CN102217542B - Method for propagating red-leaf lonicera maackii quickly - Google Patents

Method for propagating red-leaf lonicera maackii quickly Download PDF

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CN102217542B
CN102217542B CN 201110134199 CN201110134199A CN102217542B CN 102217542 B CN102217542 B CN 102217542B CN 201110134199 CN201110134199 CN 201110134199 CN 201110134199 A CN201110134199 A CN 201110134199A CN 102217542 B CN102217542 B CN 102217542B
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seedling
autumnal leaves
red autumnal
amur honeysuckle
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CN102217542A (en
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石进朝
陈兰芬
左利娟
张耀川
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Beijing Vocational College of Agriculture
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Abstract

The invention discloses a method for propagating red-leaf lonicera maackii quickly, and belongs to the technical field of the quick propagation of plants. The method comprises the following steps of: collecting an explant; performing subculture; performing rooting culture; and performing hardening-seedling culture and field transplanting. The obtained red-leaf lonicera maackii has the advantages of multiple seedling quantity and high propagating coefficients, and can be propagated quickly within a short period; and the method has the advantages of keeping the character of red laminae of the red-leaf lonicera maackii stable.

Description

A kind of red autumnal leaves Amur honeysuckle method for quickly breeding
Technical field
The invention belongs to the micropropagation of plants technical field, specifically be meant a kind of red autumnal leaves Amur honeysuckle method of breeding fast.
Background technology
Amur honeysuckle (Lonicera maackii) is Caprifoliaceae (Caprifoliaceae) Lonicera one machaka seeds, has that happiness light, drought resisting, adaptability are strong, the characteristics of extensive management, makes extensive use in the scape in the northern gardens of China.See flower spring, see fruit autumn, and complete stool is used as medicine, and has higher viewing and admiring and medical value, the document that sees reference [1]: Ceng Lingjie, Xing Junbo, Li Ping. the pre-test of the plant chemotaxonomy research of Caprifoliaceae. and CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2003,25 (3): 184-187; List of references [2]: Lee's Fang. wild plant Flos Lonicerae maackii Determination of nutritional components [J]. chemistry and biotechnology, 2007,24 (1): 77-78; List of references [3]: Chen Yuekai. the Flavonoid substances analysis [J] of Amur honeysuckle. University Of Shanxi's journal: natural science edition, 2006,29 (3): 305-307.The red autumnal leaves Amur honeysuckle is Amur honeysuckle one a natural variation type, and characteristic is: blade is redness in 3～April of spring, and 5～November, new leave was red, and Lao Ye fades to red green, kermesinus, green.Observe the black leaf characteristic have stronger stability for years, have higher ornamental value, be Beijing area one color tree species of one's native land.
At present mainly adopt cottage method for the breeding of red autumnal leaves Amur honeysuckle, because maternal quantity is few, factor such as planting percent is few, and reproduction coefficient is little, limited these fine color seeds---the production of red autumnal leaves Amur honeysuckle is promoted.
Summary of the invention
High among the present invention in order to solve in the prior art red autumnal leaves Amur honeysuckle ornamental value; But the problem that is difficult for breeding; A kind of red autumnal leaves Amur honeysuckle method for quickly breeding is provided, and this propagation method is that scale is promoted fast to produce and utilized this color tree species of one's native land to lay a good foundation.
Red autumnal leaves Amur honeysuckle method for quickly breeding provided by the invention, when red autumnal leaves Amur honeysuckle tissue culture, suitable explant material is the red autumnal leaves Amur honeysuckle young sprout semi-lignified band bud branch section of growth in the greenhouse by solar heat, induces the generation of indefinite bud.Earlier with 70% C 2H 5OH handles 1min, uses HgCl again 2Sterilization 4-5min is best, and the explant germination rate reaches as high as 60-63.3%.The all unsuitable material of lignification band bud branch section, mature leaf, petiole as explant.The appropriate media of the different cultivation stages of red autumnal leaves Amur honeysuckle tissue culture is respectively: subculture medium is MS+6-BA 0.3mgL -1+ NAA0.1mgL -1, root media is 1/2MS+IBA0.2mgL -1Behind the uncork refining seedling 3d, select the red autumnal leaves Amur honeysuckle test-tube seedling transplanting of diameter >=0.5mm for use, can obtain 76.7% survival rate.Different times transplantation rooting test-tube plantlet survival rate is 73.3%-86.7% in 1 year, and the best period that red autumnal leaves Amur honeysuckle test-tube plantlet is transplanted is May September to next year, and survival rate was minimum when transplanted July.Described propagation method specifically realizes through the following steps flow process:
The first step, explant collection;
Be chosen in the red autumnal leaves Amur honeysuckle young sprout semi-lignified band bud branch section of growth in the greenhouse by solar heat, earlier with 70% C 2H 5OH handles 1min, uses HgCl again 2Sterilization 4-5min, sterile distilled water carries out disinfection.
