CN103782906B - A kind of fragrant camphor tree kind gushes the method for golden plant regeneration - Google Patents
A kind of fragrant camphor tree kind gushes the method for golden plant regeneration Download PDFInfo
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- CN103782906B CN103782906B CN201310737749.XA CN201310737749A CN103782906B CN 103782906 B CN103782906 B CN 103782906B CN 201310737749 A CN201310737749 A CN 201310737749A CN 103782906 B CN103782906 B CN 103782906B
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Abstract
The method of a kind of fragrant camphor tree kind ' gushes gold ' plant regeneration, comprise the following steps: the young sprout that (1) gathers fragrant camphor tree kind and ' gushes gold ' and take out then, blade is removed after taking back laboratory, be cut into the stem section with 1 ~ 2 band axillalry bud, after asepticize, be seeded to the formation of inducing axillalry bud in the MS medium containing 6-benzyl aminoadenine and indolebutyric acid; (2) subculture and Multiplying culture is carried out in ' gushing gold ' axillalry bud grown fine being seeded to containing 6-benzyl aminoadenine and heteroauxin MS medium together with stem section; (3) formation of inducing adventitious root in the 1/2MS medium containing indolebutyric acid is seeded to after the Multiple Buds scalpel cutting long about the 2.0cm of being of tender, the average bud of growth children, the propagation that grows fine formed.Instant invention overcomes axillary bud deriving and growth difficulty, subculture and propagation young leaves black extremely, come off and phenomenon that growth potential is weak, make sterilizable material long-term subculture, thus can breed in a large number.The Multiple Buds growing situation that induction produces is vigorous, genetic character is consistent with female parent.
Description
Technical field
The invention belongs to field of tissue culture, be specifically related to a kind of method that fragrant camphor tree kind gushes golden plant regeneration.
Background technology
Camphor tree (Cinnamomumcaphora) has another name called fragrant camphor tree, is the subtropical evergreen broad-leaved arbor of Lauraceae Cinnamomum.Rapidly, flourishing, the four seasons are evergreen in its growth, leaf beautiful and distribute giving off a strong fragrance, are precious fragrant oils seeds and the view that city is important and green tree species.Fragrant camphor tree new varieties gush gold (Cinnamomumcamphora ' Yongjin ', National Botanical new varieties power numbers 2009009) be fragrant camphor tree seed variation kind, limb cerise, young leaves, flower are in golden yellow, pericarp is in yellow, and limb, leaf, pericarp color have Seasonal dynamics change, as tall and big color leaf arbor, there is very high ornamental plantation and apply potentiality and value.But it is unstable to gush gold seeds genetic material, nursery offspring degradation ratio, up to more than 99%, has carried out Grafting experiments to obtaining the filial generation keeping maternal merit at present for obtaining choiceness.Adopt cottage propagation, due to elite stand branch limited amount, be difficult to the demand meeting reality.
Tissue cultures is the asexual quick propagating technology most with application potential, can keep maternal excellent hereditary capacity.By plant tissue culture technique, not only can provide the breeding nursery stock of neat and consistent in a short time, and can also seed improvement and utilization and genetic transformation lay the first stone, in many botanical seedling culturings, obtain application.Domestic and international at present the training of common fragrant camphor tree group to be paid much attention to, since training achievement in research from the group of the fragrant camphor tree of company side's green grass or young crops report in 1992, Chinese scholars utilizes the stem section, stem apex, seed, In vitro Embryo, spire, tender shoots etc. of stump coppice shoot, different larval instar to carry out Study on tissue culture work as explant and have induced whole plant.Gush the mutational variety of gold as common fragrant camphor tree, there is not been reported both at home and abroad in its group training research work.It is common fragrant camphor tree seed variation that fragrant camphor tree new varieties gush gold, and huge as the following value potentiality applied of important Colored-leaf Plants, current rare numbers, the research work of its Fast-propagation is very urgent.Therefore, explore the method for gushing golden plant regeneration, realize its biological control, not only can enrich Foliage plant kind, meet the need of market, also to the tissue culture technology research of other plant of Lauraceae, there is important reference.
Summary of the invention
For solving above-mentioned prior art Problems existing, the present invention mainly fills up existing fragrant camphor tree new varieties and gushes golden tissue culture technology blank, come off for the induction of gushing axillalry bud in golden plant regeneration process being explant with stem section and subculture and the breeding young leaves of growth difficulty, axillalry bud and black extremely, Multiple Buds takes root the problems such as difficulty is large, research is applicable to the condition of culture gushing golden each stage of tissue-culturing rapid propagation, and being intended to provides effective help for realizing gushing golden Fast-propagation.
