CN105165618B - The method that Picea Mongolica somatic embryo occurs - Google Patents
The method that Picea Mongolica somatic embryo occurs Download PDFInfo
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Abstract
A kind of method that the present invention occurs for Picea Mongolica somatic embryo, belongs to field of plant cultivation seedling technology, is concretely the method given birth to using Picea Mongolica mature embryo somatic embryos fetal hair.The present invention uses full ripe Picea Mongolica seed, mature embryo is stripped for explant material, it is inoculated on calli induction media and cultivates, twice embryo callus subculture is produced after subculture, above-mentioned callus propagation is expanded into numerous purpose so as to reach, somatic embryo is produced by embryo callus subculture differentiation again, somatic embryo is normally sprouted after drying and other treatment：It is an advantage of the invention that being given birth to using Picea Mongolica mature embryo somatic embryos fetal hair of the present invention, substantial amounts of Picea Mongolica somatic embryo is obtained, the seasonal bottleneck produced using rataria as explant has been broken away from, has realized the quick breeding of Picea Mongolica.
Description
Technical field
The invention belongs to field of plant cultivation, concretely, it is related to the one of Picea Mongolica mature embryo somatic embryo generation
The method of kind.
Background technology
Picea Mongolica（Picea mongolica）It is distributed across China of the domestic white tone pile of stones nature reserve area in the Inner Mongol
Peculiar novel species, belongs to rare plant.Picea Mongolica, as a unique afforestation in sandy land seeds, is that energy is set up in Desert system
The sustainable systematic protection woods engineering for enough maintaining sand ground and developing to Oasis Process direction provides a new thinking.Therefore, grind
Study carefully Picea Mongolica fast numerous with great economic implications and ecological significance；Due to Picea Mongolica seed production be vulnerable to biennial bearing,
The insect pest influence such as cold spell in later spring and weight tooth bark beetle, generative propagation is hindered, and carries out somatic embryo using mature embryo and have
There are many merits, such as quantity is more, reproduction speed fast, not by seasonal effect, shoot regeneration frequency height, as plant vegetative propagation
A kind of Main Means.Developing all in the preservation, the numerous and production practices of expansion to Picea Mongolica germ plasm resource has important
Meaning.
The content of the invention
The object of the invention is in order to solve to realize the fast numerous problem of Picea Mongolica with the Development of Somatic Embryogenesis, with sand ground cloud
China fir mature embryo is explant, using LM as the 6-BA of the additional various concentrations of minimal medium, 2, and 4-D, KT, ABA, PEG4000 are lured
Lead embryo callus, embryo callus propagation, embryonic differentiation, drying and other treatment and plant regeneration, and provide a kind of induction,
Method and condition of culture when subculture and seedling, with realize plant regeneration with it is fast numerous.
The object of the invention is realized and completed by following technical scheme：
The present invention includes following production stage：
