CN106688895A

CN106688895A - Method for carrying out generation of hybridized liriodendron chinense somatic embryos by utilizing salicylic acid

Info

Publication number: CN106688895A
Application number: CN201710189443.3A
Authority: CN
Inventors: 施季森; 施帅男; 陈金慧; 成铁龙; 杨立明; 陈婷婷; 陆叶
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2017-03-27
Filing date: 2017-03-27
Publication date: 2017-05-24
Anticipated expiration: 2037-03-27
Also published as: CN106688895B

Abstract

The invention discloses a method for carrying out generation of hybridized liriodendron chinense somatic embryos by utilizing salicylic acid. The method comprises the following steps: establishing a hybridized liriodendron chinense suspension system, obtaining liquid single cells, transiting and inducing the generation of the hybridized liriodendron chinense somatic embryos; in a process of inducing the generation of the hybridized liriodendron chinense somatic embryos, adding the salicylic acid and ABA (Abscisic Acid) into a culture medium at the same time or independently adding the salicylic acid. According to the method for carrying out the generation of the hybridized liriodendron chinense somatic embryos by utilizing the salicylic acid, disclosed by the invention, a plant growth regulator SA is added in a generation process of the hybridized liriodendron chinense somatic embryos, so that the inductivity of the somatic embryos and the synchronism of development are effectively improved; benefits of breeding hybridized liriodendron chinense somatic cell engineering germchits are improved.

Description

A kind of method that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid

Technical field

The invention belongs to tissue-cultured seedling technical field, and in particular to one kind carries out hybridized Chinese tuliptree body embryo using salicylic acid (SA) The method of generation.

Background technology

In recent years, people constantly increase the research occurred to plant somatocyte embryo, it is obtained within decades Quickly development, and obtain very big achievement.Because the body embryo that somatic induction is produced possesses generation, quantity is more, the speed of growth Hurry up, structural integrity, reproductive efficiency high the features such as, make body embryo generation technique turn into micropropagation of plants effective means, be conducive to Preserve excellent forest kind, shorten improved seeds cultivation time, the development for accelerating industrialization etc., have in the development of forestry Significance.

Plant somatocyte embryo is the effective way of plant regeneration in cell engineering, and it can be in some plants especially It is to play a great role during the xylophyta growth and breeding cycle is more long, or the vegetable seeds having is difficult to sprout.Body embryo is led It is divided into the formation of induced embryonic callus and the morphogenesis of body embryo.First stage selection explant, selection immature embryo, Root, stem, leaf, floral organ official rank tissue induced synthesis embryo callus；Second stage induced embryonic callus are differentiated to form body Blast.Cells,primordial forms globular embryo by a series of development, ultimately forms cotyledonary embryos, and cotyledonary embryos are again through further culture Form complete plant.The direct somatic embryo of plant is in the explant direct development such as including organ, tissue, cell It is divided into somatic embryo.And body embryo is further to develop into somatic embryo after explant forms callus callus indirectly, Or form somatic embryo after suspending and cultivating after forming embryo callus.

Forest somatic embryo research origin is in 20 century 70 Rao etc. to grinding that the somatic embryo of santal occurs Study carefully.Although the body embryo of most of xylophyta has certain difficulty, the effort of decades, xylophyta is sent out by body embryo Life forms regeneration plant and has reached more than 200 kinds, and the research that also body embryo of some commerical tree species occurs also obtains newly to enter Exhibition.The parts such as masson pine, torch pine somatic embryo generation system gymnospermous is more ripe, being capable of tissue culture technique Realize plant regeneration.Chen Jinhui etc. is successfully established hybridized Chinese tuliptree somatic embryo and system occurs first, and an one-step inducing of going forward side by side goes out Body embryo seedling, accelerates the industrialized development of hybridized Chinese tuliptree.

When body embryo occurs, extraneous factor induction inside changes or internal oneself factor expresses controlling gene, so that Complete the generation of plant body embryo.Its main influence factor has：Explant selection, genotype, the selection of culture medium, environment bar Part, growth regulatory substance etc..Wherein plant hormone plays irreplaceable effect in body embryo generation.

1) selection of genotype and explant

As long as plant tissue or organ living all can be used as the material of tissue cultures.Body embryo occurs can be because of the shape of explant The factors such as state, type, genotype difference and exist very big difference.In tissue culture procedures are carried out, the explant device of selection Official's differentiation degree is low and physiological status is preferable, can greatly promote body embryo and inductivity occurs.Therefore when tissue induction is carried out, Good, the best in quality material of physiological status is generally selected, the success rate of plant embryonal induction can be increased.

Due to the difference of plant genotype so that during somatic embryo inducement hormone the aspect such as use, condition of culture all Have differences.There is larger difference in the somatic embryo inducement efficiency of different genotype, it is possible to because different genotype is most Good condition of culture is different.When the induced embryonic callus such as Huang Lu and body embryo occur, the body embryo inductivity of different genotype is found Have differences.

2) selection of culture medium

Be generally incubated base it is main by carbohydrate, nitrogen source, inorganic salts, vitamin etc. into being grouped into, each part All play an important role.Wherein nitrogen source has large effect to somatic embryo.Cui Kairong etc. adds difference in the medium When nitrate nitrogen and ammoniacal nitrogen the induction matrimony vine body embryo of ratio occur, find when only adding ammoniacal nitrogen to be not added with nitrate nitrogen in culture medium, Somatic Embryo of Lycium Barbarum fetal hair gives birth to inductivity highest.

