CN102726302B - Dark culture method for banana tissue culture - Google Patents
Dark culture method for banana tissue culture Download PDFInfo
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- CN102726302B CN102726302B CN201210248030.5A CN201210248030A CN102726302B CN 102726302 B CN102726302 B CN 102726302B CN 201210248030 A CN201210248030 A CN 201210248030A CN 102726302 B CN102726302 B CN 102726302B
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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 that Banana Tissue is cultivated.
Background technology
Banana (Musa balbisiana Colla) is one of important tropical fruit (tree) of south China, is also the Important Economic revenue source in many areas simultaneously, and many peasant households rely on plantation banana to shake off poverty and set out on the road to prosperity.Most cultivation edible banana is all unisexuality triploid AAA, there is the sterile gene of height, main by vegetative propagation, in production, conventionally utilize suction bud to breed, when seedling is not enough, also can adopt the subterranean stem stripping and slicing of banana to breed seedling, can accumulate much virus but the method is used for a long time, as: bunchy top, mosaic disease etc.Cause serious harm.The problems such as in addition, bad banana seedling can cause undergrowth, and fruit is not good, results disunity.Banana Tissue culture technique at short notice Fast-propagation goes out the health seedling without damage by disease and insect of the high-quality of a large amount of uniform specification, and, the cultivation of new varieties and introduce and also need to utilize virus-free and group culturation rapid propagating technology to accelerate the development of famous-brand and high-quality kind.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, the application of this technology has been played great progradation to developing rapidly of China's banana industry, also make kind of seedling industrialized production be achieved, by improved seeds spread, reach large-scale commercial banana production at short notice.But this industry was through the application of more than 20 years, and we find this technology Shortcomings, and in production application, subject matter is:
Time productive culture monocycle long, in producing to cultivate (being inoculated into new medium to the cultivation) time be more than 30 days the general monocycle for banana group training.
2. produce power consumption large, in cultivation process, need to supplement illumination, light filling is time 10h/ days, intensity of illumination 1500～2000lx.
3. the energy consumption that light filling produces is high, conventionally adopt the fluorescent lamp of 40W owing to supplementing illumination, in the time of work, can produce a large amount of heats, in order to reduce the cultivation temperature of culturing room, just necessary air-conditioning temperature-reducing, will produce no small electricity charge annual summer thus, increase operation costs to enterprise, be unfavorable for very much batch production production.
4. because the production cycle is long, cause 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 meet the required illumination of growing of each blake bottle, culture layer frame can only be put 108 bottles of subculture materials conventionally, take up room large, adopt dark cultural method of the present invention, every layer of culturing rack can be put 200 ~ 220 bottles, effectively utilized space.It is many that the group training seedling occupation of land of equivalent is cultivated in artificial lighting, is also the major reason that plant area increases.
Above shortcoming is ubiquitous problem during traditional group training is produced, in the torrid zone and the subtropical zone of southern china, tissue culture seedlings of bananas has become large seedling kind, there is every year the market demand that exceedes more than one hundred million strains, but because production cost is in (the labour cost that increases progressively year by year, the electricity charge, place rent etc.) hinder greatly the development of planting seedling industrialized industry, therefore develop a kind of new tissue culture technology to overcome the shortcoming of original training method, guarantee that further developing of banana seedling industry is imperative.
In Banana Tissue cultivation process of the present invention, adopt dark cultural method can effectively make up the many deficiencies in light training method, for the stable development of banana seedling industry provides technical support.
The advantage that dark cultural method in Banana Tissue cultivation has has following 4 points:
1. the production cycle shortens, the mode that Banana Tissue is cultivated as adopted light to cultivate, 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 the mode of dark cultivation instead, incubation time can foreshorten to 15 ~ 18 days.
In vitro tissue and test-tube plantlet suitable at the specific temperature of one g and D.As: one section of suitable temperature is (25 ± 2) ℃.In the time that room temperature does not meet this temperature, should start air-conditioner, regulate as requested temperature, until meet the requirements.Traditional light cultivation formula tissue is cultivated and is generally made intensity of illumination reach 1500～2000lx, reach this intensity of illumination, and every layer of culturing rack needs to fill respectively two fluorescent lamps, for illumination.Can produce thus a huge illuminating power expense.As: the culturing room of 15 ㎡ more can hold five layers of culturing rack of 15 wide 35cm of long 130cm.Calculate by every layer of two 40W fluorescent lamp, the day power consumption of each culturing room light filling is:
W=40×2×5×15×10=60kw/h
Year power consumption is so: 365 × 60=21900kw/h
A plant tissue culture factory that produces 3500～4,000 ten thousand strain group training seedlings per year is because the electricity expense that light filling produces can be up to 547500 degree/years.
