture. One month after the outset of the culture, a green shoot primordium conglomerate having a diameter o~ 20 mm was obtained. Every one month thereafter, this shoot primordium conglomerate was divided into 5-10 mm in diameter, which was then planted into a fresh fundamental culture medium as described above.
Young plant forming method:
The same solid culture medium as in Example 1 was used as the culture medium for forming young plant.
About 80 mQ of this culture medium was poured into a 300 m~ conical flask, and the shoot primordium conglomerate of about 15 mm in diameter was placed on the thus poured culture medium. The culture was carried out under the same conditions as in Example 1. Two to three weeks after the culture, 10-20 mm in ]ength of the cormuses were formed and roots were formed from the base portions -thereof. About two months after the stational culture, about 2-3 juvenile young plants each having a length of 100-150 mm were formed per one shoot primordium conglomerate.
~21'7339 In the case of the hybrid vigor Indian corn, the propagation rate is about four times per month, reaching the rate of 412~6xl06 per year. That is, about l~,000,000 juvenile young plants per year can be 05 produced from one parent plant. Thus, the annual plant can be produced industrially according to the present invention. Further, it was confirmed that the thus obtained shoots indicated the same hybrid vigor as the parent plant. Therefore, the population having the same hybrid vigor can be industrially mass-produced.
Example 3: Hybrid vigor rice plant (Oryza sativa) Propagation method:
As the fundamental culture medium for the hybrid vigor rice plant was employed the same modified culture medium as in Example l except that 0.25-l.0 mg/Q
of naphthaleneacetic acid was employed. Pursuant to the method in Example l, about l mm in length of the shoot apex was cut off, and cultured in the above fundamental culture medium to obtain 5 mm in diameter of a light green shoot primordium conglomerate in one month after the outset of the culture. Every one month thereafter, this shoot primordium conglomerate was divided into about 3-4 mm in diameter, which w~s then planted into a fresh culture medium as described above and was propagated.
Young plant forming method:
As the culture medium for forming young plant was employed a solid c-ulture medium as prepared in ~217339 ~xample 1 in such a manner that the fundamental culture medium excluding sucrose, naphthaleneacetic acid and 6-benzylaminopurine was diluted 5 times, and then 20 g/Q of sucrose and 8 g/Q of gelatine were added 05 thereto to obtain the medium as adjusted at pH 5.7-5.8.
About 80 mQ of the thus prepared culture medium was poured into a 300 mQ conical flask, and the shoot primordium conglomerate ~f about 3-5 mm in diameter was placed thereon. The culture was carried out under the same conditions as in Example 1. Two to three weeks after the culture, the cromuses each having a length of 5-100 mm in length were formed and roots were formed from the base portions thereof. One to two pieces of the juvenile young plants each having a length of about 70 mm were formed per one shoot primordium conglomerate two months after the culture.
In the case of the hybrid vigor rice plant the propagation rate is rather high, reaching the rate of about five times per month, i.e., the rate of 512~2.5x108 per year. That is, about 250,000,000 juvenile young plants can be produced per year from one parent plant. It was confirmed that the thus obtained shoots indicated the same hybrid vigor as the parent plant. Thus, the population of the same hybrid vigor can be industrially mass-produced.
Example 4: Hybrid morning glory (Ipomoea nil R _ ) As the fundamental culture medium for the hybrid morning glory was employed the same modified i217339 cu1ture medium as in Example 1 except that 0.25-1.0 mg/Q
of 2,4-dichlorophenoxyacetic acid was used instead of naphthaleneacetic acid and 6-benzylaminopurine. Pursuant to the method in Example 1, about 0.5-1.0 mm in length 05 of the shoot apex was cut off and cultured in the above fundamental culture medium. One month after the outset of culture, a green shoot primordium conglomerate of about 5 mm in diameter was obtained. Every one month thereafter, the thus obtained shoot primordium conglomerate was div-ded into about 3-5 mm in diameter and was propagated in a fresh culture medium as described above.
Young plant forming method:
As the culture medium for forming young plant was employed the same solid culture medium as in Example 1 prepared in such a manner that the above culture medium excluding sucrose and 2,4-dichlorophenoxyacetic acid was diluted 5 times, and 20 g/Q of sucrose, 0.05-0.5 ppm of 6-benzylaminopurine and 8 g/Q of gelatine were added thereto to obtain the culture medium as adjusted at pH 5.7-5.8.
