CN102318558A - Rapid propagation method for improving tissue culture seedling quality of Agave sisalana perrine - Google Patents
Rapid propagation method for improving tissue culture seedling quality of Agave sisalana perrine Download PDFInfo
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Abstract
The invention relates to a rapid propagation method for improving tissue culture seedling quality of Agave sisalana perrine. The method comprises the steps of: (1) explant treatment and disinfection; (2) initiation and inoculation; (3) induction of adventitious bud cultivation; (4) induction of clumpy bud cultivation; (5) multiplication of subculture; (6) rooting culture; (7) seedling adaptation; (8) temporary planting and transplanting; (9) bagged dispersal breeding seedling cultivation. The method of the invention adopts SH as a basic medium and more active cytokinin TDZ (thidiazuron), in cooperation with other cultivation conditions. The method provided in the invention also employs a different tissue culture seedling cutting method and changes the original seedling culture method of Agave sisalana perrine, so that the seedling culture time can be saved by 6 months and the problems like variety degeneration and sickness of Agave sisalana perrine can be solved effectively, thus effectively guaranteeing seedling quality and promoting health development of the Agave sisalana perrine industry.
Description
Technical field
The present invention relates to a kind of sisal hemp group and cultivate the method for seedling, be specifically related to a kind of sisal hemp group that improves and cultivate the quick-breeding method of seedling quality and belong to the tissue cultivating technical field.
Background technology
Sisal hemp (
Agave sisalana perrine) the tongue the orchid family of being born in the year of dragon (
Agavaceae) Agave (
Agave), perennial unifacial leaf draft hard fibre crop.The sisal hemp growth cycle reached more than 12 years, and economic worth is high, and comprehensive purposes is wide, was maximum, the widest in area a kind of hard fibre of world today's consumption.It is strong that it has a pulling force; Sea water resistance soaks, rub resistance, high resilience, characteristic such as be difficult for skidding, and is that synthetic fibers are irreplaceable, can be made into required rope such as fishing boat industrial and mineral, transportation, the cord of vehicle tyre; Can be woven into gunnysack, wallpaper cord, carpet, batts again; Also can mix being pressed into hardboard with plastics, make fibered plaster hawk, cement tile, can be made into fine paper with cement.Especially sisal fiber has characteristics such as free from environmental pollution, difficult generation static, is widely used in making life and environmental supplies.And besides sisal fiber outer leaves about 95 percent of sisal hemp byproduct slag juice enormous potential that can be developed from sisal sisal residue extracted suds prime product, 8-9 ten thousand U.S. dollars per tonne in the international market, from ticogenin extracted again in single and double pregnenolone pregnenolone products can be formulated more than 100 kinds of medicines, the current price per ton up to 120-130 yuan.Sisal hemp is extremely valuable tropical crop resource as the economic crops of China's torrid zone characteristic, and the strong industry of wide development prospect of tool and huge export capacity.
Along with the fast development of sisal hemp industry, healthy sisal hemp seedling quality is required growing area and demand also improves day by day and increases.In sisal hemp seedling production process in the past, the conventional seedbed system technology of taking has bulbil breeding, unbearable method and unbearable stripping leaf method.Conventional seedbed system exists subject matter to have: the cultivation time is long, the provenance supply is restricted, kind of a sexual involution can occur, and very different, output reduces, and of poor quality, disease is many, and is prone to cause undesirable elements such as the seedling disease is popular.The sisal hemp tissue cultivating seedling is short because of possessing growing-seedling period, and reproduction rate is high, emerges can artificially control with growth neat and consistent, condition of culture soon, does not receive weather, seasonal effect, and characteristics such as human and material resources are saved in practicable batch production production.Has the incomparable advantage of conventional seedling.Application sisal hemp tissue cultivating seedling is imperative, and this also is to solve one of problems that conventional seedling plantation brings and best approach of drawback.
