CN106613981A - Paeonia rockii primary culture callus induction method - Google Patents
Paeonia rockii primary culture callus induction method Download PDFInfo
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- CN106613981A CN106613981A CN201611213363.9A CN201611213363A CN106613981A CN 106613981 A CN106613981 A CN 106613981A CN 201611213363 A CN201611213363 A CN 201611213363A CN 106613981 A CN106613981 A CN 106613981A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a paeonia rockii primary culture callus induction method. The paeonia rockii primary culture callus induction method comprises the following steps: (1) explant selection: selecting bulbel of paeonia rockii as an explant, washing, and pre-cooling for 24 to 48 hours; (2) sterilizing and inoculating: stripping bud scales off from the bulbel obtained in step (1) under an aseptic condition, sterilizing, and then inoculating the bulbel to a callus induction culture medium; (3) callus induction culture: putting the inoculated explant in a culture room for culturing, and culturing for 7 to 10 days by using red light. Compared with the prior art, the paeonia rockii primary culture callus induction method disclosed by the invention has the advantages that an explant pre-cooling method is adopted, specific culture medium components are combined, a red-light culture method is finally adopted, the browning rate of the paeonia rockii can be effectively reduced to be 0, no browning phenomenon happens, the callus induction rate is increased to be 100 percent, and cluster buds which are large in quantity and good in quality can be finally obtained, so that the requirements of multiplication can be met, and the establishment of a rapid propagation system is promoted.
Description
Technical field
The present invention relates to a kind of Paeonia papaveracea Initial culture callus induction method, belongs to tree peony reproduction technique field.
Background technology
' purple plague purpura ' tree peony be wild species Paeonia ostii production in commonly used cultivar.' purple plague purpura ' tree peony not only has
View and admire well and pharmacologic action, also higher oil value.In recent years, related investigation finds that ' purple plague purpura ' tree peony is alternatively arranged as
Woody grain and oil plant resources is developed and utilized, and oil is 144 pounds/acre with the general oil production of tree peony, significantly larger than sesame, oil tea etc.
Oil production.Containing abundant unrighted acid in ' purple plague purpura ' peony seed oil, leukotrienes, linoleic plus oleic acid, wherein, α-Asia
Numb acid content is up to 42.34%～49%.Alpha-linolenic acid is to constitute human body brain cell and histiocytic important component, is human body
It is indispensable itself to synthesize and irreplaceable polyunsaturated fatty acid, have again " hemotrophic nutrition element ", " vitaminF " and
The title of " plant docosapentaenoic acid ", has antitumor, anti-inflammatory, improves the functions such as cardiovascular and regulation immunity.Peony seed oil many index is all
Exceed with high-grade famous olive oil, be genuine high-end edible oil.
Tree peony generative propagation coefficient is big, but seedling variation is big, it is impossible to preferably preserve maternal merit, and seed
The permeable and gas permeability of kind skin also affect the epicotyl of the sprouting of seed, seed also to take root with dormancy, i.e. autumn, the spring
Season just germinates, and seed sprouts also irregular, there is in several years of Some seeds always resting state.Thus adopt tissue to train
Foster technology breeding branded oil becomes important stock breeding approach with tree peony.
But at present due to using easily browning during bulbil callus induction, causing callus induction rate low.
The content of the invention
Goal of the invention：In order to solve above-mentioned technical problem, the invention provides a kind of Paeonia papaveracea Initial culture callus group
Knit abductive approach.
Technical scheme：In order to realize foregoing invention purpose, the invention discloses a kind of Paeonia papaveracea Initial culture callus group
Abductive approach is knitted, is comprised the following steps：
(1) explant is selected：Select Paeonia papaveracea bulbil to be explant, clean, precooling 24-48 hours；
(2) sterilize and be inoculated with：Aseptically, step (1) gained bulbil is peelled off into scale, is sterilized, be then inoculated in
On callus inducing medium；
(3) induction of callus：Postvaccinal explant is placed in culturing room and is cultivated, ruddiness culture 7-10 days.
It is preferred that, the Paeonia papaveracea uses tree peony for " purple plague purpura " oil.
It is preferred that, cleaning method is in the step (1)：Soaked 2 hours with washing powder solution, then gently scrub bulbil appearance
Face, is then placed under running water flowing water and rinses 24 hours.
It is preferred that, the precooling temperature in the step (1) is 0-4 DEG C.
It is preferred that, sterilization method is in the step (2)：First with 75% alcohol disinfecting 20-30 seconds, then disappeared with 0.1% mercuric chloride
Malicious 10-15 minutes, then with aseptic water washing 5-6 time.
It is preferred that, in the step (2) basal medium of culture medium be MS culture mediums, wherein 6-BA1-2mg/L,
NAA0.3-0.5mg/L, silver nitrate 4-5mg/L, 7g/L agar, 30g/L sucrose, pH is adjusted to 5.8.
It is preferred that, the temperature in the step (3) during culture is 2 DEG C of 25 scholar.
