CN101720668B - Rapid propagation of sugarcane tissue culture by using intermittent immersion type bioreactor - Google Patents
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Abstract
The invention provides a method for carrying out tissue culture and rapid propagation of sugarcane by using an intermittent immersion bioreactor, which is characterized in that the sugarcane stem tip tissue subjected to detoxification treatment is used as a material after induced bud culture for several generations, and the material is transferred to the intermittent immersion bioreactor for propagation culture and rooting culture. Culturing in an intermittent immersion bioreactor at 29 deg.C under 1500Lx illumination for 16 hr for 3 min every 3 hr, and culturing for 3 min every 6 hr for rooting for 40 times of first generation, performing appropriate seedling exercising treatment after rooting, and transplanting with survival rate of about 85%. The system has high automation degree, the proliferation speed is far higher than that of the traditional tissue culture method, the labor cost is saved, and the economic benefit is increased.
Description
Technical field
The present invention relates to a kind of intermittent immersion bioreactor that utilizes and carry out the secure good health method of cane seedling of sugarcane (new platform sugar 22, new platform sugar 16, Badila) tissue culture quick breeding.
Background technology
Sugarcane (Saccharum officenarum L.) monocot genus plant guiding principle (Liliopsida), grain husk flower order (Gluimflorae), grass family (Gramineae) saccharum (Saccharum).Sugarcane is one of 10 genus in the subtribe crowd sugarcane subtribe of grass family another name for Sichuan Province.It and Plumegrass, awns belong to, hard fringe belongs to and river eight kings five belong to the nearer saccharum complex bigger with the cane breeding relation of composition relationship.It mainly contains five kinds of noble cane, India's kind, China seed, thin stem wild species, big stem wild species, and first three is a cultivar, and back two kinds is wild species.
The plant of sugarcane is made up of root, stem, leaf, flower and fruit (seed), and flower and seed are meaningful in the sugar cane breed improvement, and most sugar cane breeds are difficult for blooming usually in sugarcane production, does not also want sugarcane simultaneously in the sugarcane production and blooms.The sugarcane stem is the sugaring raw material, is made up of several joints and internode.The joint of sugarcane stem and internode what because of kind different and different with the phase of planting.
Sugarcane originates in the torrid zone, subtropical zone, and the extensively plantation in these areas.Sugarcane acreage maximum country in the world's is Brazil, secondly is India, the 3rd of China's rank.Sugarcane is for happiness temperature, happiness light crop, and is more extensive to the adaptability of soil, better with clay loam, red soil, sandy loam.Soil pH value is 4.5～8.0, and sugarcane can both grow, but with soil pH value 6.5～7.5 for suitable.
Sugarcane occupies critical role in China's agricultural economy, its output and the output value are only second to grain, oil plant and cotton, occupies the 4th.Sugarcane still is a kind of bioenergy crop of high yield, and sugarcane is high light efficiency carbon four crops, and biological yield is high, contains sugar 11%～17%, not only can supply sugaring, but Direct Production alcohol also is hybridly prepared into a kind of Fuel Petroleum that mixes with gasoline.Particularly abounding with sugarcane as Brazil etc. and the country of oil shortage adopts fermentation alcohol as one of new forms of energy, is a kind of inexhaustible bioenergy, is applied to motor vehicle fuel and can produce pollution, is called as green gasoline.Can predict,, have very big development potentiality as the sugarcane of one of important energy source meaning crop along with the continuous development of science and technology.
Sugarcane is the main sugar [yielding of China; Area account for the long-term sugared charge level of China long-pending 85% or more (the Li Qiwei volume. modern sugarcane improving technology. Guangzhou: publishing house of South China Science & Engineering University; 2001); Output of sugar accounts for more than 90% of sugar gross yield, and the sugarcane industry has become the mainstay of main producing region economic development and the main source of increasing peasant income.China and India, New Guinea are the three big sugarcane centres of origin, the world.China's sugarcane production in existing more than 3000 year record (Li Yangrui. the Chinese sugarcane sugar industry [J] that is emerging. Guangxi agricultural science, 2005,36 (1): 79-81).Be positioned at Guangxi Zhuang Autonomous Region of south subtropics monsoon climatic region, illumination is sufficient, has plenty of rain, suitable sugarcane production.Guangxi sugar output was 9,410,000 tons in 2008, accounted for 68.6% of national cane suger output, and 63.4% of sugar gross yield is genuine " Chinese sugar all ".