About 80 mQ of the thus obtained culture medium was poured into a 300 mQ conical flask, on which the shoot primordium of 3-5 mm in diameter was placed.
One week after the culture, projections considered as fine cormus in a size of 1-2 mm in length were formed.
In the case of the hybrid morning glory~ the propagation rate was two times per month, reaching the ~2~7339 rate of 212_4x103 per year. Accordingly~ about 4,000 juvenile young plants can be annually produced from one parent plant. Thus, the mass-production can be fully industrially.
Fig. 3 is a view showing the shoot primordium conglomerate o~ the hybrid vigor rice plant as magnified 6 times;
Fig. 4 is a view showing the shoot primordium conglomerate of the hybrid morning glory as magnified 8 times;
Fig. 5 is a vertically sectional view showing the primary shoot primordium of the triploid water melon as magnified 30 times at the central project portion thereof;
Fig. 6 is a vertically sectional view showing the secondary primordium of the triploid water melon as magnified 84 times; and Fig. 7 is a view showing the chromosomes 2n=33 in the shoot primordium of the triploid water melon during the meiosis middle stage in which the right and left side views are photographis of the same image taken with different focuses.
The invention is widely applicable to the useful annual plants including the medical annual plants and the agricultural and horticultural plants, parti-cularly to the triploid water melon (Citrullus battich), 12~7339 ~ 7 -the hybrid vigor Indian corn (Zea mas) and the hybrid vigor rice plant (Oryza sativa) and the hybrid morning glory (Ipomoea _il Roth) for the purpose of the mass-production while maintaining the ~riploid, hybrid vigor 05 and hybrid thereof. In addition to the above water melon, Indian corn, rice plant and morning glory, the invention is also applicable to the medicinal plant such as Japanese green gentian (Swertia japonica), poppy (Paraver somniferum), the useful edible plants such as wheat (Triticum Salivum), soy bean (Glycine max), the useful industrial plant such as rape (Brassica campestris) and safflower (Carthamus tinctorius), the useful horticultural plant such as petunia (Petunia hybrida) and the like.
The method according to the present invention for vegetatively and perennially mass~propagating the useful annual plants over a long period of time by use of the shoot primordia will be explained more in detail with reference to the specific features thereof.
First, after the shoot apex of the useful annual plant is sterilized with a sterilizing solution and washed with sterilized water, the shoot apex portion is cut off under observation using a stereomicroscope.
Then, the thus obtained shoot apex is transplanted into an artificial culture medium containing an inorganic salt composition and a plant growth hormone and subjected to the culture under rotation (hereinafter referred to as "rotation culture") under the conditions that the ~217339 temperature is 15-30C, the illumination intensity is 2,000-9,000 luxes and the number of revolution is 0.5-10 rpm to obtain shoot primordia.
In preparing the shoot primordia, the composi-05 tion and concentration t.hereof in the artificial culturemedium should be slightly varied depending upon the intended plants. As the inorganic salt composition in the artificial culture medium, the composition contained in the known culture media such as Murashige-Skoog (hereinafter referred to briefly as "MS") and Gamborg B5 (hereinafter referred to briefly as "B5") and the like may be used with a slight change in the ingredients thereof. As the plant growth hormone, auxins such as indolylacetic acid, naphthaleneacetic acid, 2,4-dichloro-phenoxyacetic acid, cytokinins such as kinetin,benzylaminopurine may be employed. The culture temper-ature is preferred to be a constant temperature of 15-30C. If it is lower than 15C, the rate of the propagation is low, while if the temperature is too high, the growth is not good and is unstable.
In order to culture the shoot primordium, strong light beams are necessary. The illumination intensity of continuous light beams of 2,000-9,000 luxes is preferable. In the illumination intensity beyond this range, the growth of the shoot primordi~lm is poor.
With respect to the culture, the rotation culture is suitable for the present invention rather than the :~Z17339 g stational culture in which the culture medium is not rotated but kept still. According to the present invention, the good results can be obtained at the low number of revolution from 0.5-10 rpm. In case the 05 stational culture is employed, the growth is slow and it is difficult to form the tissue of the shoot primordium, while if the number of the revolution is too large, much more callus portions are produced and therefore, no excellent results can be obtained.
