JPH02222627A - Production of banana seedling by culture of plant tissue - Google Patents

Abstract

PURPOSE:To obtain a number of healthy seedlings by culturing cell tissue of banana in a liquid medium under aeration and agitation and rooting the obtained shoot. CONSTITUTION:A banana cell tissue containing leaf primordium is extracted from the shoot apex of a collected sucker bud. The tissue is planted on a medium containing coconut water and cultured at 20-30 deg. for 2-3 weeks under illumination of 1,000-3,000 lux all the day long to obtain grown cell. The cell is cultured in a medium free from coconut water under the same condition as above to differentiate a number of buds. The agglomerate of the bud cells is divided into tissues each containing preferably 2-3 buds and continuously cultured in an aerated jar fermentor for 20-40 days while supplying 0.3-1.0l/min of air per 1l of the medium to obtain elongated shoots. The shoot is rooted by culturing in a medium having a nutrient source concentration decreased to a half to obtain the objective seedling.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing banana seedlings by culturing cell tissue in a liquid medium.

The banana seedlings produced according to the present invention are healthy and free from plant diseases caused by the seedlings, and can be used for banana cultivation.

[Technical background and explanation of conventional technology]

Bananas are widely distributed from the tropics to the subtropics! Banana is a perennial herbaceous plant belonging to the family, and its fruit is edible as is, and its fruit is used for ornamental purposes.1
There is a banana as an 11-leaf plant.

Up until now, bananas have been propagated by growing suckers (5uckers) that develop from the tubers of the seedlings and using them as the current plant, or by removing the suckers and transplanting them to grow and use them as the parent plant. It was propagated asexually, but if the seedlings are infected by pests, the pests will be transferred to the asexually propagated offspring, and the offspring will also be infected by pests.
Also, the occurrence of suckers is irregular and the number thereof is limited, making it impossible to produce a large number of banana seedlings.

Up to now, it has been reported that banana seedlings are obtained by planting and culturing the plant tissue of the shoot apex of suckers that develop from banana tubers in an artificial medium. Bust
amaat*: Phytopathology
) N6411 g320-322 (1974) ),! ! (7) Plant tissue at the top of the stem,
[J. C. Vessey & J. A. Rlv.
era: Turrralbm) JE 31, pp. 162-163 (1981)] [Nis Nis Cronauer and N.D. Krikonian: Horde Science (S, S, Cronauer & A, D, Kr
1konlan: Hort, 5elenee) Vol. 19, No. 2, pp. 234-235 (1984)], banana plant cell tissues were further cultured on a solid medium to form shoots, which were cultured with shaking in a limb medium. It has been reported that multiple shoots are formed by
Fadobibria, Tee Hermelin, H Yagura Brunner and Biednini: Nook Chic Vitro Cult Plant Kai Improp (F, J, NovmkJ,
Afts++V, Phadvlbulya+T+Her
Melin, H. Brunoer & B+Donln
1: Nuel.

Tech, Vitro, Cult, Plant, Imp
rov, ) pp. 167-174 (1986)].

The present inventors have been conducting research on the cultivation of cell tissues in the aI form for many years, and in their research, they found that when banana cell tissues were cultured in a liquid medium with aeration and agitation, bud differentiation in the banana cell tissues and By promoting the formation of shoots and rooting them, a large number of banana seedlings that can be planted can be produced. When the scales and cell tissues of banana suckers are cultured in a medium containing coconut water, An enlarged cell tissue is obtained, and it has been discovered that when this enlarged cell tissue is cultured in a medium containing no coconut water, a cell tissue with a large number of differentiated buds can be obtained.Based on these findings, the present invention has been made. Reached.

[Object of the invention and summary of the invention]

An object of the present invention is to provide a method for producing banana seedlings that can produce a large number of healthy banana seedlings.
Specifically, the purpose is to provide a method for producing banana seedlings that can produce a large number of healthy banana seedlings with high productivity.

The present invention provides a method for producing banana seedlings by culturing banana cell tissues and rooting the shoots. A method for producing banana seedlings characterized by increasing formation and thereby obtaining a large number of healthy banana seedlings.

In the method of producing banana seedlings by culturing banana cell tissues of the present invention, banana cell tissues are cultured in a medium containing coconut water to enlarge the cell tissues, and the enlarged cell tissues are injected with coconut water. A large number of healthy banana seedlings can be produced by culturing in a medium containing no banana to produce differentiated cell tissues of buds, and culturing the differentiated cell tissues of the buds in a liquid medium with aeration and agitation.