Second step, successive transfer culture;
With the red autumnal leaves Amur honeysuckle young sprout semi-lignified band bud branch section of above-mentioned sterilization at subculture medium MS+6-BA 0.3mgL -1+ NAA0.1mgL -1Cultivated 30 days.
The 3rd step, culture of rootage;
Behind the successive transfer culture 30 days, change root media 1/2MS+IBA0.2mgL over to -1Last cultivation.Begin to grow radical bud after 13 days, through cultivation in 30 days, root reached 3.6cm, and the root of 6 prosperities, stalwartness is arranged, and can refine seedling and cultivate transplanting.
In the 4th step, practice seedling and cultivate;
After 30 days, the test-tube plantlet of robust growth is opened bottleneck in culture of rootage, after 3 days, the red autumnal leaves Amur honeysuckle test-tube seedling transplanting of selecting diameter >=0.5mm can obtain 76.7% survival rate in the seedling-cultivating tray that vermiculite is housed.The test-tube seedling transplanting initial stage, particularly moisture requirement was strict to environmental condition, and air humidity will be controlled at about 90%, and intensity of illumination 5000～10000lx could guarantee higher transplanting survival rate.
The 5th step, field-transplanting;
Greenhouse by solar heat refining seedling was cultivated after 40～45 days, when the seedling seedling height is 25～30cm in the seedling-cultivating tray, can be transplanted to outdoor cropping.The seedling growth height can reach 50-70cm then.
The invention has the advantages that:
(1) to have a Cheng Miaoliang many in the present invention, and the advantage that reproduction coefficient is high can be bred red autumnal leaves Amur honeysuckle nursery stock in a short time fast.
(2) the present invention has the stable advantage of the red leaf characters of the red autumnal leaves of maintenance Amur honeysuckle.
Description of drawings
Fig. 1 is a red autumnal leaves Amur honeysuckle propagation method flow chart of the present invention.
Embodiment
Below in conjunction with accompanying drawing and embodiment red autumnal leaves Amur honeysuckle method for quickly breeding provided by the invention is elaborated.
The present invention provides a kind of red autumnal leaves Amur honeysuckle method for quickly breeding, and method flow is as shown in Figure 1, specifically realizes through following steps:
The first step, explant collection;
Be chosen in the red autumnal leaves Amur honeysuckle young sprout semi-lignified band bud branch section of growth in the greenhouse by solar heat, earlier with 70% C 2H 5OH handles 1min, uses HgCl again 2Sterilization 4-5min, sterile distilled water carries out disinfection.
Second step, successive transfer culture;
With the red autumnal leaves Amur honeysuckle young sprout semi-lignified band bud branch section of above-mentioned sterilization at subculture medium MS+6-BA 0.3mgL -1+ NAA0.1mgL -1Cultivated 30 days.
The 3rd step, culture of rootage;
Behind the successive transfer culture 30 days, change root media 1/2MS+IBA0.2mgL over to -1Last cultivation.When red autumnal leaves Amur honeysuckle subculture seedling length to 5～6cm, change in the root media, begin to grow radical bud after 13 days, through the cultivation of taking root in 30 days, root reached 3.6cm, and 6 prosperities, healthy and strong root are arranged, and can carry out acclimatization and transplants.