When the propagation method according to the invention is applied to, particularly, the triploid water melon, the hybrid vigor Indian corn, hybrid vigor rice plant and hybrid morning glory, the shoot primordia which propagate actively can be obtained. Tables 1-4 show examples of the optimum culture media prepared by varying the composition and the concentrations thereof where the shoot primordia of these plants are produced.
The artificial culture medium for obtaining the most rapid and stable propagation of the shoot primordium is one consisting of B5 and benzylaminopurine (BAP 20 ppm) for the triploid water melon (Table 1); one consisting of B5 and naphthaleneacetic acid (NAA, 0.25 ppm) and benzylaminopurine (BAP, 0.125 ppm) for the hybrid vigor Indian corn (Table 2); one consisting of B5 and naphthaleneacetic acid (NAA 0.25 ppm) and benzylamino-purine (BAP 0.125 ppm) for the hybrid vigor rice plant (Table 3); and one consisting of B5 and 2,4-dichloro-phenoxyacetic acid (2,4-D, 0.25 ppm) for the hybrid ~2~7339 morning glory (Table 4).
In the following Tables, NAA, 2,4-D, BAP and K
denote naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, benzylaminopurine and kinetin, respectively.
Table 1 mg/Q)¦ B P K
auxin ~ 0.125 2.0 0.125 2.0 juvenile shoot ~; juvenile juvenile O withered plant primordium primordium plant shoot shoot shoot 0.25 withered . . . . . . withered NAA prlmordlum prlmordlum prlmordlum 1.0 withered withered withered withered withered _ 2,4-D 0.25 withered withered withered withered withered 1 0 w thered withered withered withered withered Note: * the optimum culture medium for the formation of the shoot primordium of trifloid water melon ~Z1733~
Table 2 ~ , \ ytokinin _ BAP K
~ mg/Q) O
(mg/Q) \ 0.125 2.0 O.l25 2.0 ~ . _ O juvanile juvanile withered juvanile withered plant plant plant 0 25 juvanile shoot ~* juvanile juvanile withered . plant primordium~plant plant NAA _ l O juvanile shoot juvanile¦juvanile withered plant primordium plant Iplant 0 25 juvanile juvanile juvanile juvanile withered . plant plant plant plant 2,4-~
1 0 juvanile juvanile juvanile juvanile withered . plant plant plant plant I . I . I
Note~ the optimum culture medium for the formation of the shoot primordium of hybrid vigor Indian corn Table 3 ~/ i O Bn r = K
auxine \ 0.125 2.0 0.125 2.0 O Juvanile withered withered withered withered 0 25 callus shoot '~ shoot _ . primordium primordium NAA _ 1.0 callus shoot withered _ __ ~ prim~rdium 0 25 withered shoot shoot callus callus . primordium primordium 2,4-D _ _ 1.0 withered callus withered jwithered callus Note~ the optimum culture medium for the formation of the shoot primordium of hybrid vigor rice plant ~Z~73;~9 Table 4 ( ~ j --T 'P K
aux ne ~ 0.125 2.0 O.l~ ; O
O Withered liUvanile juvanile juvanile¦juvanile r---- plant plant plant Iplant 0.25 callus ~ callus callus callus ~ callus NAA . !
1 o shoot callus callus callus callus primordium _ _. _ 0.25 shoot '~ '; callus eallus callus callus 2,4-D primordium _ 1.0 shoot callus callus callus calus J primordium , L i l Note: lJ,~.t~J,; the optimum culture medium for the formation of the shoot primordium of hybrid morning glory The resulting shoot primordia are in a form of hemispherical conglomerate. The conglomerates of the triploid water melon (see Fig. 1) and the hybrid morning glory (Fig. 4) indicate light green color or dark green color and have the callus at the base portions thereof. Meanwhile, the conglomerates of the hybrid vigor Indian corn (Fig. 2) and the hybrid vigor rice plant ~Fig. 3) indicate light green color. These shoot primordia prepared in the invention have been actively propagating now for 6-7 months.
Next, when these shoot primordia are trans-planted to solid culture media for forming young plant and the culture media are subjected to stationary :lZ~7339 culture at 15-30C and at the illumination intensity of 1,000-4,000 luxes, a large number of fine cormuses are firstly ormed and roots are formed from the base portions thereof. Two to four months after the culture, 05 the plant having the same genotype, chromosomal type and phenotype as the parent plant can be obtained.
