CN107258336A - A kind of method that RNAi technology of grafting mediation promotes chrysanthemum Blooming - Google Patents

Abstract

The invention belongs to field of plant growing technology, it is related to a kind of regulation and control method at plant florescence, more specifically, is related to a kind of method that RNAi technology mediated by grafting promotes chrysanthemum Blooming.This method includes：The steps such as stock, selection scion, grafting operation are prepared, the stock is using the transgenosis chrysanthemum material after RNAi technology silence methylated genes.In the application, because transgenosis chrysanthemum material is only as stock, it is not used in and blooms, thus it can preferably avoid the bio-safety risk of genetic drift, in addition, by the application of graft technology, it is that opening in advance for different Dendranthema morifolium Varieties can be achieved only to need a transgenosis Dendranthema morifolium Varieties material, the transgeneic procedure of each kind is avoided, applicability is preferable.Promotion chrysanthemum provided herein shifts to an earlier date opening method, preferably realizes the purpose of Blooming on the basis of normal plants and chrysanthemum stay in grade are ensured, has preferable application value for the early blossoming system cultivated improved seeds.

Description

A kind of method that RNAi technology of grafting mediation promotes chrysanthemum Blooming

Technical field

The invention belongs to field of plant growing technology, it is related to a kind of regulation and control method at plant florescence, more specifically, is related to A kind of method that RNAi technology mediated by grafting promotes chrysanthemum Blooming.

Background technology

Chrysanthemum is because of autumn Flowering Characteristics, and the production upper nurturing period is long, and the application of chrysanthemum is have impact on to a certain extent.Example Such as, most of Dendranthema morifolium Varieties florescences have impact on its application in the tourist season on National Day after National Day, just.Thus promote chrysanthemum Blooming, has especially important meaning for reducing cultivation cost.

Chrysanthemum florescence study on regulation is shown, passes through short-day, methylation inhibitor（5-azac, curcumin, Decitabine Deng）The methods such as processing can make multiple kind advance flowering periods of chrysanthemum, just take time that 11 ~ 19 can be shifted to an earlier date wherein after short-day sunshine treatment My god, and methylation inhibitor can make advance flowering period 7 ~ 10 days.But in general, existing florescence control method more takes time and effort, And easily taken a turn for the worse by such environmental effects, it is impossible to stable heredity, therefore will be handled every year, it is difficult to obtain effectively Popularization and application.

The development of molecular biotechnology, new method, new way are provided for chrysanthemum rearing new variety, and also making can Blooming The stable heredity of chrysanthemum character is provided with possibility.In the prior art, patent《When one kind is bloomed using RNAi carrier regulation chrysanthemum Between method》（Application number：201710049401X）In, start with from molecular level, DNA methylation is disturbed using RNAi technology The expression of gene, makes the reduction of DNA methylation level so as to have impact on flowering time, so enable can Blooming character obtain To stable heredity.But the patented technology is in the application there is also certain defect, mainly：RNAi transfer-gen plants may lead to Cross pollination approach and occur natural hybrization with the chrysanthemum under surrounding natural growthing condition, and then there is the risk of genetic drift, this Outer this method regulating florescence of chrysanthemum, often regulates and controls a kind, it is necessary to carry out a transgenic protocol, waste time and energy.

In a word, new florescence control method and control technique is developed, developing for Floristry and promote flowers should With with highly important significance of scientific research and application value, still need and further furtherd investigate.

The content of the invention

The application purpose is to provide a kind of method that RNAi technology of grafting mediation promotes chrysanthemum Blooming, this method Based on existing RNAi interference promotes chrysanthemum Blooming technology, with reference to graft technology application, ensureing transgenic line On the premise of security, by by different Dendranthema morifolium Varieties graftings to transgenic line, regulating and controlling the florescence of different cultivars chrysanthemum, promote Make chrysanthemum Blooming, new technical thought and reference are provided for the development of chrysanthemum cultivation technique.

Details are as follows for the technical scheme that the application is taken.

A kind of method that RNAi technology of grafting mediation promotes chrysanthemum Blooming, including following operating procedure：

（1）Stock is prepared, the stock is using the transgenosis chrysanthemum material after RNAi technology silence methylated genes, concrete example Such as it is that concrete operations are for example using the chrysanthemum plant after RNAi technology silence transmethylase MET1 genes《One kind utilizes RNAi The method that carrier adjusts chrysanthemum flowering time》（Application number：201710049401X）, i.e. RNAi is disturbed turns base after MET1 genes Because of " purpurine spirit "；

The transgenic line, when growing to plant height for 20 ~ 25 cm or so, extracts top bud, breaks apical dominance, make it Grow side shoot；After it grows 2 ~ 3 side shoots, extracing can be standby as stock after unnecessary lateral bud, is further entered with facilitating Row grafting is handled, and stock is can be used as when side shoot length is to 10 ~ 15 cm and carries out grafting；

