CN1230337A - Method for producing arbor crops by photoautotrophic culture - Google Patents

Abstract

The invention provides a method of efficiently producing small plant bodies, having a genetically homogeneous state. The small plant bodies are produced by transplanting a tissue of an arboreous plant preferably of Acasia, Coffea or Garcinia into a sugar free medium, and culturing by using carbon dioxide with a high concentration under illumination. Seedling develops root better than traditional polyculture of light. The seedling growth can be improved by further using a fibriform or porous support and enhancing the permeability in the culture container.

Description

Cultivate the method that produces woody plant by photoautotrophy

The present invention relates to cultivate the method for the sapling that produces woody plant, especially Acacia, Coffea and Garcinia by photoautotrophy.

Generally speaking, woody plant (arbor crop) can produce by the sexual reproduction mode with seminal propagation, and by with cuttage, sow connect, the asexual reproduction mode of press strip or root spread produces.The disadvantage of sexual reproduction mode is:

1) thus since plant mutually between inconsistent in heredity can not obtain stable output, and the proterties of the seedling that produces with grow also different;

2) low germination rate causes low productivity ratio;

3) because its poor growth and heterozygosity height determine that the proterties of seedling needs considerable time.

4) to obtain seedling, need 2 years at least.

In contrast, asexual process for example the disadvantage of cuttage be:

1) amount from the last available spray of a maternal plant (pistillate parent) is limited;

2) asexual reproduction for example cuttage can not be applied to many seeds; And

3) to produce seedling and need 2 years at least.

The method of breeding Acacia mangium generally adopts seminal propagation, and Acacia mangium is the representative seeds in Southeast Asia.Hereditary difference widely on the seedling that the problem of this method is to be brought by seed.Therefore, need the new method that mass efficient ground produces hereditary merit clone.

For the Coffea plant, especially accounted for the fruitlet coffee of coffee bean output 70%, because it has produced genetic stability from the body pollination, adopt seed propagation method.But other coffee bean output of 30% then comprises from various coffee beans.The overwhelming majority in them is cross pollination, even and they can breed by the mode of seminal propagation, they are still heredity and go up discrepant.What therefore, generally adopt when producing these kinds is cuttage.But this cuttage also has problem same when being used for other seeds, and for example take root difficulty and reproduction rate are low.

For belonging to the tropical fruit (tree) mangosteen of Garcinia, because this kind is by the unisexual reproduction breeding, so generally adopt the seminal propagation mode.Yet,, need a kind of method of alternative seminal propagation because the grain weight that obtains by this method is limited.

Recently, when this kind in seminal propagation with for example cuttage of nourishing and generating is had any problem or these methods when impracticable, produced clone seedling (micropropagation) with the method for group training.Producing the clone seedling with tissue culture method comprises and selects skill and prepare maternal plant (step 0), set up aseptic culture (step 1), breeding (step 2), externally take root and regulate (step 3), and in the external environment that exsomatizes, tame rataria (step 4) (Hartman, H.T. etc., 1990, the principle 459-495 page or leaf of the tissue culture of micropropagation, the plant propagation principle and put into practice (waiting five editions)).This method is particularly suited for being used to produce the consistent rataria of a large amount of heredity.Again these seedling are bred this plant by successive transfer culture and breeding through the tissue of cultivating each organ gained of plant.Utilize this method successfully to produce for example clone rataria of carnation and orchid of many herbaceous plant at present by group training.

But in woody plant, method used in the above-mentioned woody plant but seldom is used for woody plant, and this is because slow, rooting rate of these (woody) plant growings and reproduction rate are low etc.Though they have industrial value, also successfully do not produce the clone embryo of Acacia, Coffea or mangosteen at present by traditional group training method (cultivating) with double the supporting of the luminous energy of gel medium.Therefore, need set up a kind of method and produce the consistent clone embryo of a large amount of heredity.

One of purpose of the present invention provides a kind of method and is used for producing effectively the consistent woody plant of heredity, and especially those belong to the method for the plant seedlings of Acacia, Coffea and Garcinia.

Its shortcoming of traditional tissue culture technology (luminous energy hold concurrently support cultivate) is because use contains the microbial contamination that sugar culture-medium causes, and the leaf of the seedling that grows in the incubator and root development are insufficient.Especially; for several mostly; it usually is difficult oneself will being stretched in the agar of gelation, so the root of growing in the agar of gelation is in domestication stage regular meeting's stasi (smith and spomer, 1995; container; gel, liquid nutrient medium and back-up system, 371-404 page or leaf; the automation of plant tissue culture and environment control (ed.:Kozai and Smith), Kluwer Academic Publisher).

Traditional pass through luminous energy hold concurrently support cultivate be used to produce woody plant for example the method for the seedling of Acacia, Coffea and mangosteen have the problems referred to above.Therefore just be difficult to produce effectively the clone seedling.In these cases, the inventor has designed in the sugar-free culture-medium under the illumination condition and has cultivated the method that produces seedling by the photoautotrophy of seedling.Therefore the inventor has selected conduct to have Acacia, Coffea and the mangosteen of high industrial value woody plant, and has studied and how to have cultivated the method for producing seedling from these plants by photoautotrophy.They find and will transfer to the sugar-free culture-medium and cultivate under light and when keeping high carbon dioxide concentration in the culture vessel from the explant that these plants obtain as a result, root development of these seedling and growth support than holding concurrently all that institute may reach when cultivating by traditional luminous energy better.They also find can improve growth of seedlings with the air permeability in porous holder and the raising culture vessel.

