CN109717078A - A kind of bletilla striata cormel in vitro growth cultural method - Google Patents
A kind of bletilla striata cormel in vitro growth cultural method Download PDFInfo
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Abstract
The present invention relates to a kind of bletilla striata cormel in vitro to grow cultural method, provides a kind of bletilla striata cormel in vitro growth cultural method for growing culture including balling culture and cormel in vitro by the regulation to medium component using plant tissue culture technique.Cultural method of the invention, the effect for promoting bletilla striata cormel in vitro to grow is very good, is solved in bletilla striata conventional organization incubation for the first time, the problem that the bulb of test tube seedling is light-weight, diameter is small.The nutrient of cormel in vitro storage can provide a large amount of nutrition for seedling development, therefore available cormel in vitro substitution test tube seedling is transplanted, so as to effectively solve the problems, such as after transplanting overground part be easy to wither, survival rate it is low.Further, since not being subject to seasonal restrictions, it can according to need and induced, grown at any time, and have many advantages, such as not to be affected by the external environment, the rapid propagation in vitro material of a large amount of high quality can be obtained in a short time, be conducive to breeding and large-scale production.This cultural method is easy to operate, practical, generalization is good.
Description
Technical field
The present invention relates to a kind of cormel in vitro to grow cultural method, and in particular to bletilla striata cormel in vitro grows cultural method.
Background technique
The bletilla striata (Bletilla striata) is orchid family bletilla striata category herbaceos perennial, large flower and brilliant color, collection it is ornamental and
Medical value is in one (Shi Jing, 2010).Since wild bletilla striata resource is excessively excavated, in addition bletilla striata seeds are in natural conditions
Lower breeding is difficult, and wild resource is sharply reduced, therefore the bletilla striata is classified as rare endangered plants (Lu Junbo by China
Deng 2011).So far, in relation to bletilla striata platymiscium research report focus primarily upon cultivation technique (be filled often etc., 2015；Chen Run
Pool, 2018), pharmacology and it is clinical (Wang Xiao etc., 2017；Hu Changyu etc., 2018), tissue cultures and fast breeding technique inquire into (Guan Changdong
Deng 2010；Zou Na etc., 2013) etc., also there is part about resource and (Shi Jing, 2010) and cell suspension system is utilized to build
The report of vertical (Liu Junkai, 2012).Based on mitogenetic breeding, mitogenetic coefficient is small for the breeding of the bletilla striata, and the plant strain growth period is longer, and
Consumption kind amount is big, should not carry out large-scale commercial production (Zhang Yicheng, 2007).Tissue culture technique have breeding coefficient it is high, by
The features such as external environmental condition limitation is small, can obtain in a short time lot of materials and accomplish scale production.In the routine of the bletilla striata
In tissue culture procedures, the bulb of test tube seedling is light-weight, diameter is small, and overground part is easy that withered, survival rate is low after transplanting, most of
It also fails within the 2nd year after transplanting reach commercial production standards, and is transplanted with cormel in vitro substitution test tube seedling, because of cormel in vitro
The nutrient of storage can provide a large amount of nutrition for seedling development, and sterile shoot survival percent is low after can effectively solving the problems, such as transplanting
(Liu Xuxin etc., 2015), but there is not been reported for the research at present in relation to bletilla striata cormel in vitro growth screening of medium formula.Cause
This, screening bletilla striata cormel in vitro grows culture medium prescription, is of great significance to the rapid propagation in vitro and industrialized production of its seedling.
Establish and grow technology (Ren Yaping, 2008) in the cormel in vitro that test tube seedling is cultivated on basis, in lily and
Achieved in the plants correlative study such as short-tube lycoris certain achievement (Zhang Yanlong etc., 2006；Ren Yaping etc., 2011；Zhang Yanni etc., 2016；
Liu Xuxin etc., 2018).Numerous test results show that plant hormone directly takes part in the induction and development of lily test tube bulbs,
Type and concentration, which grow lily bulb, has certain promotion (Su Jinghua, 2014).Varshney etc. (2000), Zhu Jin etc.