In the method for producing banana seedlings by culturing banana tissue of the present invention, shoots grown from buds with differentiated cell tissue are cultured in a medium containing half the concentration of nutrients, and the shoots are generated. It is possible to produce plantlets (seedlings) that can take root and thereby be planted.

(Detailed Description of the Invention) A sucker (5ucker) grows at the growth edge of the banana column. A sucker is a plant that grows from a banana tuber.

In the present invention, suckers are collected from the field, thoroughly washed with water, a part of the outer skin is peeled off, and plant pieces containing mainly the shoot apex are extracted. The plant pieces are sterilized with a disinfectant C (e.g., benzalkonium chloride bath solution, sodium hypochlorite aqueous solution, and ethanol), and in a clean bench, using a physical microscope, the plant pieces are removed from the stems. The banana cell tissue, including the top and leaf progenitors around the shoot apex, is removed. This cell tissue is a banana cell tissue in the method for producing banana seedlings by culturing plant tissue of the present invention.

Banana m cell tissues are planted in primary ponds containing coconut water and cultured for 2 to 3 weeks at 20 to 30°C under 24 hour illumination of 1000 to 3000 lux. The primary medium containing coconut water contains 100~TOOpp of myo-inositol (preferably 300~50~
0 p9 Otori), Nicotine #0.2-0.711 PII
(preferably 0.4-0.a ppm), pyridoxine hydrochloride 0.2-0.7 ppm (preferably 0.4-0.a ppm)
0.6pp■), thiamine hydrochloride 0.5-2. Oppm
(preferably! 60-1.59p), glycine 1-3
ppm (preferably 1.5-2.59p), coconut water 50-25Qd/j! (preferably 100 to 200-/l) and shi! IM30000~5
0000 pp ugly (preferably 35000-4500
0 p9 irises) and further benzyladenine 1-16
ppm (preferably 465-11-3 pp+a
) or kinetin r ~ 22 ppm (preferably 4.3
~15.0 ppm), and agar powder 0.7~1.0
Use a solid medium obtained by adding % (by weight). Coconut water is water contained in the hollow space of a coconut. Banana cell tissue grows from the start of culture to approx. The color changes from white to yellow-green within a week, and in about 3 weeks it becomes an aggregate of cellular tissue that has enlarged to around 5H.

This enlarged cell tissue aggregate is planted in a growth medium that does not contain coconut water and cultured for 2 to 3 weeks at 20 to 30°C under 24-hour light. This is a medium obtained by removing coconut water from the primary medium of . The enlarged cell tissue aggregate differentiates into many buds and becomes a cell tissue aggregate with many buds, and some of these buds form shoots.

A collection of cell tissues with numerous buds is composed of at least one (
Divide into cell tissues containing II (preferably 2 to 3) buds and place them in a liquid medium in an aerated jar fermentor.
0.3 to 1.0% per liter of limb culture medium. OtZ min (preferably 0
．． The cells are cultured at 20 to 30° C. under illumination for 24 hours while aerating and agitating the cells by blowing air at a rate of 4 to 0.7/min). This liquid medium changes the concentration of benzyladenine or kinetin in the growth medium to 0 to 0.
．． 2-2.2 lap theory (preferably 0.6-1.5
ppm) or kinetin 0.1-2.1 ppm
(preferably 0.4~!, 5pp■), agar is removed, and coconut water is not included.

The aggregate of cell tissues with buds moves up and down near the liquid surface, multiplying the buds, forming shoots from the buds, and elongating the shoots.

The aeration agitation culture in this jar fermenter is 2
It can be continued for 0 to 40 days, thereby obtaining a collection of m-cell tissue that has formed an elongated shoot.

The shoot is removed from the base of the aggregate of cell tissue that formed the elongated shoot, planted in a rooting medium, and cultured for 3 to 4 weeks at 20 to 30°C under 24-hour light. Roots emerge from the base and elongate, yielding a small M object (plantlet).

The rooting medium does not contain coconut water or the refined hormones benzyladenine and kinetin, and has a dilute concentration of other nutrients, and in the composition of the primary medium, coconut water, benzyl Adenine and kinetin are excluded, but the amount of agar is the same as the primary medium, 0.7 to 1.0%, and other inorganic salts and cumin are diluted 1 to 4 times (preferably about 2 times). The plantlets obtained using the 1 rooting medium are seedlings with roots attached to the plants, which are acclimatized in order to be able to withstand the external environment of 1IIn.