The shoot primordium is a projection having a diameter of 50-80 ~m, of which the surface is smooth at the initial stage (Fig. 5). The constituting cells are uniform samll polygonal cells which effect polyaxial division in which the axis of the division is vertical, parallel, oblique or the like. When the shoot primordium (primary shoot primordium) gradually enlarged and the diameter becomes 100-1,000 ~m (see Fig. 6), two strata of an epidermal system and a cortexal system are formed.
The outermost stratum consists of one or two cell strata in which the axis of the cell division is only parallel. While the cortexal system in -the inner side from the outermost stratum consists of a number of more or less larger cell conglomerate, and at inside of this inner side cortexal system are observed a number of well-developed chloroplast, vacuole, and storage substance grains. The latter shoot premordium (secondary shoot primordium) grows up in a form of the trapizoidal projection having a diameter of not less than 500 ~m, and in the cells of the epidermal system of the outermost stratum at this stage, these are large oil bodies and in the cells of the endodermal system of the inner 1;~17339 stratum, the number of chloroplast is increased and the vacuole is highly grown. ~t this stage, several primary shoot primordia are newly produced around this trapizoidal projection. Through this route, the shoot primordium 05 continously propagates and one cycle of this route terminates in about 7-14 days. Accordingly, one shoot primordium propagates about four times within about 7-14 days.
The observation of the chromosomes in the triploid water melon (2n=33) was made for the purpose of the inspection of the stability in inherency of the shoot primordia thereof, and it was confirmed that all the shoot primordia examined had the same chromosome number of 2n=33 as the parent plant (Fig. 7). It is now therefore possible to vegitatively propagate the triploid water melon while perennially maintaining the triploid. Thus, it is possible to mass-propagate the useful annual plants with the same genotype and chromosomal type over a number of years by using the shoot primordia.
On the other hand, the plastids, vacuoles, oil body, storage substance grain (starch, protein, etc.) are actively produced in the shoot primordia.
To the contrary, in the conventional callus cell method, substantially no visual secondary metabolites are produced. Meanwhile, according to the present invention, there is likelihood that the useful substances can be industrially mass-produced from the living cells by ~Z~7339 using the shoot primordia.
It is also possible to vegitatively propagate the shoot primordia in an inherently and chromosomaly stable state through the subculture at l time per 05 0.5-2 months.
The effects obtained by the invention are that it is possible to vegitatively and perennially propagate the useful annual plants by using the shoot primordia and it is also possible to obtain a large quantity of the population thereof while perennially maintaining the genotype and chromosomal type in the useful annual plants. Moreover, it is possible to manufacture the medical, edible and industrial useful substances using those produced by the living cultured cells.
The propagation rate of the shoot primordia is extremely fast to such an extent that only one shoot apex can produce annually 412_452 shoot primordia, thus enabling the mass-propagation in a large scale.
The invention will be explained more in detail with reference to the specific examples which are never intended to restrict the scope of the invention.
Example l: Triploid water melon (Cirrullus battich) Propagation method:
A modified Gamborg ~5 was employed as the fundamental culture medium for culturing water melon, the composition of which is shown in Table 5.
~Z~7339 Table 5 Gamborg B5 modified culture medium for -triploid water melon mg/Q
NaH2PO4 2H2O 170.0 KNO3 '~L 2,500.0 (NH4) 2 S04 134.0 MgSo4-7H2o '` 250.0 CaCl2 113.0 Fe-EDTA 40,0 MnSO4 4H2O 13.0 H3BO3 3.0 ZnSO~ 7H2O 2.0 Na2MoO4 2H2O 0.25 CuS04 5H2O 0.025 CoCl2 6H2O 0.025 nicotinic acid 1.0 thiami~e, HCl 10.0 pyridoxine, HCl 1.0 myoinositol 100.0 sucrose 20,000.0 naphthaleneacetic acid ~L0-0 . 25 6-benzylaminopurine ~ 0.125-2.0 pH: 5.7-5.8 In Table 5, "~" markecl compounds are used for the modification.
