CN101536629A - Method for causing seeds of cypripedium macranthum to sprout - Google Patents
Method for causing seeds of cypripedium macranthum to sprout Download PDFInfo
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Abstract
The invention discloses a method for causing seeds of cypripedium macranthum to sprout, which is as follows: seeds of cypripedium macranthum is cultivated before endothelium does not form dry films from embryo growth to spherical embryo to obtain germinal seeds. The method for causing seeds of cypripedium macranthum to sprout comprises the following steps: by selecting appropriate seeding time, namely, unripe seeds with agreeable development degree are selected to be grown in an appropriate temperature and a culture medium so that the seeds of cypripedium macranthum sprout in the condition of artificial culture, and the sprouting rate is higher and can reach as high as 60%; the method overcomes the shortcoming of non-success in the past artificial propagation; in addition, the method has real operability, key function on the success or failure of the scale propagation, and reasonable cost; therefore, the method has wide application prospect in the field of propagating cypripedium macranthum by labor and has important significant for nursing research and cultivation of cypripedium macranthum.
Description
Technical field
The present invention relates to a kind of method of the Da Hua of making lady's slipper seed germination, particularly relate to the method for a kind of Da Hua of making lady's slipper seed by non-symbiosis axenic germination.
Background technology
The Da Hua lady's slipper mainly is distributed in the North, Northeast China area in China, owing to excessively gather, is in serious Critical Condition at present, presses for to take to protect on the spot and protect with moving the effective protection measure that combines, promotes repopulate.Cultivate if can carry out artificial propagation, studying with rational exploitation and utilization in the future for the child care of Da Hua lady's slipper all has crucial meaning.
The usual manner that present non-symbiosis axenic germination is orchid seminal propagation is all widely used in research and commerce.The seed of most tropical orchids is sprouted in the axenic germination experiment easily, comparatively speaking, the sprouting of the living blue seed in ground is much more difficult, often need special culture medium prescription and condition of culture, and different genus, even seed germination desired culture conditions not of the same race is all different in the same genus, is difficult to as the easy collective media that finds of tropical epiphytic orchid.Therefore, it is not very successful that blue aseptically sowing seeds breeding is given birth on ground, temperate zone, has only the part kind to sprout smoothly at present.The axenic germination of Cypripedium plant is blue more much more difficult than give birth to commonly again, and especially the big premium under the Da Hua lady's slipper is spent series.Mudanjiang Teachers College had once carried out aseptic seeding test in 3 years, the measure of having taked many promotions to sprout, but none piece seed germination to the Da Hua lady's slipper.
Summary of the invention
An object of the present invention is to provide a kind of method of the Da Hua of making lady's slipper seed germination.
The method that makes Da Hua lady's slipper seed germination provided by the present invention is to get kind of embryonic development to spheroidal embryo period but the Da Hua lady's slipper immature seed that secundine does not begin to form before the dry film is cultivated the seed that obtains sprouting.
Described seed specifically can be a Da Hua lady's slipper prematurity green pods seed.
Wherein, described cultivation is carried out under dark condition.
The temperature of described cultivation can be 18-22 ℃, is preferably 20 ℃.
The used medium of described cultivation can be improvement VW+ (0.5mg/L-2.0mg/L) 6-BA+ (50ml/L-200ml/L) nutrients, and described nutrients is coconut palm breast, murphy juice or bananas juice; Be preferably improvement VW+2.0mg/L6-BA+100ml/L coconut palm breast.
The used medium of described cultivation also can be improvement VW+ (50ml/L-200ml/L) nutrients, and described nutrients is coconut palm breast, murphy juice or bananas juice; Be preferably improvement VW+100ml/L coconut palm breast.
The used medium of described cultivation also can be MS+ (0.5mg/L-2.0mg/L) 6-BA+ (50ml/L-200ml/L) nutrients, and described nutrients is coconut palm breast, murphy juice or bananas juice; Be preferably MS+2.0mg/L 6-BA+100ml/L coconut palm breast.
The used medium of described cultivation also can be MS+ (50ml/L-200ml/L) nutrients, and described nutrients is coconut palm breast, murphy juice or bananas juice; Be preferably MS+100ml/L coconut palm breast.
The pH value of above-mentioned arbitrary described medium can be 5.6-5.8.
