CN1806512A - E. tirucalli cuttage propagation and tissue-culturing quick-propagation method - Google Patents
E. tirucalli cuttage propagation and tissue-culturing quick-propagation method Download PDFInfo
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Abstract
The invention provides a method for breeding emerald tree through cutting and grafting, comprising: choosing grafting material and preparing cutting seed bed base material, pre-treating grafting material with hormone, replanting grafting material into cutting seed bed base material, and seedling stage managing; immersing grafting material in root-growing powder, IBA or NAA, and then for cultivating process for 25-30 days with root growth rate being over 80%. The invention also provides a method for preparing large quantity of complete strain of emerald tree through tissue culture and fast breeding technology, comprising: pre-treating or not treating emerald explant, inducing explant in differentiation culture medium to differentiate adventitious bud, inducing adventitious bud in clump bud breeding culture medium for clump bud, inducing clump growing root in root growing culture medium to get germ-free tube sprout, training sprout and replanting tube sprout. Adding ZT, 6-BA, NAA, IBA or GA3 with different concentration according to different stage. The tissue culture time is short, the k-factor of bud is over 4.5 and root growth rate is over 95%.
Description
One, technical field
The invention belongs to technical field of bioengineering, the production method that relates to a kind of pencil tree micropropagation of plants seedling, specifically provide a kind of method, and a kind of method that obtains a large amount of pencil tree whole plants by tissue culture rapid propagation technique is provided by a large amount of whole plant of cottage propagation acquisition pencil tree
Two, background technology
Pencil tree (Euphorbia tirucalli) is the Euphorbiaceae euphorbia, has another name called the rubber root of Beijing euphorbia, green coral, Sapium japonicum; Be upright stingless shrub or dungarunga, high 2～10m; Be integrate medicinal, energy supply, view and admire, the raw material of industry, environmental greening, water and soil conservation, multi-functional, multipurpose seeds with the energy, ecology, economical synthesis benefit.Ground such as China Hainan, Guangzhou, Xishuangbanna are as the fragmentary introducing and planting of flower garden ornamental tree species (Zang Runguo, " spike mountain range, Hainan Island arboretum is mainly constructed type tropical tree register ", forest-science, 2002 years, Vol38 (1)).The pencil tree alcohol that from pencil tree, extracts in stem, leaf, root and the milk, have clearing heat and detoxicating, swelling and pain relieving and stronger discharge function (Li Tongqin, " discussion of euphorbia plant medicinal history evolution and value ", traditional Chinese medicine academic periodical, 2003, Vol21 (8)).In addition, the juice constituent class of pencil tree secretion is similar to diesel oil, need not or a little the worker can become biodiesel, enjoy people to pay close attention to (Kong Xianwen, " the development biomass energy can obtain many-sided benefit ", energy-conservation, 2003 the 2nd phases, total the 247th phase).At present, the medicinal and energy of pencil tree is worth just progressively to be familiar with, and beginning is carried out introducing and planting to it as the energy, protection forest afforestation seeds.
Because pencil tree is only the original producton location seeding, so other milpa mainly adopts vegetative propagation.And in vegetative propagation, cottage propagation is a kind of agricultural, forestry, cheap simple asexual reproduction method of being widely used in.The maximum characteristics of its of this method are that simple to operate, filial generation simultaneously can keep maternal good hereditary capacity.But, at present the open report of pencil tree cultured in vitro is mainly concentrated on the route of synthesis of oil body and phytosterin compound in cell and protobiont suspension culture and the cell, disclose as German publication (DE3126001A) the pencil tree cell formula that leaves standstill has been cultivated, selecting high temperature resistance or cryophylactic callus or cell aggregation, it does not relate to grow thickly inducing of bud and taking root of pencil tree.To being the both not relevant patent disclosure of vegetative propagation pencil tree cuttage of purpose with propagation, there is not the non-patent literature report yet.And for the research by the tissue-culturing quick-propagation pencil tree, Japan's publication (JP57074086A) discloses the lux (LX at 100-10000 (preferred 100-3000), illumination condition down together) is suitable for the acquisition of euphorbia callus, but also do not relate to inducing and taking root of the bud of growing thickly, so prior art can not reach the purpose of efficient quickly tissue culture breeding pencil tree.
Therefore, need provide a kind of methods that can efficiently breed high-quality pencil tree seedlings fast in a large number now.