~217339 First, the shoot apex portion having a length of about 15 mm was out off from a juvenile young plant of an actively growing water melon. After washing with sterilizing solution, the leaves were consecutively 05 peeled off from the outer side to the inner side under observation using a stereomicroscope with a pair of tweezers and a knife to pick up a shoot apex of about 1-1.5 mm with two or three fine leaf primordia at the innermost side. This picked-up shoot apex was cultured in the above foundamental culture medium. The rotation culture was effected in a test tube of 27 mm in diameter x 200 mm in length into which 25 mQ of the foundametal culture was poured, under the conditions that the temperature was 15-30C, the illumination intensity was 2,000-9.000 luxes, and the number of revolution was 0.5-10 rpm. The green shoot primordium conglomerate of about 10 mm in diameter was obtained one month after the outset of the culture. Thereafter, this conglomerate was cut every one month to obtain a subdivision of about 5-10 mm in diameter, which was then transplanted into a fresh culture medium as described above and propagated.
Young plant forming method:
As the fundamental culture medium for forming young plant, there was employed a solid culture medium prepared in such a manner that the above fundamental culture medium excluding sucrose, naphthaleneacetic acid and 6-benzylaminopurine was diluted 5 times, and lZ17339 20 g/Q of sucrose, 0.05-0.5 ppm of 6-benzylaminopurine, and 8 g/Q of gelatine were added thereto to obtain the culture medium as adjusted at pH 5.7-5.8. About 80 mQ
of this culture medium was poured into a 300 mQ conical 05 flask, on which was placed stationally a subdivided shoot primordium conglomerate of 3-5 mm in diameter.
The culture was effected under stationary conditions that the temperature was 15-30C, and the illumination intensity was 4,000 luxes (illuminated for 16 hours and non-illuminated for 8 hours). Two to three weeks after the stationary culture, 4-6 pieces of the dark green cormuses each having a length of 3-4 mm were formed per one shoot primordium conglomerate.
According to the invention, it is possible to perennially propagate the annual plants in a large amount. In the case of the triploid water melon, the proparation rate thereof is about three times per month, reaching the propagation rate of 3l2~-5xlo5 per year. That is, about 500,000 juvenile young plants can be produced in year from one parent plant. Thus, according to the present invention, the annual plants can be produced satisfactorily industrially. It was confirmed that the shoots thus obtained had the same triploid as the parent plant. Accordingly, the popula-tion having the same chromosomal type and genotype asthe parent ones can be industrially mass-produced.
Example 2: Hybrid vigor Indian corn (Zea mays) ~Z1~339 Propagation method:
As the fundamental culture medium for the hybrid vigor Indian corn was employed the same solid culture medium as in Example 1 except that 0.25-1 mg/Q
05 of naphthaleneacetic acid and 0.125 mg/Q of 6-benzyl-aminopurine were employed. Pursuent to the method in Example 1, about 1 mm of the shoot apex was cut off and subjected to the culture in the above fundamental culture. One month after the outset of the culture, a green shoot primordium conglomerate having a diameter o~ 20 mm was obtained. Every one month thereafter, this shoot primordium conglomerate was divided into 5-10 mm in diameter, which was then planted into a fresh fundamental culture medium as described above.
Young plant forming method:
The same solid culture medium as in Example 1 was used as the culture medium for forming young plant.
About 80 mQ of this culture medium was poured into a 300 m~ conical flask, and the shoot primordium conglomerate of about 15 mm in diameter was placed on the thus poured culture medium. The culture was carried out under the same conditions as in Example 1. Two to three weeks after the culture, 10-20 mm in ]ength of the cormuses were formed and roots were formed from the base portions -thereof. About two months after the stational culture, about 2-3 juvenile young plants each having a length of 100-150 mm were formed per one shoot primordium conglomerate.
~21'7339 In the case of the hybrid vigor Indian corn, the propagation rate is about four times per month, reaching the rate of 412~6xl06 per year. That is, about l~,000,000 juvenile young plants per year can be 05 produced from one parent plant. Thus, the annual plant can be produced industrially according to the present invention. Further, it was confirmed that the thus obtained shoots indicated the same hybrid vigor as the parent plant. Therefore, the population having the same hybrid vigor can be industrially mass-produced.
Example 3: Hybrid vigor rice plant (Oryza sativa) Propagation method:
As the fundamental culture medium for the hybrid vigor rice plant was employed the same modified culture medium as in Example l except that 0.25-l.0 mg/Q
of naphthaleneacetic acid was employed. Pursuant to the method in Example l, about l mm in length of the shoot apex was cut off, and cultured in the above fundamental culture medium to obtain 5 mm in diameter of a light green shoot primordium conglomerate in one month after the outset of the culture. Every one month thereafter, this shoot primordium conglomerate was divided into about 3-4 mm in diameter, which w~s then planted into a fresh culture medium as described above and was propagated.