As far back as the later stage seventies; South China hotwork research institute, Fujian hotwork institute and the Dongfanghong institute of agricultural sciences adopt sisal leaves, axillalry bud and sexual hybridization seed to come evoked callus to differentiate whole plant more respectively; Obtained a small amount of sisal hemp tissue cultivating seedling; But the stage survival rate is low heeling in, and field planting is not succeeded.Subsequently two during the last ten years; Guangxi subtropical crop research institute, Tropical Crop Variety Resource Institute of Chinese Academy of Tropical Agricultural Sciences, Southern Asia Institute of Tropical Crop, Chinese Academy of Tropical Agricultural Science have also carried out the research of sisal hemp tissue culture technique; Reported respectively and utilized sisal hemp stem apex and blade evoked callus to cultivate whole plant, but all failed application on producing.
Summary of the invention
The purpose of this invention is to provide a kind of quick-breeding method that the sisal hemp group is cultivated the seedling quality that improves; The present invention is through a large amount of tests and research; Have aspect applying that bottle seedling vitrification phenomenon is serious, pollution rate is higher, reproduction coefficient is low and heeling in realistic situations such as transplanting survival rate is low to the production of present sisal hemp seedling; A kind of method that the sisal hemp group is cultivated the fast breeding technique of seedling quality that improves being provided, it is characterized in that adopting the numerous bud approach of bud, is minimal medium with SH; Adopted active stronger basic element of cell division TDZ; Cooperate other condition of culture that is fit to, and adopted different tissue cultivating seedling cutting methods, seedling plantation specification changes field planting on 1～2 kilogram of bag seedling into by 4～6 kilograms.
In order to address the above problem, the technical scheme that the present invention adopted is:
A kind ofly improve the quick-breeding method that sisal hemp group is cultivated the seedling quality, may further comprise the steps:
(1) processing of explant and sterilization
Bulbil with preferred sisal hemp is an explant material, chooses canopy and plants the high bulbil of 20～30cm, carries out the sterilization of explant; Excision snippings and the old stem of a part are rinsed well with running water earlier, remove outer blade with the knife blade band girdle; 2～3 of the lobus cardiacuses of the long 0.5cm of reservation; Stem keeps 1～1.5cm, rinse well and slightly airing be placed on UV-irradiation sterilization 20min in the desinfection chamber, then material is used 75% alcohol disinfecting 2min respectively; With 0.1% mercuric chloride sterilization 15min, use aseptic water washing again 5～6 times;
(2) start and inoculation
Medium preparation back branch installs in the blake bottle, places in the HTHP power autoclave, and in 121 ℃, the strict down sterilization 20min of 0.1～0.15MPa supplies inoculation to use after the sterilization, in aseptic artificial infection chamber, be 4 with the material rip cutting of handling well, is connected in the medium;
(3) evoking adventive bud is cultivated
The medium that is made into phenylurea derivative TDZ 1.0mg/L, 1-naphthoic acid NAA0.1mg/L, sucrose 5% and the agar 0.6% of SH medium, synthetic is as medium; At 28 ± 2 ℃; Illumination 10～12h/d; Cultivate under the condition of intensity of illumination 1800 ~ 2000Lx, induce the generation indefinite bud after 20 ~ 25 days;
(4) the induced bundle bud is cultivated
After turning out indefinite bud; Stay 1cm stem and rip cutting to be 2 and be inoculated in the medium that SH, TDZ0.5mg/L, NAA0.05mg/L, sucrose 5% and agar 0.6% is made into, cultivation temperature is 27 ± 1 ℃, illumination 12h/d; Intensity of illumination 1800 ~ 2000Lx cultivates to induce after a week and produces the clump bud;
(5) shoot proliferation is cultivated
Induce through the cutting of constantly carrying out indefinite simple bud to produce the bud of growing thickly, obtain to grow thickly in a large number bud, reach shoot proliferation about 8 days;
(6) culture of rootage
The selection above bud seedling of height 3cm in the bud of growing thickly; The bud of will growing thickly separates into simple bud and is inoculated into the medium that SH, NAA1.0mg/L, IBA2.0mg/L, active carbon 0.1% and sucrose 3% is made into and carries out root induction; Cultivation temperature is 28 ± 2 ℃, illumination 10～12h/d, intensity of illumination 3000Lx; Cultivated 10-15 days, and cultivated and induce whole plant;
