CN101869076A - Method for inoculating stem tip of banana sucker - Google Patents
Method for inoculating stem tip of banana sucker Download PDFInfo
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- CN101869076A CN101869076A CN 201010218424 CN201010218424A CN101869076A CN 101869076 A CN101869076 A CN 101869076A CN 201010218424 CN201010218424 CN 201010218424 CN 201010218424 A CN201010218424 A CN 201010218424A CN 101869076 A CN101869076 A CN 101869076A
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Abstract
The invention provides a method for inoculating a stem tip of a banana sucker. The method comprises the following steps of: pre-processing the banana sucker; cutting the sucker into sucker cylinders; ringing leaf sheath of the banana sucker along the junction of the leaf sheath and corm by using a scalpel sterilized at a high temperature layer by layer in sequence until all enwrapped leaf sheath is peeled; and then easily picking the sterile steam tip and inoculating the steam tip in MS inductive culture medium with 4 to 5mg/L of BA, 0.05 mg/L of NAA and 35g/L of white sugar for inductive culturing. The method overcomes the disadvantages of need of disinfector, complex pre-processing process of the material, low efficiency and the like in a conventional method and achieves the effects of convenience, easiness, practicability, high efficiency and environmental friendliness.
Description
Technical field
The present invention relates to field of plant tissue culture technique, be specifically related to the method for inoculation of banana explant and acquisition sterilizable material, i.e. the method for inoculating stem tip of banana sucker.
Background technology
The banana of broad sense has comprised banana (Banana) and plantain (Plantain) two big classes, belongs to Musaceae (Musaceae) Musa (Musa).Banana cultivated species (Musa sp.) belongs to the unisexuality triploid, and (AAA, AAB ABB), have the height sterility.
Banana is the important fruit in the world, also is one of China south of the Five Ridges four big good fruits.Because banana does not have seed, so traditional propagation method is to inhale the bud plantation by taking, can bring many problems, as causing disease spread, germplasm degeneration, production management difficulty or the like easily and inhale the bud plantation.In the early 1980s, China has just begun the research of banana tissue culture technique, and the high-quality test-tube plantlet has obtained great popularizing planting.Up to the present, the banana master plants the district and adopts the plantation of banana test-tube plantlet substantially all, and main varieties of plant has Brazil, William Si, No. 2, Guangdong, No. 1, wide powder, Shuande mini-bus plantain, new northern any of several broadleaf plants etc.
Banana tissue culture explant mainly contains inhales bud stem apex, rhachis and male flower meristematic tissue etc.Produce grow thickly bud as the cultural method of explant by the evoked callus approach with rhachis and male flower meristematic tissue, carry out enrichment culture then.These two kinds of methods are all passed through the process of plant tissue cell's " dedifferentiation---differentiation " again, cause easily that in this process gene changes and produces higher aberration rate.Therefore the method that produces the bud of growing thickly by the evoked callus approach is not suitable for being used for breeding economic crops seedlings such as banana.Inhale the direct method of indefinite clump bud that produces of bud stem apex without " dedifferentiation---differentiation again " process and induce, can not have much impact to gene, in low concentration 6-BA concentration (under 2～2.0mg/L), subculture number was controlled at for 12 generations with under the interior condition, and the test-tube plantlet aberration rate of producing is lower.Therefore the production of banana test-tube plantlet batch production at present all is to adopt to inhale the method that the bud stem apex is directly induced indefinite clump bud basically.
On the inoculating stem tip of banana sucker method, traditional way is: earlier the banana of choosing is inhaled bud and be cut into 3 centimetres of sizes of about 5 cm x, 3 cm x, on superclean bench, carefully stem apex is cut into 1～2 centimetre of size then again, approximately soak with 0.1% mercuric chloride solution, carried out surface sterilization 15～20 minutes, use aseptic water washing again 3 times, the surface texture of sterilizing through pruning is inoculated at last and carries out inducing culture on the inducing culture.