The present invention relates to cultivate the generation woody plant, especially belong to the method for the plant of Acacia, Coffea and Garcinia by photoautotrophy.More specifically, it relates to

(1) method of production woody plant seedling, it comprises transfers to the tissue of woody plant in the sugar-free culture-medium and illumination cultivation under the situation of supplying carbon dioxide gas;

(2) method of (1), wherein said woody plant belongs to the plant of Acacia;

(3) method of (1), wherein said woody plant belongs to the plant of Coffea;

(4) method of (1), wherein said woody plant belongs to the plant of Garcinia;

(5) (1) each method in (4), wherein said cultivation are carried out in the culture vessel of 500 micromoles/mole of carbon dioxide concentration keeping 350;

(6) each method in (1) to (4), the scope of wherein cultivating its photosynthesis photon flow is 150 to 300 micromole/rice ²Carry out under the illumination of/second;

(7) each method in (1) (6) is wherein used multifilament or porous holder or their mixture in medium;

(8) method of (7), holder wherein are the mixtures of cellulose fiber peacekeeping vermiculite; And

(9) each method in (1) to (8), cultivation is wherein carried out in the culture vessel of tool height gas permeability.

Brief Description Of Drawings

Fig. 1 has shown the time graph of Acacia seedling net photosynthesis rate (after this being called " Pn ") under light.Indicated average ± SD.Handle in the code name, " S30 " represents the multitudinous sugar of 30 grams per liters; " S0 " refers to sugar-free, and " GR " refers to that growth regulator is arranged; " NR " refers to there is not growth regulator; " CE " refers to be rich in carbonic acid gas; " NE " is not rich in carbonic acid gas.

Fig. 2 has shown the time graph of the Pn that the coffee seedling was cultivated in 45 days.Handle in the code name, " L " on the left side and " H " refer to 150 and 350 micromole/rice respectively ²The photosynthesis photon flow (after this being referred to as " PPF ") of/second, " N " on the right and " E " are represented low and high carbon dioxide concentration respectively.

Fig. 3 has shown that photon density and gas concentration lwevel are to the influence of coffee growth of seedling in photoautotrophy is cultivated.Handle in the code name, " L " " M " on the left side and " H " representative are respectively 150,250 and 350 micromole/rice ²The PPF of/second, " N " on the right and " E " represent low and high carbon dioxide concentration respectively.

Fig. 4 has shown the system of exploitations such as Niu.Among the figure, 1 represents fluorescent lamp; 2 data loggers; 3 monitor the capnograph of gas concentration lwevel in little growth room; 4 PPF sensors; 5 little growth rooms; 6 thermocouples; 7 plastic cups; 8 culture vessels; 9 flow speed controllers; The NaCl saturated solution of 10 control relative moisture; 11 compressed gas cabins; And 12 CO ₂The cabin, source.

Fig. 5 shows the estimated result of Pn on the every leaf area that the system (Fig. 4) with Niu etc. obtains.Handle in the code name, " L " on the right and " H " represent low and high gas exchange number respectively, and " S " on the left side and " F " represent respectively and contain sugar and sugar-free.

Fig. 6 shows the estimated result of the Pn of the every leaf area that obtains with portable photosynthesis (PP) system.

Noun used herein " xylophyta " thus refer in root and stem to form xylem and to make its most cells wall lignifying make the plant of its curing. The meaning of this noun and draft or succulent plant are opposed, in these plants and insufficient formation xylem.

The present invention relates to cultivate to produce by photoautotrophy the method for xylophyta seedling. The method comprises that the tissue with xylophyta forwards in the sugar-free culture-medium and replenishes and cultivate under illumination condition with carbon dioxide.

To coming from the plant of Acacia, the branch with leaf can be used as source tissue's (explant) of cultivating usefulness. The branch with leaf that is used for cultivating is usually formed and can be come from the multiple branch that forms at the culture medium that contains plant growth regulating substance by a branch and multi-disc leaf. For belonging to the plant of Coffea, unijunction can be saved leafy butt as explant. Each joint often there is one to two blade. Can obtain unijunction and save leafy butt by at the internode place branch of isolated growth being cut apart. For belonging to the plant of Garcinia, for example can be with the branch cut apart from the aseptic germination seedling as explant.

