CN103749310A - Method for promoting pinus massoniana tissue-cultured subcultured bud to root - Google Patents
Method for promoting pinus massoniana tissue-cultured subcultured bud to root Download PDFInfo
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Abstract
The invention discloses a method for promoting a pinus massoniana tissue-cultured subcultured bud to root. The method comprises the following steps: selecting the robust and half lignified pinus massoniana tissue-cultured subcultured bud with a seedling height of 2-3 cm; by adopting different culture media in a two-step rooting method, inoculating the subcultured bud in a 1/2 modified MS culture medium IV that contains indoleacetic acids, indolebutyric acids and sucrose; conducting induction treatment on an adventitious root primordium 21-28 d; transplanting the subcultured bud to a 1/2 modified MS culture medium V that contains activated carbon or ascorbic acids, and sucrose, without auxins, in order to promote the adventitious root to express and extend; transplanting a rooted regeneration plant to a mixed matrix that contains yellow soil or peat soil, vermiculite and perlite for cultivating. According to the invention, the method can tremendously promote and improve the rooting condition of the subcultured bud, the rooting rate of the pinus massoniana subcultured bud is above 93.1%, and the highest rooting rate is as high as 97.6%; the root system quality is higher; the survival rate of transplanting is above 85.1%, and the highest survival rate of transplanting is as high as 93.0%; the method has good economic benefits and social benefits.
Description
Technical field
The invention belongs to plant propagation technical field, relate to tissue cultivating and seedling technology, especially a kind of promotion masson pine group training subculture blastogenesis method for root.
Background technology
Masson pine (Pinus massonianan) originates in China, is one of important species of the important green barren hill of south China, paper making raw material woods and resin tapping woods, natural land woods, very wide in China's distribution.The seeds high as a kind of comprehensive utilization value, promotion potential is large, masson pine not only can be used to produce three plates, papermaking and chemical fibre industry manufacture, also can be used to produce rosin, the turpentine wet goods raw material of industry.In recent years, along with the intensification of contradictions such as peter out of the non-renewable resources such as the growing life requirement of people and timber resource shortage and oil, ecological, economic and social sustainable development is more and more subject to social concerns.The problems such as, resource security peaceful based on China's ecology and social safety, masson pine, as one of the main industrial cut stock seeds of China and Tree Species as Bio-energy, is greatly developed Masson Pine Plantation necessary.
China, since " six or five " National Key Research Programs, has obtained compared with quantum jump at aspects such as masson pine fine-variety breeding and Fast-growing Cultivation of Cinnamomuns.At present, masson pine breeding mainly be take the sexual propagation of seed orchard and seed production stand as main.Yet sexual propagation exists the problem of differentiation, can not improve to greatest extent genetic improvement gain, and vegetative propagation can overcome the above-mentioned deficiency of sexual propagation.But the current still difficulty of traditional vegetative propagation technique of masson pine (cuttage and grafting) meets the demand of afforestation, restricted the process of Fine Variety Breeding of Pinus massoniana.Modern forestry is particular about fast growing, high-quality and efficient, and the use of plant tissue culture technology is the effective way that realizes masson pine choiceness nursery stock fast breeding.
Horse hair Pinus tissue is cultivated the comparatively seeds of difficulty that take root.Numerous scholar's research showed in the past, take root and conventionally can obtain comparatively desirable rooting rate, and after subculture, its simple bud rooting rate is obviously on the low side with the masson pine simple bud of first culture.As take masson pine mature seed as explant source, and Wu little Qin etc. (2009) and season Kong Shu etc. (2010) are directly taken root simple bud after the first culture through the induction of clump bud and elongation, and its rooting rate is respectively 85% and 92.5%; And Xue Ying (2006) is taken root getting simple bud after subculture is cultivated, its rooting rate is only 26.7%.Realize the seedling industrialized production of masson pine group training, must break through subculture blastogenesis root difficulty bottleneck factor.
Summary of the invention
Object of the present invention, mainly for the low present situation of masson pine group training subculture bud rooting rate, for realizing the large-scale production of masson pine group training seedling, provides a kind of promotion masson pine group training subculture blastogenesis method for root.
