CN104126505B - For the somatic embryo in-vitro regeneration method that Lilium tenuifolium genetic transformation is numerous soon with planting ball - Google Patents
For the somatic embryo in-vitro regeneration method that Lilium tenuifolium genetic transformation is numerous soon with planting ball Download PDFInfo
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Abstract
The invention provides a kind of method of the somatic embryo Regeneration in Vitro numerous soon with planting ball for Lilium tenuifolium genetic transformation, its processing step comprises gets Lilium tenuifolium tests for sterility, inoculated in MS+BA? 1.0mgL
-1+ NAA? 0.5mgL
-1middle cultivation 35 days, obtains non embryogenic callus.Subsequently by its turn in MS+BA? 0.5mgL
-1+ NAA? 1.0mgL
-1does middle cultivation 45 days, proceed to MS+NAA after obtaining embryo callus? 0.5mgL
-1in, adjustment embryo sex ratio, is then used alternatingly this two kinds of medium successively, can obtains the embryo callus culture that embryo sex ratio is high, germination rate is low.Finally turn in MS, every 1cm
3embryo callus culture can be formed more than 21 individual cells embryos.The present invention is explant with aseptic seedling, reduces the consumption to lily original seed; Frequency of embryonic callus induction is high, and preservation effect is good, and naked eyes can distinguish, is the good receptor of genetic transformation; Comparatively previous methods, the somatic embryo regenerating system that the present invention sets up improves 40% and 3-4 times respectively in callus induction rate and seedling coefficient.
Description
Technical field
The invention belongs to flowering bulb cultured in vitro and genetic breeding research field, particularly relate to the somatic embryo Regeneration in Vitro technical field for Lilium tenuifolium genetic transformation and bulb propagation.
Background technology
Somatic embryo is a kind of embryoid by unicellular growth, adopt in this way breeding have draw materials less, genetic stability is high, aberration rate is low and regeneration rate advantages of higher, effectively can alleviate transfer-gen plant later stage heavy screening operation, be considered to, in the field such as gene engineering, Somatic Fusion, cell mutant induction, artificial seed preparation, hybrid zygote rescue culture, preserving seed and nursery stock Fast-propagation, there is wide application prospect, become the focus of the gene engineering aspect research such as genetic transformation in recent years.
Lilium tenuifolium (
liliumpumilumDC.Fisch.), another name morningstar lily is that China originates in Wild bulbar flower, natural distribution in northeast, Qinghai, Shaanxi, the high and cold area such as Ningxia, be one of the widest, distribution latitude the is by north lily kind that distributes, self-sow is under Schattenseite sparse woods.Lilium tenuifolium better resistance, Salt And Alkali Tolerance, cold-resistant, drought-enduring, resistance to the moon, introduce a fine variety and need not tame, pattern is scarlet in addition, flower pattern is graceful, can directly apply to family potted plant, garden and urban landscaping, is the good plant material of forcing culture.Lilium tenuifolium bulb eats enriching yin, calms the nerves, effect of moistening lung, and stem, leaf have the effect of anti-inflammatory analgetic.
But Lilium tenuifolium needed for 3 years from seminal propagation to plant blossom, kind ball growth cycle is long, and in recent years because the deterioration of the ecological environment and people are that unauthorized and excessive mining phenomenon is serious, its initial species ball natural distribution amount falls sharply, endangered.Therefore, Lilium tenuifolium originates in Wild bulbar flower as a kind of excellent China, its Germ-plasma resources protection and bulb reproduction extremely urgent.In addition, Lilium tenuifolium plants stems stalk is comparatively thin and delicate, and fancy points awaits further improvement.Somatic embryo regeneration as a kind of modern vegetative propagation technique, be hopeful to solve lily ball breeding most, high-quality kind ball is cultivated, lily embryo morphology builds up, organize the important technology field of the aspect theory and practice problems such as differentiation and plant regeneration.Embryo callus is the good receptor material of genetic transformation, evoked callus and long-term keep its embryo sexual state to based on the fancy points improvement of transgenic technology and germplasm innovation of far-reaching significance.The application of this technology will produce significance to fields such as lily preserving seed, artificial seed development, screening mutant, polyploid breeding and transgenic breedings.
