JP2005245332A - Artificial aseptic culture for orchid (yoania flava) - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optimal culture medium for aseptic artificial culture of saprophytic orchids which have no chlorophyll, particularly Yoania flava, and provide a growing method for dense seedlings under the aseptic culture by using aseptic budding and aseptic proliferation to grow cultured seedlings needed for protection of Yoania flava and further by enlarging the proliferated seedlings according to the method of this invention. <P>SOLUTION: This aseptic artificial cultivation method has the step where the seeds of Yoania flava are aseptically germinated on the aseptic germinating culture medium, and the step where the germinated and differentiated seedlings are proliferated on the aseptic proliferation medium. The proliferated seedlings are aseptically enlarged and made dense. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an artificial culture of wild orchids, and more particularly to a method for aseptic artificial culture of linoleum orchids (humus orchids).

  Shinanosho Kiran is a humic orchid that has no chlorophyll inhabiting the southern part of Nagano Prefecture, and was described as a new species in May 2002. Until now, two kinds of genus of Shokilan, Yoannia japonica and Yoania amagiensis, have been confirmed in Japan, but artificial growth has not been successful. Shinosho Kiran also needs immediate protection, but even its ecological details were unclear.

  In the life history of orchidaceae plants, it is well known that they have a symbiotic relationship with filamentous fungi generally called orchid fungi. These filamentous fungi (symbiotic fungi) have been elucidated to have an important role in orchid germination and growth, but especially humic orchids such as linden shrimp have no chlorophyll, so they depend on symbiotic fungi. It is considered that it is difficult to germinate without the presence of commensal bacteria.

On the other hand, “a method for mass production of wild orchids” in which wild orchids are cultivated aseptically has been introduced (Patent Document 1). This method is a method of mass-producing wild orchids such as shrimp, shung orchid, and hekkaran, which are designated as endangered species, without using symbiotic bacteria.
A pretreatment step of performing germination inhibitor removal treatment and sterilization treatment on artificially pollinated seeds;
A culture process for growing seedlings by providing a first culture process for seeding and cultivating seeds in a medium containing a germination promoter, and a second culture process for culturing the seed in a medium containing a root promoter;
This is a method for mass production of wild orchids, which includes the process of transferring cultured seedlings from the medium to the culture soil, acclimatizing and raising them.

  According to Non-Patent Document 1, germination of aseptic orchids has become possible in many species and has developed as a horticultural plant.

  On the other hand, similar attempts have been made for terrestrial orchids including humic orchids, and the details are summarized in Non-Patent Document 2.

However, among the humic orchids, no successful examples of artificial germination or artificial culture have yet been reported for the genus Shokilan, particularly Shinano shokiran.
JP 2003-189750 A Joseph Ardity edited by "Orchid Biology I" (Edited by Shoichi Ichihashi, Japanese version of Seikodo Shinkosha, 1991, p. 13) “Terrestrial Orchid” (Hanne N. Rasmussen CAMBRIDGE UNIVERSITY PRESS 1995, pp. 17, 77, 142, 227)

  Therefore, the present invention provides a medium optimal for aseptic artificial culture of humic orchids that do not have chlorophyll, especially linden guts, and also provides cultured seedlings necessary for protection of linden guts by sterilized germination and sterilization. It aims to nurture. Further, the present invention provides a method for growing a solid cultured seedling under aseptic culture by aseptically growing the seedling grown according to the present invention.

  In order to solve the above-mentioned problems, the present inventor has developed an aseptic artificial culture method as described below as a result of intensive research.

  The aseptic artificial culture method of the present invention includes a step of germinating humic orchid seeds on a sterile germination medium and a step of growing germinated and differentiated seeds on a sterile growth medium.

  In addition, if necessary, after growing in a sterile growth medium, it can be further enlarged using a sterile enlargement medium.