Three, summary of the invention
First purpose of the present invention has been to provide a kind of method that obtains a large amount of whole plant of pencil tree by cottage propagation, specifically, method comprises and selects the plugged ear material and prepare cuttage seeding bed substrate, hormone preliminary treatment plugged ear material, the plugged ear material is transplanted in the process of cuttage seeding bed substrate, seedling management.
Therefore, pencil tree branch cutting propagation method comprises:
1. select the plugged ear material and prepare the cuttage seeding bed substrate
The plugged ear material is selected from the annual lignification in the open environment that carries disease germs and/or the pencil tree live body branch of semi-lignified.
The seedbed is arranged on well lighted, the place that has abundant water resources, and cutting bed length and width, moderate deeply (long 2～3 meters, wide 1～2 meter, dark 30～40 centimetres), and carry out weeds cleaning etc. in advance, matrix is by detritus soil and the river sand and the perlite of equal-volume (being 1: 1: 1 ratio of volume ratio, together following), or by isopyknic detritus soil and river sand, or by isopyknic purple soil and river sand, or by isopyknic loess and river sand, or effluent sand or detritus soil preparation separately.
2. hormone preliminary treatment plugged ear material
After choosing the pencil tree live body branch of healthy and strong carry disease germs pencil tree live body branch or healthy and strong annual lignification and/or semi-lignified, be cut into the long segment of 10～20cm, the segment base portion be immersed in handled in the hormone solution 1～12 hour with secateurs; Described hormone is selected from the root-inducing powder ABT of 0.05mg/L～0.15mg/L
6In solution or 0.1mg/L～0.2mg/L indolebutyric acid (IBA) solution or methyl (NAA) solution of 0.01mg/L～0.2, preferred hormone concentration: ABT
60.15mg/L, IBA0.1mg/L, NAA0.15mg/L.
With the cuttage of plugged ear material in the cuttage seeding bed substrate
With hormone preliminary treatment plugged ear material, with 20/m
2The density cuttage on the seedbed, carry out seedling management after permeable spraying for the first time, cuttage is after 25～30 days, cuttage rooting forms whole plant.
4. seedling management
After cuttage finishes, comprise the conventional seedling management of water spray fertilising, weeding deinsectization, pruning branch according to Changes in weather and soil regime.
Second purpose of the present invention provides a kind of method that obtains a large amount of pencil tree whole plants by tissue culture rapid propagation technique.
Therefore, the method for tissue culture quick breeding pencil tree comprises:
1. explant selection and preliminary treatment, surface sterilization, inoculation
Choose the stipes section or the stem top of live plant in the open environment that carries disease germs, after cleaning repeatedly with running water, polyethylene glycol with 15%～25% (PEG6000) soaked base portion 3～6 hours, after cleaning repeatedly with running water again, in ultra-clean or gnotobasis, handled 0～5 minute with 70% Ethanol Treatment 0～30 second or with 5%～10% hydrogen peroxide, after sterile water washes repeatedly, carry out 4～10 minutes disinfecting with 5%～10% hypochlorite solutions or 0.1%～1% mercuric chloride solution again, wash repeatedly to be used for inoculated and cultured with sterile water at last.
Perhaps, explant also can be selected from stipes section or stem top or the live body aseptic seedling in the gnotobasis, and is directly used in the inoculated and cultured.
2. the inoculated and cultured of explant---be inducing of pencil tree indefinite bud
The differentiation adventitious buds medium is meant that the top explant that can induce stipes section or stem is divided into the medium of indefinite bud, and explant just can be able to the induced bud differentiation through cultivation in 10～20 days in this medium.Detailed process is: explant is inserted in the medium cultivate, medium is the MS minimal medium, solid or semisolid, added in the gelatin of 0.7%～1.0% agar or 0.8%～2.0% or the liquid medium within and directly added solid particle (bead, perlite, materials such as vermiculite), the zeatin (ZT) of additional 0.5～1.0mg/L and the methyl (NAA) of 0.01～0.1mg/L, or the methyl (NAA) of the 6-benzylaminopurine (6-BA) of 0.1～1.0mg/L and 0.01～0.1mg/L, preferred hormone scope is 3/4MS+ZT0.5mg/L+NAA0.02mg/L and 1/2MS+6-BA 0.5mg/L+NAA0.02mg/L, inductivity is more than 80%, condition of culture is that intensity of illumination is 800～1600LX, every day, light application time was 10～16 hours, and cultivation temperature is at 18～26 degree.But through the just generation of evoking adventive bud of 10～20 days cultivations.