Summary of the invention
The object of the invention is with Lilium tenuifolium tests for sterility for explant, the dedifferentiation of induction explant forms embryo callus, through the adjustment of embryo sex ratio and the stage of preservation, obtain embryo sex ratio higher than 90%, germination rate lower than 5% embryo callus, keep for a long time embryo callus effectively being preserved in the basis of embryo sexual state and inducing globular embryo at control callus, obtain embryo sex ratio up to 100%, germination rate be 0 good embryo callus culture, wherein 1cm
3callus tissue culture contains the globular embryo of more than 20.And sprout the stage at somatic embryo, improve germination rate, promote globular embryo to sprout, by the growth course going through similar zygotic embryo, finally successfully obtain the somatic embryo of maturation germination, 1cm
3embryo callus culture can be formed more than 21 individual cells embryos.Thus expand the somatic embryo regenerating system that numerous and preserving seed provides efficient stable for the kind ball of Lilium tenuifolium, and for providing embryo callus this excellent transformation receptor based on genetically modified genetic transformation.
Technical scheme provided by the invention, comprises following processing step:
1. the explant dedifferentiation stage (non embryogenic callus induction period)
Choose the Lilium tenuifolium aseptic seedling that clove diameter is 1-1.2cm, blade is cut to long 0.5cm segment, faces up, tiling is inoculated in dedifferentiation medium MS+BA1.0mgL
-1+ NAA0.5mgL
-1in (wherein BA/NAA is 2), light is cultivated.Containing sucrose 30gL in this medium
-1, agar (bar) 7gL
-1, pH5.8,121 DEG C of autoclavings 20 minutes.Culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1; When inoculating 15 days, blade, petiole top layer shrinkage form trickle bright yellow particle, rear formation callus.When inoculating 35 days, form yellow green, sparkling and crystal-clear degree is large, humidity is high, the non embryogenic callus of surface smoothing.
2. the cells,primordial transformation stage (embryonic callus induction stage): cultivate the 45th day in dedifferentiation medium, the non embryogenic callus that explant dedifferentiation in step 1 obtains is cut to 0.5cm
3fritter, transfers in embryonic callus induction medium MS+BA0.5mgL
-1+ NAA1.0mgL
-1in (namely BA/NAA is 1/2), make non embryogenic callus insert 2-3mm in medium, light is cultivated.Culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1.Inoculate 30 days, form glassy yellow, the obvious embryo callus of graininess on yellow green non embryogenic callus surface.
3. embryo sex ratio and the graininess adjusting stage: cultivate 45 days through step 2, remove yellow green non embryogenic callus part, jonquilleous embryo callus is cut to 1cm
3fritter, transfers in embryo callus adjustment medium MS+NAA0.5mgL
-1in, light is cultivated.Culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1.Can obtain after same medium squamous subculture 1 time embryo sex ratio higher than 90%, germination rate lower than 5%, the good embryo callus of graininess.
4. globular embryo induction period: select the embryo callus obtained in step 3, remove the bud of green non embryogenic callus and sprouting, be used alternatingly embryonic callus induction medium (MS+BA0.5mgL successively
-1+ NAA1.0mgL
-1) and embryo sex ratio adjustment medium (MS+NAA0.5mgL
-1), culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1.Within every 45 days, change a subculture, be used alternatingly 2 times.Can obtain embryo sex ratio up to 100%, germination rate be 0 good embryo callus culture, wherein 1cm
3callus tissue culture contains the globular embryo of more than 20.
5. somatic embryo is sprouted and the seedling stage: turn in MS minimal medium by the embryo callus culture containing globular embryo in step 4, culturing room's temperature is 25 ± 1 DEG C, adopt airtight sealed membrane, ensure that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1.After 15-20 days, globular embryo is sprouted, and experiences heart-shape embryo, peltate embryo and cotyledonary embryos stage successively and sprouts formation mature somatic embryo, 1cm
3embryo callus culture can be formed more than 21 individual cells embryos.