Technique effect：Relative to the method that prior art, the present invention adopt explant precooling, and combine specific culture
Based component, finally again using the method for ruddiness culture, it is 0 that can effectively reduce Paeonia papaveracea melting brown rate, is occurred without browning,
It is 100% to improve callus induction rate, can finally obtain that quantity is more, the measured Multiple Buds of matter, so as to meet shoot proliferation
Need, promote the foundation of rapid propagation system.
Specific embodiment
Embodiment 1
1st, explant is selected：Select " purple plague purpura " oil to be explant with tree peony bulbil, soaked 2 hours with washing powder solution, then
Banister brush gently scrubs bulbil outer surface, is then placed under flowing water and rinses 24 hours, is then wrapped with gauze and is placed in refrigerator
(0-4 DEG C) precooling 24 hours.
2nd, explant sterilization and inoculation：Explant is gone on aseptic operating platform, bulbil is peelled off into scale, first use 75% wine
Essence sterilization 20 seconds, then with 0.1% mercuric chloride sterilize 10 minutes, then with aseptic water washing 5 times after, be inoculated in induction of callus
On base, per vial a stem section is connect.Callus inducing medium composition be MS culture mediums, 6-BA1mg/L, NAA0.3mg/
L, silver nitrate 4mg/L, 7g/L agar, 30g/L sucrose；Medium pH is adjusted to 5.8.
3rd, induction of callus：Postvaccinal explant is placed under culturing room's suitable condition and is cultivated.Condition is temperature
2 DEG C of 25 scholar is spent, ruddiness culture is carried out 7 days.
4th, result：Callus induction rate 100%, melting brown rate is Differentiation ration of adventitious buds after 0,20 days：85%, and adventitious bud
Robust growth.
Embodiment 2
1st, explant is selected：Select " purple plague purpura " oil to be explant with tree peony bulbil, soaked 2 hours with washing powder solution, then
Banister brush gently scrubs bulbil outer surface, is then placed under flowing water and rinses 24 hours, is then wrapped with gauze and is placed in refrigerator
(0-4 DEG C) precooling 48 hours.
2nd, explant sterilization and inoculation：Explant is gone on aseptic operating platform, bulbil is peelled off into scale, first use 75% wine
Essence sterilization 30 seconds, then with 0.1% mercuric chloride sterilize 15 minutes, then with aseptic water washing 5 times after, be inoculated in induction of callus
On base, per vial a stem section is connect.Callus inducing medium composition be MS culture mediums, 6-BA2mg/L, NAA0.3mg/
L, silver nitrate 5mg/L, 7g/L agar, 30g/L sucrose；Medium pH is adjusted to 5.8
3rd, induction of callus：Postvaccinal explant is placed under culturing room's suitable condition and is cultivated.Condition is temperature
2 DEG C of 25 scholar is spent, ruddiness culture is carried out 10 days.
4th, result：Callus induction rate 100%, melting brown rate is Differentiation ration of adventitious buds after 0,20 days:88%, and adventitious bud
Robust growth.
Embodiment 3
1st, explant is selected：Select " purple plague purpura " oil to be explant with tree peony bulbil, soaked 2 hours with washing powder solution, then
Banister brush gently scrubs bulbil outer surface, is then placed under flowing water and rinses 24 hours, is then wrapped with gauze and is placed in refrigerator
(0-4 DEG C) precooling 36 hours.
2nd, explant sterilization and inoculation：Explant is gone on aseptic operating platform, bulbil is peelled off into scale, first use 75% wine
Essence sterilization 25 seconds, then with 0.1% mercuric chloride sterilize 12 minutes, then with aseptic water washing 5 times after, be inoculated in induction of callus
On base, per vial a stem section is connect.Callus inducing medium composition be MS culture mediums, 6-BA1.5mg/L,
NAA0.4mg/L, silver nitrate 4.5mg/L, 7g/L agar, 30g/L sucrose；Medium pH is adjusted to 5.8
3rd, induction of callus：Postvaccinal explant is placed under culturing room's suitable condition and is cultivated.Condition is temperature
2 DEG C of 25 scholar is spent, ruddiness culture is carried out 9 days.
4th, result：Callus induction rate 100%, melting brown rate is Differentiation ration of adventitious buds after 0,20 days:87%, and adventitious bud
Robust growth.
Reference examples 1
1st, explant is selected：Select " purple plague purpura " oil to be explant with tree peony bulbil, soaked 2 hours with washing powder solution, then
Banister brush gently scrubs bulbil outer surface, is then placed under flowing water and rinses 24 hours, is then wrapped with gauze and is placed in refrigerator
(0-4 DEG C) precooling 12 hours.
