CN102428872B - Culture medium and culture method for cultivating immature embryo of double-petal paeonia lactiflora - Google Patents

Abstract

The invention specifically relates to a culture medium prescription for cultivating immature embryo of double-petal paeonia lactiflora and a method for cultivating the seedlings of the paeonia lactiflora by using the culture medium. The culture medium comprises adventitious bud induction culture medium, adventitious bud strengthening culture medium and rooting culture medium; and the culture medium is formed by adding TDZ, cane sugar and agar based on improved MS or 1/2 improved MS. The culture method comprises the following steps: selecting the seedling embryo; preparing the culture medium; and inoculating to cultivate, wherein the step of inoculating to cultivate is the step of adopting the three culture mediums to induce the adventitious bud, strengthen the adventitious bud and root. Because of the invention, the seedling rate of the immature embryo reaches over 60%, and the technical obstacle that the double-petal paeonia lactiflora cannot establish the seedlings caused by the dysplasia of the seedling embryo. Each germination seedling embryo can establish 3-8 adventitious buds and 5-10 fleshy roots, so the effect that the seedlings of the normal seed differentiate the buds after three years is realized, the growth speed of the seedlings of the paeonia lactiflora is improved, the hybrid seedlings of the paeonia lactiflora flowers at early stage, and the breeding period of the paeonia lactiflora is shortened.

Description

Medium and cultural method that polyphyll Chinese herbaceous peony rataria is cultivated

Technical field

The present invention relates to field of culturing tissues of plants, be specifically related to the culture medium prescription that a kind of polyphyll Chinese herbaceous peony rataria is cultivated, and the method for utilizing this medium culture Chinese herbaceous peony seedling.

Background technology

The good fancy breed of the existing Chinese herbaceous peony of China mainly obtains through the seed selection of growing directly from seeds naturally, does not all show their parental source in the relevant books of Chinese herbaceous peony kind introduction.Though many R&D institutions all on purpose the apolegamy of the selection through the Chinese herbaceous peony parent carried out the Chinese herbaceous peony crossbreeding, progress is very slow for many years, trace it to its cause, this be owing to Chinese herbaceous peony ( Paeonia lactifloraPall.) most important polyphyll proterties is to form by the lobe variation takes place behind its gynoecium or the staminody in the ornamental quality; Therefore; When utilizing the good Chinese herbaceous peony double variety of fancy points to do father and mother originally to carry out crossbreeding; It is solid to be difficult to fertilization, though or can solid many embryo depauperations and can not insemination and emergence; Work according to us is experienced, and in the hybrid seed that obtains, normally the seeds germinated ratio accounts for about 70% because the seed endosperm growth is not enriched, and therefore, the planting percent that improves hybrid seed is the key technology of decision Chinese herbaceous peony crossbreeding operating efficiency.

Plant rataria culture technique is to utilize modern tissue culture means the plant rataria to be separated the technology that under the aseptic condition that exsomatizes, develops into seedling from seed; Be primarily aimed at can not normal development seed carry out the embryo rescue, this on crops such as precocious peach, apricot, Zante currant, cherry by successful Application.Work about tree peony, Chinese herbaceous peony embryo culture; In " Genetical Breeding of Ornamental Plants " teaching material the 32nd chapter " peony breeding " of Cheng Jinshui chief editor; Mr. Li Jiayu points out to use for reference the hybrid embryo culture technique and improves peony distant hybrid planting percent, but not quoting relevant document specifies; Equally, the concrete citations that in " breeding method of tree peony and tree peony and Chinese herbaceous peony " literary composition of Lv Zhenwei, does not also have the peony embryo culture.　　　　　　　　

About the existing many work of the research of Chinese herbaceous peony tissue culture, mainly concentrate on the fast breeding technique research that utilizes band internode stem section, axillalry bud induced bundle to sprout, these researchs effect on the Chinese herbaceous peony crossbreeding is used is little.In the master thesis " Primary Study of Chinese herbaceous peony tissue culture and genetic transformation " of Zhou Meiyan (2007), the author has carried out the cultured in vitro work of Chinese herbaceous peony embryo, and Cheng Miao; But aspect drawing materials; This research " 1., the kind selected for use ' the beautiful slave of powder ' is the single-lobe kind, 2., embryo is the mature seed in September ", these 2 are inconsistent for " kind of 1. selecting for use is mainly the polyphyll type; 2. embryo depauperation; can not normal mature " condition in the Chinese herbaceous peony breeding, therefore, the research does not have direct application value to Chinese herbaceous peony crossbreeding work yet.