Advantage of the present invention and good effect are:
1. be explant with tests for sterility, reduce the consumption of lily original seed, can available protecting Wild ornamental resources, and also lossless to test tube bulbs, do not affect acclimatization and transplants, for scientific breeding Lilium Germplasm provides new approaches;
2. embryo callus and non embryogenic callus can distinguish with color naked eyes.In the present invention, surface smoothing, yellowish green be non embryogenic callus, and glassy yellow, be embryo callus in obvious particle proterties;
3. frequency of embryonic callus induction is high, and preservation effect is good, for genetic transformation provides good receptor.In the embryonic callus induction stage, transform with the non embryogenic callus of dedifferentiation induction cells,primordial, inductivity is high; The stage is preserved at embryo callus, improved culture medium is filled a prescription, keep for a long time embryo callus is effectively preserved in the basis of embryo sexual state at control callus, obtain embryo sex ratio higher than 90%, germination rate lower than 5% embryo callus, and globular embryo is induced on the basis of effectively preserving embryo callus, obtain embryo sex ratio up to 100%, germination rate be 0 good embryo callus culture, wherein 1cm
3callus tissue culture contains the globular embryo of more than 20, lays the foundation for significantly improving regeneration efficiency; In addition, this embryo callus is strong to common antibiotics susceptibility, for Lilium tenuifolium provides embryo callus this excellent transformation receptor based on genetically modified genetic transformation;
4. regeneration efficiency is high.The present invention sprouts the stage at somatic embryo, improves germination rate, promotes that globular embryo is sprouted, by the growth course going through similar zygotic embryo, finally successfully obtains the somatic embryo of maturation germination, 1cm
3embryo callus culture can be formed more than 21 individual cells embryos, with forefathers about other kind lily somatic embryo Regeneration in Vitro result of study compared with, frequency of embryonic callus induction and seedling differentiation coefficient all improve 20%, coefficient of differentiation improves 5 times, achieve the object of highly efficient regeneration, thus expand the somatic embryo regenerating system that numerous and preserving seed provides efficient stable for the kind ball of Lilium tenuifolium; And compare the existing research of Lilium tenuifolium, the present invention establishes somatic embryo regenerating system first, and compared with the Regeneration in Vitro approach adopted with forefathers, callus induction rate improves 40%, coefficient of differentiation improves 3-4 doubly, can be used as the regenerating system of the fast numerous efficient stable of Lilium tenuifolium kind ball;
5. reduce production cost.The novel hormonals such as current TDZ, PIC, DIC, KT are widely used in induction (TangYP, 2010 of other kind lily callus or somatic embryo; TribulatoA, 2006; KhosraviS, 2007), but this parahormone price is generally higher.And the Lilium tenuifolium somatic embryo generation system set up in the present invention, only use BA and NAA two kinds of frequently seen plants hormones, commercially available price is only the 1/10-1/5 of novel hormonal, and the basis ensureing regeneration efficiency effectively reduces production cost; In addition, sprout the stage at somatic embryo, need additionally to add NAA, IBA or BA in forefathers' research, to promote that somatic embryo is sprouted and take root (Zhai Yan etc., 2011; Yuan Xue etc., 2012), and the present invention adopts the MS minimal medium not adding any hormone, ensures somatic embryo differentiated coefficient and while normally taking root, effectively can reduce production cost, enhance productivity.
Accompanying drawing explanation
Fig. 1 is explant dedifferentiation (Bar:5mm);
Fig. 2 is embryonic callus induction (Bar:5mm);
Fig. 3 is non embryogenic callus (Bar:1mm);
Fig. 4 is the embryo callus (Bar:5mm) after adjustment;
Fig. 5 is globular embryo induction (Bar:2mm);
Fig. 6 is that somatic embryo sprouts (Bar:5mm);
Fig. 7 is indefinite bud seedling (Bar:5mm);
Fig. 8 is somatic embryo seedling (Bar:5mm);
Fig. 9 is globular embryo (Bar:2mm);
Figure 10 is heart-shape embryo (Bar:2mm);
Figure 11 is peltate embryo (Bar:2mm);
Figure 12 is cotyledon shape postembryonic period (Bar:5mm);
Figure 13 is non embryogenic callus sections observation (Bar:200 μm);
Figure 14 is embryo callus sections observation (Bar:200 μm);
Figure 15 is adventitious shoot regeneration;
Figure 16 is somatic embryo regeneration.
Embodiment
In order to more describe processing step of the present invention in detail, give an actual example explanation below.
