CN101720667B - Tissue culture rapid propagation method for crocus - Google Patents
Tissue culture rapid propagation method for crocus Download PDFInfo
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- CN101720667B CN101720667B CN2008102010214A CN200810201021A CN101720667B CN 101720667 B CN101720667 B CN 101720667B CN 2008102010214 A CN2008102010214 A CN 2008102010214A CN 200810201021 A CN200810201021 A CN 200810201021A CN 101720667 B CN101720667 B CN 101720667B
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Abstract
The invention discloses a tissue culture rapid propagation method for crocus. The method comprises the steps of explant sterilization, callus induction, clustered shoot induction culture, aventitious shoot subculture, corm induction culture and the like so as to finally obtain the induced corm. The invention adopts the tissue culture method to perform the callus induction and corm induction culture of crocus, establish the reproductive system of crocus superior varieties and perform industrial seedling production of seedlings and can fast propagate the superior varieties in large.
Description
Technical field
The present invention relates to tissue culture and the propagation method of plant, be specifically related to the quick breeding method for tissue culture of Crocus sativus.
Background technology
Crocus sativus (Crocus sativus L) has another name called safflower, west safflower, is Iridaceae (Iridaceae) crocus herbaceos perennial.Originate in ground such as various countries, southern Europe and Iran, after India changes Tibet over to and imports China again into, the introducing and planting success is so have another name called safflower or west safflower.Because Crocus sativus is that column cap is used as medicine, output is extremely low, the time and effort consuming of gathering; Simultaneously provenance is few, and factors such as the suitable cultivation area is limited, condition harshness cause it to cost an arm and a leg, and is being classified as rare rare traditional Chinese medicine, and is described as " plant gold ".The pistil stigma of Crocus sativus is a rare Chinese medicine, has the effect of promoting blood circulation and removing blood stasis, removing pattogenic heat from the blood and toxic material from the body, resolving stagnation for tranquilization, is traditional gynaecology, traumatology good medicine.Also be mainly used in the treatment of stomach trouble, menstruation regulating, measles, heating, hepatosplenomegaly etc. clinically.Especially in recent years obtaining curative effect preferably aspect the treatment cardiovascular and cerebrovascular disease.
Because Crocus sativus can not be carried out sexual reproduction, can only carry out vegetative propagation through bulb, and its bulb degradation phenomena is serious under the cultivation condition, causes the germ plasm resource famine.China is since introducing a fine variety, and cultivation Crocus sativus output is lower always, can not satisfy needs of people far away.Tissue culture technique has obtained using widely on medicinal plant in recent years, and this preservation and production for the relatively poor rare Chinese medicine medicine resource of fertility provides new approach.Utilize the method for biotechnology, tissue culture to carry out seedling production, on a lot of plants, become the commodity production of batch production, and brought abundant economic benefit.
Therefore, adopting the method for tissue culture, carry out the Crocus sativus callus induction, the inducing and the regeneration of bulb of the bud of growing thickly, is the approach of exploring fast, obtain in a large number, constantly effective cormel.Utilize the method for tissue culture to set up the breeding system of major clique Crocus sativus, carry out the factorial seedling growth production of seedling, have the advantages of a large amount of fast breeding major clique kinds, can solve the germ plasm resource quality problems of Crocus sativus.In order to explore the approach that enlarges provenance, adopt tissue culture technique to carry out Crocus sativus callus and bottom set and induce and study, in the hope of establishing certain basis for the preservation of its germ plasm resource and utilization.
Summary of the invention
Technical problem to be solved by this invention is that research is set up the aseptic fast traditional font of Crocus sativus and is, and improves the rate of increase, suitable for mass production high quality seedling.
The present invention provides a kind of Crocus sativus quick breeding method for tissue culture.
The present invention utilizes the method for tissue culture, has carried out Crocus sativus callus and bottom set and has induced, and sets up the breeding system of major clique Crocus sativus, carries out the factorial seedling growth production of seedling, a large amount of fast breeding major clique kinds.