Young plant forming method:
As the culture medium for forming young plant was employed a solid c-ulture medium as prepared in ~217339 ~xample 1 in such a manner that the fundamental culture medium excluding sucrose, naphthaleneacetic acid and 6-benzylaminopurine was diluted 5 times, and then 20 g/Q of sucrose and 8 g/Q of gelatine were added 05 thereto to obtain the medium as adjusted at pH 5.7-5.8.
About 80 mQ of the thus prepared culture medium was poured into a 300 mQ conical flask, and the shoot primordium conglomerate ~f about 3-5 mm in diameter was placed thereon. The culture was carried out under the same conditions as in Example 1. Two to three weeks after the culture, the cromuses each having a length of 5-100 mm in length were formed and roots were formed from the base portions thereof. One to two pieces of the juvenile young plants each having a length of about 70 mm were formed per one shoot primordium conglomerate two months after the culture.
In the case of the hybrid vigor rice plant the propagation rate is rather high, reaching the rate of about five times per month, i.e., the rate of 512~2.5x108 per year. That is, about 250,000,000 juvenile young plants can be produced per year from one parent plant. It was confirmed that the thus obtained shoots indicated the same hybrid vigor as the parent plant. Thus, the population of the same hybrid vigor can be industrially mass-produced.
Example 4: Hybrid morning glory (Ipomoea nil R _ ) As the fundamental culture medium for the hybrid morning glory was employed the same modified i217339 cu1ture medium as in Example 1 except that 0.25-1.0 mg/Q
of 2,4-dichlorophenoxyacetic acid was used instead of naphthaleneacetic acid and 6-benzylaminopurine. Pursuant to the method in Example 1, about 0.5-1.0 mm in length 05 of the shoot apex was cut off and cultured in the above fundamental culture medium. One month after the outset of culture, a green shoot primordium conglomerate of about 5 mm in diameter was obtained. Every one month thereafter, the thus obtained shoot primordium conglomerate was div-ded into about 3-5 mm in diameter and was propagated in a fresh culture medium as described above.
Young plant forming method:
As the culture medium for forming young plant was employed the same solid culture medium as in Example 1 prepared in such a manner that the above culture medium excluding sucrose and 2,4-dichlorophenoxyacetic acid was diluted 5 times, and 20 g/Q of sucrose, 0.05-0.5 ppm of 6-benzylaminopurine and 8 g/Q of gelatine were added thereto to obtain the culture medium as adjusted at pH 5.7-5.8.
About 80 mQ of the thus obtained culture medium was poured into a 300 mQ conical flask, on which the shoot primordium of 3-5 mm in diameter was placed.
One week after the culture, projections considered as fine cormus in a size of 1-2 mm in length were formed.
In the case of the hybrid morning glory~ the propagation rate was two times per month, reaching the ~2~7339 rate of 212_4x103 per year. Accordingly~ about 4,000 juvenile young plants can be annually produced from one parent plant. Thus, the mass-production can be fully industrially.
Claims (4)
1. A method for perennially mass-propagating a useful annual plant by use of shoot primordium, comprising the steps of:
cutting off a shoot apex from the useful annual plant and transplanting the thus cut off shoot apex into an artificial culture medium containing an inorganic salt composition and a plant growth hormone;
rotation-culturing the shoot apex under the condi-tions that a temperature is 15-30°C, an illumination intensity is 2,000-9,000 luxes and a number of revolution is 0.5-10 rpm to propagate the shoot primordium; and stationally culturing the shoot primordium to form young plants, whereby the useful annual plant is perennially mass-propagated in a short period of time, while maintaining the genotype and chromosomal type for a number of years.
cutting off a shoot apex from the useful annual plant and transplanting the thus cut off shoot apex into an artificial culture medium containing an inorganic salt composition and a plant growth hormone;
rotation-culturing the shoot apex under the condi-tions that a temperature is 15-30°C, an illumination intensity is 2,000-9,000 luxes and a number of revolution is 0.5-10 rpm to propagate the shoot primordium; and stationally culturing the shoot primordium to form young plants, whereby the useful annual plant is perennially mass-propagated in a short period of time, while maintaining the genotype and chromosomal type for a number of years.