Coconut palm breast: be liquid content contained in the coconut.
Murphy juice can be prepared as follows and obtain: potato decortication, be cut into small pieces, and potato is mixed with the mass ratio of distilled water with 1: 1, rub, obtain murphy juice, standby.
Bananas juice can be prepared as follows and obtain: the banana peeling, banana is mixed with the mass ratio of water with 1: 1, and rub, obtain bananas juice, standby.
The present invention makes the method for Da Hua lady's slipper seed asepsis sprouting, promptly selecting to be in embryo opportunity by the appropriate sowing of accurate selection has grown to having the sprouting ability, but germination inhibitor also inchoate one narrow growth window period (is promptly planted embryonic development to spheroidal embryo period, and before secundine does not begin to form dry film) immature seed sow, add the suitable temperature and the use of medium, Da Hua lady's slipper seed is sprouted under the artificial culture condition, and germination rate is than higher, reach as high as 60%, having overcome artificial propagation does not in the past have the successful shortcoming of an example; In addition, the inventive method has actual operation, and cost is reasonable, and especially the success or failure to the scale breeding have key effect.Therefore, the inventive method will have broad application prospects in artificial propagation Da Hua lady's slipper field, and the child care of Da Hua lady's slipper and scale breeding are had important society and economic implications.
Description of drawings
Fig. 1 is the growth course figure of Da Hua lady's slipper embryo and integument.
Fig. 2 is seed germination microexamination figure.
Fig. 3 is Da Hua lady's slipper immature seed developmental process and germination rate relation.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. among the following embodiment if no special instructions, all can be bought from commercial channels and obtain.
The immature seed cultured in vitro of embodiment 1, Da Hua lady's slipper (Cypripedium macranthum) is grown
Sprout research for the seed cultured in vitro of carrying out the Da Hua lady's slipper, need to understand the developmental process of its seed from being fertilized ripe each period.
At the florescence Da Hua lady's slipper (Baihua Shan Mountain forest farm, Beijing) is carried out artificial pollination, pollination back 4-12 week, weekly, the ovary (capsule) of gathering differing maturity.After adopting FAA fixer vacuum suction to fix 24 hours, go to normal temperature preservation in 70% alcohol.The paraffin method film-making, the Nikon microscopically is observed, and CCD takes pictures.
3 repetitions are established in experiment, and the result is as shown in table 1, and is specific as follows:
1, the growth of after fertilization embryo
The Da Hua lady's slipper needs 3.5 months from the seed maturity of pollinating.Be to grow to break up very vigorous period about Da Hua lady's slipper pollination back 4 weeks (28 days), blastular is reached maturity and is in critical condition after prefecundation, have multiple developmental process to mix in thousands of the ovules in same ovary and occur, more egg cell has been fertilized and has been produced zygote (fertilized egg).Follow the formation of zygote, synergidae is degenerated gradually, and zygote polarity is strengthened.Cell nucleus is positioned at chalazal end, and nuclear is big, and a big kernel is arranged.Micropylar end is a big vacuole (Fig. 1-A).
Pollinated back 35 days, the division of the horizontal lack of balance of zygote develops into a 2-blast, forms basal cell (Fig. 1-B) of a less terminal cell and a big vacuole of tool.It is the primary endosperm nucleus of free type and the chalazal end of shifting to blastular that the polar core after fertilization is grown, and degenerates the disappearance of endosperm when causing seed maturity with regard to just rapid extremely in early days what embryo germinated.Terminal cell is grown and is formed idiosome, and the basal cell grows the formation suspensor.Terminal cell is behind a horizontal split, and the cell on top vertically splits into two cells again, adds the basal cell of micropylar end, and formation T shape 4-blast (Fig. 1-C).
Pollinated back 42 days, idiosome continues division, and cell quantity increases, and volume is filled blastocyst cavity gradually, grows to form cellulous early stage ball embryo.The basal cell is through once division, and the suspensor of bud into 2-cell long and narrow is positioned at position (Fig. 1-D) of endostome.Pollinated back 49 days, the idiosome cell quantity continue to increase, and fills up blastocyst cavity substantially, forms globular embryo, and suspensor renews, but no longer continues division, and the external integument cell be microscler cell (Fig. 1-E) of highly vacuolization.
