CN102726302A - Dark culture method for banana tissue culture - Google Patents
Dark culture method for banana tissue culture Download PDFInfo
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Abstract
The invention discloses a dark culture method for banana tissue culture, and the method comprises the following steps of: (1) selecting and treating an explant; (2) performing dark culture of inducing a dedifferentiation tissue; (3) performing dark culture of rooting and differentiation; and (4) performing rooting acclimatization and transplanting. The method provided by the invention is characterized in that dark light or weak light is adopted for culture in the process of the dark culture of inducing the dedifferentiation tissue and the dark culture of rooting and differentiation, in stead of adding light (1500-2500 lx) for mulitiplication culture, strong seedling culture and culture at early stage of rooting in traditional banana tissue culture; besides, the dark culture way is adopted so that power consumption caused by supplementary lighting of a daylight lamp in traditional light culture is avoided and heat generated by the supplementary lighting can be counteracted; and therefore, the cost taken for cooling is reduced. As a result, by utilizing the method of the invention, the production efficiency can be improved; the production cost can be reduced; and the development of industry can be promoted.
Description
Technical field
The invention belongs to field of plant tissue culture technique, specifically relate to the dark cultural method of banana tissue culture.
Background technology
Banana (Musa balbisiana Colla) is one of southern important tropical fruit (tree) of China, also be simultaneously the important income source in many areas, and many peasant households relies on the plantation banana to shake off poverty and set out on the road to prosperity.Most cultivation edible banana all is unisexuality triploid AAA; Sterile gene with height mainly leans on vegetative propagation, utilizes the suction bud to breed in the production usually; When seedling is not enough; Also can adopt the subterranean stem stripping and slicing of banana to breed seedling, but this method is used the many viruses of meeting accumulation for a long time, as: bunchy top, mosaic disease etc.Cause serious harm.In addition, bad banana seedling can cause undergrowth, and fruit is not good, problems such as results disunity.The banana tissue culture technique can breed the health seedling of no damage by disease and insect of the high-quality of a large amount of uniform specification at short notice fast, and, the cultivation of new varieties and introduce and also need utilize virus-free and group culturation rapid propagating technology to accelerate the development of famous-brand and high-quality kind.The banana tissue culture technology is that China agri-scientific research worker is in the research and development popularization of the later stage eighties 20th century; This The Application of Technology has played great progradation to developing rapidly of China's banana industry; Make that also kind of seedling industrialized production is achieved; At short notice the improved seeds large tracts of land is promoted, reached the large-scale commercial banana production.But this industry was through the application in more than 20 years, and we find and should technology have deficiency that subject matter is in production application:
1. it is long that time monocycle is cultivated in production, and (promptly being inoculated into new medium to the cultivating) time of general monocycle cultivation during the training of banana group is produced is more than 30 days.
2. it is big to produce power consumption, owing in the incubation, need to replenish illumination, light filling is time 10h/ days, intensity of illumination 1500～2000lx.
3. the energy consumption of light filling generation is high; Owing to replenish the fluorescent lamp that 40W is adopted in illumination usually, when work, can produce great amount of heat, in order to reduce the cultivation temperature of culturing room; Just necessary air-conditioning temperature-reducing; Will produce no small electricity charge annual thus summer, increased operation costs to enterprise, is unfavorable for very much batch production production.
4. because the production cycle is long, cause the production site turnover slow, take a large amount of culturing racks, thereby cause plant area to increase.
5. adopt artificial lighting to cultivate, in order to satisfy the required illumination of each blake bottle growth, then the culture layer frame can only be put 108 bottles of subculture materials usually; It is big to take up room; Adopt dark cultural method of the present invention, every layer of culturing rack can be put 200 ~ 220 bottles, effectively utilized the space.It is many that the tissue cultivating seedling occupation of land of equivalent is cultivated in artificial lighting, also is the major reason that plant area increases.
Above shortcoming is a ubiquitous problem during the traditional group training is produced; The torrid zone and subtropical zone at southern china; Tissue culture seedlings of bananas has become large seedling kind, and annual all have a market demand that surpasses more than one hundred million strains, but since production cost in the year by year (labour cost that increases progressively; The electricity charge; Place rent etc.) hindered the development of planting seedling industrialized industry greatly, therefore developed a kind of new tissue culture technology, guaranteed that further developing of banana seedling industry is imperative to overcome the shortcoming of original training method.
Adopt dark cultural method then can effectively remedy the many deficiencies in the light training method in the banana tissue culture procedures of the present invention, for the stable development of banana seedling industry provides technical support.