The method of the invention comprises the following steps:
(1) explant sterilization: bulb is washed, remove epithelium, place sterilization inoculation on the superclean bench; Prior to rinsing 30-40 second in 70% alcohol, use 0.1%HgCl again
2Sterilized 8-10 minute, and used aseptic water washing at last 4～5 times, thoroughly the remaining HgCl of flush away
2, the bulb after the sterilization is cut into 1-1.5cm
3, be inoculated on the medium;
(2) callus induction: will pass through during above-mentioned bulb behind the sterilization is inoculated on the inducing culture MS+0.5mg/L NAA+5.0mg/L6-BA medium in gnotobasis, and cultivate 20 days results callus;
(3) inducing clumping bud is cultivated: callus is inoculated on the MS+0.5mg/L NAA+2.0mg/L6-BA medium, and under dark condition (the unglazed photograph of 24h), 20 ℃ of cultivations can induce the bud of growing thickly in a large number after 20 days;
(4) indefinite bud successive transfer culture: the bud of will growing thickly is transferred on the MS+0.5mg/LNAA+2.0mg/L6-BA medium after cutting into single bud, under dark condition, and 20 ± 2 ℃ of cultivations, bud is long to 2-3cm after 15 days;
(5) bulb inducing culture: when the high 2-3cm of bud, be transferred to MS+0.5mg/L NAA+3.0mg/L6-BA medium, (light application time 12h/d induces bulb under the intensity of illumination 1500～20001x) at illumination condition.The said medium MS of the inventive method, NAA and 6-BA all can obtain from commercially available.
The inventive method is based on following experimental study, and its result is following:
(1) method:
(1) explant sterilization: bulb is washed, remove epithelium, place sterilization inoculation on the superclean bench; Prior to rinsing 30-40 second in 70% alcohol, use 0.1%HgCl again
2Sterilized 8-10 minute, and used aseptic water washing at last 4～5 times, thoroughly the remaining HgCl of flush away
2, the bulb after the sterilization is cut into 1-1.5cm
3, be inoculated on the medium;
(2) callus induction: will pass through during above-mentioned bulb behind the sterilization is inoculated on the inducing culture MS+0.5mg/L NAA+5.0mg/L6-BA medium in gnotobasis, and cultivate down at dark condition (the unglazed photograph of 24h); MS, B have been investigated
5, three kinds of different medium of White are to the influence of Crocus sativus callus induction rate, different plant growth regulator and illumination condition are to the influence of Crocus sativus callus induction and growth.
(3) inducing clumping bud is cultivated: callus is inoculated on the MS medium of variable concentrations hormone combination, is used for induced bundle and sprouts; Investigate the influence that illumination and temperature condition are sprouted to induced bundle.
(4) indefinite bud successive transfer culture: the MS medium that is transferred to the variable concentrations hormone combination after the bud of will the growing thickly cutting
In, carry out the bud proliferation experiment.Investigate the influence of illumination and temperature condition to adventitious bud proliferation.
(5) bulb inducing culture: when the high 2-3cm of bud, carry out bulb and induce experiment.The seedling of will growing thickly is transferred in the MS medium of variable concentrations hormone combination, under illumination and dark condition, induces bulb respectively.
(2) result:
1. explant sterilization: sterilization time is short, and explant edge brownization degree is light, but pollution rate is higher; Sterilization time is oversize, and pollution rate is lower, but explant can be by calcination, and great majority can not form callus.Saffron corm Disinfection Effect through the sterilization of step 1 method is good, can form callus.
2. callus induction: MS is suitable evoked callus medium (seeing table 1), in the variable concentrations hormone combination, with MS+0.5mg/LNAA (methyl)+5.0mg/L6-BA (6-benzyl aminoadenine) be advisable (seeing table 2).(the unglazed photograph of 24h) helps callus Growth (seeing table 3) under the dark condition.
