CN100998315B - Direct sometic embryogenesis of sea-buckthorn, and regeneration of sea-buckthorn plants - Google Patents
Direct sometic embryogenesis of sea-buckthorn, and regeneration of sea-buckthorn plants Download PDFInfo
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- CN100998315B CN100998315B CN200610000196XA CN200610000196A CN100998315B CN 100998315 B CN100998315 B CN 100998315B CN 200610000196X A CN200610000196X A CN 200610000196XA CN 200610000196 A CN200610000196 A CN 200610000196A CN 100998315 B CN100998315 B CN 100998315B
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Abstract
A technique for directly generating the somatic embryo of seabuckthorn and regenerating its plant includes such steps as choosing round seeds, disinfecting, culturing in germinating culture medium until euphylis is fully grown, taking hypocotyl, cotyledon and the leaf of test-tube plantlet, and inducing culture in culture medium SH containing agar powder, KT and IAA.
Description
Technical field
The present invention relates to a kind of direct generation and plant regeneration technique of sea-buckthorn somatic embryo, belong to forestry cell engineering breeding field.
Background technology
Sea-buckthorn (Hippophae rhamnoides L.) is Elaeangnaceae (Elaeagnaceae) Hippophne (Hippophae L.) perennial deciduous fruit tree, shrub or a dungarunga.Its fruit, pericarp, blade, bark and fabricated product thereof contain 280 several physiological active substances, have the special efficacy of strong anti-inflammatory, sterilization, pain relieving and promotion regeneration.Wherein many compositions kill and suppress tumour cell, radioresistance, anticoagulation, hypotensive, prevent that aspects such as blood vessel embolism, anti-ageing, antifatigue, enhancing human body vigor and immunity from all demonstrating magical result of treatment.
The sea-buckthorn branches and leaves are luxuriant, the lateral root prosperity, and root turion is extremely strong, and the root of going here and there rapidly forms intensive colony from tiller, and can form a large amount of root nodules with symbiosis such as actinomycetes, bacterium, mycobacteriums, has the nitrogen fixing capacity stronger than soybean (but per hectare fixed nitrogen 180kg).In addition, has growth rapidly, drought-resistant, salt tolerant alkali is barren, drive in freezing winter and in sultry summer, having and conserve water and soil, check winds and fix drifting sand, improve the soil and the adaptability obvious effect that promotes the ecological balance such as strong, be not only the pioneer tree species of dry wind sand ground district's afforestation, also is important firewood forest and fodder forest seeds.So in current conceding the land to forestry, agricultural industry restructuring, the plantation sea-buckthorn has important nutritive value, the ecological value and economic worth.
At present, sea-buckthorn mainly adopt grow directly from seeds, grafting (bud grafting) and cuttage and seedling culture, but also division propagation.But because sea-buckthorn is dioecian plant, the offspring of seminal propagation easily morphs, and the seedling sex is difficult to distinguish, can only be used for the construction of the ecological forest.And carry out vegetative propagation with seedling cuttage, grafting and root turion, and exist that planting percent is low, speed waits shortcoming slowly, be difficult to adapt to the demand of market to nursery stock; Continuous expansion along with the sea-buckthorn cultivated area, for bury having drought resisting, cold-resistant, salt tolerant alkali, desertification in satisfy producing, the wilderness demand of the new lines nursery stock of disease-resistant worm, output height, merit such as big fruit is stingless, the in-vitro propagate technology of sea-buckthorn has just obtained research more deeply in short more than ten years.
At present, be explant about the scholar with cotyledon, hypocotyl, blade, bud (tender shoots and sleeping bud) etc. both at home and abroad, obtained test-tube plantlet by adventitious organogenesis, but series of technical such as the explant brown stain is serious, reproduction coefficient is low, the difficulty of taking root that ubiquity have hindered sea-buckthorn factorial seedling growth and the choiceness large-scale application on producing.And somatic embryo (is called for short the body embryo, SE) has two complete electrode structures, energy primary recycling whole plant; And in heredity, has a relative advantages of higher stability.When the body embryo can also produce secondary embryo repeatedly when condition of culture is suitable, thus organizator embryo clone, and this body embryo generation system provides new approach for the in-vitro propagate of sea-buckthorn.Relevant sea-buckthorn somatic embryo is induced and the research of plant regeneration, yet there are no special report at home and abroad yet up to now.Therefore, hypocotyl, cotyledon and test-tube plantlet blade that the present invention sprouts with hippophae rhamnoides seed are that material has carried out cultured in vitro, the indefinite tip and somatic embryo have been obtained, for the genetic transformation of indefinite organ and choiceness variation screening provide scientific basis and technical support.