Traditional sugarcane mating system is to utilize the axillalry bud on the cane stalk section to breed, yet all vegetative crops deterioration of variety very easily occurs after the plantation for many years continuously as potato, sweet potato, banana, grape, strawberry with other.And, cause sugarcane yield and quality seriously to descend because artificial origins such as production management technology usually cause sugarcane district sugarcane disease large tracts of land popular.
Sugarcane is to embody one of totipotent crop of cell the earliest.1969, people such as the Heing.DJ plant that from sugarcane callus, regenerated.The a large amount of rapidly breeding test-tube plantlets of sugarcane can pass through dual mode.A kind of is to induce the tender leaf explant to produce callus, by callus differentiation plant; Another kind is to cultivate near the bud (terminal bud, axillalry bud) of stem top growing point, makes it the breeding of tillering in a large number.After the breeding some, induced bud sends out roots, and forms whole plant.At present, with the stem apex cultured in vitro technology combination thermal treatment of the tender stem-tip tissue of children as explant, the cane seedling of the detoxification that secures good health; And be used for producing, can make sugarcane production-increasing 20%～40%, cane sugar content increase (absolute value) (You Jianhua, He Weizhong more than 0.5%; Ceng Hui etc. talk the application and the benefit prospect of detoxification health seedling sugarcane production in Guangxi. the sugarcane sugar industry; 2001, (1): 13～17). but this method still comes with some shortcomings, and is embodied in:
1, appreciation rate is low; Because traditional tissue culture technology reproduction coefficient is lower, is generally for 2.0～4.0 per generations, and breed the propagation that a large amount of tissue cultivating seedling needs higher algebraically; Be generally about 10～12 generations; But along with the aberration rate that increases the while tissue cultivating seedling of subculture number is also increasing, the quality and the resistance of tissue cultivating seedling then reduce, and just can not obtain enough economic interests but do not improve the subculture number in producing; So for the economic pursuit interests, batch production is produced the sugarcane tissue-culture seedling and is put on market because of the seedling inferior that subculture too much causes with regard to some are arranged.
2, automatization level is low, and labour intensive is owing to need the propagation of higher algebraically in traditional sugarcane tissue-culture production process; Will transfer once in general about 20 days, need a large amount of human relay working subcultures, thereby increase labour cost; Cause the increase of production cost; And a large amount of tissue culture bottle of traditional method needs, not only take a large amount of space arrangements, and a large amount of manual operations of needs in the process of using operation.
3, the production cycle long, traditional tissue culture method since reproduction coefficient lower, so through the general needed time of cultivation in 10～12 generations be about 10 months.
4, production cost is high; The major part of the production cost of tradition sugarcane tissue culture is labourer's expense; Account for about 65% of whole production cost greatly, and the major part in the labour cost is the expense of transit working, owing to need the cultivation of higher generation; So the labourer's expense in transit working accounts for very big ratio, cause production cost to increase.
What time be the several topmost problem that is run on the present sugarcane production below; Now in the process saliency of producing, gradually in the process that the production of sugarcane tissue-culture seedling develops in a healthy way its to inhibition, at first; Because growth coefficient is not high; Cause breeding the increase of algebraically, thereby cause the quality of tissue cultivating seedling to can not get guaranteeing easily, render to market to some seedlings inferior easily; Secondly, automatization level is low, and labour intensive needs a large amount of laboring fees to spend in the transit working of tissue cultivating seedling, thereby causes the increase of production cost; At last, because the increase of subculture number, thereby cause the prolongation of production cycle, can not reasonable distribution in the supply of tissue cultivating seedling, can not tackle the demand in market flexibly and produce.Because growth coefficient is low; The amount of labour is big; Automatization level is low, and production cost is than higher reason, and traditional method is difficult in the production that adapts to following large-scale sugarcane tissue-culture seedling; Use the gap immersion bioreactor and carry out the defective that the production of sugarcane tissue-culture seedling can produced in conventional processes, provide safeguard for producing high-quality sugarcane tissue-culture seedling cheaply.