The different medium of table 1 are to the influence of callus induction
The different hormone combinations of table 2 are to the influence of callus growth
Table 3 illumination condition is to the influence of callus induction
3. inducing clumping bud is cultivated: through 30 days cultivation, and callus differentiation and bud formation in the part medium.Wherein, shorter with the time that the MS+0.5mg/LNAA+2.0mg/L6-BA medium sprouts, and growth rate is very fast.Callus differentiates budlet, and the young shoot number increases gradually and forms the bud clump.Carried out illumination condition to the inducing of the bud of growing thickly, the result shows under the dark condition can induce the bud of growing thickly in a large number, and illumination condition is unfavorable for the callus differentiation and bud formation.The too high induced bundle that is unfavorable for of temperature is sprouted, and preference temperature is 20 ± 2 ℃.
4. indefinite bud successive transfer culture: on the MS medium of 2.0mg/L6-BA (6-benzyl aminoadenine), change the concentration of NAA, bigger to the shoot proliferation influence of young shoot.When not adding NAA (methyl), the clump bud number of formation is less, but formed young shoot internode is longer, and subculture once more easily.NAA concentration is during greater than 1.0mg/L, and the clump bud number of formation is more, and the more thin and weak subculture once more that is unfavorable for of bud, and NAA concentration is big more, and formed young shoot is short more, and height of seedling also reduces gradually, and the callus lines that base portion forms increases gradually.Wherein still with MS+0.5mg/LNAA+2.0mg/L6-BA medium bud cultivation effect for well.Carried out the propagation of illumination condition to the bud of growing thickly, the result shows that the dark condition bud of growing thickly can breed, and illumination condition is unfavorable for adventitious buds proliferation.The too high adventitious buds proliferation that is unfavorable for of temperature, preference temperature is 20 ± 2 ℃.
5. bulb inducing culture: the seedling of will growing thickly is transferred in the MS medium of variable concentrations hormone combination, and it is short that the MS+0.5mg/LNAA+3.0mg/L6-BA medium induces the bulb required time, and the bulb growth is fast.Can not induce bulb under the dark condition, after 40 days under the illumination condition bottom set inductivity of indefinite bud can reach 65.2%.
Influence the fast numerous factor of Crocus sativus:
Hormone concentration: the tissue culture key of success is culture condition, and wherein the concentration of the growth hormone and the basic element of cell division is wherein very important factor.
Illumination condition: all should under dark condition, carry out at Crocus sativus callus induction, inducing clumping bud and bud enrichment culture, induce bulb under illumination condition, to carry out.
Temperature effect: the annual growth in April November to next year of Crocus sativus, May is to the dormancy in October.In the group training, temperature is too high, and the growing way of seedling is poor, and with brownization generation.During 20 ℃ of left and right sides, the seedling growth is vigorous.In the time of more than 30 ℃, seedling is can brownization dead, and this is consistent with Crocus sativus field growing habit, and the cultivation Crocus sativus also shows as entering in the May resting state of withering.
Embodiment
Below according to embodiment to further explain of the present invention
Embodiment 1
(1) is explant with the bulb of uncivilized nation's safflower then, bulb is rinsed well in running water, remove epithelium, placing sterilization inoculation on the superclean bench.Prior to rinsing 30s in 70% alcohol, use 0.1%HgCl again
2Sterilization 8-10min uses aseptic water washing 5 times at last, thoroughly the remaining HgCl of flush away
2
(2) bulb after will sterilizing is cut into 1cm
3Size is inoculated on the MS+0.5mg/L NAA+5.0mg/L6-BA medium, cultivates down at dark condition (24 hours unglazed photographs) and can gather in the crops callus in 20 days.Callus is long-term subculture with this understanding, not brownization.
(3) callus is transferred on the MS+0.5mg/L NAA+2.0mg/L6-BA medium, under dark condition, 20 ℃ of cultivations can induce the bud of growing thickly in a large number after 20 days.
(4) will grow thickly and be transferred on the MS+0.5mg/L NAA+2.0mg/L6-BA medium after bud cuts into single bud, under dark condition (24 hours unglazed photographs), 20 ℃ of cultivations, bud is long to 2cm after 15 days.