Summary of the invention
Main purpose of the present invention is to overcome serious, the series of technical such as reproduction coefficient is low, the difficulty of taking root of explant brown stain in the sea-buckthorn group training process, and a kind of direct generation and plant regeneration method of sea-buckthorn somatic embryo is provided.
Technical scheme of the present invention is: (1) selects full hippophae rhamnoides seed, be placed on after the sterilization on the germination medium, and be that 25 ± 2 ℃, intensity of illumination are 1500～2000lx in temperature, the photoperiod is to cultivate under the condition of 18h/d.The germination medium mainly comprises 6.5g/L agar powder and 30g/L white granulated sugar, regulates pH value to 5.8; (2) treat that true leaf grows fully after, clip hypocotyl, cotyledon and test-tube plantlet blade carry out indefinite bud and somatic embryo inducing culture, minimal medium is SH (Schenk and Hildebrandt, be called for short a SH) medium, mainly comprises following composition: NH
4H
2PO
4300mg/L, KNO
32500mg/L, CaCl
22H
2O 200mg/L, MgSO
47H
2O 400mg/L, KI 1mg/L, H
3BO
35.0mg/L, MnSO
4H
2O 7.6mg/L, ZnSO
47H
2O 1mg/L, Na
2MoO
42H
2O 0.1mg/L, CuSO
45H
2O 0.2mg/L, CoCl
26H
2O 0.1mg/L, FeSO
47H
2O 15mg/L, Na
2-EDTA 20mg/L, inositol 1000mg/L, nicotinic acid 5.0mg/L, thiamine hydrochloride 5.0mg/L, puridoxine hydrochloride 0.5mg/L; Additional plant hormone is: the heteroauxin (indole-3-acetic acid is called for short IAA) of the kinetin of 0.5～1.0mg/L (kinetin is called for short KT) and 0.2～0.5mg/L; The somatic embryo inducing culture also comprises 6.5g/L agar powder and 20～30g/L white granulated sugar, and the pH value is 5.8; Cultivation temperature is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx, and the photoperiod is 18h/d.Statistics blade body cell stage inductivity, blade body cell stage rooting rate, cotyledon somatic embryo inductivity, cotyledon somatic embryo rooting rate, hypocotyl somatic embryo inductivity, hypocotyl somatic embryo rooting rate when being cultured to 50 days.
The invention has the beneficial effects as follows series of technical such as having solved in the sea-buckthorn group training process reproduction coefficient is low, the difficulty of taking root, by plant hormone and carbon nutrition concentration in the control agent cell stage inducing culture, hypocotyl, cotyledon and test-tube plantlet blade that hippophae rhamnoides seed is sprouted carry out cultured in vitro, the indefinite tip and somatic embryo have been obtained, for the genetic transformation of the indefinite organ of sea-buckthorn and choiceness variation screening provide scientific basis and technical support.
Embodiment
Selecting full hippophae rhamnoides seed, be placed on after the sterilization on the germination medium, is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx in temperature, and the photoperiod is to cultivate under the condition of 18h/d; The germination medium mainly comprises 6.5g/L agar powder and 30g/L white granulated sugar, regulates pH value to 5.8.After treating that true leaf grows fully, clip hypocotyl, cotyledon and test-tube plantlet blade carry out indefinite bud and somatic embryo inducing culture, and minimal medium is the SH medium; Additional plant hormone is: KT0.5～1.0mg/L and IAA0.2～0.5mg/L; The somatic embryo inducing culture also comprises agar powder 6.5g/L and white granulated sugar 30g/L, and the pH value is 5.8; Cultivation temperature is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx, and the photoperiod is 18h/d.Statistics blade body cell stage inductivity, blade body cell stage rooting rate, cotyledon somatic embryo inductivity, cotyledon somatic embryo rooting rate, hypocotyl somatic embryo inductivity, hypocotyl somatic embryo rooting rate when being cultured to 50 days.