3. grow thickly the inducing of bud
The inducing clumping bud medium is meant the medium that can induce the indefinite bud that broken up to form the bud of growing thickly, and explant just can be able to the induced bundle generation of sprouting through 25～30 days cultivation in this medium.Detailed process is that the indefinite bud that well-grown 1～3cm is high inserts MS minimal medium (the same), the methyl (NAA) of the zeatin of 0.5～2.0mg/L (ZT), 0.01～0.1mg/L and the gibberellin (GA of 0.01～0.1mg/L
3), preferred hormone scope is MS+ZT0.5mg/L+NAA0.05mg/L+GA0.05mg/L.Condition of culture is the same.Through 15～25 days cultivation, can make adventitious bud inducing be the bud of growing thickly, growth coefficient can reach more than 4.5.
4. culture of rootage and obtain the sterile test tube seedling
Root media is meant can induce the stem section with band differentiation bud to take root and forms the medium of whole plant, and the stem section of band differentiation bud is able to root induction, formation aseptic seedling after cultivating through 15～20 days in this medium.Detailed process is: 1/2MS minimal medium (the same) is gone in bud grafting, and methyl (NAA) or the 0.1～1.0mg/L indolebutyric acid (IBA) of additional 0.05～0.5mg/L, preferred hormone scope is NAA0.4mg/L, IBA0.2mg/L.Cultivated through 10～20 days in this medium, the stem section of differentiation bud just can be taken root (rooting rate reaches 90%), forms aseptic seedling.
5. hardening and transplant test-tube plantlet
Deng the aseptic seedling length to 1 after taking root～when 3cm is high, opening the blake bottle lid continues to cultivate 4～8 days, take out seedling carefully with tweezers, after the clean residual media of running water, plant in by isopyknic detritus soil, river sand and the perlite preparation and matrix sterilization in advance, place culturing room, greenhouse, outdoor being cultured in succession to survive, survival rate reaches 70%.
The component such as the table 1 of employed medium:
The component of table 1 medium
| Medium | Basis | Hormone |
| The differentiation adventitious buds medium | The 3/4MS minimal medium | 0.5 the methyl (NAA) of the zeatin of～1mg/L (ZT) and 0.01～0.1mg/L, the zeatin (ZT) of preferred 0.5mg/L and the methyl of 0.02mg/L |
| The 1/2MS minimal medium | 0.1 the 6-benzylaminopurine (6-BA) of～1.0mg/L and the methyl (NAA) of 0.01～0.1mg/L, the 6-benzylaminopurine (6BA) of preferred 0.5mg/L and the methyl (NAA) of 0.02 mg/L | |
| The inducing clumping bud medium | The MS minimal medium | 0.5 the methyl (NAA) of the zeatin of～2.0mg/L (ZT), 0.01～0.1mg/L and the gibberellin (GA of 0.01～0.1mg/L 3), zeatin (ZT), the methyl (NAA) of 0.05mg/L and the gibberellin (GA of 0.05mg/L of preferred 0.5mg/L 3) |
| Root media | The 1/2MS minimal medium | 0.05 the methyl of～0.5mg/L (NAA), preferred 0.4mg/L methyl (NAA) |
| The 1/2MS minimal medium | 0.1～1.0mg/L indolebutyric acid (IBA), preferred 0.2 mg/L indolebutyric acid (IBA) |
Compared with prior art, more than invention has following outstanding advantage:
1. the method for quickly breeding of pencil tree cuttage provided by the invention and tissue culture can not be subjected to the influence in season, increases substantially plant propagation coefficient (survival rate reaches more than 75%), obtains a large amount of high-quality pencil tree seedlings at short notice.
2. the screening and the directive breeding that help the cold-resistant variety of pencil tree.
3. provide high quality seedling in enormous quantities for the large-scale development pencil tree.The enforcement of this technology is promoted, and will produce positive impact to ecology, economy and energy aspect.
Description of drawings
Accompanying drawing 1: by the test-tube plantlet of pencil tree tissue rapid propagation technology acquisition.