Because the present invention has used dark culture technique instead, aspect illumination, savable expense is appreciable every year, and great advantage is provided in the competition in market for enterprise.
3. enter after summer, the heat that utilizes light culture technique light filling illuminating lamp tube to come out is larger, make culturing room's temperature surmounted far away banana crops tissue can the frozen limit temperature 33 ℃ to reach 28～30 ℃ of desirable cultivation temperature above, must need to utilize air conditioner cooling.Calculate with air conditioner of 15～20 ㎡ (power: 750w/h), a year cooling electric weight is about 600 degree (by 6 months/years), and the production scale cooling electric weight of year production 4,000 ten thousand strains is 15000 degree.Improve training mode: after dark cultivation, its cooling expense can significantly decline, and reduces production cost.
4. along with the raising year by year of space expenses cost, originally the subculture monocycle is that the production model of 25 days 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.Dark mode of cultivating becomes monocycle time shorten after 15 days, and this problem just can significantly slow 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
Object of the present invention is in order to solve the deficiencies in the prior art, and the dark cultural method that provides a kind of Banana Tissue to cultivate,
Cultivate in induction dedifferentiation and the atomization of taking root and adopt dark cultural method at tissue, avoid Traditional Man illumination cultivation, both shortened cultivation cycle, also reduced energy consumption, and when assurance group training seedling quality, trained seedling Propogation and culture for group and reduced production cost.
Technical scheme of the present invention is as follows:
Dark cultural method in Banana Tissue cultivation, it comprises the steps:
(1) the choosing and processing of explant;
(2) induction dedifferentiation tissue is dark cultivates;
(3) take root and break up dark cultivation;
(4) take root and tame and transplant;
Described explant choose be choose early spring and autumn (particularly arid season) the virus-free banana plant that growing way is vigorous suction bud; The processing method of explant: suction bud is cleaned up, progressively peel off outer bract, excise unnecessary stem part, retain the little scapus with terminal bud and the former base of lateral bud, controlling scapus diameter of phi is 5～10cm, and scapus is put on superclean bench, with 75 ~ 80% alcohol-pickled 30 ~ 35s sterilizations, use again 0.1 ~ 0.2%HgCl solution to soak after 10 ~ 15min sterilizing, use aseptic water washing 3～4 times, for subsequent use.
Inhaling bud is that abiogenous cripetura between plant rhizosphere or terrestrial stem axil, plumpness are rosula brachyplast.Inhaling bud bottom can take root naturally, therefore can separate and plant separately and cultivate into new plant from maternal plant.
The dark cultivation of described induction dedifferentiation tissue is that the explant banana scapus that step (1) is disinfected is inoculated in the blake bottle of splendid attire inducing culture, is then placed at 28～30 ℃ and carries out half-light cultivation 25～30 days, produces clump bud tissue; Then the clump bud tissue cutting obtaining is carried out to subculture cultivation, subculture is cultivated and is also adopted inducing culture to carry out half-light cultivation, and cultivation temperature is 28～30 ℃, and subculture cultivation cycle is 15 ~ 18 days, reproduction coefficient is 3 ~ 4, and subculture is cultivated the banana clump bud tissue that obtains requirement; Described inducing culture is take MS as basal medium, 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 is 5.8～6.0.This possess good subculture and the ability of the differentiation of taking root from bud tissue.
To coming from culture (comprising cell, tissue or its segment) that explant breeds by changing fresh culture and constantly cutting or separate, carry out the cultivation of continuous multi-generation, be just called subculture and cultivate.Also refer to that callus grows after a period of time on medium, nutrients exhaustion, moisture loss, and accumulated some metabolites, and now needing these tissues to transfer on new medium, this transfer is called subculture and cultivates or the cultivation of going down to posterity.
Reproduction coefficient (propagationcoefficient) refers to the number that is obtained new talent in a subculture is cultivated by a seedling (bud or bud clump).