(2002) He Wangguang duckweed etc. (2005) can provide nutriment for plant, have the study found that sucrose is the carbon source in culture medium
Promote the effect of plant growth, the sucrose of higher concentration can stimulate lily and short-tube lycoris bulb to be formed and grown.Zhang Yanlong etc.
(2006), it opens clean etc. (2010) and Zhang Yanbo (2013) research is thought, the training for inducing lily test tube bulbs to be formed and grown
Feeding base is mainly MS, but in culture medium various concentration a great number of elements also can formation to bulb and growth have an impact.Su Jing
The result of study of magnificent (2014) is shown, wet red and david lily clove when 6-BA and NAA concentration is 0.5mg/L in culture medium
It is best to grow effect.In addition, in the medium add Optimum Contents active carbon (AC), to lily bulb development also have centainly at
Effect (Bong et al., 2005；Hu Fengrong etc., 2006；Wang Xiaoli etc., 2011；Qin Xinhui etc., 2015).Existing research shows
Receive 3 kinds of plants of golden flower category (Lachenalia) and vitro of Lycoris albiflora (Lycoris albiflora) and Lycoris (Lycoris
) etc. chinensis the survival rate of its test tube bulbs is directly related with the size of test tube bulbs after transplanting, the bulb that test tube seedling is formed
It is bigger more can improve transplanting survival rate (Slabbert&Niederwieser, 1999；Yang Xi, 2013), but have not yet to see the bletilla striata
Bulb grows culture and is reported with the correlative study that bulb substitution test tube seedling is transplanted.
Plant growth regulator type and concentration proportioning, minimal medium, sucrose and activated carbon content is inquired into try the bletilla striata
The influence that pipe bulb grows, it is intended to explore the method for promoting bletilla striata cormel in vitro to grow in the short time, filter out most suitable bletilla striata ball
Stem grows culture medium prescription, and then is transplanted with cormel in vitro substitution test tube seedling, is rapid propagation in vitro, breeding and the industry of the bletilla striata
Metaplasia, which produces, provides reference frame.
Summary of the invention
The object of the present invention is to provide a kind of bletilla striata cormel in vitro to grow cultural method.Using method for plant tissue culture,
By the regulation to medium component, a kind of bletilla striata cormel in vitro growth cultural method is provided.
The purpose of the present invention is what is be realized by the following method.
Bletilla striata cormel in vitro of the invention grows cultural method, it is characterised in that incubation step is as follows:
(1) balling culture: the sterile adventitious bud of the bletilla striata is divided into simple bud, is transferred in root media and carries out strong plantlets and rootage
And balling culture, cultivation temperature are 24~26 DEG C, intensity of illumination 2000LX, light application time 12h/d, balling incubation time is
30~70d；The root media is 1/2MS+0.5mg/L IBA+1.0mg/L GA3+ 20g/L sucrose+3.0g/L plant is solidifying
Glue；Wherein 1/2MS culture medium refers in the MS culture medium disclosed in Murashige and Skoog (1962), by a great number of elements
Dosage halves, remaining ingredient and dosage are constant；The a great number of elements is KNO3、NH4NO3、CaCl2·2H2O、MgSO4·7H2O、
KH2PO4；The IBA is 3- indolebutyric acid；The GA3For gibberellin；
(2) cormel in vitro grows culture: after taking step (1) balling culture, test tube seedling similar in bulb size cuts its leaf
Each bulb is inoculated in bulb and grown in culture medium by piece and root system；Cultivation temperature be 23~27 DEG C, intensity of illumination be 1500~
2500LX, light application time 12h/d, it is 30~150d that bulb, which grows incubation time,；The bulb grow culture medium be 1/2~
+ 0~0.5g/L of 2MS+0.5~1.5mg/L 6-BA+0.1~1.0mg/L NAA+0~120g/L sucrose+3.0g/L plant gel
AC；Wherein 1/2~2MS culture medium refers in the MS culture medium disclosed in Murashige and Skoog (1962), a great number of elements
Dosage is 1/2~2 times of original formulation dosage, remaining ingredient and dosage are constant；The a great number of elements is KNO3、NH4NO3、
CaCl2·2H2O、MgSO4·7H2O、KH2PO4；The 6-BA refers to the fast cry of certain animals of 6- benzyl amino；The NAA, refers to α-naphthylacetic acid；It is described
AC refers to active carbon.