The present invention is achieved in that
Promote a masson pine group training subculture blastogenesis method for root, comprise two step rooting methods of clump bud induction, the elongation of clump bud, subculture cultivation, root induction, adventitious root extension, test-tube seedling transplanting, obtain masson pine group training subculture blastogenesis offspring, its operating procedure is as follows:
(1) clump bud induction: by after masson pine seed sterilization, carry out seed germination under aseptic condition, then the long hypocotylar germ free apical bud of intercepting band 0.5cm is explant, carries out clump bud induction in inducing culture I;
(2) clump bud extends: the Multiple Buds inducing is extended in elongation medium II to cultivation;
(3) subculture is cultivated: the single terminal bud that cuts elongation bud is inoculated in shoot proliferation medium III and is bred, obtain subculture bud I; Subculture bud I repeating step (2) is extended, singlely cut the subculture bud I of elongation and be divided into terminal bud and band lateral bud bud section, then in step (3) shoot proliferation medium, continue propagation and obtain subculture bud II, by subculture bud II repeating step (2) and step (3) again, obtain new proliferation and subculture bud; Repetitive cycling step (2) and (3), until obtain a large amount of enough culture of rootage subculture buds;
(4) root induction: the single subculture bud that cuts robust growth, semi-lignified, high 2-3cm, be seeded in the 1/2 modified MS medium IV that contains heteroauxin (IAA), indolebutyric acid (IBA), sucrose, adventitious root primordia 21-28d is processed in induction;
(5) adventitious root extension: will there is the visible adventive root explant of naked eyes and proceed in the 1/2 modified MS medium V that contains active carbon (AC) or ascorbic acid (Vc), sucrose, and promote expression and the elongation of adventive root;
(6) test-tube seedling transplanting: the Transplantation of Regenerated Plantlets of having taken root is cultivated in the mixing light ground mass that yellow soil or peat soil, vermiculite and perlite volume ratio are 1: 1: 1 to water spray moisturizing.
Above-described its material content of medium I is: 6-BA2.5-4.0mgL
-1, NAA0.1-0.3mgL
-1, sucrose 30000mgL
-1and modified MS medium.
Above-described its material content of medium II is: NAA0.25-0.4mgL
-1, sucrose 30000mgL
-1and modified MS medium.
Above-described its material content of medium III is: ZT1.0-3.0mgL
-1, KT0-1.0mgL
-1, NAA0-0.3mgL
-1, sucrose 30000mgL
-1and modified MS medium.
Above-described its material content of medium IV is: IAA1.0-2.5mgL
-1, IBA0.1-0.5mgL
-1, sucrose 1.0-2.0% and 1/2 modified MS medium.
Above-described its material content of medium V is: AC3000-5000mgL
-1or Vc10-30mgL
-1, sucrose 2.0% and 1/2 modified MS medium.
Basic composition is of above-described modified MS medium: KNO
31580mgL
-1; NH
4nO
3800mgL
-1; CaCl
22H
2o260mgL
-1; MgSO
47H
2o370mgL
-1; Ca (NO
3)
24H
2o200mgL
-1; KH
2pO
4170mgL
-1; MnSO
4h
2o22.3mgL
-1; ZnSO
47H
2o8.6mgL
-1; CuSO
45H
2o0.025mgL
-1; H
3bO
36.2mgL
-1; Na
2moO
42H
2o0.025mgL
-1; KI0.83mgL
-1; CoCl
26H
2o0.025mgL
-1; Cobastab
11.0mgL
-1; Cobastab
60.5mgL
-1; Nicotinic acid 0.5mgL
-1; Glycine 2.0mgL
-1; Inositol 200mgL
-1.
Above-described cultivation controlled condition is: cultivation temperature 20-30 ℃, illumination 1500-2000lx, illumination every day 12-16h.