Summary of the invention

In order to solve the polyphyll Chinese herbaceous peony because embryo depauperation and the normal technical barrier of insemination and emergence; The invention provides the culture medium prescription that a kind of polyphyll Chinese herbaceous peony rataria is cultivated; And the method for utilizing this medium culture Chinese herbaceous peony seedling, for providing key technology, the Chinese herbaceous peony crossbreeding supports.

The invention discloses the medium that one group of polyphyll Chinese herbaceous peony rataria is cultivated, it comprises:

1. evoking adventive bud medium, filling a prescription is: improvement MS+TDZ 0.5～1.5 mg/L, sucrose 100gL ^-1, agar 6gL ^-1, pH is 5.8;

2. indefinite bud strong seedling culture base, filling a prescription is: improvement MS+TDZ 0.01～0.05 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, pH is 5.8;

3. root media, filling a prescription is: 1/2 improvement MS+IAA, 0.5～1.0 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, active carbon 0.1%, pH are 5.8;

Wherein said improvement MS is that the improvement of the macroelement content in the MS medium is KNO ₃125 mg/L, NH ₄NO ₃250 mg/L, MgSO ₄7H ₂O 125 mg/L, KH ₂PO ₄275 mg/L, CaCl ₂2H ₂O 250 mg/L, other trace element, molysite and organic component content are constant;

Improveing 1/2 MS is meant the macroelement content among the said improvement MS is reduced by half other components unchanged.

The invention also discloses the method with above-mentioned medium culture polyphyll Chinese herbaceous peony rataria, concrete steps are:

(1) draw materials: early July is got the prematurity polyphyll Chinese herbaceous peony seed of healthy growth, picking embryo.

(2) culture medium prescription:

1. evoking adventive bud: improvement MS+TDZ 0.5～1.5 mg/L, sucrose 100gL ^-1, agar 6gL ^-1, pH is 5.8.Place 2 months (accompanying drawing 1) of 25 ℃ of dark cultivations;

2. indefinite bud strong sprout: change above-mentioned material over to improvement MS+TDZ 0.01～0.05 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, pH is 5.8.Place 25 ℃, cultivate 2 months (accompanying drawing 2) under the 1200lx light intensity;

3. take root: change the bud of above-mentioned stalwartness over to 1/2 improvement MS+IAA, 0.5～1.0 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, active carbon 0.1%, pH are 5.8.Continue to place 25 ℃, cultivate under the 1200lx light intensity by 2-3 month (accompanying drawing 3).

Effect: (1) immature embryo planting percent reaches more than 60%, overcome polyphyll Chinese herbaceous peony seed because of the embryo depauperation can not Cheng Miao technology barrier.

(2) each embryo of sprouting can form 3-8 indefinite bud, and 5-10 fleshy root reached the effect of 3 years ability of normal planting seed seedling differentiation seedling, improved the growth rate of Chinese herbaceous peony seedling, helps Chinese herbaceous peony hybrid seedling early flowering, shortens the Chinese herbaceous peony breeding cycle.

Description of drawings

Fig. 1 is that wound one side forms the indefinite bud of growing thickly.

Fig. 2 is indefinite bud growing state in the strong seedling culture base.

Fig. 3 is the seedling situation of taking root.

Embodiment

Employed in the present invention term only if other explanation is arranged, generally has the implication of those of ordinary skills' common sense.

Below in conjunction with concrete preparation embodiment and application implementation example, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit scope of the present invention by any way.

In following embodiment, various processes and the method do not described in detail are conventional methods as known in the art.The source of agents useful for same, trade name and be necessary to list its constituent person indicate when occurring first that all used thereafter identical reagent is like no specified otherwise, and is all identical with the content of indicating first.

Embodiment 1:

With polyphyll Chinese herbaceous peony kind ' the red gorgeous brightness of striving ' is the examination material, carries out this research work:

(1) draw materials: early July gathers ' the red gorgeous brightness of striving ' pericarp and begins the pod of annesl, soaks 10 min with cleaning solution, and running water washes clean cleaning solution; Soaking after 20 minutes running water with 1% rinses well; Peel off the fruit pod, take out seed, earlier with 75% alcohol surface sterilizing, 30 s; Again with the mercuric chloride of 0.1 % sterilization 8min, aseptic water washing 5～6 times.Place on the aseptic filter paper, cut seed open, choose embryo with dissecting needle with scalpel.