The dark cultivation of the described differentiation of taking root is inoculated in the blake bottle that is loaded with root media cultivate the banana clump bud tissue obtaining through subculture, at 28 ~ 30 ℃, carrying out half-light cultivates 10～15 days, induction is turned out after root system, then the banana seedlings that grows root system is transferred to sun light green house and carry out natural lighting hardening; After natural lighting hardening 20 days, the sturdy health of the cane of banana seedlings, blade turns dark green by bright yellow, well developed root system, root hair is abundant; Described root media is take MS as basal medium, 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 is 5.8～6.0.
Hardening is in the situation that protection is grown seedlings; take to leak informaton, lower the temperature, suitably control the process that the measures such as water are taken exercise by force to seedling; make can adapt to rapidly after its field planting the unsuitable environmental condition of open country, shorten transplanting seedling time, strengthen the resistivity to low temperature, strong wind etc.
Described half-light is cultivated and is referred in Plant Tissue Breeding chamber, blake bottle is placed on culture layer frame, and blake bottle diameter is Φ 5 ~ 6cm, blake bottle spacing≤1 cm, cultivate by the natural daylight that incides common culturing room, each blake bottle is in half-light cultivation conditions.Described 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 close-packed arrays is placed on culturing rack, the placing distance of blake bottle is little, when natural lighting on daytime is mapped on blake bottle, due between blake bottle mutually block and transparent blake bottle between diffuse reflection effect, therefore blake bottle is in half-light or low light level state.Evening, each blake bottle was also in half-light state because available light is very small and weak.Of the present invention is exactly to organize in this state cultivation, do not need to carry out shading as traditional dark cultivation, and also without carrying out extra artificial light filling,, and the training of traditional group need to be carried out artificial light filling.Traditional Banana Tissue is cultivated in early days not irradiation, but after bud sprouting, needs illumination 12h left and right every day, and intensity of illumination is 2000～3000lx, and energy consumption is large.; The every 40 genius divisible switching of stem apex clump sorite 1 time when subculture is cultivated, cultivation cycle is long.Plant Tissue Breeding of the present invention laboratory is without direct sunlight.
Described take root domestication and transplanting refer to the banana seedlings after step (3) natural daylight hardening, clean root medium, are transplanted in nutrition cup, grow seedlings 50 ~ 60 days, then be transplanted to large Tanaka's field planting and can obtain banana seedling in booth.
Cultural method of the present invention is also adapted in the tissue cultivation of banana of dwarf banana or other kind.
Advantage of the present invention:
1. adopt method of the present invention, the stage that the dedifferentiation cultivation stage of cultivating at tissue and differentiation are taken root secretly cultivates, under the state of tissue in the low light level or half-light, grow, the fissional speed of dedifferentiation is accelerated, avoid the inhibitory action of illumination in people's light light filling process, cultivation cycle is shortened, enhance productivity.
2. adopt dark training method, make to emerge neat healthy and strong, fast growth, aberration rate is low, and the differentiation of taking root is fast, and light culture technique has larger advantage relatively.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 significantly reduce light and cultivate the power consumption producing in production, 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 place, greatly improve the availability of production site.
5. improve production efficiency, reduced labour intensity.The dark cultural method that the present invention adopts, the stage that group training is taken root in dedifferentiation cultivation stage and differentiation is grown by natural daylight, therefore do not need to control the intensity of its illumination, do not need the intensity of manual adjustment illumination, reduce labour, be conducive to enhance 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 Banana Tissue cultivation, it comprises the steps:
(1) the choosing and processing of explant
Choose the suction bud of the virus-free banana plant that banana variety osmanthus any of several broadleaf plants No. 6 arid season growing way in early spring is vigorous; Then suction bud is cleaned up with running water, clean 2～3 times with commercially available cleaning solution again, progressively peel off outer bract, excise unnecessary stem part, retain the little scapus with terminal bud and the former base of lateral bud, controlling scapus diameter Ф is 5～10cm, scapus is put on superclean bench, with 75% alcohol-pickled 30s, then soak 10 min with 0.1%HgCl solution, shake abundant sterilizing, outwell sterilization liquid.Use again aseptic water washing 3～4 times, for subsequent use.