The advantage that dark cultural method in the banana tissue culture has has following 4 points:
1. the production cycle shortens; The banana tissue culture is as adopting the light best cultivation, because light has certain inhibitory action, being inoculated into the general monocycle incubation time of inoculation next time from this is 20 to 25 days; Used dark best cultivation instead, then incubation time can foreshorten to 15 ~ 18 days.
2. in vitro tissue and the test-tube plantlet g and D under a kind of specific temperature that suits.Like: one section suitable temperature is (25 ± 2) ℃.When room temperature does not meet this temperature, then should start air-conditioner, regulate temperature as requested, up to meeting the requirements.Traditional light is cultivated the formula tissue culture generally makes intensity of illumination reach 1500～2000lx, reach this intensity of illumination, and every layer of culturing rack then need be adorned two fluorescent lamps respectively, supplies illumination to use.Can produce a huge illuminating power expense thus.As: the culturing room of one 15 ㎡ can hold five layers of culturing rack of 15 wide 35cm of long 130cm more.Calculate by every layer of two 40W fluorescent lamp, then the day power consumption of each culturing room's light filling is:
W=40×2×5×15×10=60kw/h
Year power consumption is so: 365 * 60=21900kw/h
One family produces the plant tissue culture factory of 3500～4,000 ten thousand strain tissue cultivating seedling per year because the electricity expense that light filling produced then can be up to 547500 degree/years.
Because the present invention has used dark culture technique instead, every year, savable expense aspect illumination was appreciable, for enterprise great advantage was provided in the competition in market.
3. after getting into summer; The heat that utilizes light culture technique light filling illuminating lamp tube to come out is bigger; Make culturing room's temperature surmount the banana crops tissue far away and can the frozen limit temperature will reach 28～30 ℃ of desirable cultivation temperature more than 33 ℃, must utilize the air conditioner cooling.(power: 750w/h) calculate, a year cooling electric weight is about 600 degree (by 6 months/years), and the production scale cooling electric weight of producing 4,000 ten thousand strains year is 15000 degree with air conditioner of 15～20 ㎡.Improve training mode: after dark the cultivation, then its cooling expense can descend significantly, reduces production cost.
4. along with the raising year by year of space expenses cost, originally the subculture monocycle is that 25 days production model has shown turnover slowly, takies a large amount of culturing room, and the decline of the output of unit are has increased the cost of operation.After dark best cultivation shortened into 15 days with time monocycle, this problem just can significantly be slowed down.
5. the increase of the unit culture density of culturing rack has improved the quantum of output of unit are.
Summary of the invention
The object of the invention is in order to solve the deficiency that exists in the prior art, and a kind of dark cultural method of banana tissue culture is provided,
Tissue culture induce dedifferentiation and the atomization of taking root in adopt dark cultural method; Avoided traditional artificial lighting to cultivate; Both having shortened cultivation cycle, and also reduced energy consumption, is that tissue cultivating seedling breeding cultivation has reduced production cost when guaranteeing the tissue cultivating seedling quality.
Technical scheme of the present invention is following:
Dark cultural method in the banana tissue culture, it comprises the steps:
(1) explant selection and processing;
(2) induce the dark cultivation of dedifferentiation tissue;
(3) take root that differentiation is dark cultivates;
(4) take root domestication and transplant;
Said explant selection be choose early spring and autumn (particularly arid season) the virus-free banana plant that growing way is vigorous the suction bud; The processing method of explant: will inhale bud and clean up, and progressively peel off outer bract, and excise unnecessary stem part; Keep the little scapus with terminal bud and lateral bud original hase, control scapus diameter of phi is 5～10cm, and scapus is put on the superclean bench; With 75 ~ 80% alcohol-pickled 30 ~ 35s sterilizations; After using 0.1 ~ 0.2%HgCl solution soaking, 10 ~ 15min sterilization again,, subsequent use with aseptic water washing 3～4 times.
Inhaling bud is that abiogenous cripetura between plant rhizosphere or terrestrial stem axil, plumpness are rosula brachyplast.Inhaling the bud bottom can take root naturally, so can and plant separately and cultivate into new plant from the maternal plant separation.