(5) be transferred to MS+0.5mg/L NAA+3.0mg/L6-BA medium, under illumination condition (light application time 12 hours/day, intensity of illumination 15001x), induce bulb.
Claims (3)
1. a Crocus sativus quick breeding method for tissue culture is characterized in that this method comprises the following steps:
(1) explant sterilization: bulb is washed, remove epithelium, place sterilization inoculation on the superclean bench; Prior to rinsing 30-40 second in 70% alcohol, with 0.1% HgCl2 sterilization 8-10 minute, use aseptic water washing at last 4～5 times again, thoroughly the remaining HgCl of flush away
2, the bulb after the sterilization is cut into 1-1.5cm
3, be inoculated on the medium;
(2) callus induction: will pass through during above-mentioned bulb behind the sterilization is inoculated on the inducing culture MS+0.5mg/L NAA+5.0mg/L 6-BA medium in gnotobasis, and under dark condition, cultivate 20 days results callus;
(3) inducing clumping bud is cultivated: callus is inoculated on the MS+0.5mg/L NAA+2.0mg/L 6-BA medium, and 20 ℃ of cultivations induced the bud of growing thickly under dark condition in 20 days;
(4) indefinite bud successive transfer culture: the bud of will growing thickly is transferred on the MS+0.5mg/L NAA+2.0mg/L 6-BA medium after cutting into single bud, under 24 hours unglazed photographs of dark condition, and 20 ± 2 ℃ of cultivations, bud is long to 2-3cm after 15 days;
(5) bulb inducing culture: when the high 2-3cm of bud, be transferred to MS+0.5mg/L NAA+3.0mg/L 6-BA medium, under illumination condition, induce bulb.
2. a kind of Crocus sativus quick breeding method for tissue culture according to claim 1, the dark condition that it is characterized in that said step (2), (3) and (4) is 24 hours unglazed photographs.
3. a kind of Crocus sativus quick breeding method for tissue culture according to claim 1, the illumination condition that it is characterized in that said step (5) is light application time 12h/d, intensity of illumination 1500～2000 lx.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103875432B (en) * | 2014-04-09 | 2016-02-24 | 南京工业大学大丰海洋产业研究院 | The method that son plants ball is prepared in the stripping and slicing of low temperature treatment safflower |
| CN104855289B (en) * | 2015-05-21 | 2017-03-22 | 江苏丰收大地种业发展有限公司 | Method for culturing and producing micro-bulbodium of crocus sativus L. through superficial layer |
| CN105359973B (en) * | 2015-11-25 | 2017-10-27 | 浙江大学 | The cultivation of the detoxicating cuvette bulb of No. 1 west safflower of sarranine |
| CN105519436A (en) * | 2016-01-09 | 2016-04-27 | 佛山市金蓝领教育科技有限公司 | Culture medium and induction method for improving saffron callus induction rate |
| CN105660394A (en) * | 2016-01-09 | 2016-06-15 | 佛山市金蓝领教育科技有限公司 | Strong seedling medium for improving saffron transplanting survival rate |
| CN108419679B (en) * | 2018-05-31 | 2020-10-09 | 浙江大学 | Tissue culture method of saffron |
| CN115554288B (en) * | 2022-04-28 | 2024-01-26 | 江苏省中国科学院植物研究所 | Rapid propagation method of crocus sativus corms |
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| CN1954668A (en) * | 2005-10-25 | 2007-05-02 | 中国科学院过程工程研究所 | Scale tissue culture quickly reproducing method of saffron crocus seedball (sprout) |
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| CN1954668A (en) * | 2005-10-25 | 2007-05-02 | 中国科学院过程工程研究所 | Scale tissue culture quickly reproducing method of saffron crocus seedball (sprout) |
Non-Patent Citations (3)
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| 何 凯等.藏红花球茎诱导丛生芽及球茎再生.《四川大学学报(自然科学版)》.2002,第39 卷(第6 期),摘要. * |
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