In the method for the invention, the explant of gained like this is transferred in the sugar-free culture-medium and illumination cultivation under replenishing with the condition of carbon dioxide. Because different from whole plant, plant tissue cultures can not produce carbohydrate by photosynthesis, so all think in the past and should replenish carbohydrate at the culture medium that is used for Plant Tissue Breeding. In the method for the invention, therefore plant culture can carry out photosynthesis additional with carbon dioxide and under illumination condition. Therefore carbohydrate is not added in the culture as carbon source. Because carbohydrate may bring out microbial contamination, so should not add carbohydrate in the culture medium, this Pollution on Plant has a negative impact. Used culture medium contains the inorganic nutrients thing and comprises a great number of elements for example nitrogen, phosphorus, potassium, magnesium and calcium, and trace element for example iron, manganese, copper and zinc. In addition, culture medium can contain vitamin for example nicotinic acid and thiamine hydrochloride, organic nutrient substance is amino acid for example, and growth regulator for example 6-benzyl ammonia purine, kinetin, indole-3-acetic acid, NAA, 2, the 4-dichlorphenoxyacetic acid, the N6-[2-isopentene group] adenine, indole-3-butyric acid etc. But they are always not necessary. The preferred embodiment of culture medium is Murashige and Skoog (MS) culture medium (Murashige, T. and F.Skoog, 1962, physiol.Plant 15:473-497) of half strength.

Especially preferred holder or their mixture with multifilament or porous improves the growth of taking root with the explant of cultivating in basal culture medium. When using traditional culture medium for example may be to taking root and growth of seedling have a negative impact during Agar Gel. It is believed that thereby the multifilament of tool high-air-permeability or porous holder can increase in the culture medium concentration of oxygen and improve and take root and grow. And the use of these holders also helps pH, EC of control culture medium etc. The example of holder includes but not limited to vermiculite, perlite, cellulose fibre, polyester fiber, ceramic fibre, rock wool and their mixture. But the shortcoming of polyester fiber, ceramic fibre and rock wool is to cultivate when going in the soil and may sustain damage when the root with plant separates to transfer to holder, and supporter itself can not be degraded. When independent use vermiculite or perlite, its granular form can not stable support plant wherein. So the especially preferred not vermiculite of the above-mentioned shortcoming of tool and the mixture of cellulose fibre (for example Florialite (Nisshinbo Industries, Inc)).

For condition of culture, for example belong to the plant of Acacia, its luminous intensity of shining is generally at 150 to 300 micromole/rice²/ second PPF. Can reduce photosynthetic efficient if luminous intensity is too low. Excessive luminous intensity equally also is disadvantageous, because growth and increase that it will suppress plant expend. CO in the culturing room₂Concentration generally remains in the scope of 1000 to 2000 micromole/moles. If CO₂Concentration is low to be disadvantageous, because this will not reach effective photosynthesis. On the contrary, the CO that has surpassed degree of saturation₂Concentration is unwanted equally, because it can not increase photosynthetic efficient. Generally in 25 to 30 ℃ scope, humidity is generally between 50 to 70% for cultivation temperature. Circulation of air in the culture vessel generally is to be undertaken by per hour changing 3 to 6 times. Thereby CO in especially preferably combining above-mentioned each condition in above-mentioned scope with culture vessel₂Concentration remain in the scope of 350 to 500 micromole/moles. Because CO₂Concentration be subjected to the impact of the photosynthetic rate of plantling in the container, also should regulate the outer CO of container₂Concentration is so that the interior CO of container₂Concentration remain in the desirable scope.

Can under aforesaid similarity condition, cultivate the plant that belongs to Coffea. Yet, since the CO of Coffea plant photosynthetic rate₂Saturation point is higher than sallee, can be with CO in the culturing room₂Concentration bring up to 5000 micromole/moles.

Also can under aforesaid similarity condition, cultivate the plant that belongs to Garcinia. In illumination period, they can be 100 to 300 micromole/rice at PPF²/ second lower the cultivation. Between the seedling culture period, light (secretly) phase generally is 12 to 16 (12 to 8) hour/day. And for improving gas permeability, culture vessel should be ventilated. For example, can be stained with at the vessel port place filter paper (for example Milli-Seal, Millipore) of gas permeability to improve aeration. The preferred at least part of container be printing opacity so that seedling can carry out photosynthesis. For example, can use transparent lid.

The invention provides a kind ofly for cultivate producing xylophyta by photoautotrophy, especially belonging to the method for the plant of Acacia, Coffea and Garcinia. Method of the present invention is so that people can produce the upper consistent xylophyta seedling of heredity at short notice in a large number.

The present invention will be described in detail but should not be construed as and be used to limit the present invention with following embodiment.The photoautotrophy micropropagation of embodiment 1 Acacia seedling

In order to determine that can the photoautotrophy micropropagation be applied on the Acacia mangium, under different condition of culture, compared the double growth of seedlings and the net photosynthesis rate (Pn) of cultivating of supporting of photoautotrophy cultivation and luminous energy.

Particularly, will be with conventional method (MS medium, replenish with 20 grams per liter sucrose, 1 mg/ml BAP, and 8 grams per liter agar) the Acacia mangham branch of successive transfer culture is as explant (50 to 100 milligrams of fresh weights (Fw)/branch), 4 explants are transferred in 370 milliliters the container that contains 60 milliliters of MS medium, as shown in table 1ly designed 9 kinds of processing.