2nd, explant sterilization and inoculation：Explant is gone on aseptic operating platform, bulbil is peelled off into scale, first use 75% wine
Essence sterilization 30 seconds, then with 0.1% mercuric chloride sterilize 15 minutes, then with aseptic water washing 5 times after, be inoculated in induction of callus
On base, per vial a stem section is connect.Callus inducing medium composition be MS culture mediums, 6-BA2mg/L, NAA0.3mg/
L, silver nitrate 5mg/L, 7g/L agar, 30g/L sucrose；Medium pH is adjusted to 5.8.
3rd, induction of callus：Postvaccinal explant is placed under culturing room's suitable condition and is cultivated.Condition is temperature
2 DEG C of 25 scholar is spent, white light culture is carried out 10 days.
4th, result：Callus induction rate 65.2%, melting brown rate is 24.8%, Differentiation ration of adventitious buds after 20 days：70.6%,
And adventitious bud growth is thin and weak.
Reference examples 2：
It is identical with the method for embodiment 2, the difference is that only：Not comprising the precooling step in step 1, cultivation results are such as
Under：Callus induction rate 63.8%, melting brown rate is 25.9%, Differentiation ration of adventitious buds after 20 days：68.5%, and adventitious bud growth
It is thin and weak.
Reference examples 3：
It is identical with the method for embodiment 2, the difference is that only：Silver nitrate is not included in culture medium, cultivation results are as follows：More
Injured tissue inductivity 64.7%, melting brown rate is 26.2%, Differentiation ration of adventitious buds after 20 days：69.4%, and adventitious bud growth is thin and weak.
Claims (7)
1. a kind of Paeonia papaveracea Initial culture callus induction method, it is characterised in that comprise the following steps：
(1) explant is selected：Select Paeonia papaveracea bulbil to be explant, clean, precooling 24-48 hours；
(2) sterilize and be inoculated with：Aseptically, step (1) gained bulbil is peelled off into scale, is sterilized, be then inoculated in callus
On tissue inducing culture；
(3) induction of callus：Postvaccinal explant is placed in culturing room and is cultivated, ruddiness culture 7-10 days.
2. Paeonia papaveracea Initial culture callus induction method according to claim 1, it is characterised in that the purple plague purpura
Tree peony uses tree peony for " purple plague purpura " oil.
3. Paeonia papaveracea Initial culture callus induction method according to claim 1, it is characterised in that the step
(1) cleaning method is in：Soaked 2 hours with washing powder solution, then gently scrub bulbil outer surface, be then placed on running water flowing water
It is lower to rinse 24 hours.
4. Paeonia papaveracea Initial culture callus induction method according to claim 1, it is characterised in that the step
(1) precooling temperature in is 0-4 DEG C.
5. Paeonia papaveracea Initial culture callus induction method according to claim 1, it is characterised in that the step
(2) sterilization method is in：First with 75% alcohol disinfecting 20-30 seconds, then sterilized 10-15 minutes with 0.1% mercuric chloride, then use sterilized water
Rinse 5-6 time.
6. Paeonia papaveracea Initial culture callus induction method according to claim 1, it is characterised in that the step
(2) in the basal medium of culture medium be MS culture mediums, wherein 6-BA1-2mg/L, NAA0.3-0.5mg/L, silver nitrate 4-5mg/
L, 7g/L agar, 30g/L sucrose, pH is adjusted to 5.8.
7. Paeonia papaveracea Initial culture callus induction method according to claim 1, it is characterised in that the step
(3) temperature in during culture is 2 DEG C of 25 scholar.
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| CN201611213363.9A CN106613981A (en) | 2016-12-23 | 2016-12-23 | Paeonia rockii primary culture callus induction method |
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| CN201611213363.9A CN106613981A (en) | 2016-12-23 | 2016-12-23 | Paeonia rockii primary culture callus induction method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107593446A (en) * | 2017-10-19 | 2018-01-19 | 寿县正阳油用牡丹种植专业合作社 | A kind of fast breeding method of the induction and regeneration of tree peony |
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| CN101548647A (en) * | 2009-05-11 | 2009-10-07 | 中国林业科学研究院林业研究所 | Tree peony callus induction method |
| CN102144563A (en) * | 2009-05-11 | 2011-08-10 | 中国林业科学研究院林业研究所 | Method for inducing calluses of peony anther |
| CN105075863A (en) * | 2015-08-10 | 2015-11-25 | 甘肃农业大学 | Rapid paeonia rockii reproduction method |
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2016
- 2016-12-23 CN CN201611213363.9A patent/CN106613981A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548647A (en) * | 2009-05-11 | 2009-10-07 | 中国林业科学研究院林业研究所 | Tree peony callus induction method |
| CN102144563A (en) * | 2009-05-11 | 2011-08-10 | 中国林业科学研究院林业研究所 | Method for inducing calluses of peony anther |
| CN105075863A (en) * | 2015-08-10 | 2015-11-25 | 甘肃农业大学 | Rapid paeonia rockii reproduction method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107593446A (en) * | 2017-10-19 | 2018-01-19 | 寿县正阳油用牡丹种植专业合作社 | A kind of fast breeding method of the induction and regeneration of tree peony |
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