(7) refining seedling
The sisal hemp tissue cultivating seedling that culture of rootage is obtained places under the natural light environment, bright and clear and do not have the place of direct light, made change temperature hardening 10～15 days;
(8) heel in transplanting
With tempering good sisal hemp tissue cultivating seedling, with the medium of the clean shoot root portion of clear water, then sterilizing for 30 seconds in the potassium permanganate liquid of the root of seedling immersion 0.1%; Again seed is planted in the seedling culture hole plate matrix; A young plant is planted in hole, a cave, puts on the seedbed planting good cave dish seedling, and the transplanting of bottle seedling must be carried out in the shading seedling growth greenhouse; At 22 ℃～27 ℃, require humidity than suitable growth temperature below 70%;
(9) packed thin planting seedling is cultivated
The sisal hemp tissue cultivating seedling just carries out the cultivation of thin planting seedling through heeling in to transplant when reaching height of seedling 20cm, cultivates 5～6 months, and seedling reaches field planting on the thin planting seedling that bag seedling that 20～23 of Zhan Ye, high 40～50 ㎝, young plant weigh 1～2kg replaces 4～6kg weight.
The present invention with respect to the beneficial effect of prior art is:
(1) the present invention adopts the numerous bud approach of bud, compares with other tissue culture technology, avoids occurring the higher problem of the strong approach induce variation of callus group rate.
(2) with SH be minimal medium; Adopted active stronger basic element of cell division TDZ; Cooperate other condition of culture that is fit to, and adopted different tissue cultivating seedling cutting methods, the inductivity, clump bud induction rate and the indefinite bud that obtain higher indefinite bud than other tissue culture technology lure the root rate.Basically solved sisal hemp tissue cultivating seedling vitrifying seedling problem.
(3) successfully carried out H.11648 numb tissue cultivating seedling and produced in batches and the demonstration popularization, the tradition rules of growing seedlings have been reformed in test.Seedling plantation specification changes field planting on 1～2 kilogram of bag seedling into by 4～6 kilograms, has saved about 6 months seedling raise period.
(4) result from constituent parts plantation tissue cultivating seedling sees, though consistently think the little fast growth of tissue cultivating seedling seedling in early stage, grows almost consistent with the conventional seedling of 4 kilogram weights through 1～2 year; The group training bag seedling of plantation 1～2 kilogram weight; Seedling raise period is the shortest, saves labor, and saves transport capacity.And can reach the standard of cutting of opening simultaneously, and can effectively solve the sisal hemp deterioration of variety, plant the seedling diseases worm and propagate, the problem that seedling quality can not guarantee etc.Scale, standardization, factorial seedling growth can be accomplished, the realistic problem of seedling lazy weight can be alleviated simultaneously effectively.
(5) a whole set of sisal hemp tissue cultivating seedling cultivation technique measure is summed up in research, makes the application in the land for growing field crops produces as early as possible of sisal hemp tissue cultivating seedling.Ended in 2009-2010; The land-reclaimable Science Institute in Zhanjiang has cultivated the H.11648 numb tissue cultivating seedling of more than 200 ten thousand strains and has carried out transplanting breeding and field demonstration plantation, promoted; Popularizing area reaches 20,000 hectares; Practice thrift about 2,000,000 yuan of cost, increasing makes profits moistens more than 6,000 ten thousand yuan, has obtained better economic and social benefit.Played positive impetus for giving full play to reclamation area, Zhanjiang at the healthy and sustainable development of the advantage aspect the sisal hemp industry, optimization industrial upgrading and promotion sisal hemp industry.
Embodiment
Through embodiment the present invention is explained further details below, these embodiment only are used for explaining the present invention, do not limit the scope of the invention.