Table 1

Handle code	Multitudinous sugared concentration (grams per liter)	Growth regulator (1 mg/litre/IBA	Rate of ventilation (hour -1)	CO in the culturing room 2Concentration (micromole/mole)	Holder	PPF (micromole/rice 2/ second)
							S30-GR-CE S30-NR-CE S0-GR-CE S0-NR-CE S30-GR-NE S30-NR-NE S0-GR-NE S0-NR-NE contrast	????30 ????30 ????0 ????0 ????30 ????30 ????0 ????0 ????30	Whether be	????6.7 ????6.7 ????6.7 ????6.7 ????0.6 ????0.6 ????0.6 ????0.6 ????0.6	????1300 ????1300 ????1300 ????1300 ????n.e. ????n.e. ????n.e. ????n.e. ????n.e.	V+CF V+CF V+CF V+CF V+CF V+CF V+CF V+CF agar	????150 ????150 ????150 ????150 ????150 ????150 ????150 ????150 ????150

In table 1, the low CO of " n.e. " representative ₂Concentration (500 to 600 micromole/mole) and " V+CF " represent 10 gram vermiculite and cellulose (Floriatile, Nisshinbo Industries, mixtures Inc) in each container.Concentration 8 grams per liters of used agar.

Control treatment is designed to carrying out with traditional photosynthetic foster cultivation under the condition of similarity of holding concurrently.Air themperature was 26 to 27 ℃ under 16 hours (8 hours) light (secretly) phases in the culturing room.Relative moisture in the culturing room maintains 60%.White fluorescent lamp is used as light source.Measure the original position Pn value of seedling weekly in illumination period according to the method for (J.Agr.Meterd., 43 (1): 21-30,1987) such as Fwjwara.(the 28th day) measures fresh weight, dry weight and rooting rate when culture period finishes.

As shown in table 2, the container of highly breathable and CO in the culturing room ₂The binding energy of enrichment increases fresh weight and the dry weight of seedling significantly.Equally, unmatchful its that have of sugar and growth regulator has no significant effect in the medium.The all low and unrooted of seedling dry weight in the control treatment and weight in wet base.

Table 2

Handle code	Fresh weight (milligram/seedling)	Dry weight (milligram/seedling)	Take root (%)
				S30-GR-CE S30-NR-CE S0-GR-CE S0-NR-CE S30-GR-NE S30-NR-NE S0-GR-NE S0-NR-NE contrast	????222±89 ????155±21 ????410±145 ????298±4 ????152±15 ????116 ????110±33 ????139±38 ????100	????40±21 ????32±1 ????62±24 ????41±14 ????22±2 ????17 ????18±4 ????20±4 ????14	????94 ????81 ????100 ????82 ????38 ????75 ????82 ????46 ????0
Variance analysis sugar growth regulator CO 2	????NS ????NS ???? **	????NS ????NS ???? **	????NS ????NS ????NS

In table 2, " NS " and " * * " represents " not remarkable " and " remarkable when P＜0.01 " respectively.

Sugar-free, gas permeability height and be rich under the CO2 disposition (S0-GR-CE and S0-NR-CE handle) photoperiodic Pn in the training period than higher (Fig. 1).The above results shows that the photoautotrophy micropropagation has improved Acacia mangham growth of seedlings, photosynthesis and takes root.The coffee (Coffea arabusfa) of photoautotrophy micropropagation (1) culture in vitro of embodiment 2 Coffea seedling belongs to the aptitude tests of seedling photosynthesis

To CO different in the culture vessel ₂Studied the photosynthesis ability of the Coffea seedling (Coffea arabusta) of culture in vitro under concentration and the PPF level.Particularly, measure Pn:(1 with three kinds of methods) during 45 days based on culture vessel in and the CO of the outer mensuration of container ₂The time graph of concentration determination Coffea seedling original position Pn.(2) (the Niu etc. of system that develop with people such as Niu, 1997, J Japanese Soc.Hort Sci., Suppl.2.300-301) in the 10th and the 30th day external evaluation and test seedling original position Pn value of cultivation period, and (3) are with the Pn of external Coffea seedling in 45 day age of portable photosynthesis (PP) system evaluation.1) according to CO ₂The net photosynthesis rate time graph of the seedling of the Coffea seedling of concentration determination.

The following cultivation of carrying out the Coffea seedling.At first, the unijunction with 3 stripped Coffeas (Coffea arabusta) seedling saves the more piece butt at 480 centimetres ³Cultivate in the container of plastic tank type.Contain 75 milliliters of sugar-frees in each container, partly measure the MS medium as holder, wherein do not contain growth regulator and Florialite (Nisshinbo Industries is Inc) as holder.Before autoclaving, medium pH is transferred to 5.70.With its aperture is that (Milli-Seal Millipore) is used for covering the pair of holes on the transparency cover on the culture vessel for 0.5 micron ventilative filter membrane.Estimate that according to the method for Kozai etc. the gas purging number of times is 2.34/ hour (Kozai, T. etc., 1986, J.Agr Meterol., 42 (2): 119-127) in the container.Experiment in culturing room in two CO ₂Carry out under concentration (Cout) level: 400 to 500 (environment) and 1,400 to 1,500 micromole/mole.Three days is 50 micromole/rice with explant in PPF ²Cultivate under/second the condition, with the white fluorescent tube of non-pyrogenicity shine (Matsushita Electric Industrial CO., Ltd.).In below seven days luminous intensity is increased to 100 micromole/rice ²/ second, subsequently 10 days are increased to 150 micromole/rice again ²/ second.To last 25 days, three kinds of other luminous intensities of level have been adopted: 150,250 and 350 micromole/rice ²/ second (table 3).In the culturing room air themperature is remained on 28 ± 2 ℃, relative moisture remains on 70 to 80%, and every day, illumination was 16 hours.