Said to induce dark cultivation of dedifferentiation tissue be that the explant banana scapus that step (1) disinfects is inoculated in the blake bottle of splendid attire inducing culture, places then to carry out half-light under 28～30 ℃ and cultivated 25～30 days, produces clump bud tissue; Clump bud tissue cutting the carrying out successive transfer culture that will obtain then; Successive transfer culture also adopts inducing culture to carry out half-light and cultivates, and cultivation temperature is 28～30 ℃, and the successive transfer culture cycle is 15 ~ 18 days; Reproduction coefficient is 3 ~ 4, and successive transfer culture obtains the banana clump bud tissue of requirement; Said inducing culture is basal medium with MS, and other each constituent contents are 6-benzyl aminoadenine 1～3mg/L, NAA growth hormone 0.2～1mg/L, and Su white granulated sugar 2%～5%, Agar agar 2 ~ 5g, pH are 5.8～6.0.This possess good subculture and the ability of the differentiation of taking root from the bud tissue.
To coming from culture (comprising cell, tissue or its segment) that explant breeds through changing fresh culture and constantly cutting or separate, carry out the cultivation of continuous multi-generation, just be called successive transfer culture.Also be meant callus after growth a period of time on the medium, nutrients is exhausted, moisture loss, and accumulated some metabolites, and need transfer to these tissues on the new medium this moment, and this transfer is called successive transfer culture or goes down to posterity cultivation.
Reproduction coefficient (propagationcoefficient) is meant in successive transfer culture the number that is obtained new talent by a seedling (bud or bud clump).
The said dark cultivation of differentiation of taking root will be inoculated in the blake bottle that is loaded with root media through the banana clump bud tissue that successive transfer culture obtains; Under 28 ~ 30 ℃, carrying out half-light cultivated 10～15 days; After inducing culture went out root system, the banana seedlings that will grow root system was again transferred to sun light green house and is carried out natural lighting refining seedling; Natural lighting was refined seedling after 20 days, the sturdy health of the cane of banana seedlings, and blade changes dark green by bright yellow, well developed root system, the root hair is abundant; Said root media is basal medium with MS, and other each constituent contents are NAA growth hormone 0.05 ~ 0.15mg/L, Su white granulated sugar 25 ~ 27g/L, and Agar agar 2 ~ 5g, pH are 5.8～6.0.
The refining seedling is under the situation that protection is grown seedlings; Take to leak informaton, lower the temperature, suitably control the process that measures such as water are taken exercise seedling by force; Make the unsuitable environmental condition that can adapt to open country after its field planting rapidly, shorten transplanting seedling time, strengthen resistivity low temperature, strong wind etc.
Said half-light is cultivated and is meant in the Plant Tissue Breeding chamber; The blake bottle arrangement is placed on the culture layer frame, and the blake bottle diameter is Φ 5 ~ 6cm, blake bottle spacing≤1 cm; Natural daylight through inciding common culturing room is cultivated, and each blake bottle is in the half-light cultivation conditions.Said culture layer frame is 4 ~ 6 layers, and every layer of culturing rack (130*35cm) can be placed 200 ~ 220 bottles of blake bottles.Because blake bottle is closely arranged and is placed on the culturing rack; The placing distance of blake bottle is little; When the natural lighting on daytime is mapped on the blake bottle and since between the blake bottle block each other and transparent blake bottle between the diffuse reflection effect, so blake bottle is in half-light or low light level state.Evening, each blake bottle also was in the half-light state because available light is very small and weak.Of the present invention is exactly to carry out tissue culture in this state, need as traditional dark cultivation, not carry out shading, also need not to carry out extra artificial light filling,, and traditional group training need be carried out artificial light filling.Traditional banana tissue culture is irradiation not in early days, but after bud sprouts, needs every day about illumination 12h, and intensity of illumination is 2000～3000lx, and energy consumption is big.; The per 40 genius divisible switchings of stem apex clump sorite are 1 time during successive transfer culture, and cultivation cycle is long.Plant Tissue Breeding of the present invention laboratory is no sunlight direct projection.
Said take root domestication and transplanting are meant through the banana seedlings behind step (3) the natural daylight refining seedling, clean the root medium, are transplanted in the nutrient cup, in booth, grow seedlings 50 ~ 60 days, are transplanted to big Tanaka's field planting again and can obtain the banana seedling.
Cultural method of the present invention also is adapted in the tissue culture of banana of dwarf banana or other kind.
Advantage of the present invention:
1. adopt method of the present invention; Stage in that the dedifferentiation cultivation stage and the differentiation of tissue culture are taken root secretly cultivates; Tissue is in the down growth of state of the low light level or half-light, makes that dedifferentiation is fissional to speed up, and has avoided the inhibitory action of illumination in people's light light filling process; Make cultivation cycle shorten, enhance productivity.
2. adopt dark training method, the feasible neat stalwartness of emerging, fast growth, aberration rate is low, and the differentiation of taking root is fast, and the relative light culture technique has bigger advantage.Adopt the annual product banana seedlings of cultural method of the present invention can reach 4,000 ten thousand strains.