There are several shortcomings in this method: (1) not enough environmental protection: the general explant surface disinfectant that adopts is a mercuric chloride, is a kind of extremely toxic substance, and the discarded liquid after having sterilized can cause environmental pollution.Simultaneously, residual have disinfectant can influence the stem apex inducing culture.(2) more loaded down with trivial details, consuming time: as before carrying out surface sterilization, stem-tip tissue need to be carried out strict cutting, cut surperficial very smoothly, must often change the cutter and the tweezers of sterilize in the cutting process, complex operation, so also ten minutes consuming time.According to observations, more skilled personnel also can only inoculate 50～60 bananas suction buds in one day.(3) simple and easy inadequately, be difficult to obtain higher success ratio of inoculation.As if this method is very simple outwardly, but practical operation gets up to be difficult on top of.Adopt this method inoculation banana explant, general personnel's success ratio of inoculation 50～60%, even lower, it has been very good that the skilled person can obtain 80～85% success ratio of inoculation.
Summary of the invention
For overcoming above-mentioned technological deficiency, the purpose of this invention is to provide a kind of method of inoculating stem tip of banana sucker, method is simple for this, efficient, environmental protection.
For achieving the above object, the present invention adopts following scheme: the aseptic stem apex of picking is inoculated into and carries out inducing culture in the inducing culture, and described aseptic stem apex is peeled off leaf sheath operation and obtained by aseptic.Specifically, be to use through the scalpel of high-temperature sterilization carefully banana to be inhaled the bud-leaf sheath along itself and the girdling of bulb boundary line, dynamics is held the leaf sheath that not make point of a knife scratch this layer leaf sheath and run into the adjacent one deck in the inside, band girdle layer by layer successively, operation just can be with the assorted cingula of the outside leaf sheath leaf sheath to the inside like this; At last, the leaf sheath that all encases is all peeled away, just the aseptic stem apex of picking is inoculated into and carries out inducing culture in the inducing culture easily.
Below the invention will be further described, the concrete grammar step is as follows:
1), banana is inhaled the bud preliminary treatment.
The banana of fetching is inhaled bud carefully cut bulb and false stem skin with cutter, cutting how many tissues will decide on banana suction bud size, the general requirement of suction bud size after treatment is each long 6～10 centimetres of bulb, false stem portion, the cylinder that base diameter is about 2～3 centimetres; Smaller suction bud is each long 2～3 centimetres of bulb, false stem portion after treatment, the cylinder that base diameter is about 1 centimetre.
Cut when inhaling the bud redundance and will note some: 1. avoid cylinder that the earth of suction bud band is made dirty and is cut into as far as possible.Vertically cut from top to bottom to bulb along leaf sheath when 2. cutting, can not cut partially to the stem apex direction, if cut too deeply partially, otch is too near to stem apex, inhales bud and just is done for.Be bending if inhale bud, keep flat inhaling bud, side direction cuts interior bight portion bulb, carefully tears leaf sheath off with hand then.3. need to detect the banana variety of floral leaf heart rot, the suction bud that cuts will be carried out grouping.
2), inhale bud stem apex aseptic inoculation.
At first use dried cloth (or newspaper) to dry and cut the juice that back suction gemmule stem portion is oozed out, in case slide hand when the inoculation operation, if the suction bud that cuts is messy, available 75% alcohol wipe is clean.
The second, the leaf sheath girdling.After waiting to inoculate cutter sterilization thoroughly, inhale bud stem apex aseptic inoculation and just can begin.Hold suction bud cylinder bulb part (if it is less to inhale bud with left hand, also available left hand thumb, forefinger and middle finger are caught), the exposed portions serve bulb, with scalpel point of a knife cutter under the false stem outermost layer leaf sheath bag mouth, along leaf sheath and the counterclockwise girdling of bulb boundary line, left hand thumb and forefinger will be ordinatedly clockwise rotate inhaling the bud cylinder during girdling, allow the standardized circle of point of a knife.Will note using the cutter dynamics during girdling, optic lobe sheath thickness is in time adjusted firmly weight, and principle is that point of a knife neither contacts the internal layer leaf sheath, and the leaf sheath after the girdling is peeled off easily.And thin more the closer to the stem apex leaf sheath, the girdling dynamics is also light more.This needs exercise a little to accomplish.