Table 3

Handle code	?CO 2Concentration (micromole/mole)	PPF (micromole/rice after the 21st day 2/ second)	The seedling number
				????LN ????MN ????HN ????LE ????ME ????HE	????400-500 ????400-500 ????400-500 ????1400-1500 ????1400-1500 ????1400-1500	??????150 ??????250 ??????350 ??????150 ??????250 ??????350	???15 ???15 ???15 ???15 ???15 ???15

In the processing code of table 3, " L " on the left side, " M " and " H " represent respectively from the 21st day 150,250 and 350 micromole/rice ²The PPF of/second, " N " on the right and " E " represent low and high CO respectively ₂Concentration.

The Pn value of seedling is calculated (J.Agr.Meterol., 43 (1): 21-30,1987) with the following equation of propositions such as Fujiwara under the 10th, 20,30 and 45 day stable state, measures CO with gas-chromatography (Gc-12A, Shimadzu Co.) ₂Concentration.

Pn=KEV (C _Outward-C _In) (1) wherein " K " be CO ₂(40.5 moles/meter of conversion coefficients from the volume to the molecular weight ³); " E, " is air exchange number of times hourly in the culture vessel (hour-1); " V, " is the volume of culture vessel; " in the C " and " C is outer " is meant the inside and outside CO of culture vessel when stable state ₂Concentration (moles/mole).

Fig. 2 shows the result of the test according to the time graph of the seedling base gained of coffee seedling between 45 days culture period.All values all are the mean value of 5 detections.Pn has a little rising in 20 days cultivation.In ensuing 25 days, high concentration CO ₂Influenced the photosynthetic capacity of Coffea seedling (Coffeaarabusta) very significantly.Be rich in CO at the 45th day ₂Pn in the environment (" LE " and " HE ") is almost than not being rich in CO ₂Pn Senior Three in the environment (" LN " and " HN ") doubly.But luminous intensity is to the almost not influence of increase of coffee seedling Pn.The Pn value is at 150 and 350 micromole/rice ²There is no big difference under the condition of/second.

Fresh weight, dry weight, stem length and the root of having measured seedling at the 45th day are long.Be rich in CO ₂Environment in all these has increased.In luminous intensity is 150 micromole/rice ²Under the situation of/second (LE), it promotes that the effect of growth is especially obvious.2) the net photosynthesis rate of external test Coffea seedling

Pn for external mensuration Coffea seedling under the situation that does not change condition of culture, having begun that just unijunction is saved leafy butt cultivates in the plastic cup (diameter is 25 millimeters, highly is 30 millimeters) of the 10 milliliter of half amount MS medium that contains sugar (20 grams per liter) and sugar-free with the system (Fig. 4) of exploitations such as Niu.Three cups are sterilely placed 480 centimetres that cover with two Millipore filter paper (Milli-Seal) ³In the container, container is placed be not rich in CO then ₂PPF be 150 micromole/rice ²In the chamber of/second.At the 10th and the 30th day, three cups during each medium handled took out, and the Merlon Magenta type container (Fig. 4) that places 4 Millipore filter paper (Milli-Seal) is to measure C _InAnd C _OutwardThe air exchange rate of estimating this container is 6.5 hours ^-1At every cover condition (C _OutwardAnd PPF) measures C in the stable state under _InAnd C _Outward, calculate the Pn value according to the equation (1) of Fujiwara etc.The temperature of growth box is remained on 28 ± 1 ℃.Measured the Pn value of seedling: 120 (L) and 247 (H) micromole/rice with two kinds of other luminous intensities of level (PPF) at the 10th day ²/ second.At the 30th day, with 320 micromole/rice ²The PPF of/second measures the Pn value of seedling.Measure and just finish, measure each dry weight of handling all seedling and leaf area again.Use gas chromatograph (GC-12A, Shimadzn CO.) to measure CO then ₂Concentration.

Fig. 5 shows the result who measures Pn with the system of exploitations such as Niu (Fig. 4) according to leaf area.From the result of this processing proved the Coffea seedling under the photoautotrophy condition, have can grow extraordinary potential.Result at the 10th and the 30th day shows the CO in container ₂Concentration is along with CO in the growth box ₂The increase of concentration and increasing.At the 10th day, when luminous intensity (PPF) increases, CO ₂Concentration has obviously influenced Pn, and (Fig. 5 a).Result at the 30th day shows the CO of external Coffea seedling ₂Saturation point is quite high (when luminous intensity is 320 micromole/rice ²During/second, be 4,500 to 5,000 micromole/moles) (Fig. 5 b).3) with portable photosynthetic use (PP) system, (PP System CO., Ltd.) mensuration is made rate based on the net photosynthesis of leaf area to model C IRAS-1

At 150 micromole/rice ²(Nisshinbo Industries Inc.) partly measures cultivation Coffea seedling on the MS medium at sugar-free to/second PPF with Floriatite down.Stretch leaf with its 3rd and be used for measuring Pn.Every leaf is all placed leaf box (2.5 centimetres of the leaf areas of PP system ²).The preset temperature point of box is 28 ℃.Three kinds of other CO of level have been studied ₂Concentration: 462,951 and 1754 micromole/moles.At each CO ₂Under the concentration, when measuring PPF is set in 10,50,100,200,300,500,700 and 1000 micromole/rice with the PPF sensor ²/ second.With the effective light source of doing of halogen lamp.