Pollinated back 56 days, idiosome still keeps globular embryo, is full of blastocyst cavity fully, and suspensor degeneration disappearance (Fig. 1-F).Idiosome is up to back 90 days seed maturities of pollination, and (Fig. 1-J) is in proembryo period and fails to enter differential period still to keep the form of globular embryo.
2, the growth of interior exosper (integument)
Growth at embryo is early stage, and interior external integument is close mutually, external integument cell height vacuolization, and inner integument is by two-layer cellularity (Fig. 1-A).Pollinated back 56 days, along with the growth of embryo, idiosome increases gradually, fills up blastocyst cavity, nucellar cell's absorption of degenerating gradually, and the inner integument cell is close to idiosome (Fig. 1-F).Pollinated back 70 days, seed is near ripe, and integument cell dewaters gradually, and the inner integument attenuation is condensed to the fine and close cell of skim, tightly surrounds idiosome, the bud into secundine.Grow and the exosper that comes forms secondary wall (Fig. 1-G) gradually by external integument.
3, the kernel texture of ripe Da Hua lady's slipper
Simple in structure during seed maturity, double-deck skin and the idiosome of planting only arranged, there is not endosperm.Inner integument finally develops into one deck can be dyed dry film matter secundine (Fig. 1-H, 1-I) for the densification of redness by sarranine.The secondary wall of exosper cell supports the fusiform long and narrow profile of exosper, is huge air chamber (Fig. 1-I, 1-J) between interior exosper.
4, the best inoculation time of Da Hua lady's slipper seed cultured in vitro is selected
At occurring in nature, the Da Hua lady's slipper grows in high height above sea level or high latitude cold district, and shortage provides the endosperm of nutrition at the sprouting initial stage, after seed maturity scatters, will 1 year in addition the longer time after, run into special symbiosis fungi, penetrate kind of a skin, provide nutrition to help its seed germination by fungal infection.But in the experiment of Da Hua lady's slipper seed asepsis sprouting, though a large amount of nutriments such as carbohydrate are provided artificially, but, fine and close membranous secundine enters in the embryo because having intercepted materials such as moisture, nutrition over a period to come, idiosome is difficult to obtain to sprout necessary material conditions, thereby causes the sprouting difficulty of mature seed.Da Hua lady's slipper seed has inside and outside two structure of planting skin; should be of a kind of protective adaptations of these species to extreme natural environment; it can be protected rataria and suppress the sprouting of embryo in one period long resting stage; avoid seed to cross premature eruption and be subjected to the injury of harsh external condition and death; this is favourable characteristic to plant itself, but becomes an obstacle when artificial propagation.
So need search out best seed collecting and sowing opportunity, promptly embryo has had the potential ability of sprouting, but also not active one period of short duration critical period of inhibiting factor.Specific to the developmental stage be exactly embryonic development to the globular embryo stage, and secundine period of drying shrinkage not as yet.The Da Hua lady's slipper needs about 90 days from the seed maturity time of pollinating.As can be seen from Table 1, the ovary that grows in the Da Hua lady's slipper of Baihua Shan Mountain develops into the early stage ball embryo of many cells the 6th week about the pollination back greatly, and to the 8th week continuing to develop into the globular embryo in late period, inner integument has become the cell that skim is close to idiosome.Until seed maturity, still keep the form of globular embryo.Consider that Da Hua lady's slipper natural distribution is wide, grow in the influence of the factors such as variation, year border fluctuation of population phenophase of zones of different and developmental rhythm, concrete fate apart from when pollination during seed collecting may be different, as long as select to have grown, it should be the best window period that seed asepsis sprouting is collected seed but inner integument does not also develop into the period of fine and close dry film matter secundine to the globular embryo stage.
The development time of table 1, Da Hua lady's slipper after fertilization seed different developmental phases correspondence
* DAP=Days after pollination, i.e. fate after the pollination
This research finds that in the sections observation of Da Hua lady's slipper seed different development stage the mature seed of Da Hua lady's slipper all has inside and outside bilayer (integument) and plants skin, inner integument exists in the whole growth course of embryo always, and the dry film matter secundine structure of formation one deck densification when seed development is ripe is close to embryo.This is all different with the structure that majority is bred more or less freely orchid mature seed.