（2）Scion is selected, the chrysanthemum sprout of the consistent target variety of growth selection health, growing way removes radical leaves and axillary bud After make scion；

The chrysanthemum sprout length is 4 ~ 5cm or so；

The target variety is, for example,：Early flower variety " National Day is red ", late flowering variety " the Huanshui River water gold autumn " etc.；

（3）Grafting operation, by step（2）Middle scion grafting is to step（1）In stock, specifically grasped for example with cleft graft Make, concrete operation method is：

By step（2）The base portion of middle scion is whittled into long 2 ~ 3 centimetres smooth wedge-like；

Cut off step（1）Middle stock major branch top, rip cutting stock major branch center forms it into long 2 ~ 3 centimetres of gaps；

Then the wedge-shaped scion cut is put into being stitched between stock, alignment is adjacent to；

Tightened from bottom to top with preservative film sturdy；

After the completion of grafting, normal field management.

Engrafting method is a kind of very conventional method in arable farming breeding, but the application in florescence control extremely has Limit, and due to chrysanthemum draft characteristic, using relatively fewer.The achievement of new RNAi silent technology regulating florescence of chrysanthemum so that The stable heredity of chrysanthemum Blooming character has the possibility realized.On this basis, using graft technology, inventor is to turn base Because chrysanthemum material is as stock, using non-transgenic Dendranthema morifolium Varieties as scion, and RNAi reticent signals are transmitted in grafting part, Make the expression of MET1 genes in its interference scion that played a role in scion so as to influence DNA methylation level, and then influence to connect The flowering time of fringe breed chrysanthemum.

In the application, because transgenosis chrysanthemum material is only as stock, it is not used in and blooms, thus can preferably avoids gene The bio-safety risk of drift, in addition, by the application of graft technology, only needing a transgenosis Dendranthema morifolium Varieties material to be that can be achieved Different Dendranthema morifolium Varieties open in advance, it is to avoid the transgeneic procedure of each kind, and applicability is preferable.

In general, promotion chrysanthemum provided herein shifts to an earlier date opening method, is ensuring normal plants and chrysanthemum The purpose of Blooming preferably is realized on the basis of stay in grade, has for the early blossoming system cultivated improved seeds and preferably should With value；This method is compared to other florescence control methods simultaneously, and less input for human and material resources, suitable for mass, scale Metaplasia is produced, and productivity effect is more objective, thus with preferable application value.

Embodiment

Technical scheme is further discussed in detail with reference to embodiments, specific embodiment is being introduced Before, it is briefly discussed below with regard to being related to partial material situation in following embodiments.

Biomaterial：

Transgenosis " purpurine spirit " chrysanthemum material, referring specifically to《A kind of method that utilization RNAi carrier adjusts chrysanthemum flowering time》 （Application number：201710049401X）Middle operation, because the material is prepared by inventor, thus remains with associated materials；

Early flower variety " National Day is red ", late flowering variety " the Huanshui River water gold autumn ", are common Dendranthema morifolium Varieties.

Embodiment

The RNAi technology of grafting mediation provided herein promotes the method for chrysanthemum Blooming, specific operation process letter It is described below.

（1）Stock is prepared, so that RNAi disturbs the transgenosis after MET1 genes " purpurine spirit " chrysanthemum material as an example, due to original Material is relatively limited, thus the appropriate amplification breeding of progress first is standby, is carried out when amplification is bred using tissue culture mode, culture used Base is：MS+1.0 mg/L 6-BA+ 0.15 mg/L NAA（pH= 5.8）, then entered with MS culture mediums as root media Row is taken root, and is formulated as the % sucrose of MS+3.Condition of culture is：Temperature（25±1）DEG C, intensity of illumination is 2000 Lux, per the daylight It is 12 according to the time.

After chrysanthemum seedling takes root, continue to carry out conventional hardening after cultivating 1 ~ 2 month, during hardening, by rooted plantlet transplant to Culture matrix in hardening, hole tray is carried out in hole tray is：Pearl salt：Vegetation charcoal=2：1（Mass ratio）；Shading culture in natural environment；

After chrysanthemum seedling grows new root, transplant to experimental plot, carry out conventional field management standby as stock；

By taking Henan area as an example, transgenic line about June at the beginning, you can grow to 20 ~ 25 cm or so, now extract top Bud is held, breaks apical dominance, it is grown side shoot；After it grows 2 ~ 3 side shoots, standby it can make after extracing unnecessary lateral bud For stock, grafting processing is further carried out to facilitate, stock is can be used as when side shoot length is to 10 ~ 15 cm and carries out grafting.

（2）Select scion, growth selection health, the chrysanthemum sprout of the consistent target variety of growing way, remove radical leaves with Make scion after axillary bud；

The chrysanthemum sprout length is 4 ~ 5cm；

The target variety is specific by taking early flower variety " National Day is red " and late flowering variety " the Huanshui River water gold autumn " as an example.