(2) culture medium prescription:

1. embryo is inoculated in the medium of following 3 kinds of evoking adventive buds earlier: 1), improvement MS+TDZ 0.5 mg/L, sucrose 100gL ^-1, agar 6gL ^-1, pH is 5.8; 2), improve MS+TDZ 1.0 mg/L, sucrose 100gL ^-1, agar 6gL ^-1, 3), improvement MS+TDZ 2.0 mg/L, sucrose 100gL ^-1, agar 6gL ^-1, pH is 5.8, pH is 5.8, places 25 ℃ of dark cultivations 2 months, about 5-10 the bud (accompanying drawing 1) of growing thickly on the cross section of wound one side, all can occur taking turns.

2. with above-mentioned grow thickly bud according to bud how much be divided into the 1-3 piece, every 3-5 bud changes 3 kinds of medium that reduce TDZ over to: 1), improvement MS+0.01 mg/L+TDZ 0.01～0.05 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, pH is 5.8; 2), improve MS+TDZ 0.02 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, pH is 5.8,3) and improvement MS+TDZ 0.05 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, pH is 5.8, places 25 ℃, carries out strong seedling culture 2 months under the 1200lx light intensity, seedling all can healthy and strong growth (accompanying drawing 2).

3. the bud with above-mentioned stalwartness changes following 2 kinds of root medias over to: 1), 1/2 improvement MS+IAA 0.5 mg/L, and sucrose 30 gL ^-1, agar 6 gL ^-1, active carbon 0.1%, pH are 5.8; 2), 1/2 improve MS+IAA 0.5 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, active carbon 0.1%, pH are 5.8; Place 25 ℃, carry out culture of rootage under the 1200lx light intensity, all can form 5-10 fleshy root after 2-3 month, can carry out acclimatization and transplants (accompanying drawing 3).

The invention is not restricted to these disclosed embodiments, the present invention will cover the scope described in the patent book, and the various modification of claim scope change with equivalence

Claims (2)

1. the medium cultivated of one group of polyphyll Chinese herbaceous peony rataria is characterized in that comprising:

1. evoking adventive bud medium, filling a prescription is: improvement MS+TDZ 0.5～1.5 mg/L, sucrose 100gL ^-1, agar 6gL ^-1, pH is 5.8;

2. indefinite bud strong seedling culture base, filling a prescription is: improvement MS+TDZ 0.01～0.05 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, pH is 5.8;

3. root media, filling a prescription is: 1/2 improvement MS+IAA, 0.5～1.0 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, active carbon 0.1%, pH are 5.8;

Wherein said improvement MS is that the improvement of the macroelement content in the MS medium is KNO ₃125 mg/L, NH ₄NO ₃250 mg/L, MgSO ₄7H ₂O 125 mg/L, KH ₂PO ₄275 mg/L, CaCl ₂2H ₂O 250 mg/L, other trace element, molysite and organic component content are constant;

Improveing 1/2 MS is meant the macroelement content among the said improvement MS is reduced by half other components unchanged.

2. a polyphyll Chinese herbaceous peony rataria cultural method comprises the step of choosing embryo, medium preparation and inoculated and cultured, it is characterized in that the step of said inoculated and cultured is:

1. evoking adventive bud: embryo is inserted the evoking adventive bud medium, place 25 ℃ of dark cultivations 2 months; Said evoking adventive bud culture medium prescription is: improvement MS+TDZ 0.5～1.5 mg/L, sucrose 100gL ^-1, agar 6gL ^-1, pH is 5.8;

2. indefinite bud strong sprout: change the indefinite bud of above-mentioned cultivation over to indefinite bud strong seedling culture base, place 25 ℃, cultivated 2 months under the 1200lx light intensity; Said indefinite bud strong seedling culture based formulas is: improvement MS+TDZ 0.01～0.05 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, pH is 5.8;

3. take root: the bud of the stalwartness that 2. above-mentioned steps is obtained changes root media over to and continues to place 25 ℃, cultivates under the 1200lx light intensity 2-3 month; Said culture of rootage based formulas is: 1/2 improvement MS+IAA, 0.5～1.0 mg/L, sucrose 30 gL ^-1, agar 6 gL ^-1, active carbon 0.1%, pH are 5.8;

Wherein said improvement MS is that the improvement of the macroelement content in the MS medium is KNO ₃125 mg/L, NH ₄NO ₃250 mg/L, MgSO ₄7H ₂O 125 mg/L, KH ₂PO ₄275 mg/L, CaCl ₂2H ₂O 250 mg/L, other trace element, molysite and organic component content are constant;

Improveing 1/2 MS is meant the macroelement content among the said improvement MS is reduced by half other components unchanged.

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