3. adopt dark cultural method of the present invention can reduce the power consumption that is produced in the light cultivating and producing significantly, effectively reduce production costs, improve the market competitiveness.
4. adopt dark cultural method of the present invention, increase the unit culture density of culturing rack, can save and cultivate the place, improve the availability of production site greatly.
5. improve production efficiency, reduced labour intensity.The dark cultural method that the present invention adopts; Make the group training grow by natural daylight, so need not control the intensity of its illumination, do not need the intensity of manual adjustment illumination in the stage that dedifferentiation cultivation stage and differentiation are taken root; Reduced the labour, helped enhancing productivity.
Embodiment
The present invention describes with the following example, but does not limit the scope of the invention.
Embodiment 1
Dark cultural method in the banana tissue culture, it comprises the steps:
(1) explant selection and processing
Choose the suction bud of the vigorous virus-free banana plant of banana variety osmanthus any of several broadleaf plants arid season growing way in No. 6 early springs; To inhale bud then and clean up, clean 2～3 times with commercially available cleaning solution again, progressively peel off outer bract with running water; Excise unnecessary stem part, keep the little scapus with terminal bud and lateral bud original hase, control scapus diameter Ф is 5～10cm; Scapus is put on the superclean bench,, use 0.1%HgCl solution soaking 10 min again with 75% alcohol-pickled 30s; Shake abundant sterilization, outwell the sterilization soup.Use aseptic water washing again 3～4 times, subsequent use.
(2) induce the dark cultivation of dedifferentiation tissue
The banana scapus that step (1) is disinfected is inoculated in the blake bottle that is loaded with inducing culture; Inducing culture is basal medium with MS; Other each constituent contents are 6-benzyl aminoadenine (6-BA) 1～3mg/L, NAA growth hormone 0.2～1mg/L, Su white granulated sugar 2%～5%; Agar agar 2 ~ 5g, pH are 5.8～6.0.Place then and carry out half-light cultivation 25～30 days under 28～30 ℃, produce clump bud tissue; Every clump bud tissue that will obtain then cuts into and carried out successive transfer culture in 3 minutes; Successive transfer culture also adopts inducing culture to carry out half-light and cultivates, and cultivation temperature is 28～30 ℃, and the successive transfer culture cycle is 15 ~ 18 days; Reproduction coefficient is 3 ~ 4, and successive transfer culture obtains the banana clump bud tissue of requirement.
In the Plant Tissue Breeding laboratory, blake bottle is arranged and is placed on the culture layer frame, and the culture layer frame is 5 layers, and every layer of culturing rack (130*35cm) can be placed 216 blake bottles; The blake bottle diameter is Φ 6cm, and blake bottle spacing 0cm cultivates through the natural daylight that incides common culturing room, and each blake bottle is in the half-light cultivation conditions.
(3) take root that differentiation is dark cultivates
To be inoculated in the blake bottle that root media is housed through the banana clump bud tissue that successive transfer culture obtains; Root media is basal medium with MS, and other each constituent contents are NAA growth hormone 0.05 ~ 0.15mg/L, Su white granulated sugar 25 ~ 27g/L; Agar agar 2 ~ 5g, pH are 5.8～6.0.Under 28 ~ 30 ℃, carry out half-light and cultivated 10～15 days, after inducing culture went out root system, the banana seedlings that will grow root system was again transferred to sun light green house and is carried out natural lighting refining seedling; Natural lighting was refined seedling after 20 days, the sturdy health of the cane of banana seedlings, and blade changes dark green by bright yellow, well developed root system, the root hair is abundant.
In the Plant Tissue Breeding laboratory, blake bottle is arranged and is placed on the culture layer frame, and the culture layer frame is 5 layers, and every layer of culturing rack (130*35cm) can be placed 216 blake bottles; The blake bottle diameter is Φ 6cm, and blake bottle spacing 0 cm cultivates through the natural daylight that penetrates culturing room, and each blake bottle is in the half-light cultivation conditions.
(4) take root domestication and transplant
Through the banana seedlings behind step (3) the natural daylight refining seedling, clean the root medium, be transplanted in the nutrient cup, in booth, grew seedlings 50 ~ 60 days, be transplanted to the banana seedling that big Tanaka's field planting can obtain high-quality again.