The gap immersion bioreactor is that a cover massive planting fabric texture is cultivated and this living metabolite research of plant is the equipment of one; Basic principle is to utilize liquid nutrient medium being that power carries out step cultivation to the plant tissue culture seedling through filtered air pressure; It mainly is made up of 2 containers and connects through glass tube or silicone tube, and gas is the syringe filters degerming of 0.22 μ m through the filter membrane aperture; Used air compressor to realize the supply of nutrient solution and drain, wherein the control of chopper frequency is controlled flowing time and the direction that magnetic valve is realized air by time-controlled switch.Its principle is to utilize liquid nutrient medium being that power carries out step cultivation to the plant tissue culture seedling through filtered air pressure; In conventional method comparatively speaking; It has not only had the advantage of liquid culture, has avoided again owing to the lopsided physiological problem that brings with this injury of giving birth to metabolite of the long vitrifying that brings of cycle in the liquid culture process.Esclona designed this cover system in 1999 and has named commodity to be called the BIT system; (ESCALONA M; LORENZO JC; GONZALEZ B.Pineapple (ananas comosusL.Merr) micropropagation in temporary immersion systems [J] .Plant CellReports, 1999 (18): 743-748.) improved called after TIBs system through Cuba sugarcane research institute afterwards.Domestic is to be introduced to improve and be applied to tissue culturing and quick propagation of sugarcane research by the Cuba sugarcane research institute that takes the lead in from international sugarcane research center of Guangxi Academy of Agricultural Sciences.
Summary of the invention
The purpose of this invention is to provide a kind of intermittent immersion bioreactor that utilizes and carry out the secure good health method of cane seedling of sugarcane (new platform sugar 22, new platform sugar 16, Badila) tissue culture quick breeding.
The production that utilizes the gap immersion bioreactor to carry out the sugarcane tissue-culture seedling has following 4 advantages:
1, appreciation rate is high; The basic principle of gap immersion bioreactor is to utilize liquid nutrient medium being that power carries out step cultivation to the plant tissue culture seedling through filtered air pressure; The characteristics of its maximum are exactly the appreciation rate height; The appreciation rate that a sugarcane tissue-culture seedling generation is cultivated can reach 30～40 times, exceeds traditional tissue culture method far away.
2, automatization level is high, the amount of labour is little; Because utilizing the gap immersion bioreactor to carry out in the process of sugarcane tissue culture is that air compressor machine provides aerodynamic force; Control the chopper frequency of cultivation by time-controlled switch, once after the inoculation, do not need to carry out again transit working in the whole incubation; Thereby can save a large amount of amounts of labour, reduce production costs.
3, reduce cultivation algebraically; Reduce production cost; The material that utilizes the gap immersion bioreactor to carry out the sugarcane tissue culture be with utilize produced in conventional processes tissue cultivating seedling the 5th generation, the 6th generation seedling, once the inoculation after no longer the switching, reduced cultivation algebraically; Reduce the expense of transit working, saved production cost.
4, the tissue cultivating seedling quality is good, and adaptability is strong, because the high-quality tissue cultivating seedling of production that is reduced to of algebraically provides assurance, can lower aberration rate to a certain extent.The gap immersion bioreactor carries out the sugarcane tissue culture owing to utilize filtered air to be power, has strengthened the gas exchange in the blake bottle in the process of cultivation, and simulating natural environment is grown, and strengthens the quality and the adaptive capacity of tissue cultivating seedling.
The technical scheme that the present invention taked is:
1, material chosen and inoculation; Utilize material source that the gap immersion bioreactor carries out the sugarcane tissue culture in through the 5th generation of traditional group culture method successive transfer culture or the 6th generation tissue cultivating seedling; At the most to select this two Dai Miao to be because just have seedling the time enough material supplies to carry out the gap immersion bioreactor to advance to cultivate when cultivated for 5～6 generations with conventional method, and be unlikely to because cultivate the problems such as variation and resistance difference of the too high and generation of algebraically; Inoculation needs to divide individual plant to carry out, and the Lao Ye part of excision sugarcane tissue-culture seedling base portion, keeps the complete of individual plant seedling basically, also can suitably excise if young leaves is long, stays young leaves 1～2cm.Inoculum density is 70ml～100ml culture fluid/strain.