Wherein, balling incubation time described in step (1) is 40d；
It is 120d that bulb described in step (2), which grows incubation time,；
It is 1/2MS+1.0mg/L 6-BA+0.1mg/L NAA+ that bulb described in step (2), which grows culture medium prescription,
20.0g/L sucrose+3.0g/L plant gel+0.5g/L AC.
The present invention also protects the bletilla striata cormel in vitro by above method culture.
Grow in Screening of Media in bletilla striata cormel in vitro of the invention, test uses 3 factors (auxin, cell division
Element and minimal medium), 3 horizontal (respectively 3 concentration of auxin, the basic element of cell division and minimal medium), carry out L9(34)
Orthogonal design, totally 9 processing combinations (table 1), screening bletilla striata bulb most preferably grow formula, culture experiment 45d.
1 plant growth regulator of table and minimal medium expand the L of influence on bletilla striata cormel in vitro9(34) orthogonal test because
Plain water-glass
Data 1,2,3 in table indicate 3 different levels of each factor, and data indicate the specific dense of different level in bracket
Angle value.
Range analysis is carried out using 2 data of table, the results are shown in Table 3, influences the examination of bletilla striata cormel in vitro fresh weight proliferation rate
The factor of testing is ordered as NAA > minimal medium > 6-BA, and the experimental factor for influencing diameter and hyperproliferation rate is ordered as training substantially
Base > NAA > 6-BA is supported, it is NAA and minimal medium that illustrating, which influences the principal element of bletilla striata bulb proliferation, and the effect of 6-BA is smaller.
Wherein (table 3), when NAA concentration is 0.1mg/L, each index average that bletilla striata cormel in vitro grows is maximum, fresh weight proliferation rate, diameter
Proliferation rate and hyperproliferation rate are respectively 44.77%, 12.99% and 8.24%, are improved compared with 0.5 with 1.0mg/L processing
141.48% and 23.37%, 103.61% and 7.98%, 57.25% and 36.88%；When minimal medium is 1/2MS, fresh weight
Proliferation rate, diameter proliferation rate and hyperproliferation rate mean value are maximum, be respectively increased 5.10% and 119.69% compared with MS and 2MS processing,
24.29% and 125.69%, 103.83% and 83.17%；The influence that 6-BA grows bulb is smaller, in concentration 1.0mg/L
Fresh weight proliferation rate mean value it is maximum.
2 plant growth regulator of table and minimal medium expand the orthogonal experiments of influence on bletilla striata cormel in vitro
Difference extremely significant (P < 0.01) and significantly (P < 0.05) is respectively indicated with the large and small mothers that write different after column data.Under
Together.
Rooting rate (%)=bulb number of taking root/inoculation bulb sum × 100；
Germination rate (%)=rudiment bulb number/inoculation bulb sum × 100；
The adventitious bud sum for Bud Differentiation number=differentiate/inoculation bulb sum；
Fresh weight proliferation rate (%)=fresh weight difference (fresh weight-initial fresh weight when off-test)/initial fresh weight × 100 (diameter,
Height is same as above).
3 plant growth regulator of table and minimal medium expand the range analysis result of influence on bletilla striata cormel in vitro
A, B, C are respectively fresh weight proliferation rate, diameter proliferation rate and hyperproliferation rate；K is the average value of each factor level；R
For the very poor of each factor level；R ' is adjusted very poor.
The results of analysis of variance is as shown in table 4, and influence of the NAA to bulb fresh weight and diameter proliferation rate reaches extremely significant level respectively
(P<0.01, similarly hereinafter) and the level of signifiance (P<0.05, similarly hereinafter), the influence to hyperproliferation rate be not significant (P>0.05, similarly hereinafter)；Base
Influence of the basal culture medium to fresh weight proliferation rate and hyperproliferation rate reaches the level of signifiance, and the influence to diameter proliferation rate reaches extremely significant
It is horizontal；And influence of the 6-BA to each index does not reach the level of signifiance.
4 plant growth regulator of table and minimal medium expand the results of analysis of variance of influence on bletilla striata cormel in vitro
*It indicates significant difference (P < 0.05), * * indicates that difference is extremely significant (P < 0.01).