（1）Ripe Picea Mongolica seed is soaked into 12~15h with clear water, the seed for floating on liquid level is given up, it is remaining
Seed with running water rinse 3~4 times, in superclean bench, by dragon spruce seed first with 70~75% alcohol-pickled 28~32s
Afterwards with sterile water wash 3~5 times, then with 2% liquor natrii hypochloritis sterilization 12min, then with aseptic water washing 3~5 times；
（2）Will be through step（1）Treated seed is stripped with scalpel after complete embryo, is inoculated in following media
In：LM culture mediums+6-BA2~5mg/L+2,4-D4~8mg/L+KT0.5~2.0mg/L+ sucrose 30g/L+ gellan gums 2g/L, pH
Control to be 22 ± 2 DEG C, carry out 20~25d of Fiber differentiation generation callus under dark condition in 5.6~5.8, temperature；
（3）By step（2）Callus be inoculated in following culture medium：1/2~1/4LM culture mediums+6-BA1~4mg/
L+2,4-D1~5mg/L+ KT0.1~1.0mg/L+ sucrose 20~30g/L+ gellan gums 2~4g/L, pH control 5.6~5.8,
Temperature, which is 22 ± 2 DEG C, 20~25d of squamous subculture is carried out under dark condition makes callus further growth；
（4）By step（3）Callus be inoculated in following media：1/2~1/4LM culture mediums+6-BA1.0~
1.9mg/L+2,4-D1.0~1.9mg/L+ KT0.1~0.5mg/L+ sucrose 20~30g/L+ gellan gums 2~4g/L, pH control
It is 22 ± 2 DEG C, 20~25d of squamous subculture is carried out under dark condition in 5.6~5.8, temperature, obtains the non-embryo of faint yellow tight structure
Property callus and the thread embryo callus subculture of white translucent；
（5）By step（4）Embryo callus be inoculated in following media：1/2~1/4LM culture mediums+6-BA0.1
~1.0mg/L+2,4-D1.0~3.0mg/L+ sucrose 20~30g/L+ gellan gums 2~4g/L, pH control are in 5.6~5.8, temperature
To carry out Multiplying culture under 22 ± 2 DEG C, dark condition, bred every 20~25d with same recipe squamous subculture；
（6）By step（5）Embryo callus is inoculated in following media：1/2~1/4LM culture mediums+ABA15~
2~4g/L of 20mg/L+PEG400040~50g/L+ sucrose 20~30g/L+ gellan gums, it is 22 that pH, which is controlled in 5.6~5.8, temperature,
± 2 DEG C, differentiation culture is carried out under dark condition, carry out growing dragon spruce after squamous subculture, 40~60d with same recipe every 20d
Somatic embryo；
（7）By step（6）Somatic embryo is inoculated in 1/2~1/4LM culture mediums+3~7g/ of sucrose 20~40g/L+ gellan gums
L+PEG400050~80g/L is cultivated on more than 7d, the filter paper that body embryo is laid in two layers of moistening, is placed on a sterile petri dish
Middle drying and other treatment 7 days, each culture dish places about 150 individual cells embryos, and above procedure is 22 ± 2 DEG C, illumination in cultivation temperature
Condition is that 1200Lx, light application time are to carry out reaching desiccation purpose under conditions of 14h/d；
（8）By step（7）The somatic embryo of described completion desiccation be inoculated in 1/4~1/2LM culture mediums+sucrose 20~
It can be sprouted after 30g/L+ agar 5~8g/L, 30d and grow up to the healthy and strong Picea asperata seedlingss with root.
It is an advantage of the invention that using Picea Mongolica mature embryo as explant, using LM as minimal medium, additional various concentrations
6-BA, 2,4-D, KT, ABA, PEG4000 come carry out embryonic callus induction, embryo callus propagation, embryonic differentiation,
Drying and other treatment and plant regeneration, and provide it is a kind of induce, subculture and method and condition of culture during seedling, to realize plant again
It is raw with it is numerous soon.