For achieving the above object, technical scheme of the present invention is:
Fragrant camphor tree kind gushes a method for golden plant regeneration, comprises the following steps:
(1) gather fragrant camphor tree kind and gush the golden young sprout taken out then, after taking back laboratory, remove blade, be cut into the stem section with 1 ~ 2 band axillalry bud, with liquid detergent cleaning 4 ~ 5min, more than running water 30min.With the alcohol disinfecting 30s of 75% on superclean bench, aseptic water washing 3 times, then with 0.1% mercuric chloride according to stem section diameter sterilization 8 ~ 12min, aseptic water washing 4 ~ 5 times.Filter paper suck dry moisture, cuts stem section brownization part, is seeded in the MS medium without hormone.Select the explant of aseptic stem section as axillary bud deriving, containing in the MS medium of 6-benzyl aminoadenine and indolebutyric acid, after 20d, axillalry bud starts to sprout and opens up leaf, elongation gradually;
(2) by grow fine gush golden axillalry bud be seeded to containing 6-benzyl aminoadenine and heteroauxin MS medium together with stem section in axillalry bud can normal growth and propagation, after 30d, average effective bud can reach 6.5 at most;
(3) by become simple bud to be seeded to containing indolebutyric acid after long for tender, the average bud of the growth children Multiple Buds scalpel cutting formed for about 2.0cm, the propagation that grows fine 1/2MS medium in the formation of inducing adventitious root, after 30d, the highest rooting rate is 41.3%, to continue Transplantation of Regenerated Plantlets to cultivate after 30d to peat: perlite=3: in the matrix of 1, transplanting survival rate can reach 85%.
The condition of culture that said process adopts is: pH value 5.8 ~ 6.0, temperature 24 ~ 26 DEG C, illumination 2000Lux, every day light application time 12h.
Wherein, in step (1), the time of explant collection is August, adds 6-BA1.0mg/L, IBA0.02mg/L, sucrose 30% in the medium of described induction axillalry bud.
The axillalry bud extended in step (2) needs together access subculture and proliferated culture medium together with former stem section, adds 6-BA3.0mg/L, IAA0.05mg/L, sucrose 30% in this medium.
Promote in the medium of root induction and elongation, to add IBA0.05mg/L, sucrose 20% described in step (3).
Relative to prior art, beneficial effect of the present invention is: the present invention gushes golden stem section for explant with fragrant camphor tree kind, regeneration plant is obtained by direct organ occurring mode, and by the regulation and control of hormone kind and concentration overcome axillary bud deriving and growth difficulty, subculture black extremely with propagation young leaves, come off and phenomenon that growth potential is weak, make sterilizable material long-term subculture, thus can breed in a large number.Induced by the present invention that the Multiple Buds growing situation of generation is vigorous, genetic character is consistent with female parent, for the extensive group of training production realizing quality material lays the foundation.
Accompanying drawing explanation
The buds are shooting forth in order to gush golden armpit for Fig. 1;
Fig. 2 extends and exhibition leaf for gushing golden axillalry bud;
Fig. 3 for gush golden axillalry bud young leaves black dead, come off;
Fig. 4 is that the lignification of gushing golden axillalry bud is difficult to propagation;
Fig. 5 is for gushing golden shoot proliferation;
Fig. 6 gushes taking root of golden Multiple Buds.
Embodiment
Be described in further detail the present invention below in conjunction with drawings and the specific embodiments: if without specified otherwise, following reagent is commercial reagent, and following equipment is this area common equipment.
Embodiment 1:
Experiment material of the present invention is taken from the fragrant camphor tree new varieties that Qiu Ai base, Ningbo State Forestry Administration, P.R. China seedling center grows 14 years and is gushed golden plant.Get grow enrich, the side tip of semi-lignified and top taper divide, remove blade, be cut into the stem section with 1 ~ 2 band axillalry bud, with liquid detergent cleaning 4 ~ 5min, more than running water 30min.With the alcohol disinfecting 3min of 75% on superclean bench, aseptic water washing 3 times, then with 0.1% mercuric chloride according to stem section diameter sterilization 8 ~ 12min, aseptic water washing 4 ~ 5 times.Filter paper suck dry moisture, cuts stem section brownization part, is seeded in the MS medium without hormone.Select the explant of aseptic stem section as axillalry bud, be seeded in the inducing culture of MS+6-BA1.0mg/L+IBA0.02mg/L, after 20d, treat that the stem section of axillalry bud starts to sprout and continued growth.
Be that the axillalry bud of about 2cm is transferred in MS+6-BA3.0mg/L+IAA0.05mg/L subculture and proliferated culture medium by the height obtained in above-mentioned condition of culture, on this medium black dead, the fallen leaves of axillalry bud young leaves, growth gradually the problem such as weak be resolved, and each axillalry bud can form 6.5 effective buds.