Fig. 6 has shown with the PP system according to Coffea leaf mensuration Pn value.Along with CO ₂The increase Pn of concentration and luminous intensity also increases.At PPF is 200 micromole/rice ²Under the situation of/second, CO ₂Concentration is that Pn under 1754 micromole/moles (triangle among the figure) is high 1.8 times during approximately than 462 micromole/moles (square frame among the figure), and is higher 1.4 times than 951 micromole/moles (circle among the figure).When PPF is increased to 1,000 micromole/rice ²During/second, CO ₂Pn when concentration is 1754 moles/mole is higher 2.1 times when 462 micromoles/mole than it, and is higher 1.7 times when 951 micromoles/mole than it.

Result's proof Coffea seedling under the photoautotrophy condition has very high growth in vitro and breeding potential.(2) by sugar, CO in the Coffea seedling (Coffea arabusta) of photoautotrophy cultivation micropropagation ₂The influence of concentration and luminous intensity

In first experiment, (20 grams per liter) being arranged or not having 70 milliliter half of (0 grams per liter) sucrose to measure two unijunctions joints of cultivation more piece butt on MS agar (9 grams per liter) medium in 300 milliliters of Merlon column jar type containers (70 millimeters high, 80 mm dias).Do not use plant growth regulator.Before autoclaving with pH regulator to 5.7.Contain that to have the aperture on the lid of container of sugar-free culture-medium be that (Milli-Seal Millipore), but does not then have filter paper to the container that contains sugar culture-medium for 0.5 micron gas permeability filter paper.In 60 days culture period, the developmental condition of seedling is 70 micromole/rice ²The PPF of/second, the illumination period of 10 hours every days.Temperature is 26 ± 2 ℃, and relative moisture is 55 to 65%.Come statistical ground to calculate the long and leaf area of fresh weight, dry weight, stem of every strain seedling by variance analysis.In second experiment, cultivate three unijunctions on 75 milliliter of half amount MS liquid sugar-free sucrose medium in 480 milliliters of Merlon column jar type containers and save leafy butt.Holder employing Florialite (NisshinboIndustries, Inc.).Have two apertures on the lid of this jar and be 0.5 micron ventilative filter paper (Milli-Seal, Millipore).In every day in 45 days, with seedling in 16 hours illumination period in two-stage CO ₂Concentration (500 to 1500 micromole/mole) and two-stage PPF (150 to 250 micromole/rice ²/ second) grows under the condition.Before autoclaving with pH regulator to 5.7.At the 45th day, collect fresh weight, dry weight, leaf area and the long value added of stem of every strain seedling, to 9 strain seedling triplicate and carry out variance analysis in each is handled.

Measured the influence of sucrose concentration at the 60th day that cultivates, the results are shown in Table 4 the coffee growth of seedling.Table 4

Handle code	Fresh weight (milligram)	Dry weight (milligram)	Stem long (millimeter)	Leaf area (centimetre 2)
					???S20 ???S0	????331 NS????251 NS	????63 NS????68 NS	????23 *????28 *	????1.73 *????2.22 *

In the processing code name in table, the cultivation of " S20 " representative in the medium that contains 20 grams per liter sucrose, and also on behalf of sugar-free, " S0 " cultivate." NS " and " * " representative " not remarkable " and " remarkable when p=0.01 ".

The result of table 4 show the Coffea seedling can be on sugar-free culture-medium well-grown.Long and leaf area is being significantly greater than containing on the sugar culture-medium at the stem of seedling on the sugar-free culture-medium, but the increase of fresh weight and dry weight does not then have significant difference between each processing.At the base portion that contains all stems of sugar culture-medium callus is arranged all, but callus on sugar-free culture-medium, then do not occur.

PPF level and CO have been studied ₂Concentration is to the influence of external Coffea growth of seedlings, and the result sees table 5.Table 5

Handle code	Fresh weight (milligram)	Dry weight (milligram)	Stem long (millimeter)	Leaf area (centimetre 2)
					????LN ????LE ????HN	????200.34 ????760.38 ????224.44	????41.03 ????132.10 ????44.38	????10.33 ????30.56 ?????8.34	????24.44 ????81.27 ????26.87

????HE	????385.21	???81.70	???17.33	????51.09
					Variance analysis CO 2Concentration (C) PPF C * PPF	????? **?????NS ????? *	???? **????NS ????NS	????? **????? *????? *	????? **?????NS ?????NS

Low PPF (150 micromole/rice represented respectively in processing code name in the table, " L " and " H " ²/ second) and high PPF (250 micromole/rice ²/ second), " N " and " E " represents low CO respectively ₂Concentration (500 micromole/mole) and high CO ₂Concentration (1500 micromole/mole)." NS " " ^*" and " ^*" representative is not remarkable respectively, " remarkable during p=0.05 " and " remarkable during p=0.01 ".