In the 4th step, practice seedling and cultivate;
In culture of rootage after 30 days; The test-tube plantlet of robust growth is opened bottleneck, and greenhouse by solar heat refining seedling was cultivated after 3 days, and the red autumnal leaves Amur honeysuckle test-tube seedling transplanting of selecting diameter >=0.5mm is in the seedling-cultivating tray that vermiculite is housed; Greenhouse by solar heat refining seedling was cultivated 40～45 days, can obtain 76.7% survival rate.The test-tube seedling transplanting initial stage, particularly moisture requirement was strict to environmental condition, and air humidity will be controlled at about 90%, and intensity of illumination 5000～10000lx could guarantee higher transplanting survival rate.
The 5th step, field-transplanting;
Greenhouse by solar heat refining seedling was cultivated after 40～45 days, when the seedling seedling height is 25～30cm in the seedling-cultivating tray, can be transplanted to outdoor cropping.The seedling growth height can reach 50-70cm then.
The red autumnal leaves Amur honeysuckle method for quickly breeding that provides for the invention described above; The present invention has also carried out following process of the test; Obtained the preferred parameter of red autumnal leaves Amur honeysuckle breeding in the process of the test, more helped the progress of the breeding of red autumnal leaves Amur honeysuckle, process of the test is following:
One, material:
For 1-5 gives birth to red autumnal leaves Amur honeysuckles (Lonicera maackii ' Hongye ') plant.Test is chosen red autumnal leaves Amur honeysuckle lignification band bud branch section, semi-lignified band bud branch section, band bud children spray section, blade, petiole as explant material.
Two, method:
1. red autumnal leaves Amur honeysuckle proper explant screening;
Choose from the red autumnal leaves Amur honeysuckle of greenhouse by solar heat in June and to be about 1.5cm and to be explant material with the lignification branch section of a pair of axillalry bud, semi-lignified band bud branch section, band bud children spray section, blade, each 30 at 5 positions of petiole; With flowing water flushing material 2h, red autumnal leaves Amur honeysuckle explant is cleaned up.Change explant over to superclean bench, use 70%C 2H 5OH cleans 0.5-1min, with sterile water wash 1-2 time, and the HgCl with 1% 2Clean 4-5min, carry out disinfection for 6-7 time with sterile water wash.On the MS medium, carry out initial culture, condition of culture is: temperature (25 ± 1) ℃, illuminance 2000lx, light application time 12hd -1After 30 days, confirm suitable explant according to the germination rate size.The result sees table 1.
Table 1 red autumnal leaves Amur honeysuckle different explants material and first relation for germination rate
Figure GDA0000148201270000031
Table 1 shows: in red autumnal leaves Amur honeysuckle lignification band bud branch section, semi-lignified band bud branch section, band bud children spray section, blade, 5 positions of petiole, have only semi-lignified band bud branch section can on the MS medium, induce the generation of axillalry bud after 30 days., all pollute as the explant position of drawing materials with red autumnal leaves Amur honeysuckle lignification band bud branch section, band bud children spray section is killed in disinfecting process, and does not germinate.Though blade, petiole pollution rate are lower, can not induce the generation of axillalry bud.Therefore, the suitable explant material of red autumnal leaves Amur honeysuckle is a semi-lignified band bud branch section.
2. the sterilizing test of explant:
2.1 different sterilization methods are to the influence of explant germination rate;
April respectively in the greenhouse by solar heat and the red autumnal leaves Amur honeysuckle plant cultivated of open country choose and be about 1.5cm semi-lignified band bud branch section as explant material, with flowing water flushing material 2h from the beginning, make the explant cleaning surfaces.Adopt 0.1%HgCl 23-8min, 70%C 2H 5OH 60s+HgCl 23-8min is different, and disinfecting time carries out disinfection, and 12 processing are set altogether.Each handles 30 in sample.Condition of culture is: temperature (25 ± 1) ℃, illuminance 2000lx, light application time 12hd -1The result sees table 2.