2. The method as claimed in claim 1, wherein the useful annual plant is one selected from the group consisting of triploid water melon, hybrid vigor Indian corn, hybrid vigor rice plant, and hybrid morning glory.
3. The method as claimed in claim 1, wherein the inorganic salt composition is Gamborg B5 culture medium, the plant growth hormone is selected from the group consisting of naphthaleneacetic acid, 2,4-dichloro-phenoxyacetic acid, kinetin and benzylaminopurine.
4. The method as claimed in claim 1, wherein the stational culture is effected under the conditions that a temperature is 15-30°C and an illumination intensity is 1,000-4,000 luxes.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6,554/83 | 1983-01-20 | ||
| JP58006554A JPS59132823A (en) | 1983-01-20 | 1983-01-20 | Method of making annus plant hapropappus perennial and mass-breeding same by shoot rudiment |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1217339A true CA1217339A (en) | 1987-02-03 |
Family
ID=11641547
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000443717A Expired CA1217339A (en) | 1983-01-20 | 1983-12-20 | Method for perennially mass-propagating the useful annual plants by use of the shoot primordia |
Country Status (4)
| Country | Link |
|---|---|
| JP (1) | JPS59132823A (en) |
| CA (1) | CA1217339A (en) |
| DE (1) | DE3401291C2 (en) |
| FR (1) | FR2539579B1 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4830966A (en) * | 1984-09-07 | 1989-05-16 | Sungene Technologies Corporation | Process for regenerating corn |
| US4666844A (en) * | 1984-09-07 | 1987-05-19 | Sungene Technologies Corporation | Process for regenerating cereals |
| US4665030A (en) * | 1984-09-07 | 1987-05-12 | Sungene Technologies Corporation | Process for regenerating corn |
| JPS6196994A (en) * | 1984-10-17 | 1986-05-15 | Hiroshima Univ | Productoin of sweet polyglycoside by stem top cultivation of stevia |
| JPS61115417A (en) * | 1984-11-12 | 1986-06-03 | 日本鉱業株式会社 | Mass breeding of horseradish seedling |
| JPH0611209B2 (en) * | 1985-09-04 | 1994-02-16 | 王子製紙株式会社 | Mass growth method for woody plants |
| US5310673A (en) * | 1986-06-26 | 1994-05-10 | Oji Paper Company, Ltd. | Mass propagation through shoot primordia and regeneration of plants from protoplasts of shoot primordia |
| GB2195656B (en) * | 1986-06-26 | 1991-04-24 | Oji Paper Co | Mass propagation through short primordia |
| GB2211204B (en) * | 1987-10-20 | 1992-05-20 | Oji Paper Co | Process for production of plant transformant |
| ES2084706T3 (en) * | 1989-09-30 | 1996-05-16 | Kirin Brewery | PLANTS PRODUCTION METHOD. |
| IT1317038B1 (en) * | 2000-06-05 | 2003-05-26 | Vitroplant Vivai Di Zuccherell | METHOD FOR THE REGENERATION OF PLANTS AND ITS USES FOR THE MULTIPLICATION AND / OR TRANSFORMATION OF PLANTS. |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1401665A (en) * | 1972-05-30 | 1975-07-16 | British Petroleum Co | Plants |
| FR2275142A1 (en) * | 1974-06-20 | 1976-01-16 | Anvar | PROCESS FOR OBTAINING LETTUCE VARIANTS WITH IMPROVED CHARACTERISTICS |
| JPS586553A (en) * | 1981-06-30 | 1983-01-14 | Fujitsu Ltd | Magnetic disc controller |
-
1983
- 1983-01-20 JP JP58006554A patent/JPS59132823A/en active Granted
- 1983-12-20 CA CA000443717A patent/CA1217339A/en not_active Expired
-
1984
- 1984-01-16 DE DE19843401291 patent/DE3401291C2/en not_active Expired
- 1984-01-17 FR FR8400689A patent/FR2539579B1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| FR2539579A1 (en) | 1984-07-27 |
| JPH038171B2 (en) | 1991-02-05 |
| DE3401291C2 (en) | 1986-10-30 |
| JPS59132823A (en) | 1984-07-31 |
| FR2539579B1 (en) | 1988-08-26 |
| DE3401291A1 (en) | 1984-07-26 |
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