2, enrichment culture is transferred to the sugarcane tissue-culture seedling that cuts and carries out enrichment culture in the intermittent immersion bioreactor, and the prescription that utilizes the sugarcane stem apex to induce the subculture seedling to carry out the medium of enrichment culture at intermittent immersion bioreactor is: the MS+6-BA0.6mg/L+NAA0.1mg/L+20g/L white granulated sugar; The medium pH value is 6.2; Condition of culture is 29 ℃ of temperature, illumination 1500Lx, illumination in 16 hours; 8 hours dark; The chopper frequency of intermittently cultivating be per 3 hours once, incubation time is 3 minutes, cultivates 30 days.
3, culture of rootage, sugarcane subculture seedling carries out the medium of culture of rootage at intermittent immersion bioreactor prescription is: MS+6-BA0.01mg/L+NA2.0mg/L+ active carbon 50mg/L+20g/L white granulated sugar, the medium pH value is 6.2; Condition of culture is 29 ℃ of temperature, illumination 1500Lx, illumination in 16 hours; 8 hours dark every days; The chopper frequency of intermittently cultivating be per 6 hours once, incubation time is 3 minutes, cultivates 20 days.
4, take root seedling domestication and transplanting, ends of taking root can be transplanted after in refining the seedling booth, putting 3～5 days; During transplanting with clear water wash clean root; Beginning to transplant is with husky training, and lid plastic film 5 days is taken off the special compound fertilizer for sugar cane that jede Woche behind the film sprays a time 0.2%; Transplant to nutritive cube after 40 days, in nutritive cube, grow seedlings and to transplant to the land for growing field crops about one month.
5, intermittent immersion bioreactor chopper frequency enrichment culture be 3 hours each, incubation time is 3 minutes; The chopper frequency of culture of rootage be 6 hours each, incubation time is 3 minutes.
Good effect of the present invention:
What (1) intermittent immersion bioreactor carried out the fast numerous utilization of sugarcane is through the tissue cultivating seedling in 5,6 generations of detoxification treatment cultivation, has guaranteed the aseptic and healthy of material, and therefore, pollution rate is very low in the process of cultivating; Use low Dai Miao also to guarantee the quality and the health degree of tissue cultivating seedling, reduce aberration rate.
(2) be intermittent cultivation owing to utilizing intermittent immersion bioreactor to carry out sugarcane fast numerous; Alleviated the injury of phenol to tissue cultivating seedling; Simultaneously owing to exchange gas in the blake bottle makes that the photosynthetic and respiration capability of tissue cultivating seedling is strengthened; Thereby strengthened the adaptive capacity of tissue cultivating seedling, ensured transplanting survival rate, survival rate of the present invention reaches about 85%.
(3) utilize intermittent immersion bioreactor to carry out the fast numerous cycle weak point of sugarcane; Need 50 days left and right sides time from being inoculated into the completion of taking root, and growth rate, appreciation rate are very high, can arrive 40 times; Be higher than traditional tissue culture method far away, make the mobility of batch production production, supply strengthen.
(4) blake bottle of use 2.5L; Once inoculate 10～15 strain subculture tissue cultivating seedling; Can obtain the tissue cultivating seedling about 500 strains through propagation and culture of rootage, improve the reproduction coefficient of sugarcane tissue-culture greatly, and the subculture number of tissue cultivating seedling was lowered within 7 generations; Reduced the subculture number, very big advantage has been arranged with respect to traditional method.
(5) this cover system automaticity is high, saves labour cost, increases economic benefit.
Embodiment
1, the foundation of intermittent immersion bioreactor, on the basic principle of traditional batch immersion bioreactor, the equipment of selecting to be fit to domestic conditions carries out low cost assembling foundation.
2, material: select for use Guangxi sugarcane research institute biotron utilize the 5th generation that the sugarcane stem apex detoxify cultivates or the 6th generation seedling.
3, method:
3.1, choose the tissue cultivating seedling sturdy, that base portion is clean as inoculation material; When cutting material, peel off the Lao Ye of tissue cultivating seedling as far as possible; Young leaves is done a little excision, stays young leaves 1～2cm, is also removed fully by the blade of the blackout of phenol infringement or flavescence; But can not destroy the growing point of tissue cultivating seedling and the bud point of base portion.