The present invention has the following advantages with respect to existing technology:
Promotion masson pine group training subculture blastogenesis method for root of the present invention, select the masson pine group training subculture bud of robust growth, semi-lignified and height of seedling 2-3cm, adopt different medium with two step rooting methods, 21-28d days is processed in first induction, then be forwarded to and there is no growth hormone, but in the medium that contains active carbon or ascorbic acid, greatly promote and improved subculture blastogenesis root situation.Adopt this rooting method, masson pine subculture bud rooting rate reaches more than 93.1%, and the highest rooting rate reaches 97.6%, and root system quality is higher, and transplanting survival rate reaches more than 85.1%, and high-servival rate reaches 93.0%, has good economic benefit and social benefit.
Accompanying drawing explanation
Fig. 1 is that masson pine group of the present invention is trained the seedling of taking root;
Fig. 2 is masson pine group training transplanted seedling of the present invention.
Embodiment
The following examples can make the present invention of those skilled in the art's comprehend, but do not limit the present invention in any way.
embodiment 1
1, experiment material
Take masson pine then ripe cone seed germination germ free apical bud be explant, cone picks up from Ningming of Guangxi portiatree provenance construction masson pine seed production stand the good masson pine family of high yield fat Ningming No. 3.
2, experimental technique
(1) clump bud induction
Aseptic explant is inoculated into improvement MS+6-BA3.0mgL
-1+ NAA0.1mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1medium on induce cultivation.Cultivate after 26d, average clump bud induction rate reaches 99.1%.
Basic composition is of described modified MS medium: KNO
31580mgL
-1; NH
4nO
3800mgL
-1; CaCl
22H
2o260mgL
-1; MgSO
47H
2o370mgL
-1; Ca (NO
3)
24H
2o200mgL
-1; KH
2pO
4170mgL
-1; MnSO
4h
2o22.3mgL
-1; ZnSO
47H
2o8.6mgL
-1; CuSO
45H
2o0.025mgL
-1; H
3bO
36.2mgL
-1; Na
2moO
42H
2o0.025mgL
-1; KI0.83mgL
-1; CoCl
26H
2o0.025mgL
-1; Cobastab
11.0mgL
-1; Cobastab
60.5mgL
-1; Nicotinic acid 0.5mgL
-1; Glycine 2.0mgL
-1; Inositol 200mgL
-1.
(2) clump bud extends
The budlet that grows thickly inducing is transferred to improvement MS+NAA0.25mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1in elongation medium, to promote the elongation growth of culture.Extend and cultivate 27d, the high 3-5cm of clump bud.
(3) subculture bud is cultivated
The simple bud of plant height 2-3cm in Multiple Buds is cut, be inoculated into modified MS medium+ZT2.0mgL
-1+ KT1.0mgL
-1+ NAA0.2mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1on carry out subculture bud propagation 23d.
Shoot proliferation bud is put in step (2) elongation medium, to promote shoot proliferation bud to extend.Extend and cultivate 28d, the high 3-5cm of shoot proliferation bud.Elongation subculture bud is divided into after the long terminal bud of 2cm and the long band of 1cm lateral bud bud section, and repeating step (3) shoot proliferation and step (2) clump bud extend.Subculture is cultivated 6 times, and average growth coefficient 6.4 obtains large quantities of for taking root subculture bud.
(4) root induction
The single subculture bud that cuts robust growth, semi-lignified, high 2-3cm, is inoculated into 1/2 modified MS medium+IAA2.5mgL
-1+ IBA0.1mgL
-1the former base of inducing adventitious root in+sucrose 1.0% medium.After induction 25d, root induction rate reaches 97.6%.
(5) adventitious root extension
To obviously there is the visible adventive root explant of naked eyes and proceed to 1/2 modified MS medium+AC5000mgL
-1in+sucrose 2.0% medium, promote adventive root express and extend, after 28d, add up adventive root incidence, record mean elements and observe root system quality (between rhizome callus a situation arises).Concrete data are in Table 1.
(6) test-tube seedling transplanting
Regeneration plant root agar is cleaned, transplanted in yellow soil and cultivate, water spray moisturizing, added up transplanting survival rate after one month.Concrete data are in Table 1.