Embodiment one:
Selecting full hippophae rhamnoides seed, be placed on after the sterilization on the germination medium, is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx in temperature, and the photoperiod is to cultivate under the condition of 18h/d.The germination medium mainly comprises 6.5g/L agar powder and 30g/L white granulated sugar, regulates pH value to 5.8.After treating that true leaf grows fully, clip hypocotyl, cotyledon and test-tube plantlet blade carry out indefinite bud and somatic embryo inducing culture, and minimal medium is SH; Additional plant hormone is: KT 0.5mg/L and IAA 0.2mg/L; The somatic embryo inducing culture also comprises 6.5g/L agar powder and 20g/L white granulated sugar, and the pH value is 5.8; Cultivation temperature is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx, and the photoperiod is 18h/d.The result shows that blade body cell stage inductivity reaches 27.6%, blade body cell stage rooting rate reaches 28.3%, cotyledon somatic embryo inductivity reaches 27.9%, cotyledon somatic embryo rooting rate reaches 35.2%, hypocotyl somatic embryo inductivity reaches 41.2%, hypocotyl somatic embryo rooting rate reaches 62.3%.
Embodiment two:
Selecting full hippophae rhamnoides seed, be placed on after the sterilization on the germination medium, is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx in temperature, and the photoperiod is to cultivate under the condition of 18h/d.The germination medium mainly comprises 6.5g/L agar powder and 30g/L white granulated sugar, regulates pH value to 5.8; (2) treat that true leaf grows fully after, clip hypocotyl, cotyledon and test-tube plantlet blade carry out indefinite bud and somatic embryo inducing culture, minimal medium is SH; Additional plant hormone is: KT 0.7mg/L and IAA 0.3mg/L; The somatic embryo inducing culture also comprises 6.5g/L agar powder and 25g/L white granulated sugar, and the pH value is 5.8; Cultivation temperature is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx, and the photoperiod is 18h/d.The result shows that blade body cell stage inductivity reaches 28.5%, blade body cell stage rooting rate reaches 30.4%, cotyledon somatic embryo inductivity reaches 32.7%, cotyledon somatic embryo rooting rate reaches 38.8%, hypocotyl somatic embryo inductivity reaches 43.1%, hypocotyl somatic embryo rooting rate reaches 65.1%.
Embodiment three:
Selecting full hippophae rhamnoides seed, be placed on after the sterilization on the germination medium, is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx in temperature, and the photoperiod is to cultivate under the condition of 18h/d.The germination medium mainly comprises 6.5g/L agar powder and 30g/L white granulated sugar, regulates pH value to 5.8; (2) treat that true leaf grows fully after, clip hypocotyl, cotyledon and test-tube plantlet blade carry out indefinite bud and somatic embryo inducing culture, minimal medium is SH; Additional plant hormone is: KT 0.9mg/L and IAA 0.4mg/L; The somatic embryo inducing culture also comprises 6.5g/L agar powder and 28g/L white granulated sugar, and the pH value is 5.8; Cultivation temperature is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx, and the photoperiod is 18h/d.The result shows that blade body cell stage inductivity reaches 28.8%, blade body cell stage rooting rate reaches 32.1%, cotyledon somatic embryo inductivity reaches 37.2%, cotyledon somatic embryo rooting rate reaches 40.0%, hypocotyl somatic embryo inductivity reaches 46.8%, hypocotyl somatic embryo rooting rate reaches 67.8%.
Embodiment four:
Selecting full hippophae rhamnoides seed, be placed on after the sterilization on the germination medium, is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx in temperature, and the photoperiod is to cultivate under the condition of 18h/d.The germination medium mainly comprises 6.5g/L agar powder and 30g/L white granulated sugar, regulates pH value to 5.8; (2) treat that true leaf grows fully after, clip hypocotyl, cotyledon and test-tube plantlet blade carry out indefinite bud and somatic embryo inducing culture, minimal medium is SH; The somatic embryo inducing culture also comprises 6.5g/L agar powder and 30g/L white granulated sugar, and the pH value is 5.8; Cultivation temperature is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx, and the photoperiod is 18h/d.The result shows that blade body cell stage inductivity reaches 29.1%, blade body cell stage rooting rate reaches 32.4%, cotyledon somatic embryo inductivity reaches 37.7%, cotyledon somatic embryo rooting rate reaches 41.0%, hypocotyl somatic embryo inductivity reaches 47.1%, hypocotyl somatic embryo rooting rate reaches 68.9%.