As shown in table 5, regardless of luminous intensity, to be rich in CO ₂The seedling of handling is than being rich in CO ₂The seedling of handling looks much better.Be not rich in CO ₂Environment under, PPF has increased growth.But, be rich in CO ₂Environment in, seedling is at 150 micromole/rice ²Ratio is at 250 micromole/rice during/second (LE) ²To grow much betterly during/second (HE).At PPF is 150 micromole/rice ²/ second and CO ₂Concentration is that fresh weight, dry weight, the stem of its seedling under the situation of 1500 micromole/moles (LE) is long, leaf area is more much higher than other processing.The result shows and is being rich in CO ₂Environment in the coffee growth of seedlings can be enhanced.(3) holder and air capacity of soils are to the influence of the micropropagation of Coffea (Coffea arabusta) seedling cultivated by photoautotrophy.

With Floriatile (cellulose fiber peacekeeping vermiculite; 10 gram/containers; Nisshinbo Industries, Inc.) and Bacto agar (being added to above-mentioned medium) to 8g/l and gelling as holder, in 480 milliliters of transparent vessels that contain 75 milliliter of half amount MS medium (20 grams per liter sucrose or sugar-frees, no growth regulator) with three strain Coffea seedling (Coffea arabusta) culture in vitro 20 days.Covering of container can not have the hole that two holes (diameter is 10 millimeters) also can be arranged, and each hole is 0.5 micron ventilative filter paper (Milli-Seal, Millipore) covering with the aperture.It is 0.24 to 2.34 hour that air exchange rate in the container is estimated ^-1All containers all remain under such condition: 28 ℃, 75% relative moisture, every day are with non-pyrogenicity white fluorescent fluorescent tube irradiation 16 hours, environment CO ₂Concentration is 400 to 500 micromole/moles.In three days, PPF is 50 micromole/rice ²/ second, be 100 in follow seven days, be 150 in get off again 10 days, last 20 days of cultivation is 200.Method with people such as Fujiwara is calculated Pn (J.AgrMeterol., 43 (1): 21-30,1987).With the inside and outside CO of gas chromatograph for determination culture vessel ₂Concentration.To test triplicate with two constant indexs and three variable indexs.Data with variance analysis test fresh weight, dry weight, stem length and leaf area increase.Least significant difference during with p=0.05 (LSO) is tested the significance between each processing.Processing sees Table 6.Table 6

Handle code	Sucrose concentration (grams per liter)	Holder	Gas exchange number of times (per hour)
				????SAL ????SFL ????SAH ????SFH ????FAL ????FFL ????FAH ????FFH	??????20 ??????20 ??????20 ??????20 ??????0 ??????0 ??????0 ??????0	Agar cellulose/vermiculite agar cellulose/vermiculite agar cellulose/vermiculite agar cellulose/vermiculite	??????0.24 ??????0.24 ??????2.34 ??????2.34 ??????0.24 ??????0.24 ??????2.34 ??????2.34

Handle in the code, " S " on the left side and " F " represent respectively and contain sugar and sugar-free culture-medium, middle " A " and " F " represents agar and cellulose/vermiculite mixture (Florialite) respectively, and " L " on the right and " H " represent the low and high number of times of air exchange respectively.

Each processing the results are shown in Table 7.Table 7

Handle code	Fresh weight (milligram)	Dry weight (milligram)	Stem long (millimeter)	Leaf area (centimetre 2)
					????SAL ????SFL ????SAH ????SFH ????FAL ????FFL ????FAH ????FFH ?LSDp=0.05	????130 ????62 ????27 ????59 ????23 ????61 ????39 ????278 ????59	????37 ????36 ????20 ????34 ????13 ????18 ????19 ????52 ????13	????4.6 ????4.6 ????2.1 ????3.9 ????2.7 ????4.2 ????3.2 ????13.0 ????1.6	????9.7 ????6.1 ????4.5 ????5.3 ????5.1 ????5.9 ????4.5 ????13.0 ????2.9
Variance analysis sucrose concentration (A) holder (B) air exchange number of times (C) A * B * C	???? *???? **???? *????NS	????NS ???? **????NS ????NS	???? **???? **???? **???? **	????NS ???? **???? **????NS

In the table, " NS ", " ^*" and " ^*" representative " not remarkable " respectively, " remarkable when p=0.05 " and " remarkable when p=0.01 ".

Whether the existence of sugar does not influence the 40th day cultivation to the increase of dry weight and leaf area, though it can influence fresh weight and the long increase of stem in the holder.The type of holder can influence all above-mentioned growth parameter(s)s.The air exchange rate influences dry weight, stem is long and the increase of leaf area.These three factors interact to long generation of stem.The recruitment of fresh weight and dry weight, stem length and leaf area is maximum in sugar-free, fibrous holder and high air exchange rate (FFH) are handled.Root growth and growth are maximum in FFH handles.In the processing of fibrous holder (FFL, SFH and FFH handle) but in root induction, but in SFL handles (containing sugar, cellulose holder and low air exchange rate) then do not induce.In containing sugared agar processing (SAL and SAH), do not observe root and stretch or grow, and produced callus at the base portion of stem.The photosynthetic rate that FFH handles in whole experiment is the highest, in the time of the 10th day every strain seedling be 0.7 micromole/hour, in the time of the 20th day, be 1.0, in the time of the 30th day, be 2.1, in the time of the 40th day, be 3.9.These results show in sugar-free culture-medium with highly porous holder and high air exchange speed and can improve external Coffea growth of seedlings and take root.The photoautotrophy micropropagation of embodiment 3 mangosteen seedling

The branch that will take off from the nursery stock of aseptic germination is as explant (640 to 870 milligrams/branch of fresh weight).Explant is forwarded in 370 milliliters of polycarbonate containers that contain 100 milliliters of MS medium.Vermiculite is used as holder (every container 25 to 35 grams).Handle coded representation such as table 8 for 5 with setting.