Accompanying drawing 2: by the seedling of pencil tree cottage propagation technology acquisition
Embodiment
Embodiment one: obtain complete pencil tree plant by cuttage pencil tree branch
1. select the plugged ear material and prepare the cuttage seeding bed substrate
The plugged ear material is selected from the annual lignification in the open environment that carries disease germs and/or the pencil tree live body branch of semi-lignified.
The seedbed is arranged on well lighted, the place that has abundant water resources, and cutting bed length and width, moderate deeply (2～3 meters, wide 1～2 meter, dark 30～40 centimetres in length), and carry out weeds cleaning etc. in advance, matrix is prepared respectively according to the ratio of table 2.
2. hormone preliminary treatment plugged ear material
After choosing the pencil tree live body branch of healthy and strong carry disease germs pencil tree live body branch or healthy and strong annual lignification and/or semi-lignified, be cut into the long segment of 10～20cm, the segment base portion be immersed in handled in the hormone solution 1 hour with secateurs;
With the cuttage of plugged ear material in the cuttage seeding bed substrate
With hormone preliminary treatment plugged ear material, with 20/m
2The density cuttage on the seedbed, carry out seedling management after permeable spraying for the first time, cuttage is after 25～30 days, cuttage rooting forms whole plant.
4. seedling management
After cuttage finishes, comprise the conventional seedling management of water spray fertilising, weeding deinsectization, pruning branch according to Changes in weather and soil regime.Add up survival rate at last, result such as table 2 are listed.
Table 2 cuttage survival rate (plant of survival rate=cutting survival/cuttage branch sum)
| Group number | (equal-volume) formed in the cuttage seedbed | Pretreated hormone (mg/L), the processing time | Survival rate (%) |
| A1 | Detritus soil, river sand, perlite | 0.05mg/LABT 6Solution, 1h | 78.2 |
| A2 | Detritus soil, river sand | 0.10mg/LABT 6Solution, 1h | 80.2 |
| A3 | River sand | 0.15mg/LABT 6Solution, 12h | 84.9 |
| B1 | Loess, river sand | 0.1mg/L indolebutyric acid solution, 12h | 85.6 |
| B2 | Detritus soil, river sand, perlite | 0.15mg/L indolebutyric acid solution, 1h | 84.9 |
| B3 | Detritus soil | 0.2mg/L indolebutyric acid solution, 6h | 83.7 |
| C1 | Purple soil, river sand | 0.01mg/L naphthalene acid solution, 1h | 81.6 |
| C2 | Detritus soil, river sand | 0.15mg/L naphthalene acid solution, 6h | 82.9 |
| C3 | Detritus soil | 0.2mg/L naphthalene acid solution, 12h | 82.7 |
| CK1 | Detritus soil, river sand | Hormone 0,1h | 65.1 |
| CK2 | Detritus soil | Hormone 0,1h | 67.8 |
| CK3 | River sand | Hormone 0,12h | 62.4 |
Embodiment two: fast numerous acquisition pencil tree plant is cultivated by explantation tissue
With axillalry bud and terminal bud section is explant, cuts into the segment of 0.5-1cm.Carry out inducing of indefinite bud according to aforesaid method, but through 10 days the just generation of evoking adventive bud of cultivation, inductivity can reach 86-90.6%.On the bud induction medium of growing thickly, obtain the bud of growing thickly then, growth coefficient 4.5, effectively bud 85-91.7% through 20 days cultivations.The bud of will growing thickly changes on the root media, begins to take root through 7 days cultivations, develops into complete plant, inoculates 20 days rooting rates and reaches 80-95%.