Among Fig. 1, each figure expression respectively:
(A) pollination is back 28 days, the blastular and the zygote of firm fertilization, engineer's scale=50um;
(B) pollination is back 35 days, and zygote develops into one 2-blast, engineer's scale=50um;
(C) pollination is back 35 days, and zygote develops into T shape 4-blast, engineer's scale=20um;
(D) pollination is back 42 days, and embryo development is early stage ball embryo, and the basal cell grows and is the suspensor of long and narrow, engineer's scale=50um;
(E) pollination is back 49 days, and embryonic development is to the spheroidal embryo stage, and interior external integument is obvious, engineer's scale=50um;
(F) pollination is back 56 days, and embryo still keeps the globular embryo form, and suspensor is degenerated and disappeared engineer's scale=50um;
(G) pollination is back 70 days, and embryo still keeps the globular embryo form near ripe, and inner integument develops into secundine and is close to idiosome, engineer's scale=50um;
(H) the back 90 days mature seed vertical section feature of pollination shows secundine and the undifferentiated globular embryo of being close to the idiosome growth, engineer's scale=50um;
(I) pollination is back 90 days, mature seed cross section, exosper and air chamber in showing, engineer's scale=50um;
(J) pollination is back 90 days, mature seed vertical section, exosper and air chamber in showing, engineer's scale=50um.
The sprouting of embodiment 2, Da Hua lady's slipper seed
Carry out artificial different friendship pollination at Da Hua lady's slipper (Cypripedium macranthum) (Baihua Shan Mountain forest farm) florescence, the pollination back is since the 4th week, gather once green prematurity kind pod weekly, carry out artificial aseptic seeding and TTC seed activity dyeing experiment, after planting the sowing seed amount in every bottle is added up, the 9th week statistics sprouting number after planting, and calculating germination rate.Judge the seed germination standard: seed expands, and the protocorm embryo is prominent to break in the seed coat, and spills the ball stem of white, the visible minimum white point of naked eyes, under Stereo microscope as Fig. 2.The number seeds of germination rate=sprouting/(the seed total number X rate of dyeing of sowing) X100%.Rate of dyeing is that embryo can be dyed the percentage of red seed by TTC after seed was measured dye liquor dyeing with the TTC seed activity, and there do not have active seed not dyed to be red.
One, sterilization and sowing: green pods is the semi-matured capsule in pollination back, and after gathering, parts such as the unnecessary carpopodium of excision earlier, withered petal, scar are stained with the Safeguard perfumed soap with toothbrush carefully to scrub kind of a pod outer surface, and flowing water washed more than half an hour; Receive solution sterilization more than 15 minutes putting into 1% hypochlorous acid on the superclean bench, with aseptic water washing for several times, put into 75% alcohol again and soak, calcination is 1-2 time on alcolhol burner.Cut the fruit pod with the scalpel after the sterilization at last, seed is trembled in media surface equably gently.
Two, cultivate:
Below all to regulate pH value before each medium sterilization be 5.6-5.8, guarantee that weakly acidic condition makes the normal seed germination growth.100 of each sowings, after planting seed counting to being broadcast in every bottle.
(1) cultivates with medium 1 (A curve in the corresponding diagram 3)
Cultivate under 20 ℃, dark condition with medium 1.
Medium 1:MS+100ml/L coconut palm breast.
3 repetitions are established in experiment, and the result takes the mean.The germination rate of the seed of different development stage is shown in the table 41.
(2) cultivate with medium 2 (B curve in the corresponding diagram 3)
Cultivate under 22 ℃, dark condition with medium 2.
Medium 2:MS+0.5mg/L 6-BA+100ml/L murphy juice.
Murphy juice is prepared as follows and obtains: potato decortication, be cut into small pieces, and potato is mixed with the mass ratio of distilled water with 1: 1, rub, obtain murphy juice.
3 repetitions are established in experiment, and the result takes the mean.The germination rate of the seed of different development stage is shown in the table 42.
(3) cultivate with medium 3 (C curve in the corresponding diagram 3)
Cultivate under 20 ℃, dark condition with medium 3.
Medium 3:MS+2.0mg/L 6-BA+50ml/L coconut palm breast.