The above results shows, by plant hormone and carbon nutrition concentration in the control agent cell stage inducing culture, hypocotyl, cotyledon and test-tube plantlet blade that Chinese hippophae rhamnoides seed is sprouted carry out cultured in vitro, the indefinite tip and somatic embryo have been obtained, series of technical such as the low and difficulty of taking root of explant reproduction coefficient have been solved in the sea-buckthorn group training process, for the genetic transformation of the indefinite organ of sea-buckthorn and choiceness variation screening provide scientific basis and technical support, significant.
Claims (2)
1. the direct generation and the plant regeneration method of a sea-buckthorn somatic embryo the steps include:
(1) selecting full hippophae rhamnoides seed, be placed on after the sterilization on the germination medium, is that 25 ± 2 ℃, intensity of illumination are 1500～2000lx, photoperiod to be to cultivate under the condition of 18h/d in temperature; The germination medium mainly comprises 6.5g/L agar powder and 30g/L white granulated sugar, regulates pH value to 5.8;
(2) treat that true leaf grows fully after, clip hypocotyl, cotyledon and test-tube plantlet blade carry out indefinite bud and somatic embryo inducing culture, minimal medium is SH medium: NH in the inducing culture
4H
2PO
4300mg/L, KNO
32500mg/L, CaCl
22H
2O 200mg/L, MgSO
47H
2O 400mg/L, KI 1mg/L, H
3BO
35.0mg/L, MnSO
4H
2O7.6mg/L, ZnSO
47H
2O 1mg/L, Na
2MoO
42H
2O 0.1mg/L, CuSO
45H
2O 0.2mg/L, CoCl
26H
2O 0.1mg/L, FeSO
47H
2O 15mg/L, Na
2-EDTA 20mg/L, inositol 1000mg/L, nicotinic acid 5.0mg/L, thiamine hydrochloride 5.0mg/L, puridoxine hydrochloride 0.5mg/L, 6.5g/L agar powder and 20～30g/L white granulated sugar; Additional plant hormone is the kinetin of 0.5～1.0mg/L and the heteroauxin of 0.2～0.5mg/L; Regulating the pH value is 5.8.
2. the direct generation and the plant regeneration method of sea-buckthorn somatic embryo according to claim 1 is characterized in that: the somatic embryo inducing culture temperature in the step (2) is 25 ± 2 ℃, and intensity of illumination is 1500～2000lx, and the photoperiod is 18h/d.
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| CN200610000196XA CN100998315B (en) | 2006-01-09 | 2006-01-09 | Direct sometic embryogenesis of sea-buckthorn, and regeneration of sea-buckthorn plants |
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|---|---|---|---|
| CN200610000196XA CN100998315B (en) | 2006-01-09 | 2006-01-09 | Direct sometic embryogenesis of sea-buckthorn, and regeneration of sea-buckthorn plants |
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| CN100998315B true CN100998315B (en) | 2011-06-01 |
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| CN107155891A (en) * | 2017-06-13 | 2017-09-15 | 新疆阿勒泰地区园林场 | A kind of abundant kind big fruit sea-buckthorn stem tip tissue culture based formulas of the Liao Dynasty |
| CN107041311A (en) * | 2017-06-13 | 2017-08-15 | 新疆阿勒泰地区园林场 | A kind of sea-buckthorn blade method for tissue culture |
| CN108617297A (en) * | 2018-04-28 | 2018-10-09 | 新疆阿勒泰地区林业工作管理站 | The cuttage transplantation method of sea-buckthorn tissue-cultured seedling |
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Non-Patent Citations (4)
| Title |
|---|
| 孙兰英等.沙棘组织培养培养基筛选试验.沙棘11 3.1998,11(3),14-16. |
| 孙兰英等.沙棘组织培养培养基筛选试验.沙棘11 3.1998,11(3),14-16. * |
| 徐虹等.沙棘组织培养技术研究现状及存在问题.沙棘12 1.1999,12(1),11-13. |
| 徐虹等.沙棘组织培养技术研究现状及存在问题.沙棘12 1.1999,12(1),11-13. * |
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