Concrete outcome is referring to table 3
The pencil tree plant is cultivated by table 3 explantation tissue
| Group number | Medium | Medium and hormone are formed | Efficient (%) |
| A1 | The differentiation adventitious buds medium | 3/4MS+ZT(0.5/mg/L)+NAA(0.01mg/L) | 86 |
| A2 | 3/4MS+ZT(1/mg/L)+NAA(0.02mg/L) | 90.6 | |
| A3 | 1/2MS+BA(0.5mg/L)+NAA(0.02mg/L) | 90.3 | |
| A4 | 1/2MS+BA(1mg/L)+NAA(0.1mg/L) | 74.2 | |
| B1 | The inducing clumping bud medium | MS+ZT(1.0mg/L)+NAA(0.05mg/L)+GA 3 (0.01mg/L) | 85 |
| B2 | MS+ZT(1.0mg/L)+NAA(0.1mg/L)+GA 3( 0.05mg/L) | 89 | |
| B3 | MS+ZT(0.5mg/L)+NAA(0.05mg/L)+GA 3 (0.01mg/L) | 90 | |
| B4 | MS+ZT(0.5mg/L)+NAA(0.05mg/L)+GA 3 (0.05mg/L) | 91.7 | |
| C1 | Root media | 1/2MS+NAA(0.4mg/L) | 95 |
| C2 | 1/2MS+NAA(0.05mg/L) | 90 | |
| C3 | 1/2MS+IBA(0.2mg/L) | 92 | |
| C4 | 1/2MS+IBA(0.5mg/L) | 84 |
Claims (10)
1, the production method of the plant pencil tree (E.tirucalli) of a kind of Euphorbiaceae Euphorbia (Euphorbia), described greenstone sapling is characterized in that comprising following process by a large amount of cottage breedings acquisitions in the land for growing field crops or in the greenhouse under the condition of bacterium are being arranged:
(1) select plugged ear material and prepare the cuttage seeding bed substrate: the plugged ear material is selected from the annual lignification in the open environment that carries disease germs and/or the pencil tree live body branch of semi-lignified; The cuttage seedbed is by isopyknic detritus soil and river sand and perlite, or by isopyknic detritus soil and river sand, or by isopyknic purple soil and river sand, or by isopyknic loess and river sand matrix prepared, or independent effluent sand or detritus soil matrix prepared;
(2) hormone preliminary treatment plugged ear material: after choosing the healthy and strong pencil tree live body branch that carries disease germs, be cut into the long segment of 10～20cm with secateurs, the segment base portion is immersed in handled in the hormone solution 1～12 hour, described hormone is selected from the root-inducing powder ABT of 0.05mg/L～0.15mg/L
6In solution or 0.1mg/L～0.2mg/L indolebutyric acid solution or the naphthalene acid solution of 0.01mg/L～0.2mg/L;
(3) with the cuttage of plugged ear material in the cuttage seeding bed substrate: with hormone preliminary treatment plugged ear material, with 20/m
2The density cuttage on the seedbed, carry out seedling management after permeable spraying for the first time, cuttage is after 25～30 days, cuttage rooting forms whole plant;
(4) seedling management: after cuttage finishes, comprise the conventional seedling management of water spray fertilising, weeding deinsectization, pruning branch according to Changes in weather and soil regime.
2, the production method of the plant pencil tree (E.tirucalli) of a kind of Euphorbiaceae Euphorbia (Euphorbia), described greenstone sapling produces acquisition in a large number by the tissue cultivating mode of the aseptic quick breeding in greenhouse, comprise preliminary treatment or not preliminary treatment pencil tree explant, induce explant differentiation indefinite bud, indefinite bud induced bundle on the inducing clumping bud proliferated culture medium is sprouted, induced bundle is sprouted and taken root, obtains sterile test tube seedling, hardening and transplant test-tube plantlet on root media process, it is characterized in that at differential medium:
(1) described differentiation adventitious buds medium is meant the medium that can induce stipes section or stem top explant to be divided into indefinite bud, explant just can be able to the induced bud differentiation through cultivation in 10～20 days in this medium, this medium by in the 3/4MS minimal medium, adding 0.5～1mg/L zeatin ZT and the methyl of 0.01～0.1mg/L, or in the 1/2MS minimal medium, add the 6-benzylaminopurine of 0.1～1.0mg/L and the methyl of 0.01～0.1mg/L is prepared;
(2) described inducing clumping bud medium is meant the medium that can induce the indefinite bud that has broken up to form the bud of growing thickly, explant just can be able to induced bundle through 25～30 days cultivation and sprout and produce and bred in this medium, the methyl NAA of zeatin ZT, the 0.01～0.1mg/L of this medium by adding 0.5～2.0mg/L in the MS minimal medium and the gibberellin GA of 0.01～0.1mg/L
3Prepared;
(3) described root media is meant to induce and has the medium that the stem section of being with the differentiation bud is taken root and formed whole plant, the stem section of band differentiation bud is able to root induction and forms aseptic seedling after cultivating through 15～20 days in this medium, and this medium is prepared by methyl or the 0.1～1.0mg/L indolebutyric acid that adds 0.05～0.5mg/L in the 1/2MS minimal medium.
