JP2008074752A - Method for using rare saccharide for promotion or control of growth of vegetable shoot - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To establish an effective clone proliferation method for a bulbous crop by taking a tissue of a horticultural crop, especially a bulbous crop as an object for tissue culture and using a rare saccharide as an agent for promoting or controlling the growth of a tissue culture material of the bulbous crop. <P>SOLUTION: The invention provides a method for using a rare saccharide for the promotion or control of the growth of a shoot in the case of using the tissue of a horticultural crop as an explant, and planting and culturing the plant on a medium. The tissue of the horticultural crop is the tissue of a bulbous crop. A thickened bulbous base of the shoot of Gloriosa Lutea is vertically divided into two and used as the explant. The rare saccharide is D-psicose. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  In a plant tissue culture method, the present invention relates to a technique for promoting growth or adjusting a culture of a horticultural crop, particularly a bulb crop, using a rare sugar.

  In plant tissue culture, the plant growth regulator, which is a plant hormone-like substance, is used to examine the culture conditions so that the totipotency to regenerate individual plants from parts of the tissues and organs of the plant is maximized. To be done. Conventionally, in the tissue culture of tulip of a stalk plant which is a kind of bulbous plant, several conditions have been studied in addition to plant growth regulators in the medium for the efficiency of adventitious bud formation. Nishiuchi (Non-patent Document 1) examined the high temperature treatment of the mother sphere before culturing and the temperature at the time of culturing, and PGAlderson et al. The efficiency of adventitious bud formation is being investigated. However, in these conventional methods, the rate of differentiation of adventitious buds is low, and further, the rate of adventitious buds is slow, so that the adventitious buds can be extended to the shoot length necessary for bulb differentiation treatment for 16 weeks from the start of culture. It took a long period of time.

  On the other hand, Custes and Ergervoet (Non-patent Document 3) also examined the concentration of plant growth regulators during cultivation in tissue culture of tuberous plant Gloriosa, a kind of the same bulbous plant. Takamura et al. (Non-patent Document 4) Is investigating the influence of culture temperature, but it is considered that there is a need for further improvement in its growth efficiency and the like. In addition, regarding addition of sugars other than sucrose, there has been little study on sugars added to the medium used for the growth of Gloriosa in vitro. Therefore, an improvement in the tissue culture method of plants in which many shoots are formed in a shorter period of time in bulb crops has been desired.

  D-psicose is one of the monosaccharides called rare sugars. Conventionally, rare sugars were difficult to obtain because they could not be mass-produced, and therefore, little research was conducted on their physiological activity and pharmacological activity. Recently, a mass production method using enzymes has been established by Imori et al., Faculty of Agriculture, Kagawa University, and some of the rare sugars have been established, and their physiological activity has attracted attention. Regarding the use of rare sugars for plants or microorganisms, inventions such as plant growth regulators containing rare sugars as active ingredients have been made (Patent Document 1), but these rare sugars are used for tissue culture of bulbous plants, especially for clonal propagation. There are no examples of effective use.

International Publication Number WO2005 / 112638 Bulletin of Hokkaido University of Education, Vol. 33, No. 1, pp. 49-65, 1982. Acta Horticulturae, 109, 263-270, 1980. Scientia Horticulturae, 57 (4), 323-334, 1994. Academic report, Faculty of Agriculture, Kagawa University, 54, 41-44, 2002.

An object of the present invention is to clarify the types and concentrations of rare sugars that promote or suppress plant regeneration from cells, tissues, and organs of horticultural crops, particularly bulbous crops, and to use them as growth promoters or regulators of cultures. And
More specifically, the present invention takes a horticultural crop, particularly a bulb crop tissue as a tissue culture target crop, and utilizes rare sugars as growth promoters or regulators of the bulb crop tissue culture. The aim is to establish an effective clonal propagation method.

  SUMMARY OF THE INVENTION The present invention provides a method for utilizing a rare sugar, preferably D-psicose, for promoting or adjusting the growth of shoots when planting and cultivating a horticultural crop tissue, preferably a bulbous crop tissue, as an explant in a medium. And

  In the present invention, when the base of a globulosa 'Lutea' shoot, which is a bulbous crop tissue, swelled in a spherical shape is divided into two vertically and used as an explant, it is planted in a culture medium, a rare sugar, preferably D-psicose The gist of the method is to use the shoot for promoting or adjusting the shoot growth.