Example one, Lilium tenuifolium somatic embryo Regeneration in Vitro technology
1. non embryogenic callus induction period (explant dedifferentiation stage)
Choose the Lilium tenuifolium aseptic seedling that clove diameter is 1-1.2cm, blade is cut to long 0.5cm segment, faces up, tiling is inoculated in dedifferentiation medium MS+BA1.0mgL
-1+ NAA0.5mgL
-1in (namely BA/NAA is 2), light is cultivated.Wherein containing sucrose 30gL
-1, agar strip 7gL
-1, pH5.8,121 DEG C of autoclavings 20 minutes.Culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1.
Test link finds, if be explant with scale, all occurs with indefinite bud Direct Regeneration approach.And with blade and petiole for not forming indefinite bud during explant, but when inoculation 15 days, there is shrinkage and form trickle bright yellow particle in blade, petiole top layer, rear formation callus.Inoculate 35 days, form yellow green, sparkling and crystal-clear degree is large, humidity is high, the non embryogenic callus of surface smoothing, occur through the indirect Regeneration Ways of callus.
Callus situation and callus degree are different because hormone concentration is different.BA concentration is lower is 1.0mgL
-1, NAA concentration is higher is 0.5mgL
-1time, the callus degree of blade and petiole is the highest.I.e. medium MS+BA1.0mgL
-1+ NAA0.5mgL
-1the optimum medium that (when BA/NAA is 2) is Lilium tenuifolium tests for sterility and petiole evoked callus, callus induction rate reaches 30%, and the non embryogenic callus of induced synthesis is yellow green, sparkling and crystal-clear degree is large, humidity is high and surface smoothing (see accompanying drawing 1, Fig. 3).
2. embryonic callus induction (cells,primordial transformation stage)
Cultivate the 45th day in dedifferentiation medium, i.e. non embryogenic callus Fiber differentiation the 45th day, the non embryogenic callus that explant dedifferentiation in step 1 obtains is cut to 0.5cm
3fritter, transfers in embryonic callus induction medium MS+BA0.5mgL
-1+ NAA1.0mgL
-1in (namely BA/NAA is 1/2), make non embryogenic callus insert 2-3mm in medium, light is cultivated.Culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1.Inoculate 30 days, form glassy yellow, the obvious embryo callus of graininess (see accompanying drawing 2) on yellow green non embryogenic callus surface.
Test link find, if by explant dedifferentiation produce non embryogenic callus be connected to not containing BA, NAA MS medium in, yellow green non embryogenic callus brownization gradually, does not induce and occurs embryo callus; If the concentration of adding BA in MS medium is higher is 1.5 or 2.0mgL
-1, NAA concentration is lower is 0.1 or 0.5mgL
-1, when namely traditional addition manner-BA concentration is higher than NAA concentration, non embryogenic callus differentiation produces indefinite bud, and induction does not produce embryo callus.
With studied in the past unlike, BA and the NAA concentration of adding in MS medium is all adjusted to 0.5 or 1.0mgL by the present invention
-1, and make NAA concentration higher than BA, all can obtain embryo callus.Especially when NAA adds 2 times that concentration is BA concentration, when namely BA/NAA is 1/2, the frequency of embryonic callus induction of acquisition is the highest, can reach 88%.
3. embryo sex ratio and the graininess adjusting stage
Cultivate 45 days through step 2, remove yellow green non embryogenic callus part, jonquilleous embryo callus is cut to 1cm
3fritter, transfers in embryo callus adjustment medium MS+NAA0.5mgL
-1in, light is cultivated.Culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1.Through the adjustment of embryo sex ratio and the stage of preservation, in same medium after squamous subculture 1 time, keep for a long time embryo callus is effectively preserved in the basis of embryo sexual state at control callus, can obtain embryo sex ratio higher than 90%, germination rate lower than 5%, the good embryo callus (see accompanying drawing 4) of graininess.
Embryo callus graininess easily disappears, if medium is uncomfortable, the obvious embryo callus of glassy yellow, graininess can be converted into green non embryogenic callus.Therefore, how in embryo callus preservation process, both ensured that embryo callus kept its embryo sexual state for a long time, embryo callus can be suppressed again to sprout the key link become in process of the test.Test finds, when BA and NAA concentration is all higher, is respectively 1.0 and 0.5mgL
-1time, embryo callus ratio is up to 65.67%, but the embryo callus now more than 50% is converted into green by yellow, and namely embryo is lost serious, and in this medium, embryo callus germination rate is also very high, reaches more than 30%.