  First, the seeds of shinanoshoquiran to be aseptically cultured are collected. The seeds of orchidaceae are contained in the fruit called “fruits” (fruits). However, since the fruiting rate is unstable in wild strains, it is desirable to produce them by artificial pollination. For artificial pollination, either self-pollination or cross-pollination can be used. The fruited fruit must be sterilized after collection. As a sterilization method, for example, the surface of the sorghum is washed under running water while rubbing with absorbent cotton or the like, and immersed and stirred in 70% ethanol for 30 seconds. Then, after immersing and stirring in a 1% sodium zincate aqueous solution for 10 minutes, rinse with sterilized water for 5 minutes × 3 times. Thereafter, it is further immersed in 70% ethanol for 1 minute, and the surface is baked with a gas burner for complete sterilization.

Subsequently, the tip of the fruit is cut off and divided into half halves, and the seeds inside are scraped and placed in a sterile germination medium. The component composition of the sterile germination medium is usually 0.5 to 8.0 kgm ^{−3 of} a fertilizer containing inorganic nitrogen, phosphorus and potassium in a ratio of about 1: 1: 3, and a nitrogen source of 0.5 to 5.0 kgm. ^-3 , carbon source 2.0 to 60.0 kgm ^-3 , coagulant 0 to 20.0 kgm ^-3 . Desirably, compound fertilizer containing inorganic nitrogen, phosphorus, and potassium in a ratio of about 1: 1: 3, 1.0 to 5.0 kgm ⁻³ , nitrogen source 1.0 to 3.0 kgm ⁻³ , carbon source 5. 0 to 40.0 kgm ⁻³ and coagulant 0 to 15.0 kgm ⁻³ .

  As the composite fertilizer, for example, fine powder HONEX (Hyponex Japan Co., Ltd. registered trademark) can be suitably used.

  Examples of the nitrogen source include peptone, protease peptone, and protease peptone no. 3, Hydrolyzate of protein such as tryptone, tryptase, neopeptone, proton, casitone, casamino acid or yeast extract can be used (Non-patent Document 1, page 95). In particular, casamino acid can be preferably used.

  Further, as the carbon source, one or more kinds from the group of monosaccharides such as glucose (fructose), fructose (fructose), mannitol, disaccharides such as sucrose (sucrose) and maltose (maltose), or oligosaccharides In particular, it is desirable to use glucose (monosaccharide) and sucrose (non-reducing disaccharide). Furthermore, it is desirable to use sucrose. The present inventors have confirmed that the use of sucrose has a better germination rate than glucose.

  The seed is collected and then sown in a sterile germination medium. As the medium, a solid medium is preferable to a liquid medium. This is because the germination rate is better when the seeds are placed in an air atmosphere. Therefore, it is desirable to add a coagulant to obtain a solid medium. As the coagulant, coagulants such as agar, gellite (gellan gum), thickening polysaccharides and the like can be used, and agar and gellite can be particularly preferably used. Further, the sterile germination medium usually has a pH of about 5.3 to 6.0, but preferably has a pH of about 5.5 to 5.8.

  Further, the form of the sterile germination medium for seeding the seed is not necessarily a solid medium. It is also possible to infiltrate a water-absorbing support such as sponge, non-woven fabric, paper, etc. with a liquid aseptic germination medium without adding the coagulant and sow on the surface thereof to make a sterile germination medium. In this case, however, it is necessary to sterilize the water-absorbing support used.

  When seeds are sown on the germination medium, germination occurs in 2 to 3 months, and a protocomb (original block) is formed. The seed that formed the protocomb further grows into a lysosome (rhizome). When the seed grows into a protocomb or a lysozyme, it is transferred to a sterile growth medium which is the next step.

The component composition of the sterile growth medium is usually 0.5 to 8.0 kgm ^{−3 of} a fertilizer containing inorganic nitrogen, phosphorus and potassium in a ratio of about 1: 1: 3, and a nitrogen source of 0.5 to 5.0 kgm. ^-3 , carbon source 2.0 to 60.0 kgm ^-3 , coagulant 0 to 20.0 kgm ^-3 . Desirably, compound fertilizer containing inorganic nitrogen, phosphorus, and potassium in a ratio of about 1: 1: 3, 1.0 to 5.0 kgm ⁻³ , nitrogen source 1.0 to 3.0 kgm ⁻³ , carbon source 5. 0 to 40.0 kgm ⁻³ and coagulant 0 to 15.0 kgm ⁻³ .