Fill the planter with well-drained soil and use this planter.
Plant the plantlets in the
Temperature of C, 60-90% relative humidity and 5000-30
When cultivated for 2 to 3 hours under lighting conditions of 000 lux, 2 to 3 leaves will emerge from the plantlet, forming a plant with roots, stems, and leaves. In cultivating this plantlet, a plant nutrient solution containing a small amount of chili, phosphoric acid, potash and trace elements is applied. Well-drained soil to put in the planter is vermiculite, perlite, akadama soil,
Use single or mixed soils such as swamp soil, humus, and sand.

This plant is planted in a pot filled with the same well-drained soil, applied with the same plant nutrient solution, placed in a regular net house, and cultivated for 2 to 3 months. The above-ground part grows to 20 to 30 meters and banana seedlings are obtained. These banana seedlings can be planted in a field and harvested as bananas.

In the following, the present invention will be explained in more detail by means of test examples and examples.

Test Example In growing banana seedlings by tissue culture of banana sucker cells, a test was conducted to examine the influence of the culture method on the formation of multiple shoots of cultured banana cells.

(1) Sample mtl (Formation of aggregate of llllN cod with differentiated buds) Suckers of Shimabanana, a native species of Okinawa (originating from the Ogasawara Islands), were collected in the field, washed with water, and the outer skin was peeled off. Grave apex centering around the stem apex [20jIjIC vertical) X2
0 (width) x 40 + w (height)] was cut out. The shoot apical fibers were sterilized by being immersed and stirred in a 70% ethanol aqueous solution, a 0.2% benzalkonium chloride aqueous solution, and a 1% hypochlorite se# sodium aqueous solution, and then washed with sterilized water. A circular tissue piece [0.8 mm (diameter) Xlsua (long)] containing the shoot apex and leaf primordia was excised from the shoot apex tissue using a stereomicroscope in a clean bench.

The circular tissue piece was placed in a test tube (25
m diameter), and cultured in 25 layers at 2°C for 21 days at 2500 lux for 24 hours to turn yellowish green.
A piece of cell tissue enlarged to 5mm diameter was obtained. (Composition of the primary site) MgSO or 7HO CaC1e2HO 370pH 440 ppm 1900 ppm 1650 11$11! ? Oppm 27.8 pp Proverbs 37.3 pP Peng 22.3 ppm 8.6 ppm 0.025 Ppm No NHNO NHPO FeSO-7H0 Na EDTA Mn50 4HO ZnSO Bottle 7H0 CuSO 58 0 Co C1-6) 1 0 0
．． 025 ppm I
O,83++9PengHBO
6,299■ Na MoO embroidery 2H00, 25ppm myo-inositol 400 ppm nicotinic acid
0.5 ppm pyridoxine hydrochloride
0.5 pH Thiamine hydrochloride l・3
ppm glycine 2・o pp theory vs 1140000 Ppm coconut water 150d/l benzyladenine 6.7 pHml agar
700099■ The enlarged lllIgI tissue piece was planted in a test tube (25sw diameter) containing 10-mL of growth medium, and cultured in 25 layers at 2°C for 18 days under the same lighting as above to differentiate a large number of buds. A collection of cell tissues was obtained.

(Composition of growth medium) MgSO・7■03
70 pp Otori CaC1・2H0 N03 NHN.

HPO FeSO・7H0 Na EDTA Mn50・411 0 ZnSO-7HO 4゛2 CuSO-5H0 CoC1-680 BQ Ha MoO-2H0 Myo-inositol Nicotinic acid hydrochloride Pyridoxine Hydrochloride Thiamine Glysine Sugar benzyladenine・440 ppm 1900ppm 1650 pp+m 170 ppm 27.8 pp Zen 37.3 p9 Peng 22.3 Ppm 8+6 99 layer 0.025 ppw+ 0.025 pp proverb 0.83 pHl1 6+2 ppm 0.25 ppm 400 ppm 0.5pp proverb 0.59 9■ 1.3 pp 2, Opp 40QO099 6φ7 99-layer agar 7000 pllll (Note) The growth medium is a primary medium without coconut water.

(2) Test method (2-A) Test using II body @ ground (2-^-1) Formation of multiple shoots (1) above
The aggregate of differentiated cell tissues of the buds obtained in step 1 is divided into cell tissues containing two buds, and the divided cell tissues are divided into cell tissues as described in (1) above.
Cells with buds that formed multiple shoots were planted in 50 test tubes (25 m diameter) containing 10 - of growth medium and cultured for 18 days at 25 ± 2°C under the same lighting as in (1) above. Tissue pieces were formed, and the number of cell tissue buds and the number of shoots that had elongated to two or more in each test tube were counted.