The condition of culture of said process: temperature 20-30 ℃, illumination 1500-2000lx, illumination every day 12-16h.
embodiment 2
1, experiment material
Take masson pine then ripe cone seed germination germ free apical bud be explant, cone picks up from Ningming of Guangxi portiatree provenance construction masson pine seed production stand the good masson pine family of high yield fat Ningming No. 4.
2, experimental technique
(1) clump bud induction
Aseptic explant is inoculated into improvement MS+6-BA4.0mgL
-1+ NAA0.1mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1medium on induce cultivation.Cultivate after 26d, average clump bud induction rate reaches 98.4%.
The basic composition of described modified MS medium is with embodiment 1.
(2) clump bud extends
The budlet that grows thickly inducing is transferred to improvement MS+NAA0.3mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1in elongation medium, to promote the elongation growth of culture.Extend and cultivate 28d, the high 3-5cm of clump bud.
(3) subculture bud is cultivated
The simple bud of plant height 2-3cm in Multiple Buds is cut, be inoculated into modified MS medium+ZT2.5mgL
-1+ KT0.5mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1on carry out subculture bud propagation 26d.
Shoot proliferation bud is put in step (2) elongation medium, to promote shoot proliferation bud to extend.Extend and cultivate 25d, the high 3-5cm of shoot proliferation bud.Elongation subculture bud is divided into after the long terminal bud of 2cm and the long band of 1cm lateral bud bud section, and repeating step (3) shoot proliferation and step (2) clump bud extend.Subculture is cultivated 5 times, average growth coefficient 6.0.
(4) root induction
The single subculture bud that cuts robust growth, semi-lignified, high 2-3cm, is inoculated into 1/2 modified MS medium+IAA1.5mgL
-1+ IBA0.3mgL
-1the former base of inducing adventitious root in+sucrose 1.0% medium.After induction 22d, root induction rate reaches 93.1%.
(5) adventitious root extension
To obviously there is the visible adventive root explant of naked eyes and proceed to 1/2 modified MS medium+Vc10mgL
-1in+sucrose 2.0% medium, promote adventive root express and extend, after 28d, add up adventive root incidence, record mean elements and observe root system quality (between rhizome callus a situation arises).Concrete data are in Table 1.
(6) test-tube seedling transplanting
Regeneration plant root agar is cleaned, transplanted in yellow soil and cultivate, water spray moisturizing, added up transplanting survival rate after one month.Concrete data are in Table 1.
The condition of culture of said process: temperature 20-30 ℃, illumination 1500-2000lx, illumination every day 12-16h.
embodiment 3
1, experiment material
Take masson pine then ripe cone seed germination germ free apical bud be explant, cone picks up from Ningming of Guangxi portiatree provenance construction masson pine seed production stand the good masson pine family of high yield fat Ningming No. 7.
2, experimental technique
(1) clump bud induction
Aseptic explant is inoculated into improvement MS+6-BA4.0mgL
-1+ NAA0.1mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1medium on induce cultivation.Cultivate after 26d, average clump bud induction rate reaches 99.0%.
The basic composition of described modified MS medium is with embodiment 1.
(2) clump bud extends
The budlet that grows thickly inducing is transferred to improvement MS+NAA person and says 0.4mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1in elongation medium, to promote the elongation growth of culture.Extend and cultivate 28d, the high 3-5cm of clump bud.
(3) subculture bud is cultivated
The simple bud of plant height 2-3cm in Multiple Buds is cut, be inoculated into modified MS medium+ZT2.5mgL
-1+ KT0.5mgL
-1+ NAA0.25mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1on carry out subculture bud propagation 26d.
Shoot proliferation bud is put in step (2) elongation medium, to promote shoot proliferation bud to extend.Extend and cultivate 25d, the high 3-5cm of shoot proliferation bud.Elongation subculture bud is divided into after the long terminal bud of 2cm and the long band of 1cm lateral bud bud section, and repeating step (3) shoot proliferation and step (2) clump bud extend.Subculture is cultivated 5 times, average growth coefficient 6.6.