And in the present invention, non embryogenic callus is transferred in MS+NAA0.5mgL
-1middle cultivation 45 days, embryo callus ratio is 55.81%, and turns green rate lower than 30%, and germination rate controls below 20%.
4. globular embryo induction period
Select the embryo callus obtained in step 3, remove the bud of green non embryogenic callus and sprouting, be used alternatingly embryonic callus induction medium MS+BA0.5mgL successively
-1+ NAA1.0mgL
-1with embryo sex ratio adjustment medium MS+NAA0.5mgL
-1, culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1.Within every 45 days, change a subculture, be used alternatingly 2 times.Can obtain embryo sex ratio up to 100%, germination rate be 0 good embryo callus culture, wherein 1cm
3callus tissue culture contains the globular embryo (see accompanying drawing 5) of more than 20.
5. somatic embryo is sprouted and the seedling stage
In the existing research about the generation of lily somatic embryo, additionally need add NAA, IBA or BA in the somatic embryo Germination And Seedling stage, to promote that somatic embryo is sprouted and take root (Zhai Yan etc., 2011; Yuan Xue etc., 2012), and the present invention sprouts the stage at somatic embryo, adopts the MS minimal medium not adding any hormone, ensures somatic embryo differentiated coefficient and while normally taking root, effectively can reduce production cost.
Concrete implementation step is: turn in MS minimal medium by the embryo callus culture containing globular embryo in step 4, culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensure that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1.After 15-20 days, globular embryo sprouts (see accompanying drawing 6), experiences heart-shape embryo (see accompanying drawing 10), peltate embryo (see accompanying drawing 11) and the sprouting of cotyledonary embryos (see accompanying drawing 12) stage successively form mature somatic embryo, 1cm by globular embryo (see accompanying drawing 9)
3embryo callus culture can be formed more than 21 individual cells embryos.
6. antibiotics sensitivity screening
With the embryo callus culture containing globular embryo for explant, in the optimum medium MS that the somatic embryo screening acquisition is in the present invention sprouted, add variable concentrations Kan, Hyg antibiotic and carry out sensitive concentration screening.Test link finds, embryo callus provided by the invention is responsive to common antibiotics Kan and Hyg, and the sublethal concentration that screening obtains is respectively 150mgL
-1and 30mgL
-1.
The qualification of example two, non embryogenic callus and embryo callus
Visible through stereomicroscope observation, non embryogenic callus is smooth surface then, and embryo callus has aobvious nodular texture.Observation paraffin section finds, two type callus have notable difference, wherein non embryogenic callus is inner without obvious cell structure (see accompanying drawing 13), and embryo callus mostly is outer origin, inner cell structure is obvious, becomes spheroidal more, outside by internal layer, cell diminishes gradually, and arrangement also more tight (see accompanying drawing 14).
The high efficiency of example three, checking somatic embryogenesis pathway
Although somatic embryo regenerating system has unicellular origin, reproduction coefficient advantages of higher, but due to induction relative difficulty, only have a few lily to obtain somatic embryo at present both at home and abroad, and mostly be cutting flower variety, but the numerous Lilium Germplasms comprising Lilium tenuifolium all rarely have report.At present about the research of Lilium tenuifolium Regeneration in Vitro mainly concentrates on adventitious organogenesis, to take from the bulb of strain, the Lilium tenuifolium scale of non-sterile seedling is explant when carrying out adventitious bud inducing, and derivative coefficient is 3-4.8; With Lilium tenuifolium filigree for explant, callus induction rate is 40%, and when callus carries out differentiation adventitious buds, coefficient of differentiation is the refined plum of 10.8(gold etc., 2006; Ge Bei comet etc., 2010); In the research that the relevant somatic embryo of other lilies occurs, oriental hybrid lily cutting flower variety frequency of embryonic callus induction is about 70%, and seedling differentiation rate opens skill duckweed etc. lower than 70%(, and 2008); The inductivity 79% of OT series cut-flower ' Manissa ' embryo callus, number of seedling is 5.6(Zhai Yan etc., 2011); Lilium longiflorum frequency of embryonic callus induction is 65.74%, and seedling differentiation rate reaches 81.48%, and number of seedling is 2.04(Yuan Xue, 2012).