According to the present invention, there is provided a method for promoting or adjusting the growth of shoots (stems and leaves) by adding a rare sugar in the tissue culture method of plants, in the case of tissue culture of horticultural crops, particularly bulb (tuber) crops. be able to.
More specifically, according to the present invention, it is confirmed by experiments that D-psicose can be used as a growth promoting or regulating agent for tissue cultures in tissue culture of Gloriosa, and provides an effective clolio propagation method of Gloriosa. be able to.

  Plant tissue culture is a technique for cultivating a plant in a sealed aseptic space such as a glass container in which a medium containing nutrients necessary for plant growth is placed. In the present invention, horticultural crops are taken as the target plant (crop) for tissue culture, particularly Gloriosa as a bulb crop, but it is not limited thereto. We believe that any horticultural crop, in particular a bulb crop, that can be tissue cultured by the plant tissue culture method is applicable.

Gloriosa is a bulbous (tuber) plant of the canine saffron family (sometimes referred to as the lily family) and has recently been very popular as a cut flower. Gloriosa superba 'Lutea' is a horticultural variety and is usually called Gloriosa 'Lutea'. The flowers of Gloriosa superba are yellow and red.

  In other words, Gloriosa is an unusual flower bud with wavy and slender petals that warps. It is a plant with high market value that adds gorgeous colors to ikebana and bouquets and circulates widely as pots and cut flowers. There are two methods of propagation: seedling and tuber splitting. In the case of seedlings, it takes more than 3 years to bloom. Furthermore, growing with seeds does not always give rise to individuals with the same traits (features) as the original plant, but they are often not. In the method of dividing the tuber, the tuber is produced, so that it can be vegetatively propagated (clone). However, since only one bifurcated bulb can be produced from one individual, the growth rate is extremely low and there is a risk of virus infection.

Gloriosa explants were used in the tissue culture of the examples of the present invention. The explants in this case refer to those usually used for tissue culture, and can be explants in a wide range from cells / tissues to plants. The original tissue used in Experiment 1 of the embodiment of the present invention is a base section obtained by vertically dividing a spherically enlarged base portion of Gloriosa 'Lutea' shoot formed in vitro into two. The purpose is to form a new chute from this base section.

  For plant shoots, the stems always have leaves and the leaves always stick to the stems. Both are inseparable and can be made from the same meristem. Therefore, it may be convenient to treat one stem and its attached leaf as one unit. Such a unit is called a shoot. A shoot in an immature stage is called a bud.

  Sterilizing plant shoots with a disinfectant, taking out the tip of the buds in a clean bench, planting them in the medium, and continuing the culture while controlling the temperature and illuminance, a small tissue grows into shoots. Based on this shoot, the acupuncture buds in that node are used in a medium containing a lot of cytokinins (a kind of plant hormone that mainly promotes cell division, promotes bud differentiation, and suppresses aging). By forming shoots from shoots called shoots, or forming new shoots called adventitious shoots from each tissue of the shoot, many new shoots are obtained, and those shoots are hormone-free or auxin (plant hormones) When transplanted to a medium containing a kind of substance that exhibits actions such as cell division promotion, side bud growth inhibition, rooting promotion, etc., rooting occurs and the plant body is regenerated. The regenerated plant obtained in this way can be cultivated by transplanting to soil. This is a kind of clonal propagation method.

The present invention divides a horticultural crop tissue, preferably a horticultural crop tissue into a bulbous crop tissue, more specifically a globularly enlarged base of a shoot grown in vitro in Gloriosa 'Lutea' in two vertically. A plant is used as an explant and planted in a culture medium. At that time, a rare sugar, preferably D-psicose is added to the medium.

  The medium is MS medium supplemented with 0.5 μM NAA, 5.0 μM BA, and 0.2% gellan gum. NAA is an auxin, BA is a type of cytokinin (both artificial), and gellan gum is used to harden the medium. MS medium is the most popular medium used for plant tissue culture. These are the media normally used for tissue culture of Gloriosa. To this, a rare sugar is added alone or together with glucose, instead of the sucrose usually used. The total sugar concentration is preferably about 0.09 M.