Embodiment 1
In March, 2009, we choose canopy and plant the high bulbil of 20～30cm and do explant, and a stem rip cutting is 4, through the tissue culture quick breeding of direct induced bud approach.The evoking adventive bud medium is SH+ TDZ 1.0mg/L+NAA 0.1mg/L+sucrose 5%+ agar 0.6%; After 30 days; Its inductivity is 91.1%; Indefinite simple bud is 2 to stay 1cm stem and rip cutting then, and indefinite bud is in SH+ TDZ0.5mg/L+NAA0.05mg/L+ sucrose 5%+ agar 0.6% medium, and producing the clump bud rate that produces in the indefinite bud after the week is 88.3%.In temperature is 28 ± 1 ℃, illumination 12h/d, and, about intensity of illumination 2000Lx, relative moisture is that the vitrifying rate is 4.5% under about 70% the condition of culture.The bud of growing thickly separates into simple bud and is inoculated into the 3% medium root induction of SH+NAA1.0mg/L+IBA2.0mg/L+ active carbon 0.1%+ sucrose, and about 15 days, its rooting rate reaches 98.3%; The sisal hemp tissue cultivating seedling is transplanted the back and was cultivated 2～3 months through heeling in, height of seedling 15～20cm, and directly bag seedling cultivation more just can reach more than height of seedling 40 ㎝ through 5～6 months, and strain weighs the standard of 1 ㎏, goes out the plantation of going up a hill of Venus farm, garden.
Embodiment 2
In September, 2009, to choose canopy and plant the high bulbil of 20～30cm and do explant, a stem rip cutting is 4, through the approach of direct induced bud, carries out tissue rapid propagation.Its evoking adventive bud medium is SH+ TDZ 1.0mg/L+NAA 0.1mg/L+sucrose 5%+ agar 0.6%; After 30 days; Its inductivity is 93.1%, and indefinite simple bud is 2 to stay 1cm stem and rip cutting then, and indefinite bud is in SH+ TDZ0.5mg/L+NAA0.05mg/L+ sucrose 5%+ agar 0.6% medium; After 7 days, the clump bud rate that in indefinite bud, produces is 89.7%.In temperature is 28 ± 1 ℃, illumination 12h/d,, about intensity of illumination 2000Lx, relative moisture is lower than under 70% the condition of culture, and the vitrifying rate is 2.5%.The bud of growing thickly separates into simple bud and is inoculated into the 3% medium root induction of SH+NAA1.0mg/L+IBA2.0mg/L+ active carbon 0.1%+ sucrose, and about 15 days, its rooting rate reaches 99.4%; The sisal hemp tissue cultivating seedling is transplanted the back and was cultivated 2 months through heeling in, and more than the height of seedling 20cm, directly the bag seedling is cultivated again, just can reach more than height of seedling 38 ㎝ through 5 months, and strain weighs the standard of 1 ㎏, goes out that the garden is eastern to rise the farm plantation of going up a hill.
Embodiment 3
In February, 2010, to choose the high bulbil of 20～30cm and do explant, a stem rip cutting is 4, through the approach of direct induced bud, carries out tissue rapid propagation.Its evoking adventive bud medium is SH+ TDZ 1.0mg/L+NAA 0.1mg/L+sucrose 5%+ agar 0.6%; After 30 days; Its inductivity is 95.5%, and indefinite simple bud is 2 to stay 1cm stem and rip cutting then, and indefinite bud is in SH+ TDZ0.5mg/L+NAA0.05mg/L+ sucrose 5%+ agar 0.6% medium; After 7 days, the clump bud rate that in indefinite bud, produces is 86.2%.In temperature is 28 ± 1 ℃, illumination 12h/d,, about intensity of illumination 2000Lx, relative moisture is lower than under 70% the condition of culture, and the vitrifying rate is 3.5%.The bud of growing thickly separates into simple bud and is inoculated into the 3% medium root induction of SH+NAA1.0mg/L+IBA2.0mg/L+ active carbon 0.1%+ sucrose, and about 15 days, its rooting rate reaches 96.7%; The sisal hemp tissue cultivating seedling is transplanted the back and is cultivated 2 first quarter moons through heeling in, and more than the height of seedling 20cm, directly bag seedling cultivation more just can reach more than height of seedling 40 ㎝ through 6 months, and strain weighs the standard of 1 ㎏, goes out the plantation of going up a hill of Dongfanghong farm, garden.