And Lilium tenuifolium frequency of embryonic callus induction provided by the invention reaches more than 88%, seedling differentiation rate reaches more than 95%, and coefficient of differentiation is more than 20, with forefathers about Lilium tenuifolium Regeneration in Vitro result of study compared with, callus induction rate improves 40%, coefficient of differentiation improves 3-4 doubly (see accompanying drawing 7-8,15-16), and with forefathers about somatic embryo Regeneration in Vitro result of study compared with, frequency of embryonic callus induction improves 20%, seedling differentiation rate improves 20%, coefficient of differentiation improves 5 times, can be used as the regenerating system of the fast numerous efficient stable of Lilium tenuifolium kind ball.
Claims (1)
1., for the somatic embryo in-vitro regeneration method that Lilium tenuifolium genetic transformation is numerous soon with planting ball, it is characterized in that step is:
(1) the explant dedifferentiation stage
Blade is cut to long 0.5cm segment, faces up, tiling is inoculated in dedifferentiation medium MS+BA1.0mgL
-1+ NAA0.5mgL
-1in, light is cultivated, containing sucrose 30gL in this medium
-1, agar strip 7gL
-1, pH5.8,121 DEG C of autoclavings 20 minutes, culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1; When inoculating 15 days, blade, petiole top layer shrinkage form trickle bright yellow particle, rear formation callus, when inoculating 35 days, form yellow green, sparkling and crystal-clear degree be large, humidity is high, the non embryogenic callus of surface smoothing;
(2) the cells,primordial transformation stage
Cultivate the 45th day in dedifferentiation medium, the non embryogenic callus that explant dedifferentiation in step 1 obtains is cut to 0.5cm
3fritter, transfers in embryonic callus induction medium MS+BA0.5mgL
-1+ NAA1.0mgL
-1in, now BA/NAA is 1/2, and make non embryogenic callus insert 2-3mm in medium, light is cultivated, culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1, inoculate 30 days, form glassy yellow, the obvious embryo callus of graininess on yellow green non embryogenic callus surface;
(3) embryo sex ratio and the graininess adjusting stage
Cultivate 45 days through step 2, remove yellow green non embryogenic callus part, jonquilleous embryo callus is cut to 1cm
3fritter, transfers in embryo callus adjustment medium MS+NAA0.5mgL
-1in, light is cultivated, and culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensures that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1; After same medium squamous subculture 1 time, obtain embryo sex ratio higher than 90%, germination rate lower than 5%, the good embryo callus of graininess;
(4) globular embryo induction period
Select the embryo callus obtained in step 3, remove the bud of green non embryogenic callus and sprouting, be used alternatingly embryonic callus induction medium MS+BA0.5mgL successively
-1+ NAA1.0mgL
-1with embryo sex ratio adjustment medium MS+NAA0.5mgL
-1, adopt airtight sealed membrane, ensure that tissue culture bottle humidity reaches 80-90%, within every 45 days, change a subculture, be used alternatingly 2 times, obtain embryo sex ratio up to 100%, germination rate be 0 good embryo callus culture, wherein 1cm
3callus tissue culture contains the globular embryo of more than 20;
(5) somatic embryo is sprouted and the seedling stage
Turn in MS minimal medium by the embryo callus culture containing globular embryo in step 4, culturing room's temperature is 25 ± 1 DEG C, adopts airtight sealed membrane, ensure that tissue culture bottle humidity reaches 80-90%, every day 16 h light, 8 h dark, intensity of illumination is 36 μm of olm
-2s
-1, after 15-20 days, globular embryo is sprouted, and experiences heart-shape embryo, peltate embryo and cotyledonary embryos stage successively and sprouts formation mature somatic embryo, 1cm
3embryo callus culture is formed more than 21 individual cells embryos.
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| CN110845266A (en) * | 2019-11-22 | 2020-02-28 | 顾霆 | Embryonic germ cell dedifferentiation culture medium and preparation method thereof |
| CN110972955A (en) * | 2020-01-02 | 2020-04-10 | 齐齐哈尔大学 | Method for inducing somatic embryogenesis and plant regeneration of lily leaves |
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