(2) induction dedifferentiation tissue is dark cultivates
The banana scapus that step (1) is disinfected is inoculated in the blake bottle that is loaded with inducing culture, inducing culture is take MS as basal medium, 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 is 5.8～6.0.Then be placed at 28～30 ℃ and carry out half-light cultivation 25～30 days, produce clump bud tissue; Then every the clump bud tissue obtaining is cut into 3 points and carry out subculture cultivation, subculture is cultivated and is also adopted inducing culture to carry out half-light cultivation, and cultivation temperature is 28～30 ℃, and subculture cultivation cycle is 15 ~ 18 days, reproduction coefficient is 3 ~ 4, and subculture is cultivated the banana clump bud tissue that obtains requirement.
In Plant Tissue Breeding laboratory, blake bottle is placed on culture layer frame, and culture layer frame is 5 layers, and every layer of culturing rack (130*35cm) can be placed 216 blake bottles; Blake bottle diameter is Φ 6cm, and blake bottle spacing 0cm, cultivates by the natural daylight that incides common culturing room, and each blake bottle is in half-light cultivation conditions.
(3) take root and break up dark cultivation
Be inoculated in the blake bottle that root media is housed cultivate the banana clump bud tissue obtaining through subculture, root media is take MS as basal medium, 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 is 5.8～6.0.At 28 ~ 30 ℃, carry out half-light and cultivate 10～15 days, induction is turned out after root system, then the banana seedlings that grows root system is transferred to sun light green house and carry out natural lighting hardening; After natural lighting hardening 20 days, the sturdy health of the cane of banana seedlings, blade turns dark green by bright yellow, well developed root system, root hair is abundant.
In Plant Tissue Breeding laboratory, blake bottle is placed on culture layer frame, and culture layer frame is 5 layers, and every layer of culturing rack (130*35cm) can be placed 216 blake bottles; Blake bottle diameter is Φ 6cm, and blake bottle spacing 0 cm, cultivates by the natural daylight that penetrates culturing room, and each blake bottle is in half-light cultivation conditions.
(4) take root and tame and transplant
Banana seedlings after step (3) natural daylight hardening, cleans root medium, is transplanted in nutrition cup, grows seedlings 50 ~ 60 days, then be transplanted to large Tanaka's field planting and can obtain the banana seedling of high-quality in booth.
Embodiment 2
(1) the choosing and processing of explant
Choose the suction bud of the virus-free banana plant that No. 6 Drought in Autumn growing ways in season of banana variety osmanthus any of several broadleaf plants are vigorous; Then suction bud is cleaned up with running water, clean 2～3 times with commercially available cleaning solution again, progressively peel off outer bract, excise unnecessary stem part, retain the little scapus with terminal bud and the former base of lateral bud, controlling scapus diameter Ф is 8～10cm, scapus is put on superclean bench, with 75% alcohol-pickled 35s, then soak 15 min with 0.1%HgCl solution, shake abundant sterilizing, outwell sterilization liquid.Use again aseptic water washing 3～4 times, for subsequent use.
(2) induction dedifferentiation tissue is dark cultivates
The banana scapus that step (1) is disinfected is inoculated in the blake bottle that is loaded with inducing culture, inducing culture is take MS as basal medium, 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 is 5.8～6.0.Then be placed at 28～30 ℃ and carry out half-light cultivation 25 days, produce clump bud tissue; Then every the clump bud tissue obtaining is cut into 4 parts and carry out subculture cultivation, subculture is cultivated and is also adopted inducing culture to carry out half-light cultivation, and cultivation temperature is 28～30 ℃, and subculture cultivation cycle is 15 days, reproduction coefficient is 3 ~ 4, and subculture is cultivated the banana clump bud tissue that obtains requirement.
In Plant Tissue Breeding laboratory, blake bottle is placed on culture layer frame, and culture layer frame is 4 layers, and every layer of culturing rack (130*35cm) can be placed 200 blake bottles; Blake bottle diameter is Φ 5cm, and blake bottle spacing 1cm, cultivates by the natural daylight that incides common culturing room, and each blake bottle is in half-light cultivation conditions.
(3) take root and break up dark cultivation
Be inoculated in the blake bottle that root media is housed cultivate the banana clump bud tissue obtaining through subculture, root media is take MS as basal medium, and other each constituent contents are NAA growth hormone 0.1mg/L, Su white granulated sugar 25g/L, Agar agar 2g, pH is 5.8～6.0.At 28 ~ 30 ℃, carry out half-light and cultivate 15 days, induction is turned out after root system, then the banana seedlings that grows root system is transferred to sun light green house and carry out natural lighting hardening; After natural lighting hardening 20 days, the sturdy health of the cane of banana seedlings, blade turns dark green by bright yellow, well developed root system, root hair is abundant.