Embodiment 2
(1) explant selection and processing
Choose the suction bud of the vigorous virus-free banana plant of banana variety osmanthus any of several broadleaf plants arid season growing way in No. 6 autumns; To inhale bud then and clean up, clean 2～3 times with commercially available cleaning solution again, progressively peel off outer bract with running water; Excise unnecessary stem part, keep the little scapus with terminal bud and lateral bud original hase, control scapus diameter Ф is 8～10cm; Scapus is put on the superclean bench,, use 0.1%HgCl solution soaking 15 min again with 75% alcohol-pickled 35s; Shake abundant sterilization, outwell the sterilization soup.Use aseptic water washing again 3～4 times, subsequent use.
(2) induce the dark cultivation of dedifferentiation tissue
The banana scapus that step (1) is disinfected is inoculated in the blake bottle that is loaded with inducing culture; Inducing culture is basal medium with MS; Other each constituent contents are 6-benzyl aminoadenine (6-BA) 3mg/L, NAA growth hormone 0.5mg/L, Su white granulated sugar 5%; Agar agar 2g, pH are 5.8～6.0.Place then and carry out half-light cultivation 25 days under 28～30 ℃, produce clump bud tissue; Every clump bud tissue that will obtain then cuts into 4 parts and carries out successive transfer culture; Successive transfer culture also adopts inducing culture to carry out half-light and cultivates, and cultivation temperature is 28～30 ℃, and the successive transfer culture cycle is 15 days; Reproduction coefficient is 3 ~ 4, and successive transfer culture obtains the banana clump bud tissue of requirement.
In the Plant Tissue Breeding laboratory, blake bottle is arranged and is placed on the culture layer frame, and the culture layer frame is 4 layers, and every layer of culturing rack (130*35cm) can be placed 200 blake bottles; The blake bottle diameter is Φ 5cm, and blake bottle spacing 1cm cultivates through the natural daylight that incides common culturing room, and each blake bottle is in the half-light cultivation conditions.
(3) take root that differentiation is dark cultivates
To be inoculated in the blake bottle that root media is housed through the banana clump bud tissue that successive transfer culture obtains; Root media is basal medium with MS, and other each constituent contents are NAA growth hormone 0.1mg/L, Su white granulated sugar 25g/L; Agar agar 2g, pH are 5.8～6.0.Under 28 ~ 30 ℃, carry out half-light and cultivated 15 days, after inducing culture went out root system, the banana seedlings that will grow root system was again transferred to sun light green house and is carried out natural lighting refining seedling; Natural lighting was refined seedling after 20 days, the sturdy health of the cane of banana seedlings, and blade changes dark green by bright yellow, well developed root system, the root hair is abundant.
In the Plant Tissue Breeding laboratory, blake bottle is arranged and is placed on the culture layer frame, and the culture layer frame is 4 layers, and every layer of culturing rack (130*35cm) can be placed 200 blake bottles; The blake bottle diameter is Φ 5cm, and blake bottle spacing 1cm cultivates through the natural daylight that penetrates culturing room, and each blake bottle is in the half-light cultivation conditions.
(4) take root domestication and transplant
Through the banana seedlings behind step (3) the natural daylight refining seedling, clean the root medium, be transplanted in the nutrient cup, in booth, grew seedlings 50 ~ 60 days, be transplanted to the banana seedling that big Tanaka's field planting can obtain high-quality again.
Embodiment 3
(1) explant selection and processing
Choose the suction bud of the vigorous virus-free banana plant of banana variety osmanthus any of several broadleaf plants arid season growing way in No. 6 early springs; To inhale bud then and clean up, clean 2～3 times with commercially available cleaning solution again, progressively peel off outer bract with running water; Excise unnecessary stem part, keep the little scapus with terminal bud and lateral bud original hase, control scapus diameter Ф is 5～8cm; Scapus is put on the superclean bench,, use 0.1%HgCl solution soaking 10 min again with 75% alcohol-pickled 30s; Shake abundant sterilization, outwell the sterilization soup.Use aseptic water washing again 3～4 times, subsequent use.
(2) induce the dark cultivation of dedifferentiation tissue
The banana scapus that step (1) is disinfected is inoculated in the blake bottle that is loaded with inducing culture; Inducing culture is basal medium with MS; Other each constituent contents are 6-benzyl aminoadenine (6-BA) 2mg/L, NAA growth hormone 0.6mg/L, Su white granulated sugar 3%; Agar agar 5g, pH are 5.8～6.0.Place then and carry out half-light cultivation 28 days under 28～30 ℃, produce clump bud tissue; Every clump bud tissue that will obtain then cuts into 4 parts and carries out successive transfer culture; Successive transfer culture also adopts inducing culture to carry out half-light and cultivates, and cultivation temperature is 28～30 ℃, and the successive transfer culture cycle is 18 days; Reproduction coefficient is 3 ~ 4, and successive transfer culture obtains the banana clump bud tissue of requirement.