(4) root induction
The single subculture bud that cuts robust growth, semi-lignified, high 2-3cm, is inoculated into 1/2 modified MS medium+IAA1.0mgL
-1+ IBA0.5mgL
-1the former base of inducing adventitious root in+sucrose 2.0% medium.After induction 28d, root induction rate reaches 95.1%.
(5) adventitious root extension
To obviously there is the visible adventive root explant of naked eyes and proceed to 1/2 modified MS medium+AC3500mgL
-1in+sucrose 2.0% medium, promote adventive root express and extend, after 28d, add up adventive root incidence, record mean elements and observe root system quality (between rhizome callus a situation arises).Concrete data are in Table 1.
(6) test-tube seedling transplanting
Regeneration plant root agar is cleaned, transplanted in peat soil, vermiculite and perlite volume ratio and be: in the mixed-matrix of 1: 1: 1, cultivate, water spray moisturizing, added up transplanting survival rate after one month.Concrete data are in Table 1.
The condition of culture of said process: temperature 20-30 ℃, illumination 1500-2000lx, illumination every day 12-16h.
In above embodiment 1 to 3, masson pine group training subculture blastogenesis root and transplant survival situation, as shown in Table 1 below.
Table 1 masson pine group training subculture blastogenesis root and transplant survival situation
From table 1 result, show, adopt the present invention that each family group training seedling subculture bud rooting rate of masson pine is reached more than 93.1%, the highest rooting rate reaches 97.6%, and more than the average root of hair 2-3 of every plant bar, and the callus of group training seedling base portion is less, between rhizome, connects closely.Transplant to after in yellow soil or peat soil, perlite, vermiculite mixing light ground mass, regeneration plant well-grown, after one month, transplanting survival rate is more than 85.1%, and high-servival rate reaches 93.0%.The present invention has solved the problem of masson pine group training subculture blastogenesis root difficulty preferably, produces masson pine tissue culture regeneration plant group training subculture blastogenesis method for root is provided for batch production.
Claims (8)
1. one kind promotes masson pine group training subculture blastogenesis method for root, it is characterized in that: the two step rooting methods that comprise clump bud induction, the elongation of clump bud, subculture cultivation, root induction, adventitious root extension, test-tube seedling transplanting, obtain masson pine group training subculture blastogenesis offspring, its operating procedure is as follows:
Clump bud induction: by after masson pine seed sterilization, carry out seed germination under aseptic condition, then intercepting and being with the long hypocotylar germ free apical bud of 0.5 cm is explant, carries out clump bud induction in inducing culture I;
Clump bud extends: the Multiple Buds inducing is extended in elongation medium II to cultivation;
Subculture is cultivated: the single terminal bud that cuts elongation bud is inoculated in shoot proliferation medium III and is bred, obtain subculture bud I; Subculture bud I repeating step (2) is extended, singlely cut the subculture bud I of elongation and be divided into terminal bud and band lateral bud bud section, then in step (3) shoot proliferation medium, continue propagation and obtain subculture bud II, by subculture bud II repeating step (2) and step (3) again, obtain new proliferation and subculture bud; Repetitive cycling step (2) and (3), until obtain a large amount of enough culture of rootage subculture buds;
Root induction: the single subculture bud that cuts robust growth, semi-lignified, high 2-3 cm, be seeded in the 1/2 modified MS medium IV that contains heteroauxin (IAA), indolebutyric acid (IBA), sucrose, adventitious root primordia 21-28 d is processed in induction;
Adventitious root extension: will there is the visible adventive root explant of naked eyes and proceed in the 1/2 modified MS medium V that contains active carbon (AC) or ascorbic acid (Vc), sucrose, and promote expression and the elongation of adventive root;
Test-tube seedling transplanting: the Transplantation of Regenerated Plantlets of having taken root is cultivated in peat soil, vermiculite and perlite (1: 1: 1) mixed-matrix to water spray moisturizing.
2. a kind of promotion masson pine group training subculture blastogenesis method for root according to claim 1, is characterized in that: described its material content of medium I is: 6-BA 2.5-4.0 mgL
-1, NAA 0.1-0.3 mgL
-1, sucrose 30000 mgL
-1and modified MS medium.