As shown in Table 5, with the increase of sucrose concentration in culture medium, the fresh weight proliferation rate of bletilla striata cormel in vitro, diameter proliferation
Rate and hyperproliferation rate are and larger in 40g/L and 20g/L saccharose treatment hourly value respectively in first increasing the variation tendency subtracted afterwards.Ball
Stem fresh weight proliferation rate, diameter the proliferation rate difference in sugared 0~80g/L of concentration is not significant, hyperproliferation rate with when concentration 20g/L most
Greatly, hence it is evident that the processing for being 80,120g/L greater than not sugaring and sugared concentration, the difference of concentration 20g/L and 40g/L are unobvious.Consider
To economic cost, bletilla striata cormel in vitro grows sucrose concentration being preferred with 20g/L in culture medium.
The influence that 5 cane sugar content of table grows bletilla striata cormel in vitro
Table 6 the result shows that, bulb fresh weight, diameter and the hyperproliferation rate of bletilla striata cormel in vitro with activated carbon content increase
Also the trend for first increasing and reducing afterwards is presented, and maximum in 0.5g/L active carbon processing hourly value, relatively control improves respectively
51.86%, 1.76% and 42.28%.Thus the activated carbon dosage filtered out is 0.5g/L.Therefore, bletilla striata cormel in vitro grows
Optimum formula be 1/2MS+1.0mg/L 6-BA+0.1mg/L NAA+20.0g/L sucrose+3.0g/L plant gel+0.5g/L
AC。
The influence that 6 activated carbon content of table expands bletilla striata cormel in vitro
With the data expanded between being proliferated each index correlation is done to the rudiment of taking root of bletilla striata cormel in vitro in table 2, table 5 and table 6
Analysis, the results showed that (table 7) is positively correlated (P < 0.05) in apparent between cormel in vitro proliferation and rooting rate, and between germination rate
Apparent negatively correlated (P < 0.05) is presented.Illustrate to take root to expand bletilla striata cormel in vitro and play a driving role；Bud breaks up to test tube ball
Stem has expanded depression effect.
The correlation analysis of table 7 bletilla striata test tube seedling growth indexes and expansion of corms
Grow culture medium prescription (1/2MS+1.0mg/L 6-BA+0.1mg/L NAA+ in the above-mentioned bulb that screens
20.0g/L sucrose+3.0g/L plant gel+0.5g/L AC) on the basis of, research bletilla striata cormel in vitro grows time dynamic.As a result
As shown in Figure 1-3, cormel in vitro is in the trend that is slowly increased in the preceding 90d for growing culture, and bulb grows each index and just
Difference is not significant between corresponding index when inoculation.Fresh weight proliferation rate is linearly dramatically increased in 90-150d, diameter and height
It spends proliferation rate and also increases in 90-120d most fast, when 120-150d is still significantly increasing.Therefore, bletilla striata cormel in vitro grows
Incubation time suggests duration at least 120d.
Advantages of the present invention: a kind of bletilla striata cormel in vitro growth cultural method provided by the invention can effectively promote the bletilla striata
Cormel in vitro grows, and is solved in bletilla striata conventional organization incubation for the first time, the bulb of test tube seedling is light-weight, diameter is small asks
Topic, therefore the rapid propagation in vitro to the bletilla striata, breeding and seedling industrialized production, have great importance.Bletilla striata cormel in vitro grows
Cultural method can induce at any time, grow due to not being subject to seasonal restrictions, and have proliferation times height, not by external environmental condition
The advantages that influence, is advantageously implemented batch production and large-scale production；It is transplanted with cormel in vitro substitution test tube seedling, cormel in vitro
The nutrient of storage can provide a large amount of nutrition for seedling development, and overground part is easy withered, survival rate after can effectively solving transplanting
Low problem；And it is easy to operate, it is practical, generalization is good.