By consistent for growth, highly be seeded in the raw medium of 1/2MS+IBA0.05mg/L for the Multiple Buds of about 2cm cuts, fragrant camphor tree new varieties can be obtained after 30d gush golden whole plant in this medium.
Some concrete experimental conditions illustrate validity of gushing golden each developmental stage of leaf regeneration process formula used of the present invention by contributing to below, and all experiments at least repeat 3 times.
Test example 1, hormone is on the impact of gushing golden axillary bud sprouting and growth
In 6-BA and the IBA medium being added with suitable concentration, after asepticize gush golden stem section 20d after axillalry bud start to sprout, attenuating grow young leaves and see (Fig. 1, Fig. 2).Analytical table 1 is known, and adding in the inducing culture of 6-BA separately, the germination rate of axillalry bud improves, when 6-BA concentration is 1.0mgL with the increase of 6-BA concentration
-1and the stem Duan Junke of band axillalry bud sprouts time above, but extend difficulty and do not open up leaf, this illustrates that adding separately 6-BA is unfavorable for the sprouting of gushing Jin Jingduan.Further experiment shows, in the inducing culture being added with IBA, although can improve stem section germination rate, 6-BA is too low or be too highly all unfavorable for the growth of gushing golden stem segment with axillary buds.When IBA concentration be 0.02 ~ 0.1mg/L, 6-BA is 0.5mg/L, axillary bud growth is slow, is difficult to Zhan Ye; When 6-BA is 3mg/L, though axillalry bud is extending, but axillalry bud young leaves tip occurs downright bad, and obscission appears in young leaves, and base portion callus increases and then the situation of brownization, and squamous subculture shows, the easy decline and death of axillalry bud.In MS+6-BA1mg/L+IBA0.02mg/L medium, long from average leaf, the average bud of induced bud is long all has significant advantage with the induction of other medium, simultaneously growth potential is vigorous and accompany the differentiation (Fig. 2) of indefinite bud, and therefore comprehensive analysis show that this medium is that the optimal medium that golden stem segment with axillary buds has induction is gushed in this experiment.
Different 6-BA and the IBA concentration combination of table 1 on the impact of axillary bud sprouting 1.
1.-: without callus; +: a small amount of callus; ++: callus is general; +++: callus is serious
Test example 2, hormone is on the impact of gushing golden axillalry bud subculture and propagation
Gush at subculture, golden axillalry bud easily occurs that blade is dead, growth potential weak, lignification and cause the phenomenon (Fig. 4) that is difficult to breed gradually gradually.Experimental result is as shown in table 2, medium MS adds in the medium of 6-BA and IBA of variable concentrations, although there is incorporating aspects axillalry bud to breed, the axillalry bud then formed and Multiple Buds thereof are fallen leaves gradually in subculture process, the more and more weaker final death (Fig. 3) of growth potential.And in the medium of 6-BA and IAA being added with variable concentrations, the Multiple Buds that shoot proliferation is formed can normal growth.When IAA concentration is 0.02 ~ 0.10mg/L, when 6-BA is 0.5mg/L, axillalry bud is without propagation, simultaneously axillalry bud lignification gradually, and regeneration difficulty increases; Along with 6-BA be increased to 3mg/L by 1mg/L time, Multiple Buds breeds out (Fig. 5) from the base portion of axillalry bud, axil portion, and growing way is vigorous, and leaf look light green.When 6-BA mono-timing, along with IAA concentration increases, otch base portion callus increases, and is unfavorable for the propagation of Multiple Buds.The average effective bud number that shoot proliferation reaches in medium MS+6-BA3.0mg/L+IAA0.05mg/L is all more than other proliferated culture medium quantity, and the Multiple Buds growth conditions formed is good.Therefore, this medium is that the suitable subculture of golden axillalry bud and proliferated culture medium are gushed in this experiment.
Different 6-BA and the IBA of table 2, IAA concentration combination are on the impact of axillalry bud subculture and propagation 1.
1.-: without callus; +: a small amount of callus; ++: callus is general; +++: callus is serious
Test example 3, hormone is on the impact of gushing golden Multiple Buds and taking root
By grow to 2cm gush that golden Multiple Buds is transferred to that macroelement reduces by half be added with in the root media of variable concentrations growth hormone.As shown in Table 3, medium is not taken root without Multiple Buds during any hormone, along with the rooting rate of the increase Multiple Buds of IBA concentration increases gradually, but when IBA concentration be 0.1mg/L and above time, the incision wound tissue of Multiple Buds increases, root grows from callus, and the vascular tissue of root is not connected with the vascular tissue of stem, surviving of the seedling that is unfavorable for taking root.The adventive root great majority simultaneously formed are for single, and rarer side root forms (Fig. 6).There is advantage in Multiple Buds rooting rate, mean elements and other root medias in 1/2MS+IBA0.05mg/L, therefore this medium is applicable to the culture of rootage of gushing golden Multiple Buds in this experiment relatively.
