CN101720668A - Method for tissue culturing and quick propagation of sugarcane with intermittent immersion bioreactor - Google Patents
Method for tissue culturing and quick propagation of sugarcane with intermittent immersion bioreactor Download PDFInfo
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Abstract
The invention provides to a method for tissue culturing and quick propagation of sugarcane with an intermittent immersion bioreactor, which is characterized in that: the method performs propagation culture and rooting culture by transplanting detoxicated sugarcane plants, serving as materials, cultured by generations from induced sprouts of sugarcane stem tip tissues into the intermittent immersion bioreactor. In the intermittent immersion bioreactor, a culture temperature is 29 DEG C; the illumination intensity is 1,500 Lx and the illumination time is 16 hours; an intermittent culture condition is to culture for 3min every 3h and to root for 3min every 6h; the first generation can be propagated to 40 times; after rooting is completed, a proper hardening plantlet treatment is performed; and plantlets are transplanted and the survival rate is about 85 percent. The set of system is high in automation degree, has a much higher multiplication rate than that of a traditional tissue culture method, saves labour cost and increases economic benefits.
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 group 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 planted and is cultivar, and back two kinds is wild species.
The plant of sugarcane is made 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 in the extensively plantation of these areas.The country of world's sugarcane acreage maximum 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 the biological yield height contains sugar 11%～17%, not only can also can directly produce alcohol for sugaring, 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, the illumination abundance 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 tender stem-tip tissue of children as the stem apex cultured in vitro technology of explant in conjunction with thermal treatment, the cane seedling of detoxification 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 application and the benefit prospect of detoxification health seedling in the Guangxi sugarcane production. 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, be 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, but do not improve the subculture number in producing and just can not obtain enough economic interests, 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, labour intensive, 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 height, 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, present process saliency of having produced, 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, easily some seedlings inferior are rendered to market; 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, production cost is than higher reason, 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 by glass tube or silicone tube, and gas is the syringe filters degerming of 0.22 μ m by 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 physiological problem lopsided owing to the long vitrifying that brings of cycle in the liquid culture process and that this injury of giving birth to metabolite brings again.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 taken the lead in introducing to be improved from international sugarcane research center Cuba sugarcane research institute being applied to tissue culturing and quick propagation of sugarcane research by 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 height, 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 height, the amount of labour are 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 no longer switching after the inoculation, 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 the simulating nature environment is grown, and strengthens the quality and the adaptive capacity of tissue cultivating seedling.
The technical solution used in the present invention is:
1, the selection of material 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 problems such as the variation of the too high and generation of algebraically and resistance difference; 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 substantially, 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, the sugarcane tissue-culture seedling that cuts is transferred to carries out enrichment culture in the intermittent immersion bioreactor, 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, intermittently the chopper frequency of 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, intermittently the chopper frequency of cultivating be per 6 hours once, incubation time is 3 minutes, cultivates 20 days.
4, take root seedling domestication and transplanting, the end of taking root, in the hardening booth, put after 3～5 days and can transplant, use clear water wash clean root during transplanting, 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, ensure 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 to take root for finishing, 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, process propagation and culture of rootage can obtain the tissue cultivating seedling about 500 strains, improved 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 height is saved labour cost, is increased 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, the Lao Ye of tissue cultivating seedling is peeled off as far as possible, young leaves is done a little excision, stay young leaves 1～2cm, the blackout of being encroached on by phenol or the blade of flavescence are also removed fully, 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, intermittently the chopper frequency of 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 is; 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, intermittently the chopper frequency of cultivating be per 6 hours once, incubation time is 3 minutes, cultivates 20 days.
3.4, hardening, transplanting: culture of rootage finishes, in the hardening booth, put after 3～5 days and can transplant, use clear water wash clean root during transplanting, begin to transplant is with husky training, 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. a method of utilizing intermittent immersion bioreactor to carry out tissue culturing and quick propagation of sugarcane the steps include:
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;
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, the blackout of being encroached on by phenol or the blade of flavescence are removed fully, 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 being in 6.2 the liquid nutrient medium for MS+6-BA0.6mg/L+NAA0.1mg/L+20g/L white granulated sugar, pH value, condition of culture is 29 ℃ of temperature, illumination 1500Lx, illumination in 16 hours, 8 hours dark, intermittently the chopper frequency of 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 is; 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, intermittently the chopper frequency of 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, in the hardening booth, put after 3～5 days and can transplant, use clear water wash clean root during transplanting, begin to transplant is with husky training, 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 one month.
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| CN108849531A (en) * | 2018-09-06 | 2018-11-23 | 河南师范大学 | RHIIZOMA DIOSCOREAE from Henan of China tissue cultures expanding propagation method is carried out using interval submergence bioreactor |
| CN110972944A (en) * | 2019-12-18 | 2020-04-10 | 南京师范大学 | Method for intermittently immersing dendrobium nobile seedlings |
| CN110972944B (en) * | 2019-12-18 | 2022-12-06 | 南京师范大学 | Method for intermittently immersing dendrobium nobile seedlings |
| WO2021179527A1 (en) * | 2020-03-13 | 2021-09-16 | 广西壮族自治区农业科学院 | Method for culturing pueraria thomsonii by proliferation of single-bud stem segment and one-step seedling formation |
| CN112075299A (en) * | 2020-09-23 | 2020-12-15 | 广东省农业科学院果树研究所 | Banana hemp No. 1 transplanting method |
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