(2-A-2) Rooting of shoots The shoots formed in (2-A-1) above are cut at their bases, separated from the cell tissue, and the shoots are filled with rooting medium IO-. Plant it in a test tube (25 brocade diameter) and culture it for 4 cycles at 25±2℃ under the same lighting as in (1) above, until it has 5 or more roots and the length of the above-ground part. Plantlets of 4 cs or more were formed.

(Composition of rooting medium) MgSO・7H0 CaCIII2H0 185 ppm 220 ppm 950 ppm 825 ppm s ppm 13.9 p9 Kasumi! 8φ65 9p8 11.15 ppm 4・3 ppm 0-0125 ppm 0.0125 ppm 0.415 ppm 3・1 ppm 0.125 119 theory 20G +1911 No. 3 HNO KII PO FeSO No. 7H0 Na EDTA Mn5Oψ 4H0 ZnSO-7H0 CuSO- 5H0 CoCj order 6HO! BO Na MoO drawing 28 0 Myo-inositol nicotinic acid 0.25 ppm Pyridoxine hydrochloride 0.25 T) 9 Pus Thiamine hydrochloride 0.65 ppm Glycine
1.0 ppm2g li
2G000 ppm agar 8000
pp intestinal activated carbon too ppm (Note) The rooting medium does not use coconut water or benzyladenine in the primary medium, has a high concentration of agar, and uses activated carbon, but other modifications are as follows: The degree is expressed as a percentage.

(2-A-3) Acclimation, Potting, and Seedling Growth of Plantlets The plantlets formed in (2-A-2) above were taken out from the test tubes and thoroughly washed with tap water.

Mixed soil of vermiculite and perlite (t:
1) was placed in a planter (18 m (vertical) x 40 cR (horizontal) x 14 m (depth)], and 20 of the above-mentioned washed plantlets were planted in it. This planter was placed in a glass ax, and 25℃ temperature, 60-90% phase'
The plantlets were acclimated under M humidity and 30,000 lux for 24 hours. Three weeks later, two or three new shoots had developed, and the plantlets were lively.

The plantlets were planted in pots containing mixed soil II (2:1) of Akadama soil and humus, and seedlings were raised in a regular net house. After 80 days, the above ground part will be 257
! 11 seedlings were obtained.

These seedlings were fully capable of being planted in the field.

(2-8) Test using liquid growth medium III (kinetin) (2-8-1) Formation of multiple shoots (1) above
The aggregate of differentiated cell tissues of the buds obtained was divided into cell tissues containing two buds, and the divided cell tissues were added to the liquid growth medium C kinetin)! Plant 4 seeds per flask in five 300-capacity Erlenmeyer flasks containing 0. The cells were cultured for 18 days to form cell tissue fragments having buds forming multiple shoots, and the number of buds and the number of sheets elongated to 2 clL or more in the cell tissue fragments in each flask were counted.

[Composition of liquid enriched N culture ft1l (kinetin)] Mg5O
・7HO370ppm CaCI C2HO440ppm NO NHNO NHPG FeSO・7H0 Na EDTA M! 150 ・4H0 ZnSO・ 7H0 CuSO-5HQ CoC1φ 6H0 to IBO $900111111 1650 ppm 27・8 ppm 37.3 ppm 22.3 ppm am6 ppm 0.025 9911 0 self 0259p Turtle 0.83 ppm 6.2 ppm Na MoOj 2) 1 0
0.25 pp proverb myo-inositol
400 H1 ■ Nicotinic acid 0.
511p Proverb Pyridoxine Hydrochloride 0.5p
Thiamine hydrochloride 1.3 ppm Glycine 2. Opp Otoshi! 1 @
40000 pp■Kinetin
1. ! ppH (note) liquid growth medium c
kinetin) in the primary medium of (1) above, do not use coconut water or agar, and add 1 ppm kinetin instead of benzyladenine.
MIl+ with the same composition as the primary medium except that MIL+ was used.
It is.

(2-8-2) Rooting of shoots Using the cell tissue pieces described above, shoots were rooted in the same manner as in (2-A-2).
A plantlet similar to A-2) was obtained.

(2-8-3) Acclimatization, potting, and seedling raising of plantlets Using the above plantlet, convert the small M object into 1b and potting it in the same manner as in (2-A-3) above. Then, seedlings were raised to obtain banana seedlings that can be transplanted to am.