The 3rd, leaf sheath is peeled off.When peeling off leaf sheath, hand thumb, forefinger and middle finger are caught false stem portion, the leaf sheath bag mouth after the girdling, clockwise direction is twisted false stem, makes whole layer leaf sheath stubborn behind bulb along otch, and the leaf sheath that firmly will break away from bulb draws back, expose the inside one deck leaf sheath, peel off down one deck leaf sheath then successively.
Should be noted that in this step operation: 1. when the right hand is twisted false stem, can not overexert, in case whole false stem portion is twisted into two parts.2. should earlier an openning be turned at edge, leaf sheath outside, be twisted along this openning then, up to whole layer leaf sheath is stubborn from bulb.If 3. some locally draws too shallowly during girdling, leaf sheath just is not easy stubborn from bulb.If 4. because of carelessness whole false stems have been twisted into two parts, it is complicated loaded down with trivial details that operation will become.This is to change large size scalpel (as No. 23 cuttves) and carefully to choose out leaf sheath after girdling, and point of a knife can not touch the inside leaf sheath when choosing leaf sheath, more can not run into the outside bulb, if to the outside bulb, must change the then operation of scalpel of sterilizing.5. need to peel off what of the leaf sheath number of plies, decide by the excision leaf sheath when inhaling bud size and preliminary treatment.If the leaf sheath of excision is many more, to cut the closer to stem apex, the leaf sheath number of plies that then needs to peel off can be less relatively, generally need peel off about 3～5 layers of leaf sheath and get final product.6. after whenever peeling off one deck leaf sheath, change the cutter of sterilizing again.
The 4th, inhale bud stem apex picking and inoculation.After peeling off layer by layer leaf sheath, the stem apex (deciding) that remaining diameter is about 0.4～0.8 centimetre on inhaling the bud size.Cut described false stem with the scalpel that disinfects, stay 0.3-0.7cm long, the place of in the end peeling off leaf sheath inboard, stem apex edge then with point of a knife, oblique counterclockwise slotting tool one circle, with point of a knife stem apex is picked out at last, be inoculated into and carry out inducing culture on the inducing culture.Inducing culture is a minimal medium with the MS medium, and adding 6-benzyl aminopurine (6-BA) to its final concentration is 4～5mg/L, and adding methyl (NAA) to its final concentration is 0.05mg/L, and adding white sugar to its final concentration is 35g/L.
This step should be noted that: 1. should not insert during slotting tool too deeply, inject 5 millimeters deep approximately and get final product.2. the slotting tool direction should be 40～45 with the cylinder axis, can not be along cylinder axis slotting tool.3. firmly can not be too big during the picking stem apex, whole bulb tissues insert from after, gently stem apex is chosen.If firmly too big, be easy to stem apex is got rid of on the table top, all that has been achieved is spoiled for the result.4. the stem apex of choosing must have the bulb tissue, otherwise stem apex will lose foundation, can not successfully cultivate.
The banana stem apex is closely to be surrounded by many layer leaf sheaths, and layer upon layer of leaf sheath can be kept apart the stem apex and the external world fully, thereby plays the effect of protection stem apex.To the internal layer leaf sheath, its degree that is bacterial contamination is more and more lower from outermost leaf sheath, and stem apex and near which floor leaf sheath are not bacterial contamination.Adopt method of the present invention to go out intravaginal stem apex by direct picking, do not need just can obtain aseptic stem apex through surface sterilization.
Compared with prior art, method of the present invention mainly contains following advantage: (one) does not use disinfectant, and is free from environmental pollution.The disinfectant that the tradition inoculation method adopts is 0.1% mercuric chloride solution, has severe toxicity, and the discarded liquid after using can pollute environment such as water sources.And adopt new inoculation method not adopt any disinfectant, can not produce any harmful effect, and can not grow after the stem apex inoculation and be affected because disinfectant does not rinse well to environment.(2) simple to operate, be easy to learn and use.This method need not be prepared sterilize vessel, sterile water and disinfectant etc., do not need through loaded down with trivial details cutting process, need not remember the thing that disinfecting time or the like need be done when traditional inoculation method, here you just can begin to inoculate as long as scalpel disinfected, and along false stem and bulb intersection, band girdle is very easy layer by layer.(3) rapidly and efficiently.New inoculation method operates very simple and efficient, and general about 7～8 cuttves of skilled person just can be inoculated one and inhale bud, can inoculate banana and inhale about 100 of buds in one day.