The different sterilization methods of table 2 are to the influence of red autumnal leaves Amur honeysuckle explant germination rate
Figure GDA0000148201270000041
Figure GDA0000148201270000051
Table 2 shows: 12 processing sterilizing tests that carry out from the semi-lignified red autumnal leaves Amur honeysuckle band bud branch section of greenhouse by solar heat collection in the April in vegetative period, 0.1%HgCl 2When handling 4-5min explant pollution rate, kill ratio, germination rate be respectively 26.7%, 23.3-23.6%, 46.7-50%; Pollution rate, kill ratio, germination rate that 3min handles sample are respectively 100%, 0,0, are respectively 0,100%, 0% during 7-8min.Be up to foundation with the explant germination rate, separately with 0.1%HgCl 2When explant was sterilized, suitable disinfecting time was 4-5min.And if 70%C is used in selection earlier 2H 5The OH 60s that sterilizes uses 0.1%HgCl again 2Sterilization, the disinfecting time during explant germination rate the highest (60%-63.3%) is 4-5min.It is thus clear that the suitable sterilization method of the explant of from greenhouse by solar heat, gathering is: use 70%C earlier 2H 5OH handles 60s, uses 0.1%HgCl again 2Handle 4-5min, explant has higher germination rate.
And gather explant sample, 0.1%HgCl from open country in April 2The explant germination rate is 4% when handling 6min, and all the other germination rates of handling sample are 0.If select to use earlier 70%C 2H 5OH handles 60s, is using 0.1%HgCl 2Handle 3-8min, the disinfecting time of explant germination rate the highest (6.7-10%) is 4-5min.Therefore, when the red autumnal leaves Amur honeysuckle explant sterilization that open country is gathered, use 70%C earlier 2H 5OH handles 60s, uses 0.1%HgCl again 2Handling 4-5min is advisable.
Can find out that through above test when being the material of explant with red autumnal leaves Amur honeysuckle semi-lignified band bud branch section, the red autumnal leaves Amur honeysuckle plant that should grow in the greenhouse by solar heat is gathered, and adopts 70%C 2H 5OH 60s+0.1%HgCl 24-5min can obtain the germination rate of 60%-63.3% to the explant disinfection.
2.2 different explants is gathered the influence of period to the explant germination rate;
Respectively on August 12nd, 2009, October 13, December 14, on February 11st, 2010, April 12, June 15 are gathered 30 of red autumnal leaves Amur honeysuckle semi-lignified band bud branch sections as explant, according to 70%C from greenhouse by solar heat, open country respectively 2H 5OH 60s+0.1%HgCl 24-5min carries out disinfection to explant, and the result sees table 3.
The germination rate of table 3 different acquisition red autumnal leaves in period Amur honeysuckle explant
Figure GDA0000148201270000061
Table 3 shows: the red autumnal leaves Amur honeysuckle semi-lignified band bud branch section germination rate of from greenhouse by solar heat, gathering in continuous a year changes between 53.3%-80%.And the germination rate of open country sample has only 6%, and only when early spring, gathered April, explant just has axillary bud sprouting, and the red autumnal leaves Amur honeysuckle explant that gather other period does not have axillalry bud to bear, all because of polluting death.This and the close living pubescence of Amur honeysuckle branch, the open country sample is thoroughly more relevant than the difficult sterilization of greenhouse by solar heat sample.Therefore, when carrying out the collection of red autumnal leaves Amur honeysuckle explant, the plant that can from greenhouse by solar heat, cultivate is at all seasons gathered, and can obtain higher germination rate.Should not gather from the plant of outdoor cropping.
3. the screening of subculture medium (also being the proliferation and differentiation medium):
The variable concentrations growth regulator is to the influence of red autumnal leaves Amur honeysuckle growth.With MS is increment differentiation minimal medium, adds the 6-BA and the NAA of variable concentrations therein, and each handles 30 in sample.During near bottle cap, the segment that is cut into 2cm changes proliferated culture medium over to and cultivates when reaching 5-6cm for the seedling growing height originally, and the growing height of 30d " Invest, Then Investigate " propagation seedling, internode are apart from size and 3 indexs of growth potential.Judge that according to this suitable 6-BA and NAA add concentration.The result sees table 4.