In Plant Tissue Breeding laboratory, blake bottle is placed on culture layer frame, and culture layer frame is 4 layers, and every layer of culturing rack (130*35cm) can be placed 200 blake bottles; Blake bottle diameter is Φ 5cm, and blake bottle spacing 1cm, cultivates by the natural daylight that penetrates culturing room, and each blake bottle is in half-light cultivation conditions.
(4) take root and tame and transplant
Banana seedlings after step (3) natural daylight hardening, cleans root medium, is transplanted in nutrition cup, grows seedlings 50 ~ 60 days, then be transplanted to large Tanaka's field planting and can obtain the banana seedling of high-quality in booth.
Embodiment 3
(1) the choosing and processing of explant
Choose the suction bud of the virus-free banana plant that banana variety osmanthus any of several broadleaf plants No. 6 arid season growing way in early spring is vigorous; Then suction bud is cleaned up with running water, clean 2～3 times with commercially available cleaning solution again, progressively peel off outer bract, excise unnecessary stem part, retain the little scapus with terminal bud and the former base of lateral bud, controlling scapus diameter Ф is 5～8cm, scapus is put on superclean bench, with 75% alcohol-pickled 30s, then soak 10 min with 0.1%HgCl solution, shake abundant sterilizing, outwell sterilization liquid.Use again aseptic water washing 3～4 times, for subsequent use.
(2) induction dedifferentiation tissue is dark cultivates
The banana scapus that step (1) is disinfected is inoculated in the blake bottle that is loaded with inducing culture, inducing culture is take MS as basal medium, 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 is 5.8～6.0.Then be placed at 28～30 ℃ and carry out half-light cultivation 28 days, produce clump bud tissue; Then every the clump bud tissue obtaining is cut into 4 parts and carry out subculture cultivation, subculture is cultivated and is also adopted inducing culture to carry out half-light cultivation, and cultivation temperature is 28～30 ℃, and subculture cultivation cycle is 18 days, reproduction coefficient is 3 ~ 4, and subculture is cultivated the banana clump bud tissue that obtains requirement.
In Plant Tissue Breeding laboratory, blake bottle is placed on culture layer frame, and culture layer frame is 6 layers, and every layer of culturing rack (130*35cm) can be placed 210 blake bottles; Blake bottle diameter is Φ 6cm, and blake bottle spacing 0cm, cultivates by the natural daylight that incides common culturing room, and each blake bottle is in half-light cultivation conditions.
(3) take root and break up dark cultivation
Be inoculated in the blake bottle that root media is housed cultivate the banana clump bud tissue obtaining through subculture, root media is take MS as basal medium, and other each constituent contents are NAA growth hormone 0.1mg/L, Su white granulated sugar 25g/L, Agar agar 5g, pH is 5.8～6.0.At 28 ~ 30 ℃, carry out half-light and cultivate 12 days, induction is turned out after root system, then the banana seedlings that grows root system is transferred to sun light green house and carry out natural lighting hardening; After natural lighting hardening 20 days, the sturdy health of the cane of banana seedlings, blade turns dark green by bright yellow, well developed root system, root hair is abundant.
In Plant Tissue Breeding laboratory, blake bottle is placed on culture layer frame, and culture layer frame is 6 layers, and every layer of culturing rack (130*35cm) can be placed 210 blake bottles; Blake bottle diameter is Φ 6cm, and blake bottle spacing 0cm, cultivates by the natural daylight that penetrates culturing room, and each blake bottle is in half-light cultivation conditions.
(4) take root and tame and transplant
Banana seedlings after step (3) natural daylight hardening, cleans root medium, is transplanted in nutrition cup, grows seedlings 50 ~ 60 days, then be transplanted to large Tanaka's field planting and can obtain the banana seedling of high-quality in booth.