Embodiment 4
In May, 2010, to choose the high bulbil of 20～30cm and do explant, a stem rip cutting is 4, through the approach of direct induced bud, carries out tissue rapid propagation.Its evoking adventive bud medium is SH+ TDZ 1.0mg/L+NAA 0.1mg/L+sucrose 5%+ agar 0.6%; After 30 days; Its inductivity is 98.5%, and indefinite simple bud is 2 to stay 1cm stem and rip cutting then, and indefinite bud is in SH+ TDZ0.5mg/L+NAA0.05mg/L+ sucrose 5%+ agar 0.6% medium; After 7 days, the clump bud rate that in indefinite bud, produces is 90.2%.In temperature is 28 ± 1 ℃, illumination 12h/d,, about intensity of illumination 2000Lx, relative moisture is lower than under 70% the condition of culture, and the vitrifying rate is 4.9%.The bud of growing thickly separates into simple bud and is inoculated into the 3% medium root induction of SH+NAA1.0mg/L+IBA2.0mg/L+ active carbon 0.1%+ sucrose, and after 15 days, its rooting rate reaches 92.7%; The sisal hemp tissue cultivating seedling is transplanted the back and was cultivated 2 months through heeling in, and more than the height of seedling 17cm, directly bag seedling cultivation more just can reach more than height of seedling 40 ㎝ through 6 months, and strain weighs the standard of 1 ㎏, goes out the plantation of going up a hill of Dongfanghong farm, garden.
Claims (1)
1. one kind is improved the quick-breeding method that the sisal hemp group is cultivated the seedling quality, it is characterized in that: may further comprise the steps:
(1) processing of explant and sterilization
Bulbil with preferred sisal hemp is an explant material, chooses canopy and plants the high bulbil of 20～30cm, carries out the sterilization of explant; Excision snippings and the old stem of a part are rinsed well with running water earlier, remove outer blade with the knife blade band girdle; 2～3 of the lobus cardiacuses of the long 0.5cm of reservation; Stem keeps 1～1.5cm, rinse well and slightly airing be placed on UV-irradiation sterilization 20min in the desinfection chamber, then material is used 75% alcohol disinfecting 2min respectively; With 0.1% mercuric chloride sterilization 15min, use aseptic water washing again 5～6 times;
(2) start and inoculation
Medium preparation back branch installs in the blake bottle, places in the HTHP power autoclave, and in 121 ℃, the strict down sterilization 20min of 0.1～0.15MPa supplies inoculation to use after the sterilization, in aseptic artificial infection chamber, be 4 with the material rip cutting of handling well, is connected in the medium;
(3) evoking adventive bud is cultivated
The medium that is made into phenylurea derivative TDZ1.0mg/L, 1-naphthoic acid NAA0.1mg/L, sucrose 5% and the agar 0.6% of SH medium, synthetic is as medium; At 28 ± 2 ℃; Illumination 10～12h/d; Cultivate under the condition of intensity of illumination 1800 ~ 2000Lx, induce the generation indefinite bud after 20 ~ 25 days;
(4) the induced bundle bud is cultivated
After turning out indefinite bud; Stay 1cm stem and rip cutting to be 2 and be inoculated in the medium that SH, TDZ0.5mg/L, NAA0.05mg/L, sucrose 5% and agar 0.6% is made into, cultivation temperature is 27 ± 1 ℃, illumination 12h/d; Intensity of illumination 1800 ~ 2000Lx cultivates to induce after a week and produces the clump bud;
(5) shoot proliferation is cultivated
Induce through the cutting of constantly carrying out indefinite simple bud to produce the bud of growing thickly, obtain to grow thickly in a large number bud, reach shoot proliferation about 8 days;