3 repetitions are established in experiment, and the result takes the mean.The germination rate of the seed of different development stage is shown in the table 43.
(4) cultivate (D curve in the corresponding diagram 3) with medium 4
Cultivate under 20 ℃, dark condition with medium 4.
Medium 4: improvement VW+200ml/L coconut palm breast.
3 repetitions are established in experiment, and the result takes the mean.The germination rate of the seed of different development stage is shown in the table 44.
(5) cultivate with medium 5 (E curve in the corresponding diagram 3)
Cultivate under 18 ℃, dark condition with medium 5.
Medium 5: improvement VW+0.5mg/L 6-BA+150ml/L bananas juice.
Bananas juice can be prepared as follows and obtain: the banana peeling, banana is mixed with the mass ratio of water with 1: 1, and rub, obtain bananas juice.
3 repetitions are established in experiment, and the result takes the mean.The germination rate of the seed of different development stage is shown in the table 45.
(6) cultivate (F curve in the corresponding diagram 3) with medium 6
Cultivate under 20 ℃, dark condition with medium 6.
Medium 6: improvement VW+2.0mg/L 6-BA+100ml/L coconut palm breast.
3 repetitions are established in experiment, and the result takes the mean.The germination rate of the seed of different development stage is shown in the table 46.
Wherein, the composition of MS medium is as shown in table 2.
The composition of table 2, MS medium
The composition of improvement VW medium is as shown in table 3.
The composition of table 3, improvement VW medium
Three, experimental result
1, Da Hua lady's slipper seed germination growth overall process
The result shows that the germination process of seed is as follows:
Phase I (sprouting stage): the seed embryo begins to expand and prominent gradually breaking in the seed coat, and becomes a macroscopic minimum white bead;
Second stage (ball stem stage): white bottom set is grown up rapidly, in about 2-3 week, can grow up to the ball stem of the about 2-3mm of diameter and the white pubescence of top thin life;
Phase III (differential period): when the ball stem grows to the 3mm left and right sides, the beginning differentiate root gradually, the 2-3 bar does not wait, after gradually the differentiation sprout.
A yellow segment meristem zone can be obviously observed the tip of a root and is in quadravalence section (vegetative stage) root and bud ramp.
2, germination rate statistics
Germination rate such as the table 4 and shown in Figure 3 of different medium under different condition.
The germination rate (%) of table 4, different medium and different vaccination Da Hua in period lady's slipper seed
| Numbering | Medium | The 4th week | The 5th week | The 6th week | The 7th week | The 8th week | The 9th week | The 10th week | The 11st week | The 12nd week |
| 1 | A | 0.00 | 1.23 | 8.44 | 12.60 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| 2 | B | 0.00 | 1.25 | 9.08 | 10.86 | 2.80 | 0.08 | 0.00 | 0.00 | 0.00 |
| 3 | C | 0.00 | 7.07 | 12.32 | 23.05 | 15.63 | 1.58 | 0.43 | 0.00 | 0.00 |
| 4 | D | 0.00 | 10.35 | 28.39 | 30.83 | 37.67 | 0.00 | 2.20 | 0.00 | 0.00 |
| 5 | E | 0.00 | 15.14 | 25.16 | 27.09 | 31.13 | 4.70 | 1.38 | 0.00 | 0.00 |
| 6 | F | 0.00 | 32.49 | 39.43 | 60.54 | 48.81 | 8.10 | 5.14 | 0.00 | 0.00 |
The germination rate statistics shows that Baihua Shan Mountain Da Hua lady's slipper pollination back does not all have seed germination before the 4th week and after 11 weeks.The germination rate in the 5th thoughtful the 8th week is higher relatively, reaches peak value in the 7th week mostly, and a tangible rapid drawdown was all being arranged in the 9th week, therefore, is the seed collecting period of seed cultured in vitro the best when embryonic development to the globular embryo stage.
The result of inoculation period and the research of germination rate relation matches in the experiment of preferred seed collecting period and this sprouting shown in the Da Hua lady's slipper seed development process paraffin section.From the existence of theory and practice two aspects proofs Da Hua lady's slipper seed cultured in vitro seed collecting window phase, a lot of in the past Da Hua lady's slipper seeds exsomatize and sprout the failure of an experiment, even the reason that seed all can not be sprouted is crossed late relevant with the seed collection sowing time mostly.