Embodiment 4
Dark cultural method in the cultivation of dwarf banana tissue, it comprises the steps:
(1) the choosing and processing of explant
Choose the suction bud of the virus-free banana plant that No. 1, dwarf banana kind bronze arid season growing way in early spring is vigorous; Then suction bud is cleaned up with running water, clean 2～3 times with commercially available common cleaning solution again, progressively peel off outer bract, excise unnecessary stem part, retain the little scapus with terminal bud and the former base of lateral bud, controlling scapus diameter of phi is 5～10cm, scapus is put on superclean bench, with 75% alcohol-pickled 30s, then soak 10 min with 0.1%HgCl solution, shake abundant sterilizing, outwell sterilization liquid.Use again aseptic water washing 3～4 times, for subsequent use.
(2) induction dedifferentiation tissue is dark cultivates;
The banana scapus that step (1) is disinfected is inoculated in the blake bottle that is loaded with inducing culture, and inducing culture is take MS as basal medium, 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 is 5.8～6.0; Be placed at 28～30 ℃ and carry out half-light cultivation 25～30 days, produce clump bud tissue; Then carry out subculture cultivation, subculture cultivation cycle is 15 ~ 18 days, and reproduction coefficient is 3 ~ 4, and subculture is cultivated the dwarf banana clump bud tissue that obtains actual production requirement.
Blake bottle is placed on culture layer frame, and blake bottle diameter is Φ 6cm, and blake bottle spacing≤1 cm, cultivates by the natural daylight that penetrates culturing room, and each blake bottle is in half-light cultivation conditions.Every layer of culturing rack (130*35cm) can be placed 200 ~ 220 bottles
(3) take root and break up dark cultivation
Be inoculated in the blake bottle of splendid attire root media cultivate the banana clump bud tissue obtaining through subculture, root media is take MS as basal medium, NAA growth hormone 0.05 ~ 0.15mg/L, and Su white granulated sugar 25 ~ 27g/L, Agar agar 2g ~ 5g, pH is 5.8～6.0; Cultivate through the half-lights of 10～15 days, 28 ~ 30 ℃ of cultivation temperature, induction is turned out after root system, then banana seedlings is transferred to sun light green house and carry out natural lighting hardening; After natural lighting hardening 20 days, the sturdy health of the cane of banana seedlings, blade turns dark green by bright yellow, well developed root system, root hair is abundant.
Blake bottle is placed on culture layer frame, and blake bottle diameter is Φ 6cm, and blake bottle spacing≤1 cm, cultivates by the natural daylight that penetrates culturing room, and each blake bottle is in half-light cultivation conditions.Every layer of culturing rack (130*35cm) can be placed 200 ~ 220 bottles.
(4) take root and tame and transplant
Dwarf banana seedling after step (3) natural daylight hardening, cleans root medium, is transplanted in nutrition cup, grows seedlings 50 ~ 60 days, then be transplanted to large Tanaka's field planting and can obtain the dwarf banana seedling of high-quality in booth.
Embodiment 5
(1) the choosing and processing of explant
Choose the suction bud of the virus-free banana plant that banana variety osmanthus any of several broadleaf plants No. 6 arid season growing way in early spring is vigorous; Then suction bud is cleaned up with running water, clean 2～3 times with commercially available cleaning solution again, progressively peel off outer bract, excise unnecessary stem part, retain the little scapus with terminal bud and the former base of lateral bud, controlling scapus diameter Ф is 6～9cm, scapus is put on superclean bench, with 80% alcohol-pickled 30s, then soak 10 min with 0.2%HgCl solution, shake abundant sterilizing, outwell sterilization liquid.Use again aseptic water washing 3～4 times, for subsequent use.
(2) induction dedifferentiation tissue is dark cultivates
The banana scapus that step (1) is disinfected is inoculated in the blake bottle that is loaded with inducing culture, inducing culture is take MS as basal medium, 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 is 5.8～6.0.Then be placed at 28～30 ℃ and carry out half-light cultivation 25 days, produce clump bud tissue; Then every the clump bud tissue obtaining is cut into 3 parts and carry out subculture cultivation, subculture is cultivated and is also adopted inducing culture to carry out half-light cultivation, and cultivation temperature is 28～30 ℃, and subculture cultivation cycle is 15 days, reproduction coefficient is 3 ~ 4, and subculture is cultivated the banana clump bud tissue that obtains requirement.
In Plant Tissue Breeding laboratory, blake bottle is placed on culture layer frame, and culture layer frame is 5 layers, and every layer of culturing rack (130*35cm) can be placed 220 blake bottles; Blake bottle diameter is Φ 6cm, and blake bottle spacing 0cm, cultivates by the natural daylight that incides common culturing room, and each blake bottle is in half-light cultivation conditions.