  When D-psicose is added alone or when one-tenth of glucose is replaced with D-psicose and added, shoot formation is remarkably suppressed, and in most cases no shoot is formed. That is, addition of a high concentration of D-psicose significantly suppresses shoot formation.

  On the other hand, when 1 / 100th or 1000th of glucose is replaced with D-psicose and 1/1000 is replaced with D-psicose, shoot formation is promoted significantly more than when glucose is added alone. Is formed relatively quickly. In other words, shoot formation is remarkably promoted by adding a very small amount (1/1000 to 1/100) of rare sugar (D-psicose) to glucose, a monosaccharide, and it is currently used mainly. The effect on shoot formation equivalent to or higher than that when sucrose is used can be expected.

  Details of the present invention will be described in the examples. The present invention is not limited to these examples.

"Implementation content"
[experimental method]
In plant regeneration in Gloriosa test tubes, sucrose, glucose, fructose and D-psicose were added together as a carbon source for the medium, and the effects were investigated. In any treatment section, a shoot base section obtained by dividing a shoot base formed in a test tube into two parts was used as an explant. The basic medium was an MS medium supplemented with 0.5 μM NAA and 5.0 μM BA, and cultured at 25 ° C. for 16 hours.

  The treatment zones are 0.09 M sucrose, 0.09 M glucose, 0.09 M fructose, 0.09 M glucose + 0.09 M fructose, 0.09 M D-psicose, 0.009 M D-psicose + 0.081 M glucose, 0.0009 M D-psicose + 0.0891 M glucose The treatment group was added or 0.00009 M D-psicose + 0.08991 M glucose was added.

"result"
FIG. 1 shows changes over time in the number of shoots per explant in each treatment area. Table 1 shows the effect of sugar on shoot growth. When glucose or fructose was added alone, shoot formation was clearly suppressed as compared to sucrose that was normally used. In addition, no shoots were formed in the psicose alone addition group or in the treatment group in which one-tenth of 0.09 M glucose was replaced with D-psicose. This reveals that a relatively high concentration of D-psicose inhibits shoot formation from the shoot base section of Gloriosa. On the other hand, in the treatment group in which one hundredth of 0.09 M glucose was replaced with D-psicose and in the treatment group in which one thousandth was replaced with D-psicose, shoot formation was clearly promoted compared to the 0.09 M glucose alone group, A tendency to form shoots relatively quickly was recognized. The number of shoots per tuber was also high in the sucrose group, the treatment group in which 1 / 100th of 0.09 M glucose was replaced with D-psicose, and the treatment group in which 1/1000 was replaced with D-psicose.

  The present invention shows that rare sugars that have been rarely used for propagation in tissue culture of plants so far can be promoters or regulators of shoot growth in micropropagation of horticultural crops. That is, in the tissue culture method of plants, the growth of shoots can be promoted or adjusted by adding a rare sugar to the medium during the tissue culture of horticultural crops, particularly bulbous crops. In addition, since sucrose normally used for tissue culture is a disaccharide, glucose is a monosaccharide, so the amount can be reduced to about half at the same molar concentration. Therefore, considering the fact that the unit price of glucose and sucrose, the amount of rare sugar is very small, and the mass production of rare sugar is beginning to be realized, the present invention is also expected to contribute to the cost reduction of clonal growth. it can.

It is drawing which shows the time-dependent change of the number of shoots per explant in each treatment section.

Claims (4)

A method of utilizing rare sugars for promoting or adjusting the growth of shoots when planting and cultivating horticultural crop tissue as an explant in a medium. The method of utilizing rare sugars according to claim 1 for promoting or regulating shoot growth, wherein the horticultural crop tissue is a bulbous crop tissue. A method of using the rare sugar of claim 2 used as an explant to promote or adjust the growth of shoots, by dividing the base of the globulosa 'Lutea' shoot into a spherical shape and dividing it vertically into two. The method for utilizing the rare sugar according to any one of claims 1 to 3 for promoting or adjusting shoot growth, wherein the rare sugar is D-psicose.