Specific embodiment：
Embodiment 1
（1）Ripe Picea Mongolica seed is soaked into 12h with clear water, the seed for floating on liquid level given up, remaining kind
Son is rinsed three times with running water.In superclean bench, by dragon spruce seed first with after 75% alcohol-pickled 30s use sterile water wash 3
It is secondary, then 12min is sterilized with 2% liquor natrii hypochloritis, then with aseptic water washing 5 times；
（2）Will be through step（1）Treated seed is stripped with scalpel after complete embryo, is inoculated in following media
In：LM culture mediums+6-BA3mg/L+2,4-D6mg/L+KT1mg/L+ sucrose 30g/L+ gellan gums 2g/L, pH control are in 5.7, temperature
Spend to carry out Fiber differentiation 20d generation callus under 22 ± 2 DEG C, dark condition；
（3）By step（2）Described callus is inoculated in following culture medium：1/2LM culture mediums+6-BA（2mg/L）+
2,4-D（3mg/L）+KT（0.2mg/L）+ sucrose（20g/L）+ gellan gum（2g/L）, pH control 5.7, temperature be 22 ± 2 DEG C,
Squamous subculture 20d is carried out under dark condition makes callus further growth；
（4）By step（3）Described callus is inoculated in following media：1/2LM culture mediums+6-BA1.1mg/L+
2,4-D1.6mg/L+ KT0.2mg/L+ sucrose 20g/L+ gellan gums 2g/L, it is 22 ± 2 DEG C, dark condition that pH, which is controlled in 5.7, temperature,
Lower progress squamous subculture 20d, the non embryogenic callus embryo thread with white translucent for obtaining faint yellow tight structure is cured
Wound；
（5）By step（4）Described embryo callus is inoculated in following media：1/2LM culture mediums+6-
BA0.8mg/L+2,4-D1.5mg/L+sucrose 20g/L+ gellan gums 2g/L, it is 22 ± 2 DEG C, dark condition that pH, which is controlled in 5.8, temperature,
Lower carry out Multiplying culture, is bred every 20d with same recipe squamous subculture；
（6）By step（5）Described embryo callus is inoculated in following media：1/2LM culture mediums+ABA18mg/
L+PEG400045g/L+ sucrose（30g/L）It is 22 ± 2 DEG C, under dark condition that+gellan gum 4g/L, pH, which are controlled in 5.6~5.8, temperature,
Differentiation culture is carried out, carries out growing dragon spruce somatic embryo after squamous subculture, 50d with same recipe every 20d；
（7）By step（6）Described somatic embryo is inoculated in 1/4LM culture mediums+sucrose 20g/L+ gellan gums 5g/L+
After PEG400070g/L cultures 7d, on the filter paper that body embryo is laid in two layers of moistening, it is placed in a sterile petri dish and handles 7d,
Each culture dish places about 150 individual cells embryos, above procedure cultivation temperature be 22 ± 2 DEG C, illumination condition be 1200Lx,
Light application time is progress under conditions of 14h/d so as to reach desiccation purpose.
（8）By step（7）The somatic embryo of described completion desiccation is inoculated in 1/4LM culture mediums+sucrose 20g/L+ agar
The healthy and strong Picea asperata seedlingss with root can be grown up to after 6g/L, 30d.
It is an advantage of the invention that using Picea Mongolica mature embryo as explant, using LM as the additional various concentrations of minimal medium
6-BA, 2,4-D, KT, ABA, PEG4000 come induced embryonic callus, embryo callus propagation, embryonic differentiation, at desiccation
Reason and plant regeneration, and provide it is a kind of induce, subculture and method and condition of culture during seedling, with realize plant regeneration with it is fast
It is numerous.
The inventive method embryo callus subculture inductivity is 12%, and proliferation period is bred with 4.6 times of speed, and idiophase embryo callus subculture is put down
It is 76.8% that differentiation, which produces somatic embryo germination rate after 297/g of somatic embryo, drying and other treatment,.
Although being elaborated above to the object of the invention design and embodiment, those of ordinary skill in the art can be with
Recognize, without departing from claim limit scope precondition under, still the present invention can be made it is various improvement and
Conversion, and this modifications and variations still should belong to protection scope of the present invention.