In conclusion bletilla striata cormel in vitro of the invention grows cultural method, have the following beneficial effects:
1. bletilla striata cormel in vitro of the invention grows cultural method, bletilla striata cormel in vitro growth culture medium is provided for the first time and is matched
Side and its cultural method solve in bletilla striata conventional organization incubation, the problem that the bulb of test tube seedling is light-weight, diameter is small,
It is provided for the rapid propagation in vitro of the bletilla striata, breeding and industrialized production and substitutes the new culture that test tube seedling is transplanted with cormel in vitro
Method.
2. the fresh weight, diameter and hyperproliferation rate using the bletilla striata cormel in vitro of the method for the present invention culture can increase
2016.62%, 202.53% and 108.97%.
3. cultural method of the invention can induce at any time as needed, grow not by seasonal effect, and grow effect
Well, it is limited by external environmental condition small；It is of great significance to the rapid propagation in vitro and industrialized production of realizing bletilla striata seedling.
It is easy to operate, practical, generalization is good 4. bletilla striata cormel in vitro of the invention grows cultural method.
Detailed description of the invention
Fig. 1 is bletilla striata bulb different time sections fresh weight proliferation rate variation tendency schematic diagram.In figure, abscissa indicates bletilla striata ball
Stem grows incubation time (d), and ordinate indicates bletilla striata bulb fresh weight proliferation rate (%), and error line represents standard error SE, different
Lowercase indicates that the difference between different time points reaches the level of signifiance (p < 0.05).
Fig. 2 is bletilla striata bulb different time sections diameter proliferation rate variation tendency schematic diagram.In figure, abscissa indicates bletilla striata ball
Stem grows incubation time (d), and ordinate indicates bletilla striata bulb diameter proliferation rate (%), and error line represents standard error SE, different
Lowercase indicates that the difference between different time points reaches the level of signifiance (p < 0.05).
Fig. 3 is bletilla striata bulb different time sections height growth rate variation tendency schematic diagram.In figure, abscissa indicates bletilla striata ball
Stem grows incubation time (d), and ordinate indicates bletilla striata bulb hyperproliferation rate (%), and error line represents standard error SE, different
Lowercase indicates that the difference between different time points reaches the level of signifiance (p < 0.05).
Specific embodiment
Below with reference to embodiment, the present invention is further elaborated.
A kind of bletilla striata cormel in vitro of embodiment one grows cultural method, comprising the following steps:
(1) balling culture: the sterile adventitious bud of the bletilla striata is divided into simple bud, is transferred in root media and carries out strong plantlets and rootage
And balling culture, cultivation temperature are 24~26 DEG C, intensity of illumination 2000LX, light application time 12h/d, balling incubation time is
60d；The root media is 1/2MS+0.5mg/L IBA+1.0mg/L GA3+ 20g/L sucrose+3.0g/L plant gel；
(2) cormel in vitro grows culture: taking step (1) balling culture 60d, and test tube seedling similar in bulb size, cuts
Its blade and root system obtain bulb, and are inoculated in bulb and grow in culture medium；Cultivation temperature is 23~27 DEG C, and intensity of illumination is
1500~2500LX, light application time 12h/d, it is 45d that the bulb after inoculation, which grows incubation time,；The bulb grows culture medium
For 1/2MS+1.0mg/L 6-BA+0.1mg/L NAA+20g/L sucrose+3.0g/L plant gel+0.5g/L AC.
By embodiment one method obtain and culture bletilla striata cormel in vitro, grow culture 45d when bulb fresh weight, diameter and
Hyperproliferation rate is respectively up to 33.41%, 8.11% and 8.48%.
Two bletilla striata cormel in vitro of embodiment grows cultural method, comprising the following steps:
(1) balling culture: the sterile adventitious bud of the bletilla striata is divided into simple bud, is transferred in root media and carries out strong plantlets and rootage
And balling culture, cultivation temperature are 24~26 DEG C, intensity of illumination 2000LX, light application time 12h/d, balling incubation time is
40d；The root media is 1/2MS+0.5mg/L IBA+1.0mg/L GA3；
(2) cormel in vitro grows culture: taking step (1) balling culture 40d, and test tube seedling similar in bulb size, cuts
Its blade and root system obtain bulb, and are inoculated in each bulb and grow in culture medium；Cultivation temperature is 23~27 DEG C, intensity of illumination
For 1500~2500LX, light application time 12h/d, it is 120d that the bulb after inoculation, which grows incubation time,；The bulb grows training
Supporting base is 1/2MS+1.0mg/L 6-BA+0.1mg/L NAA+20g/L sucrose+3.0g/L plant gel.