The influence of table 4 variable concentrations growth regulator confrontation red autumnal leaves Amur honeysuckle test-tube plantlet proliferate
Figure GDA0000148201270000062
Through on the MS minimal medium, the concentration of adjusting different hormones (6-BA, NAA) can promote the growth of axillalry bud.Table 4 can be found out: when 6-BA is 0.3mgL -1, NAA is 0.1mgL -1The time (numbering A2), nursery stock is sturdy behind the 30d, internode is apart from medium, dark green leaf color, high amount of growth is big, growing way is best.When 6-BA is 0.2mgL -1, NAA is 0.2mgL -1The time (numbering A5), growing way is medium, internode is apart from less, the leaf look green.6-BA is 0.2mgL -1, NAA is 0.05mgL -1, a little less than the seedling growth gesture, high amount of growth is little, and internode is apart from little (numbering A7).Show that 6-BA helps shoot proliferation and stem increases girth growth.From propagation seedling growing height and growth potential analysis-by-synthesis, suitable proliferated culture medium is that subculture medium is MS+6-BA0.3mgL -1+ NAA0.1mgL -1
4. the screening of root media;
With the 1/2MS foundation basal culture medium of making a living, add the IBA and the NAA of variable concentrations therein, each handles 30 in sample.Culture of rootage is carried out in subculture seedling inoculation, and the rootage duration of each test-tube plantlet of handling of 30d " Invest, Then Investigate ", on average take root quantity, the length of taking root, root growth gesture judge that according to this suitable IBA and NAA add concentration.The result sees table 5.
Table 5 variable concentrations growth hormone is to the influence of rooting of vitro seedling
Figure GDA0000148201270000071
Through to the analysis-by-synthesis of 6 kinds of root medias rootage duration, the quantity of taking root, root growth amount and growth potential, IBA concentration is 0.2mgL -1, the beginning rootage duration is 13d, 6 of mean elements, and average root long maximum (being 3.6cm) is 1.0mgL than IBA concentration -1, 0.5mgL -1Good to red autumnal leaves Amur honeysuckle rooting of vitro seedling.Numbering B6 NAA concentration is 0.2mgL -1The time, the root growth amount is minimum, is 1.5cm, a little less than the growth potential, is unfavorable for transplant survival.When NAA increases to 0.5mgL -1The time, taking root becomes elongated.IBA, NAA be hestening rooting significantly, receives the influence of the Endogenous Hormones of research material own, and its suitable concentration level of taking root differs.Table 5 shows that the suitable root media of red autumnal leaves Amur honeysuckle test-tube plantlet is: 1/2MS+IBA0.2mgL -1
5. refining seedling and transplanting;
When red autumnal leaves Amur honeysuckle rooting tube plantlet is grown up to 5-6cm, when 4-5 is arranged normal blade, carry out uncork time (0-5d), different-diameter seedling (＜0.3mm, 0.3-0.5mm, 0.5-1mm) transplanting and different times and transplant test.Confirm plant diameter and planting period of suitable uncork refining seedling time, the suitable seedling of taking root with planting survival rate.
5.1 the uncork refining seedling time is to the influence of test-tube seedling transplanting survival rate;
Transplant behind design uncork refining seedling 0,3d, the 5d in the test,, confirm the suitable time of uncork refining seedling, the results are shown in Table 6 according to transplanting survival rate.
The table 6 uncork refining seedling time is to the influence of test-tube seedling transplanting survival rate
Figure GDA0000148201270000072
Figure GDA0000148201270000081
Uncork is the process of rooting tube plantlet being carried out adaptive training, and suitable uncork exercise time can improve the rooting tube plantlet planting survival rate significantly.Can find out by table 6, the transplanting survival rate 0 of not uncork refining seedling red autumnal leaves Amur honeysuckle, and open bottleneck 5d, and transplanting survival rate is 53.3%, the test-tube seedling transplanting survival rate of uncork 3d is the highest, reaches 86.6%.Thus it is clear that, optimum when carrying out red autumnal leaves Amur honeysuckle test-tube seedling transplanting behind the uncork 3d.
5.2 red autumnal leaves Amur honeysuckle test-tube plantlet diameter is to the influence of transplanting survival rate
The red autumnal leaves Amur honeysuckle test-tube plantlet of selection＜0.3mm, 0.3-0.5mm and three kinds of format diameter of 0.5-1mm is implanted in when cultivating in the vermiculite, and survival rate is seen table 7.