3.2, enrichment culture: it is in 6.2 the liquid nutrient medium for MS+6-BA0.6mg/L+NAA0.1mg/L+20g/L white granulated sugar, pH value that the sugarcane tissue-culture seedling that will cut is inoculated in prescription; Condition of culture is 29 ℃ of temperature, illumination 1500Lx, illumination in 16 hours; 8 hours dark; The chopper frequency of intermittently cultivating be per 3 hours once, incubation time is 3 minutes, cultivates 30 days.
3.3, culture of rootage: after treating that enrichment culture finishes, change root media, the prescription of sugarcane root media does; MS+6-BA0.01mg/L+NAA2.0mg/L+ active carbon 50mg/L+20g/L white granulated sugar, the medium pH value is 6.2, condition of culture is 29 ℃ of temperature; Illumination 1500Lx, illumination in 16 hours, 8 hours dark; The chopper frequency of intermittently cultivating be per 6 hours once, incubation time is 3 minutes, cultivates 20 days.
3.4, refining seedling, transplanting: culture of rootage finishes; Can transplant after in refining seedling booth, putting 3～5 days, with clear water wash clean root, beginning to transplant is with husky training during transplanting; Lid plastic film 5 days; Take off the special compound fertilizer for sugar cane that jede Woche behind the film sprays a time 0.2%, transplant to nutritive cube after 40 days, in nutritive cube, grow seedlings and to transplant to the land for growing field crops about 30 days.
Claims (1)
1. method of utilizing intermittent immersion bioreactor to carry out tissue culturing and quick propagation of sugarcane is characterized in that:
1) material selection:
Select for use the 5th generation that the sugarcane stem apex detoxify cultivates or the 6th generation tissue cultivating seedling be material, said sugarcane is meant new platform sugar 22, new platform sugar 16 and these three sugar cane breeds of Badila;
2) cut material, inoculation:
Choose the tissue cultivating seedling sturdy, that base portion is clean as inoculation material; Lao Ye tissue cultivating seedling when cutting material peels off; Stay young leaves 1～2cm; Removed fully by the blackout of phenol infringement or the blade of flavescence, but can not destroy the growing point of tissue cultivating seedling and the bud point of base portion, inoculum density is 70ml～100ml/ strain;
3) enrichment culture:
The single sugarcane tissue-culture seedling that cuts is transferred to carries out enrichment culture in the intermittent immersion bioreactor, being inoculated in prescription and for MS+6-BA0.6mg/L+NAA0.1mg/L+20g/L white granulated sugar, pH value being in 6.2 the liquid nutrient medium, condition of culture is 29 ℃ of temperature; Illumination 1500Lx; Illumination in 16 hours, 8 hours dark, the chopper frequency of intermittently cultivating be per 3 hours once; Incubation time is 3 minutes, continuous culture 30 days;
4) culture of rootage:
After treating that enrichment culture finishes, change root media, the prescription of sugarcane root media does; MS+6-BA0.01mg/L+NAA2.0mg/L+ active carbon 50mg/L+20g/L white granulated sugar, the medium pH value is 6.2, condition of culture is 29 ℃ of temperature; Illumination 1500Lx; Illumination in 16 hours, 8 hours dark, the chopper frequency of intermittently cultivating be per 6 hours once; Incubation time is 3 minutes, continuous culture 20 days;
5) take root seedling domestication and transplanting:
The end of taking root; Transplant after in refining seedling booth, putting 3～5 days, with clear water wash clean root, beginning to transplant is with husky training during transplanting; Lid plastic film 5 days; Take off the special compound fertilizer for sugar cane that jede Woche behind the film sprays a time 0.2%, transplant to nutritive cube after 40 days, in nutritive cube, grow seedlings and transplant to the land for growing field crops about one month.
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| CN102613081B (en) * | 2012-04-01 | 2013-09-18 | 广州甘蔗糖业研究所 | Device and method for rapidly propagating sugarcane tissue-culture seedlings by changing culture solution in system |
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| CN103120126B (en) * | 2013-02-04 | 2014-05-07 | 南京工业大学 | Method for carrying out anoectochilus formosanus tissue culture propagation by using intermittent immersion bioreactor |
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