（3）Grafting operation, by step（2）Middle scion grafting is to step（1）In stock, specifically enter for example with cleft graft Row is operated, and concrete operation method is：

By step（2）The base portion of middle scion is whittled into long 2 ~ 3 centimetres smooth wedge-like；

Cut off step（1）Middle stock major branch top, rip cutting stock major branch center forms it into long 2 ~ 3 centimetres of gaps；

Then the wedge-shaped scion cut is put into being stitched between stock, alignment is adjacent to；

Tightened from bottom to top with preservative film sturdy；

By taking Henan area as an example, grafting time is to carry out or so mid-July, with afternoon 16 during grafting operation:00~18:00 operation It is advisable；

The chrysanthemum scion variety the Huanshui River water gold autumn is grafted onto in 12 plants of transgenosis and 12 plants of non-transgenic Dendranthema morifolium Varieties purpurine spirits respectively, often Strain contains 2 ~ 3 scion；The same processing of red work of chrysanthemum scion variety National Day；

After the completion of grafting, normal field management.

After the completion of grafting, buddingging the date in record process of the test（The date budding to there is macroscopic petal as mark Standard is counted）, flowering dates（Flowering dates is all expanded into standard with petal and counted）, budding the time（Budding the time To budding the date-the transplanting date counted as standard）, flowering time（Flowering time is using flowering dates-transplanting date as standard Counted）.

Final statistical result is as shown in the table：

Note：Control group is the Dendranthema morifolium Varieties being grafted onto in the spirit of non-transgenic purpurine, and treatment group is to be grafted onto in the spirit of transgenosis purpurine Dendranthema morifolium Varieties.

From the above it can be seen that：Scion late flowering variety " the Huanshui River water gold autumn " control group and treatment group bud time of occurrence and Just take time to be divided into 121 d and 154 d, 109 d and 145 d；And after RNA interference transfer-gen plant is grafted in, budding Time and flowering time all significantly shorten compared with control group, respectively 12 d and 9 d in advance, flower bud development time-triggered protocol group with There is no difference between control；

Scion morning flower variety " National Day is red " treatment group with compare between almost while budding, treatment group conspicuousness shortens bud Development time, blooms so that chrysanthemum shifts to an earlier date 12 d.

Analysis as can be seen that with can using the transgenic line of Blooming as stock, grafting routine Dendranthema morifolium Varieties after, pin To different Dendranthema morifolium Varieties（Early flower variety, late flowering variety）When, time advance effect of buddingging is less consistent, but with regard to flowering time Speech, can preferably realize and to open effect in advance, it was demonstrated that the feasibility of this technology, and utilize the technology, for regulation chrysanthemum flowers Phase has highly important application value.

Claims (6)

1. a kind of method that RNAi technology of grafting mediation promotes chrysanthemum Blooming, it is characterised in that including following operation step Suddenly：

（1）Stock is prepared, the stock is using the transgenosis chrysanthemum material after RNAi technology silence methylated genes；

（2）Scion is selected, the chrysanthemum sprout of the consistent target variety of growth selection health, growing way removes radical leaves and axillary bud After make scion；

（3）Grafting operation, by step（2）Middle scion grafting is to step（1）In stock, after the completion of grafting, normal field management .

2. the method that the RNAi technology of grafting mediation as claimed in claim 1 promotes chrysanthemum Blooming, it is characterised in that step （1）In, the transgenosis chrysanthemum material is transgenosis " purpurine spirit " after silence transmethylase MET1 genes.

3. the method that the RNAi technology of grafting mediation as claimed in claim 1 promotes chrysanthemum Blooming, it is characterised in that step （1）In, the stock, seed selection step is：

When chrysanthemum Material growth to plant height is 20 ~ 25 cm, top bud is extractd, breaks apical dominance, it is grown side shoot；Treat It is grown after 2 ~ 3 side shoots, extracts unnecessary lateral bud, stock is used as when side shoot length is to 10 ~ 15 cm.

4. the method that the RNAi technology of grafting mediation as claimed in claim 1 promotes chrysanthemum Blooming, it is characterised in that step （2）In, the chrysanthemum sprout length is 4 ~ 5cm.

5. the method that the RNAi technology of grafting mediation as claimed in claim 4 promotes chrysanthemum Blooming, it is characterised in that described Dendranthema morifolium Varieties are：Early flower variety " National Day is red ", late flowering variety " the Huanshui River water gold autumn ".

6. the method that the RNAi technology of grafting mediation as claimed in claim 1 promotes chrysanthemum Blooming, it is characterised in that step （3）In, grafting operation is carried out using cleft graft, concrete operation method is：

By step（2）The base portion of middle scion is whittled into long 2 ~ 3 centimetres smooth wedge-like；

Cut off step（1）Middle stock major branch top, rip cutting stock major branch center forms it into long 2 ~ 3 centimetres of gaps；

Then the wedge-shaped scion cut is put into being stitched between stock, alignment is adjacent to；

Tightened from bottom to top with preservative film sturdy.