3, according to the production method of claim 2, wherein explant is selected from the stipes section or the stem top of live plant in the open environment that carries disease germs, or is selected from stipes section or stem top or live body aseptic seedling in the gnotobasis; Described preliminary treatment is on explant stem that carries disease germs or stem top to choosing, after cleaning repeatedly with running water, soaks base portion 3～6 hours with 15%～25% polyethylene glycol PEG6000 again, cleans repeatedly with running water again, is used for then sterilizing and inoculated and cultured.
4, according to the production method of claim 3, after wherein sterilization is meant and cleans repeatedly with running water, in ultra-clean or gnotobasis, handled 0～5 minute with 70% Ethanol Treatment 0～30 second or with 5%～10% hydrogen peroxide, after sterile water washes repeatedly, carry out 4～10 minutes disinfecting with 5%～10% hypochlorite solutions or 0.1%～1% mercuric chloride solution again, wash repeatedly to be used for inoculated and cultured with sterile water at last.
5, according to the production method of claim 3 or 4, wherein inoculated and cultured refers to cultivate explant after 2～4 weeks, can produce indefinite bud, when indefinite bud grows to 1～3cm when high, it is as the sterile explant material, and bud forms or root induction produces aseptic seedling again to be used to induce pencil tree to grow thickly.
6, according to the production method of claim 3 or 4, wherein inoculated and cultured is that explant stipes or stem top are directly produced aseptic seedling through aseptic culture.
7, according to the production method of claim 3 or 4, wherein inoculated and cultured is that the explant of will disinfect is inoculated into MS or carries out the illumination temperature control and cultivate on the solid of MS improvement or semisolid culturemedium.
8, according to the production method of claim 7, wherein to cultivate be to cultivate under the condition of illumination time day of intensity of illumination, 10-16 hour at 800～1600LX and 18～26 ℃ to the illumination temperature control.
9, according to the production method of claim 7, wherein added in the solid culture medium in the gelatin of 0.7%～1.0% agar or 0.8%～2.0% or the liquid medium within and directly added solid particle, solid particle made by bead, perlite or vermiculite material.
10, according to the production method of claim 2, wherein acclimatization and transplants is the plantlet length to 1～when 3cm is high after taking root, opening the blake bottle lid continues to cultivate 4～8 days, take out seedling carefully with tweezers, after the clean residual media of running water, transplant in that prepare by equal-volume by detritus soil, river sand and perlite and matrix sterilization in advance, place culturing room or greenhouse to be cultured in succession and survive.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101057557B (en) * | 2007-06-01 | 2010-05-19 | 中山大学 | Non-tube quick reproduction method for seedings of jatropha curcas L. |
| CN101263769B (en) * | 2008-05-08 | 2010-06-02 | 福建农林大学 | Euscaphis konishii cutting propagation method |
| CN102696386A (en) * | 2012-06-29 | 2012-10-03 | 广西大学 | Branch cuttage method for breeding seedling of aleurites moluccana by using biodiesel raw material tree species |
| CN102696386B (en) * | 2012-06-29 | 2013-09-25 | 广西大学 | Branch cuttage method for breeding seedling of aleurites moluccana by using biodiesel raw material tree species |
| CN103503685A (en) * | 2013-10-23 | 2014-01-15 | 镇江市丹徒区紫杉生态农业园 | Rapid propagation method for cajuput seedlings |
| CN104365318A (en) * | 2014-09-29 | 2015-02-25 | 梁彩英 | Standardized myrtle planting technology |
| CN104798684A (en) * | 2015-04-25 | 2015-07-29 | 玉林师范学院 | Tissue culture rapid propagation method for plukenetia volubilis L. |
| CN104938179A (en) * | 2015-06-10 | 2015-09-30 | 苏州苏农园艺景观有限公司 | Planting method for cajuput flowers |
| CN106212013A (en) * | 2016-08-04 | 2016-12-14 | 临沂市农业科学院 | A kind of overlay film Radix Salviae Miltiorrhizae simple cultivation technique of cuttage formula |
| CN111011217A (en) * | 2019-12-31 | 2020-04-17 | 青海省农林科学院 | Efficient regeneration method of heterozygous diploid potato RH |
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