  It is possible to use the same components as in the sterile germination medium for the complex fertilizer, nitrogen source, and carbon source. Furthermore, the sterile growth medium usually has a pH of about 5.3 to 6.0, but preferably has a pH of about 5.5 to 5.8.

  The protocomb or resome transferred to the sterile growth medium continues to grow and enlarge. For example, after about 2 months, if it is desired to make the protocomb or lysome a rich cultured seedling, it is transferred to a sterile hypertrophic medium.

  As a sterile enlargement medium, it is desirable to use a Norstog medium. The main components of Norstog medium are shown in Table 4.

<Sample collection>
As samples for artificial growth, flower stems, berries, and underground stems of Shinano shokiran were collected. In the habitat of Shinanosho Kiran, the temperature of flowering and fruiting is 12-26 ° C, the soil temperature is 17-18 ° C at a depth of 10 cm, the soil temperature is 15-17 ° C at a depth of 40 cm, the humidity is 66-99%, the illumination is 610-43700 lx, Measurement results with photon amounts of 11 to 658 μmolm ⁻² s ⁻¹ were obtained. The environment that Shinano Shokiran prefers does not reach high temperatures, but it can be expected that it is highly humid and difficult to reach directly.

  Also, humus is deposited on the surface layer of the habitat soil. There is a large amount of gravel at the bottom, and clayey soil fills the gap.

  When fertilized, the petal begins to turn brown immediately afterwards, and eventually the base of the flower begins to swell. After 3 weeks of flowering, the fruits become large berries (fruits) and then ripen in about a month. Many of them suffer from insect damage during this process, so the number of fruits in the natural state is very low.

Artificial pollination was also carried out in order to improve the fruiting rate.
<Aseptic germination and aseptic growth>
1) Aseptic germination The surface of the collected sap was washed with absorbent cotton under rubbing water, and immersed in 70% ethanol for 30 seconds and stirred. Next, it was immersed and stirred for 10 minutes in a 1% aqueous sodium zincate solution and rinsed with sterile water for 5 minutes × 3 times. Further, after being immersed and stirred in 70% ethanol for 1 minute, the surface was baked with a gas burner and completely sterilized.

  The tip of the fruit was cut off and split into half halves, the seeds inside were scraped and placed in a sterile germination medium. As a sterile germination medium, a compound fertilizer containing Hyponex and a diluted MS medium component were prepared, and liquid and solid media were used, respectively. An example of the component composition of the sterile germination medium is shown in Table 1.

シナノショウキランの種子は、熟度が進むと種子表面の筋模様が濃くはっきりしてくる。さく果には虫喰いなどの痛みが多く、さく果を表面殺菌しても培養中にコンタミネーション（雑菌混入）が多く発生した。しかし、コンタミネ―ションを起こした種子は、一旦培地から取り出して再度滅菌すれば、再び発芽試験に用いることが可能である。

As the seeds of Shinanosho Kiran grow, the streaks on the seed surface become darker and clearer. The berries have many pains such as insect bites, and even when the berries were sterilized, many contaminations (mixed bacteria) occurred during the cultivation. However, once contaminated seeds are removed from the medium and sterilized again, they can be used again in the germination test.

  The germination situation was observed by changing the type of sterile germination medium. In addition, the difference in the number of germination depending on the maturity of seeds in each medium was also observed. The observation results are shown below.

置床後２〜３ヶ月を経ると、一部の種子からプロトコーム状に白く膨らんだ組織ができる。この実験では、前記状態に成長した種子を発芽種子とした。種子は、未熟なもののほうが発芽率が高く、培地は、液体よりも固体のほうが発芽率は高い。

Two to three months after placement, a tissue that swells white like a protocomb is formed from some seeds. In this experiment, seeds grown in the above state were used as germinated seeds. Seeds have a higher germination rate for immature seeds, and a medium has a higher germination rate for solids than liquids.

2) Sterile growth After germination in a sterile germination medium, the seeds were transplanted to several types of sterile growth medium at the stage of growth into a protocomb or lysosome, followed by regular weight measurement, photography, etc., and the progress was observed.