In the Plant Tissue Breeding laboratory, blake bottle is arranged and is placed on the culture layer frame, and the culture layer frame is 6 layers, and every layer of culturing rack (130*35cm) can be placed 210 blake bottles; The blake bottle diameter is Φ 6cm, and blake bottle spacing 0cm cultivates through the natural daylight that incides common culturing room, and each blake bottle is in the half-light cultivation conditions.
(3) take root that differentiation is dark cultivates
To be inoculated in the blake bottle that root media is housed through the banana clump bud tissue that successive transfer culture obtains; Root media is basal medium with MS, and other each constituent contents are NAA growth hormone 0.1mg/L, Su white granulated sugar 25g/L; Agar agar 5g, pH are 5.8～6.0.Under 28 ~ 30 ℃, carry out half-light and cultivated 12 days, after inducing culture went out root system, the banana seedlings that will grow root system was again transferred to sun light green house and is carried out natural lighting refining seedling; Natural lighting was refined seedling after 20 days, the sturdy health of the cane of banana seedlings, and blade changes dark green by bright yellow, well developed root system, the root hair is abundant.
In the Plant Tissue Breeding laboratory, blake bottle is arranged and is placed on the culture layer frame, and the culture layer frame is 6 layers, and every layer of culturing rack (130*35cm) can be placed 210 blake bottles; The blake bottle diameter is Φ 6cm, and blake bottle spacing 0cm cultivates through the natural daylight that penetrates culturing room, and each blake bottle is in the half-light cultivation conditions.
(4) take root domestication and transplant
Through the banana seedlings behind step (3) the natural daylight refining seedling, clean the root medium, be transplanted in the nutrient cup, in booth, grew seedlings 50 ~ 60 days, be transplanted to the banana seedling that big Tanaka's field planting can obtain high-quality again.
Embodiment 4
Dark cultural method in the dwarf banana tissue culture, it comprises the steps:
(1) explant selection and processing
Choose the suction bud of the vigorous virus-free banana plant of dwarf banana kind bronze arid season growing way in No. 1 early spring; To inhale bud then and clean up, clean 2～3 times with commercially available common cleaning solution again, progressively peel off outer bract with running water; Excise unnecessary stem part, keep the little scapus with terminal bud and lateral bud original hase, control scapus diameter of phi is 5～10cm; Scapus is put on the superclean bench,, use 0.1%HgCl solution soaking 10 min again with 75% alcohol-pickled 30s; Shake abundant sterilization, outwell the sterilization soup.Use aseptic water washing again 3～4 times, subsequent use.
(2) induce the dark cultivation of dedifferentiation tissue;
The banana scapus that step (1) is disinfected is inoculated in the blake bottle that is loaded with inducing culture, and inducing culture is basal medium with MS, 6-BA6-benzyl aminoadenine 1～3mg/L; NAA growth hormone 0.2～1mg/L; Su white granulated sugar 2%～5%, Agar agar 2g ~ 5g, pH are 5.8～6.0; Place and carry out half-light cultivation 25～30 days under 28～30 ℃, produce clump bud tissue; Carry out successive transfer culture then, the successive transfer culture cycle is 15 ~ 18 days, and reproduction coefficient is 3 ~ 4, and successive transfer culture obtains the dwarf banana clump bud tissue of actual production requirement.
Blake bottle is arranged and is placed on the culture layer frame, and the blake bottle diameter is Φ 6cm, and blake bottle spacing≤1 cm is cultivated through the natural daylight that penetrates culturing room, and each blake bottle is in the half-light cultivation conditions.Every layer of culturing rack (130*35cm) can be placed 200 ~ 220 bottles
(3) take root that differentiation is dark cultivates
To be inoculated in the blake bottle of splendid attire root media through the banana clump bud tissue that successive transfer culture obtains, root media is basal medium with MS, NAA growth hormone 0.05 ~ 0.15mg/L, and Su white granulated sugar 25 ~ 27g/L, Agar agar 2g ~ 5g, pH are 5.8～6.0; Cultivate through 10～15 days half-lights, 28 ~ 30 ℃ of cultivation temperature after inducing culture goes out root system, are transferred to sun light green house with banana seedlings again and are carried out natural lighting refining seedling; Natural lighting was refined seedling after 20 days, the sturdy health of the cane of banana seedlings, and blade changes dark green by bright yellow, well developed root system, the root hair is abundant.