Embodiment
For making the present invention easier to understand,, further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Illustrate concrete implementation step below:
1 material and apparatus
Inhale bud and cut earlier and stay false stem 10～15 centimeter length 1.1 the material preliminary treatment will be chosen the Brazilian any of several broadleaf plants (banana variety) that returns,, outer leaf sheath is connected bulb organize together and excise then from otch toward the vertical incision of bulb direction.Be cut into each long 6～10 centimetres of false stem, bulb parts at last, base diameter is 2～3 centimetres a cylinder.
1.2 apparatus 3 No. 11 scalpels and 1 No. 23 scalpels, 1 300 ℃ of electric sterilizer (also available alcolhol burner).
2 operating procedures
Inhale the bud cylinder 2.1 leaf sheath girdling left hand is caught, the right hand is taken scalpel.Begin cutter down with the scalpel point of a knife of sterilizing from leaf sheath bag mouth, row dry a circle counterclockwise to the position of playing cutter along outer leaf sheath and bulb boundary line.
2.2 leaf sheath is peeled off the right hand and caught false stem portion, twists leaf sheath to clockwise direction, has twisted earlier the leaf sheath outward flange, and is progressively stubborn from bulb whole layer leaf sheath then.After leaf sheath broke away from bulb fully, the leaf sheath that the right hand firmly will disengaging drew back, and perhaps leaf sheath is torn one by one.Peel off other leaf sheaths (about 3～5 layers) as stated above successively.
2.3 the stem apex picking cuts false stem with the scalpel of sterilizing, only stay about 0.5cm long, the place at the leaf sheath inboard of in the end peeling off with point of a knife, stem apex edge with counterclockwise slotting tool one circle of stem apex angle at 45, will have the stem apex chear stem of part bulb tissue lightly then.
2.4 the stem apex inoculation is inoculated into the MS inducing culture that is added with 6-benzyl aminopurine (6-BA) 4～5mg/L, methyl (NAA) 0.05mg/L, white sugar 35g/L with stem apex, the inoculation degree of depth is organized as suitable just to insert bulb.
2.5 the stem apex switching was cultivated after 20～25 days, the callus that the stem apex incision is formed cuts, and then stem apex is cut into 4 equal portions, is seeded on the above-mentioned inducing culture.
3 inoculation results
The stem apex inoculation was observed after 10 days, and average success rate is more than 85%, and stem apex begins to expand.Transfer again to cultivate for 1 time after 20 days and begin to induce the bud of growing thickly.
Claims (9)
1. the method for an inoculating stem tip of banana sucker, described method are that the aseptic stem apex of picking is inoculated into and carries out inducing culture in the inducing culture, it is characterized in that, described aseptic stem apex is peeled off the leaf sheath operation and obtained by aseptic.
2. the method for a kind of inoculating stem tip of banana sucker according to claim 1, it is characterized in that, described aseptic to peel off leaf sheath operation be to use through the scalpel of high-temperature sterilization banana is inhaled the bud-leaf sheath along itself and the girdling layer by layer of bulb intersection, all peels away until the leaf sheath that all encases.
3. the method for inoculating stem tip of banana sucker according to claim 2 is characterized in that, describedly asepticly peels off leaf sheath operation and comprises the steps:
1) banana is inhaled the bud preliminary treatment: use the scalpel through high-temperature sterilization to cut bulb and the false stem skin that banana is inhaled bud, the suction bud after the processing is a cylinder;
2) leaf sheath girdling;
3) leaf sheath is peeled off;
4) inhale bud stem apex picking;
More than operation is all carried out on superclean bench.
4. the method for inoculating stem tip of banana sucker according to claim 3, it is characterized in that, described step 2 operation is as follows: hold described suction bud cylinder bulb part, expose described leaf sheath and position, described bulb boundary, with point of a knife cutter under the leaf sheath bag mouth, along described leaf sheath and the counterclockwise girdling of described bulb boundary line.