(6) culture of rootage
The selection above bud seedling of height 3cm in the bud of growing thickly; The bud of will growing thickly separates into simple bud and is inoculated into the medium that SH, NAA1.0mg/L, IBA2.0mg/L, active carbon 0.1% and sucrose 3% is made into and carries out root induction; Cultivation temperature is 28 ± 2 ℃, illumination 10～12h/d, intensity of illumination 3000Lx; Cultivated 10-15 days, and cultivated and induce whole plant;
(7) refining seedling
The sisal hemp tissue cultivating seedling that culture of rootage is obtained places under the natural light environment, bright and clear and do not have the place of direct light, made change temperature hardening 10～15 days;
(8) heel in transplanting
With tempering good sisal hemp tissue cultivating seedling, with the medium of the clean shoot root portion of clear water, then sterilizing for 30 seconds in the potassium permanganate liquid of the root of seedling immersion 0.1%; Again seed is planted in the seedling culture hole plate matrix; A young plant is planted in hole, a cave, puts on the seedbed planting good cave dish seedling, and the transplanting of bottle seedling must be carried out in the shading seedling growth greenhouse; At 22 ℃～27 ℃, require humidity than suitable growth temperature below 70%;
(9) packed thin planting seedling is cultivated
The sisal hemp tissue cultivating seedling just carries out the cultivation of thin planting seedling through heeling in to transplant when reaching height of seedling 20cm, cultivates 5～6 months, and seedling reaches field planting on the thin planting seedling that bag seedling that 20～23 of Zhan Ye, high 40～50 ㎝, young plant weigh 1～2kg replaces 4～6kg weight.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103039370A (en) * | 2013-01-29 | 2013-04-17 | 中国热带农业科学院南亚热带作物研究所 | Method for establishing golden-edge arc-leaf maguey isolated cultivation and regeneration system |
| CN104839027A (en) * | 2015-06-01 | 2015-08-19 | 福建农林大学 | Inducing method for maguey bulblet |
| CN107410033A (en) * | 2017-09-29 | 2017-12-01 | 中国科学院昆明植物研究所 | The rapid propagation method of national spice berry snippings |
| CN108812307A (en) * | 2018-05-24 | 2018-11-16 | 广西壮族自治区亚热带作物研究所 | The narrow quick mating system of leaf American aloe tissue cultures |
-
2011
- 2011-08-12 CN CN 201110230235 patent/CN102318558B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 《Plant Cell, Tissue and Organ Culture》 20021231 S.K. Hazra等 Sisal plant regeneration via organogenesis 235-240 1 , 第70期 * |
| 《安徽农学通报》 20081231 陈鸿等 生物技术在剑麻研究上的应用进展 55-58 1 第14卷, 第15期 * |
| 《热带农业科学》 20081231 陈鸿等 剑麻叶片愈伤组织诱导及再生体系的建立 11-14 1 第28卷, 第6期 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103039370A (en) * | 2013-01-29 | 2013-04-17 | 中国热带农业科学院南亚热带作物研究所 | Method for establishing golden-edge arc-leaf maguey isolated cultivation and regeneration system |
| CN103039370B (en) * | 2013-01-29 | 2014-03-19 | 中国热带农业科学院南亚热带作物研究所 | Method for establishing golden-edge arc-leaf maguey isolated cultivation and regeneration system |
| CN104839027A (en) * | 2015-06-01 | 2015-08-19 | 福建农林大学 | Inducing method for maguey bulblet |
| CN107410033A (en) * | 2017-09-29 | 2017-12-01 | 中国科学院昆明植物研究所 | The rapid propagation method of national spice berry snippings |
| CN108812307A (en) * | 2018-05-24 | 2018-11-16 | 广西壮族自治区亚热带作物研究所 | The narrow quick mating system of leaf American aloe tissue cultures |
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