Blake bottle is arranged and is placed on the culture layer frame, and the blake bottle diameter is Φ 6cm, and blake bottle spacing≤1 cm is cultivated through the natural daylight that penetrates culturing room, and each blake bottle is in the half-light cultivation conditions.Every layer of culturing rack (130*35cm) can be placed 200 ~ 220 bottles.
(4) take root domestication and transplant
Through the dwarf banana seedling behind step (3) the natural daylight refining seedling, clean the root medium, be transplanted in the nutrient cup, in booth, grew seedlings 50 ~ 60 days, be transplanted to the dwarf banana seedling that big Tanaka's field planting can obtain high-quality again.
Embodiment 5
(1) explant selection and processing
Choose the suction bud of the vigorous virus-free banana plant of banana variety osmanthus any of several broadleaf plants arid season growing way in No. 6 early springs; To inhale bud then and clean up, clean 2～3 times with commercially available cleaning solution again, progressively peel off outer bract with running water; Excise unnecessary stem part, keep the little scapus with terminal bud and lateral bud original hase, control scapus diameter Ф is 6～9cm; Scapus is put on the superclean bench,, use 0.2%HgCl solution soaking 10 min again with 80% alcohol-pickled 30s; Shake abundant sterilization, outwell the sterilization soup.Use aseptic water washing again 3～4 times, subsequent use.
(2) induce the dark cultivation of dedifferentiation tissue
The banana scapus that step (1) is disinfected is inoculated in the blake bottle that is loaded with inducing culture; Inducing culture is basal medium with MS; Other each constituent contents are 6-benzyl aminoadenine (6-BA) 1mg/L, NAA growth hormone 1.0mg/L, Su white granulated sugar 2%; Agar agar 3g, pH are 5.8～6.0.Place then and carry out half-light cultivation 25 days under 28～30 ℃, produce clump bud tissue; Every clump bud tissue that will obtain then cuts into 3 parts and carries out successive transfer culture; Successive transfer culture also adopts inducing culture to carry out half-light and cultivates, and cultivation temperature is 28～30 ℃, and the successive transfer culture cycle is 15 days; Reproduction coefficient is 3 ~ 4, and successive transfer culture obtains the banana clump bud tissue of requirement.
In the Plant Tissue Breeding laboratory, blake bottle is arranged and is placed on the culture layer frame, and the culture layer frame is 5 layers, and every layer of culturing rack (130*35cm) can be placed 220 blake bottles; The blake bottle diameter is Φ 6cm, and blake bottle spacing 0cm cultivates through the natural daylight that incides common culturing room, and each blake bottle is in the half-light cultivation conditions.
(3) take root that differentiation is dark cultivates
To be inoculated in the blake bottle that root media is housed through the banana clump bud tissue that successive transfer culture obtains; Root media is basal medium with MS, and other each constituent contents are NAA growth hormone 0.15mg/L, Su white granulated sugar 27g/L; Agar agar 3g, pH are 5.8～6.0.Under 28 ~ 30 ℃, carry out half-light and cultivated 10 days, after inducing culture went out root system, the banana seedlings that will grow root system was again transferred to sun light green house and is carried out natural lighting refining seedling; Natural lighting was refined seedling after 20 days, the sturdy health of the cane of banana seedlings, and blade changes dark green by bright yellow, well developed root system, the root hair is abundant.
In the Plant Tissue Breeding laboratory, blake bottle is arranged and is placed on the culture layer frame, and the culture layer frame is 5 layers, and every layer of culturing rack (130*35cm) can be placed 220 blake bottles; The blake bottle diameter is Φ 6cm, and blake bottle spacing 0cm cultivates through the natural daylight that penetrates culturing room, and each blake bottle is in the half-light cultivation conditions.
Claims (6)
1. the dark cultural method in the banana tissue culture, it is characterized in that: it comprises the steps:
(1) explant selection and processing;
(2) induce the dark cultivation of dedifferentiation tissue;
(3) take root that differentiation is dark cultivates;
(4) take root domestication and transplant;
Said to induce dark cultivation of dedifferentiation tissue be that the explant banana scapus that step (1) disinfects is inoculated in the blake bottle of splendid attire inducing culture, places then to carry out half-light under 28～30 ℃ and cultivated 25～30 days, produces clump bud tissue; Clump bud tissue cutting the carrying out successive transfer culture that will obtain then; Successive transfer culture also adopts inducing culture to carry out half-light and cultivates, and cultivation temperature is 28～30 ℃, and the successive transfer culture cycle is 15 ~ 18 days; Reproduction coefficient is 3 ~ 4, and successive transfer culture obtains the banana clump bud tissue of requirement; Said inducing culture is basal medium with MS, and other each constituent contents are 6-benzyl aminoadenine 1～3mg/L, NAA growth hormone 0.2～1mg/L, and Su white granulated sugar 2%～5%, Agar agar 2 ~ 5g, pH are 5.8～6.0.