Table 3IBA is on the impact of gushing golden Multiple Buds and taking root
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, and any change of expecting without creative work or replacement, all should be encompassed within protection scope of the present invention.Therefore, the protection domain that protection scope of the present invention should limit with claims is as the criterion.
Claims (1)
1. fragrant camphor tree kind gushes a method for golden plant regeneration, it is characterized in that, comprises the following steps:
(1) gather fragrant camphor tree kind and gush the golden young sprout taken out then, blade is removed after taking back laboratory, be cut into the stem section with 1 ~ 2 axillalry bud, with liquid detergent cleaning 4 ~ 5min, more than running water 30min, with the alcohol disinfecting 30s of 75% on superclean bench, aseptic water washing 3 times, again with 0.1% mercuric chloride according to stem section diameter sterilization 8 ~ 12min, aseptic water washing 4 ~ 5 times, filter paper suck dry moisture, cut stem section brownization part, be seeded in the MS medium without hormone, select the explant of aseptic stem section as axillary bud deriving, be seeded in the MS medium containing 6-benzyl aminoadenine and indolebutyric acid, after 20d, axillalry bud starts to sprout and opens up leaf gradually, extend, the time of explant collection is August, and the described interpolation proportioning containing the MS medium of 6-benzyl aminoadenine and indolebutyric acid is 6-benzyl aminoadenine 1.0mg/L, indolebutyric acid 0.02mg/L, sucrose 30%,
(2) by grow fine gush golden axillalry bud be seeded to containing 6-benzyl aminoadenine and heteroauxin MS medium together with stem section in axillalry bud can normal growth and propagation, after 30d, average effective bud can reach 6.5 at most, and containing in the MS medium of 6-benzyl aminoadenine and heteroauxin in this step adds 6-benzyl aminoadenine 3.0mg/L, heteroauxin 0.05mg/L, sucrose 30%;
(3) by become simple bud to be seeded to containing indolebutyric acid after long for tender, the average bud of the growth children Multiple Buds scalpel cutting formed for 2.0cm, the propagation that grows fine 1/2MS medium in the formation of inducing adventitious root, after 30d, the highest rooting rate is 41.3%, to continue Transplantation of Regenerated Plantlets to cultivate after 30d to peat: perlite=3: transplant month in the matrix of 1; The condition of culture that said process adopts is: pH value 5.8 ~ 6.0, temperature 24 ~ 26 DEG C, illumination 2000Lux, and every day, light application time 12h, added indolebutyric acid 0.05mg/L, sucrose 20% containing in the 1/2MS medium of indolebutyric acid in this step.
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| CN105941150B (en) * | 2016-05-20 | 2018-06-15 | 宁波城市职业技术学院 | Cinnamomum camphora gushes golden adventitious buds proliferation method |
| CN106234224A (en) * | 2016-08-16 | 2016-12-21 | 吉安市林业科学研究所 | The tissue culture of great Ye Lignum cinnamomi camphorae and method for quickly breeding |
| CN106332716A (en) * | 2016-08-22 | 2017-01-18 | 何荣福 | Camphor tree cuttage method |
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| JP2006055054A (en) * | 2004-08-19 | 2006-03-02 | Taisei Corp | Method for producing cultured seedling of tamarix genus plant |
| CN102144546A (en) * | 2011-01-12 | 2011-08-10 | 福建农林大学 | Method for subculturing camphorwood tissue culture seedlings |
| CN102860258A (en) * | 2012-09-19 | 2013-01-09 | 广东省林业科学研究院 | Clonal tissue culture breeding method for camphor tree |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006055054A (en) * | 2004-08-19 | 2006-03-02 | Taisei Corp | Method for producing cultured seedling of tamarix genus plant |
| CN102144546A (en) * | 2011-01-12 | 2011-08-10 | 福建农林大学 | Method for subculturing camphorwood tissue culture seedlings |
| CN102860258A (en) * | 2012-09-19 | 2013-01-09 | 广东省林业科学研究院 | Clonal tissue culture breeding method for camphor tree |
Non-Patent Citations (2)
| Title |
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| 樟树优良单株选择与组培研究;彭东辉;《福建农林大学硕士学位论文》;20041215;第17-36页，尤其是第3.1节至第3.2.6.1节。 * |
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