Table 7 test-tube plantlet diameter is to the influence of transplanting survival rate
Figure GDA0000148201270000082
Table 7 shows: red autumnal leaves Amur honeysuckle test-tube plantlet diameter becomes positive correlation with transplanting survival rate.After diameter was the red autumnal leaves Amur honeysuckle test-tube seedling transplanting of 0.5-1mm, not only growth was fast, and a large amount of root hair growths are arranged.Therefore, when carrying out red autumnal leaves Amur honeysuckle test-tube seedling transplanting, select for use the red autumnal leaves Amur honeysuckle of diameter >=0.5mm can obtain 76.7% survival rate.
5.3 transplanting period is to the influence of red autumnal leaves Amur honeysuckle test-tube seedling transplanting survival rate;
On September 10th, 2009, December 12, on March 14th, 2010, May 11, July 15, survival rate was seen table 8 when in vermiculite matrix, cultivating red autumnal leaves Amur honeysuckle test-tube seedling transplanting.
The different transplanting of table 8 period is to the influence of test-tube seedling transplanting survival rate
Figure GDA0000148201270000083
Table 8 shows: the size order of different times transplantation rooting test-tube plantlet survival rate is September＞March＞December＞May＞July in 1 year; The best period that red autumnal leaves Amur honeysuckle test-tube plantlet is transplanted is May September to next year; Can obtain the survival rate of 73.3%-86.7%, survival rate was minimum when transplanted July.Nursery stock transplanting survival rate and temperature, cultivation matrix, control measures etc. are closely related; Temperature in May September to the next year greenhouse by solar heat is at 10 ℃-30 ℃; Help transplant survival, stable usually the reaching more than 35 ℃ in the July greenhouse by solar heat is even adopt cooling measure; Temperature causes survival rate not high also more than 30 ℃.
Adopt above-mentioned process of the test that each item suitable parameter of the propagation method of red autumnal leaves Amur honeysuckle is screened, the survival rate of the red autumnal leaves Amur honeysuckle that is obtained is greater than 70%, and employing said method that can be a large amount of carries out the breeding of red autumnal leaves Amur honeysuckle.

Claims (3)

1. a red autumnal leaves Amur honeysuckle method for quickly breeding is characterized in that comprising the steps:
The first step, explant collection;
Be chosen in the red autumnal leaves Amur honeysuckle young sprout semi-lignified band bud branch section of growth in the greenhouse by solar heat, earlier with 70% C 2H 5OH handles 1min, uses HgCl again 2Sterilization 4-5min, sterile distilled water carries out disinfection;
Second step, successive transfer culture;
With the red autumnal leaves Amur honeysuckle young sprout semi-lignified band bud branch section of above-mentioned sterilization at subculture medium MS+6-BA 0.3mgL -1+ NAA0.1mgL -1Cultivated 30 days; Condition of culture is: temperature (25 ± 1) ℃, illuminance 2000lx, light application time 12hd -1
The 3rd step, culture of rootage;
Behind the successive transfer culture 30 days, change root media 1/2MS+IBA0.2mgL over to -1Last cultivation 30 days;
In the 4th step, the refining seedling is cultivated;
After 30 days, the test-tube plantlet of robust growth is opened bottleneck in culture of rootage, after 3 days, select the red autumnal leaves Amur honeysuckle test-tube seedling transplanting of diameter >=0.5mm in the seedling-cultivating tray that vermiculite is housed, to refine the seedling cultivation;
The 5th step, field-transplanting;
The refining seedling was cultivated after 40～45 days in seedling-cultivating tray, when seedling height in the seedling-cultivating tray is 25～30cm, promptly was transplanted to outdoor cropping.
2. a kind of red autumnal leaves Amur honeysuckle method for quickly breeding according to claim 1 is characterized in that: be May September to next year the period of red autumnal leaves Amur honeysuckle test-tube seedling transplanting in the 4th step, survival rate 73.3%-86.7%.
3. a kind of red autumnal leaves Amur honeysuckle method for quickly breeding according to claim 1 is characterized in that: during described refining seedling was cultivated, air humidity kept 90%, intensity of illumination 5000～10000lx.
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