2. the dark cultural method in the banana tissue culture according to claim 1; It is characterized in that: the said dark cultivation of differentiation of taking root will be inoculated in the blake bottle that is loaded with root media through the banana clump bud tissue that successive transfer culture obtains; Under 28 ~ 30 ℃, carrying out half-light cultivated 10～15 days; After inducing culture went out root system, the banana seedlings that will grow root system was again transferred to sun light green house and is carried out natural lighting refining seedling; Natural lighting was refined seedling after 20 days, the sturdy health of the cane of banana seedlings, and blade changes dark green by bright yellow, well developed root system, the root hair is abundant; Said root media is basal medium with MS, and other each constituent contents are NAA growth hormone 0.05 ~ 0.15mg/L, Su white granulated sugar 25 ~ 27g/L, and Agar agar 2 ~ 5g, pH are 5.8～6.0.
3. the dark cultural method in the banana tissue culture according to claim 1 and 2; It is characterized in that: said half-light cultivation is meant the blake bottle arrangement is placed on the culture layer frame; The blake bottle diameter is Φ 5 ~ 6cm; Blake bottle spacing≤1 cm is cultivated through the natural daylight that incides common culturing room, and each blake bottle is in the half-light cultivation conditions.
4. the dark cultural method in the banana tissue culture according to claim 3 is characterized in that: said culture layer frame is 4 ~ 6 layers, and every layer of culturing rack can be placed 200 ~ 220 bottles of blake bottles.
5. the dark cultural method in the banana tissue culture according to claim 3 is characterized in that: said explant selection is to choose the suction bud of early spring and the vigorous virus-free banana plant of growing way in autumn; The processing method of explant: will inhale bud and clean up, and peel off outer bract, and keep the little scapus with terminal bud and lateral bud original hase, control scapus diameter of phi is 5～10cm; Scapus is put on the superclean bench, with 75 ~ 80% alcohol-pickled 30 ~ 35s, use 0.1 ~ 0.2%HgCl solution soaking, 10 ~ 15min sterilization again after, with aseptic water washing 3～4 times, subsequent use.
6. according to the dark cultural method in claim 1 or the 5 described banana tissue culture; It is characterized in that: said take root domestication and transplanting are meant through the banana seedlings behind step (3) the natural daylight refining seedling; Clean the root medium; Be transplanted in the nutrient cup, in booth, grew seedlings 50 ~ 60 days, be transplanted to big Tanaka's field planting again and get final product.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN106172011A (en) * | 2016-08-23 | 2016-12-07 | 广西壮族自治区农业科学院生物技术研究所 | A kind of dwarf banana high temperature light culture tissue culture and rapid propagation method |
| CN106688887A (en) * | 2016-12-08 | 2017-05-24 | 广东省农业科学院果树研究所 | Fenza No.1 dwarf banana tissue culture rapid propagation method |
| CN109924123A (en) * | 2018-12-26 | 2019-06-25 | 广西壮族自治区农业科学院生物技术研究所 | A kind of hybridization pollination method improving banana Setting percentage |
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| 邓立国等: "澳大利亚香蕉品种Williams球茎再生体系研究", 《广西农业科学》 * |
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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 |
| CN106172011A (en) * | 2016-08-23 | 2016-12-07 | 广西壮族自治区农业科学院生物技术研究所 | A kind of dwarf banana high temperature light culture tissue culture and rapid propagation method |
| CN106172011B (en) * | 2016-08-23 | 2018-06-22 | 广西壮族自治区农业科学院生物技术研究所 | A kind of dwarf banana high temperature light culture tissue culture and rapid propagation method |
| CN106688887A (en) * | 2016-12-08 | 2017-05-24 | 广东省农业科学院果树研究所 | Fenza No.1 dwarf banana tissue culture rapid propagation method |
| CN106688887B (en) * | 2016-12-08 | 2019-08-20 | 广东省农业科学院果树研究所 | The tissue culture and rapid propagation method of miscellaneous No. 1 dwarf banana of powder |
| CN109924123A (en) * | 2018-12-26 | 2019-06-25 | 广西壮族自治区农业科学院生物技术研究所 | A kind of hybridization pollination method improving banana Setting percentage |
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