Table 8

Handle code	Sucrose concentration Z(grams per liter)	Growth regulator Y	The air exchange number of times X(hour -1)
				S30-GR-HV S30-NR-HV S0-GR-HV S0-NR-HV S30-GR-LV	????30 ????30 ????0 ????0 ????30	Whether be	????4.4 ????4.4 ????4.4 ????4.4 ????0.1

In the table, " Z " represents initial sucrose concentration in the medium, and " Y " represents the 2-ip of 10 mg/litre and the IBA of 1 mg/litre, " X " representative is according to (Kozai, T. etc., 1986 such as Kozai, J.Agr.Meterol., 42 (2): 119-127) the air exchange rate of the container of Ce Dinging.

The condition of culture of branch is to traditional similar in the contrast, just the holder difference.Temperature is 27.5 ℃ in the illumination period container, is 25 ℃ in dark period.Relative moisture in the culturing room remains on 60%.Provide 110 micromole/rice with fluorescent tube as light pipe ²The PPF of/second.CO in the culturing room ₂Concentration is 1300 micromole/moles.(Milli-Seal is Millipore) to provide high gas exchange rate to stick two ventilative filter membranes on culture vessel.At the 30th day, measured fresh weight and the dry weight of 6 strain seedling of each processing.Equally also measured the number of Ye Hegen.Calculated illumination period Pn value according to people's such as Fujiwara method.The results are shown in Table 9.

Table 9

Handle code	Dry weight (gram/seedling)	The number of sheets (individual/seedling)	Take root (%)	CO 2Concentration (micromole/mole)	The Pn of every leaf area (micromole/rice 2/ second)
						S30-GR-EV S30-NR-HV S0-GR-HV S0-NR-HV S30-GR-LV	?0.30±0.13 NS?0.23±0.18 NS?0.23±0.10 NS?0.20±0.11 NS?0.21±0.11 NS	?6±1.4 NS?4±0.9 NS?6±0.9 *?4±0.9 NS?5±0.9	????40 ????20 ????20 ????40 ????0	?1088±21 ?1142±72 ?1039±30 ?1092±53 ?148±29	2.1±0.35 **2.3±0.88 *2.7±0.69 **3.5±1.58 *0.3±0.14
Variance analysis sugar growth regulator	????NS ????NS	????NS ???? **	????NS ????NS	????- ????-	????NS ????NS

In the table, " NS " " ^*" and " ^*" represent " not remarkable " " remarkable when p=0.05 " and " at p=0.01 time remarkable " for control experiment (S30-GR-LV) respectively.In the variance analysis, " NS " and " ^*" representative " not remarkable " and " remarkable when p=0.01 " respectively.

As shown in table 9, throughout between the reason the 30th day the time fresh weight and the dry weight of seedling do not have significant difference.In medium, add the number that growth regulator has increased leaf.In contrast, do not observe the phenomenon of taking root, but in medium having or not of sugar or growth regulator no matter, with vermiculite as holder in the processing at highly breathable, 20% to 40% branch has showed inducing of taking root.The CO of internal tank in the control treatment ₂Concentration is minimum, the Pn of every leaf area of control treatment be highly breathable processing 1/12 to 1/7.The result proves that mangosteen is being rich in CO ₂The highly breathable container on the medium of vermiculite support, can carry out the photoautotrophy micropropagation.Under these conditions, take root and Pn under the situation that does not reduce growth of seedling speed, be improved.

Claims (9)

1. produce the method for woody plant seedling, it comprises that the tissue with woody plant is transformed in the sugar-free culture-medium and illumination cultivation under the situation of supplying carbon dioxide gas.

2. the process of claim 1 wherein that said woody plant belongs to the plant of Acacia.

3. the process of claim 1 wherein that said woody plant belongs to the plant of Coffea.

4. the process of claim 1 wherein that said woody plant belongs to the plant of Garcinia.

5. each method in the claim 1 to 4, wherein said cultivation are carried out in the culture vessel of the carbonic acid gas of 500 micromoles/molar concentration containing 350;

6. each method in the claim 1 to 4, the scope of wherein cultivating its photosynthesis photon flow is 150 to 300 micromole/rice ²Carry out under the illumination of/second.

7. each method in the claim 1 to 6 is wherein used multifilament or porous holder or their mixture in medium.

8. the method for claim 7, holder wherein is the mixture of cellulose fiber peacekeeping vermiculite.

9. each method in the claim 1 to 8, cultivation is wherein carried out in the culture vessel of tool height gas permeability.

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