  Table 3 shows examples of the component composition of the sterile growth medium.

前記無菌発芽と同様、当初はハイポネックス培地（液体）でリゾームの生育が早く、２ヶ月後に６００％の成長率を示した。ここで成長率とは、試験開始時のリゾーム又はプロトコームの質量に対して増加した質量（累積した各月の増加質量）の割合をいう。その後、Ｎｏｒｓｔｏｇ培地でのリゾームの増加量（月次の増加質量）が大きくなり、４ヶ月後にはハイポネックス培地（液体）でのリゾームの増加量を上回り旺盛な生育を続けた。ＭＳ培地を基本とした培地は、他のラン科植物で発明者等がもっとも良い結果を得た１／２０ＭＳ改変培地と、ＥＣ（電気伝導度）をハイポネックス培地（液体）と同等にした１／２ＭＳ改変培地を用いたが、どちらも生長が遅く差もなかった。またＴ処方培地は炭素源としてスクロースを用いたが、全くと言えるほどリゾームは生長しなかった。

Similar to the germination, the growth of the lysosome was fast in the Hyponex medium (liquid), and the growth rate was 600% after 2 months. Here, the growth rate refers to the ratio of the increased mass (accumulated mass for each month) to the mass of the lysosome or protocomb at the start of the test. Thereafter, the amount of increase in lysosome (monthly increase mass) in the Norstog medium increased, and after 4 months, the amount of increase in lysosome in the Hyponex medium (liquid) exceeded that of vigorous growth. The medium based on the MS medium is a 1/20 MS modified medium that the inventors have obtained the best results in other orchidaceae plants, and 1/20 MS (conductivity) equivalent to the Hyponex medium (liquid). Although 2MS modified medium was used, neither of them was slow to grow and there was no difference. The T-prescription medium used sucrose as a carbon source, but the lysozyme did not grow as much as it could be said.

  FIG. 1 shows the results of Hyponex medium (liquid), 1/20 MS modified medium, and Norstog medium among the above-described sterile growth medium.

The biggest difference due to the difference in culture medium is the morphology. Hyponex medium (liquid), which had good growth, produced many thin lysosomes, and very rarely 1 to 2 lysosomes grew thicker. On the other hand, in the Norstog medium showing the same amount of growth, one to two resomes grew thick from the beginning, and after six months the internodes were clogged, but grew to a thickness that was collected locally.
<Discussion>
There have been few studies on the genus Shokilan, and artificial pollination and aseptic culture have been addressed in order to establish protection measures for shokilan.

  In the ecological survey, linden groves were rarely found in the same place every year, and especially in places where large communities were seen, we could not see linden groves the following year. For this reason, it was hardly possible to observe the timing of emergence from the soil or to carry out insect damage control from the beginning of flowering. It is a plant that is difficult to protect and proliferate because it is difficult to grow seeds in the natural state, and there are very few seeds that reach maturity.

  With regard to artificial pollination, it is not clear whether self-pollination or cross-pollination is appropriate, but the effectiveness of artificial pollination is considered to have been demonstrated.

  If a sterile germination medium is used, germination can be achieved without symbiotic bacteria. It is desirable that the seeds used for aseptic germination are relatively immature, and in this example, good results were obtained with about one month after pollination. Although there is almost no difference depending on the basic medium, it is desirable that peptone or casamino acid is added. In the medium to which these were not added, there was no germination or growth even after germination.

  Moreover, the number of germination is larger in the solid medium. Since seeds sink in the liquid medium, it is considered that the oxygen supply amount has an influence.

  After germination, the protocomb grew into a lysosome, and plant body retention in vitro (in vitro) became possible without problems. The thinly branched lysosomes can be broken and proliferated, and the lysosomes used in the examples are also increased in the same manner. Rarely, one or two thick lysosomes grew from branched and volumey lysosomes, but a thick and solid lysosome grew without branching in Norstog medium.

  If the composition of a sterile medium is examined in more detail and the conditions for fine branching and the expansion of thick lysosomes can be clarified, there is a possibility that new knowledge can be obtained not only for the protective growth of individuals but also for the elucidation of ecology.