3. a kind of promotion masson pine group training subculture blastogenesis method for root according to claim 1, is characterized in that: described its material content of medium II is: NAA 0.25-0.4 mgL
-1, sucrose 30000 mgL
-1and modified MS medium.
4. a kind of promotion masson pine group training subculture blastogenesis method for root according to claim 1, is characterized in that: described its material content of medium III is: ZT 1.0-3.0 mgL
-1, KT 0-1.0 mgL
-1, NAA 0-0.3 mgL
-1, sucrose 30000 mgL
-1and modified MS medium.
5. a kind of promotion masson pine group training subculture blastogenesis method for root according to claim 1, is characterized in that: described its material content of medium IV is: IAA 1.0-2.5 mgL
-1, IBA 0.1-0.5 mgL
-1, sucrose 1.0-2.0% and 1/2 modified MS medium.
6. a kind of promotion masson pine group training subculture blastogenesis method for root according to claim 1, is characterized in that: described its material content of medium V is: AC 3000-5000 mgL
-1or Vc 10-30 mgL
-1, sucrose 2.0% and 1/2 modified MS medium.
7. a kind of promotion masson pine group training subculture blastogenesis method for root according to claim 1, is characterized in that: basic composition is of described modified MS medium: KNO
31580 mgL
-1; NH
4nO
3800 mgL
-1; CaCl
22H
2o 260 mgL
-1; MgSO
47H
2o 370 mgL
-1; Ca (NO
3)
24H
2o 200 mgL
-1; KH
2pO
4170 mgL
-1; MnSO
4h
2o 22.3 mgL
-1; ZnSO
47H
2o 8.6 mgL
-1; CuSO
45H
2o 0.025 mgL
-1; H
3bO
36.2 mgL
-1; Na
2moO
42H
2o 0.025 mgL
-1; KI 0.83 mgL
-1; CoCl
26H
2o 0.025 mgL
-1; Cobastab
11.0 mgL
-1; Cobastab
60.5 mgL
-1; Nicotinic acid 0.5 mgL
-1; Glycine 2.0 mgL
-1; Inositol 200 mgL
-1.
8. a kind of promotion masson pine group training subculture blastogenesis method for root according to claim 1, is characterized in that: described cultivation controlled condition is: cultivation temperature 20-30 ℃, illumination 1500-2000 lx, illumination every day 12-16 h.
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| CN201410041856.3A CN103749310B (en) | 2013-11-27 | 2014-01-28 | Method for promoting pinus massoniana tissue-cultured subcultured bud to root |
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| CN104663445A (en) * | 2015-02-25 | 2015-06-03 | 杨惠才 | In-vitro rapid propagation technology of high lipid-producing pinus elliottii |
| CN111616050A (en) * | 2020-05-19 | 2020-09-04 | 广西壮族自治区林业科学研究院 | Method for improving rooting stability of superior tree subculture buds of high-yield masson pine |
| CN113142053A (en) * | 2021-04-08 | 2021-07-23 | 中南林业科技大学 | Culture method for promoting induced proliferation differentiation and effective elongation of masson pine cluster buds |
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| CN106688885A (en) * | 2016-11-15 | 2017-05-24 | 邹少英 | Method for quickly obtaining masson pine vegetative propagation seedlings for afforestation |
| CN108967195B (en) * | 2018-08-07 | 2021-07-02 | 广西壮族自治区林业科学研究院 | Culture method for proliferation and rejuvenation of tissue culture subculture bud of pinus massoniana |
| CN111657150B (en) * | 2020-07-24 | 2021-07-27 | 广西壮族自治区林业科学研究院 | Method for improving rooting capacity of masson pine based on endogenous hormone |
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| CN113142053B (en) * | 2021-04-08 | 2023-02-24 | 中南林业科技大学 | Culture method for promoting induced proliferation differentiation and effective elongation of masson pine cluster buds |
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| CN103749310B (en) | 2015-04-08 |
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