(3) take root and break up dark cultivation
Be inoculated in the blake bottle that root media is housed cultivate the banana clump bud tissue obtaining through subculture, root media is take MS as basal medium, and other each constituent contents are NAA growth hormone 0.15mg/L, Su white granulated sugar 27g/L, Agar agar 3g, pH is 5.8～6.0.At 28 ~ 30 ℃, carry out half-light and cultivate 10 days, induction is turned out after root system, then the banana seedlings that grows root system is transferred to sun light green house and carry out natural lighting hardening; After natural lighting hardening 20 days, the sturdy health of the cane of banana seedlings, blade turns dark green by bright yellow, well developed root system, root hair is abundant.
In Plant Tissue Breeding laboratory, blake bottle is placed on culture layer frame, and culture layer frame is 5 layers, and every layer of culturing rack (130*35cm) can be placed 220 blake bottles; Blake bottle diameter is Φ 6cm, and blake bottle spacing 0cm, cultivates by the natural daylight that penetrates culturing room, and each blake bottle is in half-light cultivation conditions.
Claims (4)
1. the dark cultural method in Banana Tissue cultivation, is characterized in that: it comprises the steps:
(1) the choosing and processing of explant;
(2) induction dedifferentiation tissue is dark cultivates;
(3) take root and break up dark cultivation;
(4) take root and tame and transplant;
The dark cultivation of described induction dedifferentiation tissue is that the explant banana scapus that step (1) is disinfected is inoculated in the blake bottle of splendid attire inducing culture, is then placed at 28～30 ℃ and carries out half-light cultivation 25～30 days, produces clump bud tissue; Then the clump bud tissue cutting obtaining is carried out to subculture cultivation, subculture is cultivated and is also adopted inducing culture to carry out half-light cultivation, and cultivation temperature is 28～30 ℃, and subculture cultivation cycle is 15 ~ 18 days, reproduction coefficient is 3 ~ 4, and subculture is cultivated the banana clump bud tissue that obtains requirement; Described inducing culture is take MS as basal medium, 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 ~ 5 g/L, pH is 5.8～6.0;
The dark cultivation of the described differentiation of taking root is inoculated in the blake bottle that is loaded with root media cultivate the banana clump bud tissue obtaining through subculture, at 28 ~ 30 ℃, carrying out half-light cultivates 10～15 days, induction is turned out after root system, then the banana seedlings that grows root system is transferred to sun light green house and carry out natural lighting hardening; After natural lighting hardening 20 days, the sturdy health of the cane of banana seedlings, blade turns dark green by bright yellow, well developed root system, root hair is abundant; Described root media is take MS as basal medium, 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 ~ 5 g/L, pH is 5.8～6.0;
Described half-light is cultivated and is referred to blake bottle is placed on culture layer frame, and blake bottle diameter is Φ 5 ~ 6cm, and blake bottle spacing≤1 cm, cultivates by the natural daylight that incides common culturing room, and each blake bottle is in half-light cultivation conditions.
2. the dark cultural method in Banana Tissue cultivation according to claim 1, is characterized in that: described culture layer frame is 4 ~ 6 layers, and every layer of culturing rack can be placed 200 ~ 220 bottles of blake bottles.
3. Banana Tissue according to claim 1 dark cultural method in cultivating, is characterized in that: choosing of described explant is the suction bud of choosing early spring and the vigorous virus-free banana plant of growing way in autumn; The processing method of explant: suction bud is cleaned up, peel off outer bract, retain the little scapus with terminal bud and the former base of lateral bud, controlling scapus diameter of phi is 5～10cm; Scapus is put on superclean bench, with 75 ~ 80% alcohol-pickled 30 ~ 35s, then use 0.1 ~ 0.2%HgCl
2solution soaks after 10 ~ 15min sterilizing, uses aseptic water washing 3～4 times, for subsequent use.
4. the dark cultural method in cultivating according to the Banana Tissue described in claim 1 or 3, it is characterized in that: described in take root domestication and transplanting refer to the banana seedlings after step (3) natural daylight hardening, clean root medium, be transplanted in nutrition cup, in booth, grow seedlings 50 ~ 60 days, then be transplanted to large Tanaka's field planting.
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| 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 |
| 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 |
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