(2-c) Test using liquid growth medium (benzyladenine) (2-C-1) Formation of multiple shoots (1) above
The differentiated bud aggregates obtained were divided into cell tissues containing two buds, and the amount! The IIB cell tissue was grown for 30 minutes in liquid growth medium (benzyladenine).
Plant the seeds in 5 Erlenmeyer flasks, 3 per flask, under the same lighting as above.
While shaking at 100 rpm, cells were cultured in 25 layers at 2°C for 18 days to form cell tissue pieces with buds that formed multiple shoots, and the number of cell tissue pieces in each flask and their elongation to 2α or more were determined. The number of shots made was counted.

[Liquid growth medium Mg5ON7H0 CaC1-2H0 NO NHN.

HPo F@SOll7H0 Na EDTA Mn5O-480 Z+xSO also 7H0 CuSOj 5H0 CoCI 116HO I (benzyladenine) combination] 370 ppm 440 ppm 1900 ppm 1650 [1 Ken170 ppm 27.8 ppm 37.3 99 meals 22・3 ppm 8・6 ppm 0.025 ppm 0.02529m 0.83 99 weight 6.2 ppm 0.25 PP 400 ppm 0・5 ppm 0・5 ppm l・3 ppm 2 fist o ppm BO Na Mo0 ・2H0 Myo-inositol nicotinic acid hydrochloride pyridoxine hydrochloride thiamine glycine! I m40000 ppm Bendiflaedenine 0.9 ppwr (
Note) The liquid growth medium (benzyladenine) is as described above (2-
The medium is the same as the liquid growth layer (Kinetin) except that 0.9 ppm of benzyladenine was used instead of kinetin in the liquid growth layer (TMC kinetin) in B-1).

(2-C-23 Rooting of shoots Using the cell tissue pieces described above, rooting of shoots was carried out in the same manner as in (2-A-2) above.
A plantlet similar to -2) was obtained.

(2-C-3) Dramaticization of plantlets, potting up, and raising seedlings Using the above plantlets, -σization of the plantlets in the same manner as in (2-A-3) above, After raising pots and raising seedlings, we obtained banana seedlings that can be transplanted to the field.

(2-f+) Test by aerated agitation culture (2-D-1) Formation of multiple shoots (1) above
The aggregate of differentiated lateral tissue fibers of the buds obtained was divided into M cell tissue fragments containing 3 buds, and 20 of the divided cell tissue fragments were added with 3T of liquid proliferative layer ALL (benzyladenine). 4
were planted in a jar fermenter, and cultured at 25±2°C for 40 days under the same lighting as in (1) above, with aeration and agitation of 0.5 t/min per culture medium,
Cell tissue pieces having buds that formed multiple shoots were formed, and the number of buds and the number of shoots that had elongated to 2 cm or more in each piece were counted.

(2-o-2) Rooting of shoots Using the above-mentioned 1III rooting fragment, the above-mentioned (2-A-2)
) The shoots were rooted in the same manner as in (2-A-2), and the same plantlets as in (2-A-2) were obtained.

(2-D-3) Acclimatization of plantlets, potting up and raising seedlings Use the above plantlets! (2) The plantlet was acclimatized, potted, and raised in the same manner as in (2-8-3) above, and banana seedlings that could be transplanted to the field were obtained.

(3) Test results It was as shown in Table 1.

(Margin below) (4) Discussion According to Table 1, when the cell tissue extracted from banana suckers is grown in a liquid medium, the buds attached to the cell tissue proliferate.1.Gull tissue When the buds attached to the plant elongate and form a shoot, and the shoot takes root,
Since it becomes a banana seedling, the proliferation of buds increases the number of banana seedlings that can be easily derived from the cell tissue.

It has been found that when cell tissue extracted from banana suckers is grown in a liquid medium with aeration and agitation, the number of shoots extending from the grown buds increases. When the shoots are rooted, they become banana seedlings, so an increase in the shoot ratio will promote the growth of banana seedlings, which will shorten the period required for the formation of banana seedlings. become.

Therefore, it can be seen that aeration agitation culture in liquid culture improves the production of banana seedlings.

Procedural amendment November 15, 1989

Claims (1)

[Claims] (1) A method for producing banana seedlings by culturing banana cell tissues and rooting the shoots, which is characterized in that the banana cell tissues are cultured in a liquid medium with aeration and agitation. How to produce banana seedlings.