5. the method for inoculating stem tip of banana sucker according to claim 3 is characterized in that, described step 3 operation is as follows: catch described false stem portion, twist described false stem, twisted earlier described leaf sheath outward flange to clockwise direction, and stubborn whole layer leaf sheath then from bulb; After described leaf sheath breaks away from described bulb fully, firmly the leaf sheath that breaks away from is drawn back, perhaps leaf sheath is torn one by one; Peel off other leaf sheaths as stated above successively, whenever peel off one deck leaf sheath and need change the scalpel of sterilizing again.
6. the method for inoculating stem tip of banana sucker according to claim 3, it is characterized in that, described step 4 operation is as follows: cut described false stem with the scalpel of sterilizing, stay 0.3-0.7cm long, the place at the leaf sheath inboard of in the end peeling off, stem apex edge then with point of a knife, slotting tool one encloses counterclockwise, will have the stem apex chear stem of part bulb tissue.
7. the method for inoculating stem tip of banana sucker according to claim 1 is characterized in that, described inducing culture comprises the steps:
1) stem apex inoculation;
2) stem apex switching;
Above step is all being carried out at superclean bench.
8. the method for inoculating stem tip of banana sucker according to claim 7 is characterized in that, described step 1 is for to be inoculated into stem apex on the described inducing culture, and the inoculation degree of depth is organized as suitable just to insert bulb; Described inducing culture is a minimal medium with the MS medium, adds 6-benzyl aminopurine (6-BA) 4～5mg/L, methyl (NAA) 0.05mg/L, white sugar 35g/L.
9. the method for inoculating stem tip of banana sucker according to claim 8 is characterized in that, described step 2 is for after cultivating 20～25 days, and the callus that the stem apex incision is formed cuts, and then the stem apex rip cutting is become 4 equal portions, is seeded in the described inducing culture.
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| CN 201010218424 CN101869076A (en) | 2010-07-06 | 2010-07-06 | Method for inoculating stem tip of banana sucker |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103444535A (en) * | 2013-09-05 | 2013-12-18 | 广东省农业科学院果树研究所 | Novel method for increasing tissue culture and rapid propagation coefficients of banana |
| CN104982285A (en) * | 2015-06-17 | 2015-10-21 | 广东省农业科学院果树研究所 | Method for rapid propagation of banana seedlings based on suckers |
| CN107688021A (en) * | 2016-08-04 | 2018-02-13 | 北京林业大学 | A kind of Chinese herbaceous peony underground bud free-hand section method and its application |
| CN109247236A (en) * | 2018-11-09 | 2019-01-22 | 广西壮族自治区农业科学院园艺研究所 | More simplified sterilization method in a kind of banana tissue culture |
| CN109757316A (en) * | 2019-01-16 | 2019-05-17 | 南京农业大学 | A kind of banana health planting system based on water disinfection |
| CN113142055A (en) * | 2021-04-29 | 2021-07-23 | 广西壮族自治区农业科学院 | In-vitro proliferation preservation method for banana germplasm resources |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103444535A (en) * | 2013-09-05 | 2013-12-18 | 广东省农业科学院果树研究所 | Novel method for increasing tissue culture and rapid propagation coefficients of banana |
| CN104982285A (en) * | 2015-06-17 | 2015-10-21 | 广东省农业科学院果树研究所 | Method for rapid propagation of banana seedlings based on suckers |
| CN104982285B (en) * | 2015-06-17 | 2017-06-06 | 广东省农业科学院果树研究所 | Based on the Banana Seedlings rapid propagation method for inhaling bud |
| CN107688021A (en) * | 2016-08-04 | 2018-02-13 | 北京林业大学 | A kind of Chinese herbaceous peony underground bud free-hand section method and its application |
| CN109247236A (en) * | 2018-11-09 | 2019-01-22 | 广西壮族自治区农业科学院园艺研究所 | More simplified sterilization method in a kind of banana tissue culture |
| CN109757316A (en) * | 2019-01-16 | 2019-05-17 | 南京农业大学 | A kind of banana health planting system based on water disinfection |
| CN113142055A (en) * | 2021-04-29 | 2021-07-23 | 广西壮族自治区农业科学院 | In-vitro proliferation preservation method for banana germplasm resources |
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Application publication date: 20101027 |