Claims (1)
1. a kind of method that Picea Mongolica somatic embryo occurs, it is characterised in that comprise the following steps：
（1）Ripe Picea Mongolica seed is soaked into 12~15h with clear water, the seed for floating on liquid level given up, remaining kind
Son is rinsed 3~4 times with running water；It is rear to use by dragon spruce seed first with 70~75% alcohol-pickled 28~32s in superclean bench
Sterile water wash 3~5 times, then 12min is sterilized with 2% liquor natrii hypochloritis, then with aseptic water washing 3~5 times；
（2）Will be through step（1）Treated seed scalpel is stripped after complete embryo, is inoculated in following media：LM is trained
Base+6-BA2~5mg/L+2 is supported, 4-D4~8mg/L+KT0.5~2.0mg/L+ sucrose 30g/L+ gellan gums 2g/L, pH control exists
5.6~5.8, temperature is 22 ± 2 DEG C, 20~25d of Fiber differentiation generation callus is carried out under dark condition；
（3）By step（2）Callus is inoculated in following culture medium：1/2~1/4LM culture mediums+6-BA1~4mg/L+2,4-
D1~5mg/L+ KT0.1~1.0mg/L+ sucrose 20~30g/L+ gellan gums 2~4g/L, pH are controlled
22 ± 2 DEG C, carry out 20~25d of squamous subculture under dark condition and make callus further growth；
（4）By step（3）Callus be inoculated in following media：1/2~1/4LM culture mediums+6-BA1.0~1.9mg/
L+2,4-D1.0~1.9mg/L+ KT0.1~0.5mg/L+ sucrose 20~30g/L+ gellan gums 2~4g/L, pH control 5.6~
5.8th, temperature is 22 ± 2 DEG C, 20~25d of squamous subculture is carried out under dark condition, obtains the non-embryonic callus of faint yellow tight structure
Tissue and the thread embryo callus subculture of white translucent；
（5）By step（4）Embryo callus is inoculated in following media：1/2~1/4LM culture mediums+6-BA0.1~
1.0mg/L+2,4-D1.0~3.0mg/L+ sucrose 20~30g/L+ gellan gums 2~4g/L, pH are controlled
22 ± 2 DEG C, carry out Multiplying culture under dark condition, bred every 20~25d with same recipe squamous subculture；
（6）By step（5）Embryo callus is inoculated in following media：1/2~1/4LM culture mediums+ABA15~20mg/L
+ PEG400040~50g/L+ sucrose 20~30g/L+ gellan gums 2~4g/L, pH control 5.6~5.8, temperature be 22 ± 2 DEG C,
Differentiation culture is carried out under dark condition, squamous subculture is carried out with same recipe every 20d；Dragon spruce body cell is grown after 40~60d
Embryo；
（7）By step（6）Somatic embryo is inoculated in 1/2~1/4LM culture mediums+3~7g/L+ of sucrose 20~40g/L+ gellan gums
PEG400050~80g/L is cultivated on more than 7d, the filter paper that body embryo is laid in two layers of moistening, is placed in a sterile petri dish
Drying and other treatment 7 days, each culture dish places about 150 individual cells embryos, and above procedure is 22 ± 2 DEG C, illumination bar in cultivation temperature
Part is that 1200Lx, light application time are to carry out reaching desiccation purpose under conditions of 14h/d；
（8）By step（7）The somatic embryo for completing desiccation is inoculated in 1/4~1/2LM culture mediums+20~30g/L+ of sucrose agar 5
It can be sprouted after~8g/L, 30d and grow up to the healthy and strong Picea asperata seedlingss with root.
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| CN106718893B (en) * | 2016-12-06 | 2018-09-14 | 内蒙古农业大学 | The method that Siberia apricot somatic embryo occurs |
| CN110999785B (en) * | 2019-11-28 | 2021-08-31 | 内蒙古蒙荣园林绿化工程有限责任公司 | Synthetic method of artificial seeds of picea asperata |
| CN113502257B (en) * | 2021-08-19 | 2023-08-01 | 内蒙古蒙荣园林绿化工程有限责任公司 | Drying marking method for spruce somatic embryos before germination |
| CN114276976A (en) * | 2021-11-30 | 2022-04-05 | 内蒙古农业大学 | Dissociation method of picea mongolica root tip cell protoplast |
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| CN101228848B (en) * | 2008-02-27 | 2010-06-02 | 东北林业大学 | Method for inducing plant regeneratation using somatic embryo of picea koraiensis nakai |
| CN102792888B (en) * | 2012-07-31 | 2014-05-14 | 中国林业科学研究院林业研究所 | Method for somatic embryogenesis and plant regeneration of Lijiang spruce |
| CN103497928B (en) * | 2013-09-30 | 2016-02-03 | 中国林业科学研究院林业研究所 | Drying method for treating before the sprouting of PIECA ASPERATA somatic embryo |
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