Different medium to prematurity Da Hua lady's slipper seed cultured in vitro germination rate to influence the result as follows:
Da Hua lady's slipper seed has been grown certain phase at embryo, has the sprouting ability, and the also not period of dominate, the i.e. comparatively suitable sowing of 5-8 week of inhibiting factor.In this developmental stage, Da Hua lady's slipper seed is wide in range relatively to the selection of medium, also can sprout on the most frequently used MS (culture medium A, B) in tissue culture, and germination rate is the highest can be more than 10%; When adding the 6-BA (culture medium C) of 2.0mg/L in the MS medium, germination rate significantly improves, and can reach 23.05%; When cultivating with the medium improvement VW (medium D) that contains organic nitrogen compositions such as amino acid, germination rate reaches as high as 37.67%; When adding the 6-BA (medium F) of 2.0mg/L in improvement VW medium, germination rate also is significantly increased, and reaches as high as 60.54%; The 6-BA that 0.5-2.0mg/L is described can effectively promote the stripped sprouting of Da Hua lady's slipper immature seed.The effect that adds coconut palm breast in the nutrients is better than potato and banana.
Claims (8)
1, a kind of method that makes Da Hua lady's slipper seed germination is to choose kind of embryonic development to spheroidal embryo period but the Da Hua lady's slipper seed that secundine does not begin to form before the dry film is cultivated the seed that obtains sprouting.
2, method according to claim 1 is characterized in that: described cultivation is carried out under dark condition.
3, method according to claim 1 and 2 is characterized in that: the temperature of described cultivation is 18-22 ℃, is preferably 20 ℃.
4, according to claim 1,2 or 3 described methods, it is characterized in that: the used medium of described cultivation is improvement VW+ (0.5mg/L-2.0mg/L) 6-BA+ (50ml/L-200ml/L) nutrients, and described nutrients is coconut palm breast, murphy juice or bananas juice; Be preferably improvement VW+2.0mg/L 6-BA+100ml/L coconut palm breast.
5, according to claim 1,2 or 3 described methods, it is characterized in that: the used medium of described cultivation is improvement VW+ (50ml/L-200ml/L) nutrients, and described nutrients is coconut palm breast, murphy juice or bananas juice; Be preferably improvement VW+100ml/L coconut palm breast.
6, according to claim 1,2 or 3 described methods, it is characterized in that: the used medium of described cultivation is MS+ (0.5mg/L-2.0mg/L) 6-BA+ (50ml/L-200ml/L) nutrients, and described nutrients is coconut palm breast, murphy juice or bananas juice; Be preferably MS+2.0mg/L 6-BA+100ml/L coconut palm breast.
7, according to claim 1,2 or 3 described methods, it is characterized in that: the used medium of described cultivation is MS+ (50ml/L-200ml/L) nutrients, and described nutrients is coconut palm breast, murphy juice or bananas juice; Be preferably MS+100ml/L coconut palm breast.
8, according to arbitrary described method among the claim 5-7, it is characterized in that: the pH value of described medium is 5.6-5.8.
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| CN102630464B (en) * | 2012-04-23 | 2013-04-24 | 中国科学院昆明植物研究所 | Method for reproducing paphiopedilum armeniacum |
| CN104756643A (en) * | 2015-04-27 | 2015-07-08 | 湖南省农业生物技术研究中心 | Method for studying influences of environment factors on polypogon seed germination |
| CN105638028A (en) * | 2016-02-02 | 2016-06-08 | 广西壮族自治区农业科学院生物技术研究所 | Method for promoting maturation of ottelia acuminate seeds |
| CN105638028B (en) * | 2016-02-02 | 2018-05-15 | 广西壮族自治区农业科学院生物技术研究所 | A kind of method for promoting edible seaweed flower seed maturation |
| CN106577284A (en) * | 2016-12-15 | 2017-04-26 | 四川省自然资源科学研究院 | Method for tissue culture, rapid propagation and flowering time regulation of cypripedium flavum |
| CN110786235A (en) * | 2018-08-02 | 2020-02-14 | 北京林业大学 | Method for aseptically germinating cypripedium guttatum seeds |
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| CN101536629B (en) | 2011-07-27 |
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