By the method culture bletilla striata cormel in vitro of embodiment two, 120d bulb fresh weight, diameter and hyperproliferation rate reach respectively
1042.71%, 150.13% and 87.33%.
Claims (5)
1. a kind of bletilla striata cormel in vitro grows cultural method, it is characterised in that incubation step is as follows:
(1) balling culture: being divided into simple bud for the sterile adventitious bud of the bletilla striata, is transferred in root media and carries out strong plantlets and rootage and knot
Ball culture, cultivation temperature be 24~26 DEG C, intensity of illumination 2000LX, light application time 12h/d, balling incubation time be 30~
70d；The root media is 1/2MS+0.5mg/L IBA+1.0mg/L GA3+ 20g/L sucrose+3.0g/L plant gel；Its
Middle 1/2MS culture medium refers in Murashige and disclosed in Skoog 1962 in MS culture medium, by the dosage of a great number of elements
Halve, remaining ingredient and dosage are constant；The a great number of elements is KNO3、NH4NO3、CaCl2·2H2O、MgSO4·7H2O、
KH2PO4；The IBA is 3- indolebutyric acid；The GA3For gibberellin；
(2) cormel in vitro grow culture: after taking step (1) balling culture, test tube seedling similar in bulb size, cut its blade and
Each bulb is inoculated in bulb and grown in culture medium by root system；Cultivation temperature be 23~27 DEG C, intensity of illumination be 1500~
2500LX, light application time 12h/d, it is 30~150d that bulb, which grows incubation time,；The bulb grow culture medium be 1/2~
+ 0~0.5g/L of 2MS+0.5~1.5mg/L 6-BA+0.1~1.0mg/L NAA+0~120g/L sucrose+3.0g/L plant gel
AC；Wherein 1/2~2MS culture medium refers in Murashige and disclosed in Skoog 1962 in MS culture medium, a great number of elements
Dosage be 1/2~2 times of original formulation dosage, remaining ingredient and dosage are constant；The a great number of elements is KNO3、NH4NO3、
CaCl2·2H2O、MgSO4·7H2O、KH2PO4；The 6-BA refers to the fast cry of certain animals of 6- benzyl amino；The NAA, refers to α-naphthylacetic acid；It is described
AC refers to active carbon.
2. a kind of bletilla striata cormel in vitro according to claim 1 grows cultural method, it is characterised in that described in step (1)
Balling incubation time is 40d.
3. a kind of bletilla striata cormel in vitro according to claim 1 grows cultural method, it is characterised in that described in step (2)
It is 120d that bulb, which grows incubation time,.
4. the cultural method that a kind of bletilla striata cormel in vitro according to claim 1 grows, it is characterised in that step (2) is described
Bulb grow culture medium prescription be 1/2MS+1.0mg/L 6-BA+0.1mg/L NAA+20.0g/L sucrose+3.0g/L plant coagulate
Glue+0.5g/L AC.
5. the bletilla striata cormel in vitro of the method culture either as described in claim 1-4.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103583365A (en) * | 2013-11-19 | 2014-02-19 | 贵州省生物技术研究所 | Production method of bletilla seed stems |
| KR20150103879A (en) * | 2014-03-04 | 2015-09-14 | 고려대학교 산학협력단 | Producing method of orchid seedlings |
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2019
- 2019-03-05 CN CN201910163019.0A patent/CN109717078A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103583365A (en) * | 2013-11-19 | 2014-02-19 | 贵州省生物技术研究所 | Production method of bletilla seed stems |
| KR20150103879A (en) * | 2014-03-04 | 2015-09-14 | 고려대학교 산학협력단 | Producing method of orchid seedlings |
Non-Patent Citations (2)
| Title |
|---|
| 张艳波: ""毛百合组织培养与试管鳞茎膨大的研究"", 《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》 * |
| 马小萍等: ""组培条件对试管鳞茎膨大的影响"", 《南方农业》 * |
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Application publication date: 20190507 |