  It is expected that the sterile artificial culture method according to the present invention can not only protect Shinano shokiran, but can also be applied to protect wild orchid strains belonging to the genus Shokilan, and further to orchid orchids. Is done.

The composition of the aforementioned Norstog medium is shown below.

It is a histogram which shows the kind of culture medium and the growth of aseptic lysosome.

Claims (17)

  In aseptic artificial culture of shinanoshokiran, sterilization of shinanoshoquiran characterized by comprising a step of germinating seeds aseptically on a germination medium and a step of propagating germinated and differentiated materials in a sterile growth medium Artificial culture method.   The method for aseptic artificial culture of shinanoshoquiran according to claim 1, wherein after the step of growing in the sterile growth medium, the mixture is further enlarged in a sterile enlargement medium. The component composition of the germination medium is 0.5 to 8.0 kgm ^{−3 of} a fertilizer containing inorganic nitrogen, phosphorus and potassium at a ratio of about 1: 1: 3, and a nitrogen source of 0.5 to 5.0 kgm ^{−. 3.} The aseptic artificial culture method for linden shrimp according to claim 1, wherein the composition requirements of carbon source 2.0 to 60.0 kgm ⁻³ and coagulant 0 to 20.0 kgm ⁻³ are satisfied.   4. The aseptic artificial culture method for shinanoshoquiran according to claim 3, wherein, among the component composition of the sterile germination medium, the compound fertilizer is fine powder Hyponex (registered trademark of Hyponex Japan).   5. The aseptic artificial culture method for shinanoshoquiran according to claim 3 or 4, wherein a nitrogen source is a protein hydrolyzate among the component composition of the sterile germination medium.   6. The aseptic artificial culture method of shinanoshoquiran according to claim 5, wherein the protein hydrolyzate is casamino acid.   7. The linden show according to any one of claims 3 to 6, wherein among the component composition of the germination medium, the carbon source is one or more selected from the group of monosaccharides, disaccharides and oligosaccharides. A sterile artificial culture method for Kiran.   The sterilized artificial culture method of shinanoshoquiran according to any one of claims 2 to 7, wherein the coagulant is gellite (gellan gum) among the components of the germination medium.   9. The aseptic artificial culture method for shinanoshoquiran according to any one of claims 3 to 8, wherein the sterile germination medium has a pH of about 5.3 to 6.0. The component composition of the sterile growth medium is 0.5 to 8.0 kgm ^{−3 of} a complex fertilizer containing inorganic nitrogen, phosphorus and potassium in a ratio of about 1: 1: 3, and a nitrogen source of 0.5 to 5.0 kgm ^{−. 3.} The aseptic artificial culture method for linden shrimp according to claim 1, wherein the composition requirements of carbon source 2.0 to 60.0 kgm ⁻³ and coagulant 0 to 20.0 kgm ⁻³ are satisfied.   The aseptic artificial culture method for shinanoshoquiran according to claim 10, wherein the compound fertilizer of the component composition of the sterilized growth medium is fine powder hyponex.   The aseptic artificial culture method of shinanoshoquiran according to claim 10, wherein the nitrogen source of the component composition of the sterile growth medium is a protein hydrolyzate.   The aseptic artificial culture method of Shinanoshokiram according to claim 12, wherein the protein hydrolyzate is casamino acid.   The linden show according to any one of claims 10 to 13, wherein the carbon source in the composition of the sterile growth medium is one or more selected from the group of monosaccharides, disaccharides and oligosaccharides. A sterile artificial culture method for Kiran.   The aseptic artificial culture method for shinanoshoquiran according to any one of claims 10 to 14, wherein the sterile growth medium has a pH of about 5.3 to 6.0.   The aseptic artificial culture method for shinanoshoquiran according to any one of claims 2 to 15, wherein the aseptic enlargement medium is a Norstog medium. In the aseptic artificial culture of the genus Shochiran, the method comprises the steps of germinating seeds aseptically on a sterile germination medium, and proliferating the germinated and differentiated ones in an aseptic growth medium. Artificial culture method.

Images