3) environmental condition

Be conducive to the induction of xylophyta embryo callus under usual light culture environment, when body embryo carries out maturation culture Illumination, illumination is then needed to be also beneficial to reduce abnormal rate.Although it is dark with illumination condition under can induced embryonic callus, But induced under illumination condition generation embryo callus may browning or somatic embryo regeneration frequency it is not high.Chen Jinhui etc. exists When research hybridized Chinese tuliptree somatic embryo occurs, it is found that dark situation is conducive to embryo callus to grow and body embryo occurs, body Embryo is cultivated under moving to illumination condition after occurring in time, is conducive to body embryonic development and plant regeneration.

Temperature is also the key factor for influenceing body embryo to occur.High temperature or low temperature are likely to influence the body of embryo callus Embryo generating ability.Chen little Peng has found that high temperature can cause body embryo quantity to reduce in the body embryo of cucumber is studied, and reduces body embryo and lures Conductance.Space etc. is opened in the body embryo of research Manchurian ash occurs, and after finding low-temperature treatment body embryo can be promoted to synchronize.

4) effect of the exogenous hormone in body embryo generation

Plant hormone can regulate and control plant physiology reaction under finite concentration, and growing, the aspect such as ripening and senscence sends out Wave most important functions.In plant body embryo generating process plant hormone selection it is critical that.Needed for plant body embryo occurs The plant hormone and content wanted are different, even if kindred plant plant hormone needed for the different phase during induction body embryo occurs It is also different.

From most report, auxin is that somatic embryo division is necessary, can promote the increase of Endogenous auxin, And then promote cell division and Endogenous hormone balance, finally promote cell to enter embryonism.For most plants, except Auxin, body embryo also needs to the basic element of cell division.6-BA (basic element of cell division) is conducive to calli induction.KT is then helped In the quality for improving callus.In recent years studies have found that TDZ activity is higher, body embryo has been induced, the body embryo of such as peanut is lured Lead.Many researchs all confirm ABA in all certain facilitation of the different phase of many plant body embryos.To the ABA of some plants Mutant is studied, and finds to play critical function in plant body embryo stage of ripeness ABA, is conducive to the hair in somatic embryo later stage Educate.ABA can stimulate somatic embryo maturation in the body embryo Induction Process of many plants, can effectively improve body embryo incidence. Due to the difference of tree characteristics, some plants are added Exogenous ABA in somatic embryo generation and are unfavorable for that body embryo occurs or presses down Body embryo processed occurs, such as torch pine, white clouds pine.

Salicylic acid (salicylicacid, SA) is a kind of simple small molecule phenols chemical combination of generally existing in plant Thing, chemical name is " septichen ", is the derivative of cinnamic acid.It is the active component isolated from willow bark. This group of active principle is named as salicylic acid by RaffaelePiria within 1938.1992, Raskin proposed SA can be regarded as A kind of new plant endogenous hormones.After the sixties in 20th century, people start to find that SA has important physiological action in plant. In plant growth and development process, SA can effectively regulate and control the development of plant, ripe and aging.And in some adverse circumstance environment In, under the conditions of such as arid, saline and alkaline, heavy metal, low temperature, it can effectively slow down these abiotic stress.Research discovery includes The crops such as paddy rice, soybean, barley contain salicylic acid in 34 kinds of interior plant leaf blades and reproductive organs, and content is more than 1 μ g/g fresh weights, Illustrate that it is generally existing in plant kingdom.SA is present in plant with free state and with reference to two kinds of forms of state.Free state SA is in crystalline lens, is combined to form in nucleosides, glycolipid, methyl or amino acid by SA with reference to state SA, is soluble in polarity organic molten Agent, is slightly soluble in water, and the pH of saturated aqueous solution is in that acid its value is 2.4.

Salicylic acid can adjust plant physiology reaction as a kind of novel plant hormone, in plant growth, development and resistance etc. Aspect has material impact.In recent years, more to salicylic Resistence research, the research of other field is less, is especially planting The aspect effects such as cell growth differentiation, induction body embryo generation are promoted to be rarely reported under the conditions of thing tissue or colony-formation assays.

The content of the invention

Goal of the invention：For the deficiencies in the prior art, entered using salicylic acid it is an object of the invention to provide one kind The method that row hybridized Chinese tuliptree body embryo occurs, improves body embryo inductivity, the synchronism of development, improves hybridized Chinese tuliptree body cell work The benefit of journey seedling breeding.

Technical scheme：In order to realize foregoing invention purpose, the technical solution adopted by the present invention is：

A kind of method that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid, including hybridized Chinese tuliptree suspension system is built The single celled acquisition of vertical, liquid and transition, the generation step of inducement crossbreeding Liriodendron body embryo, in inducement crossbreeding Liriodendron body embryo During generation, salicylic acid and ABA are added simultaneously in the medium, or individually add salicylic acid.

Described salicylic consumption is 2mg/L for the consumption of 0.01~1.25mg/L mol/L, ABA.

Described culture medium is fluid nutrient medium or solid medium.

The genotype of described hybridized Chinese tuliptree is C38, C138,243012,253010.

The genotype of described hybridized Chinese tuliptree is C138, and culture medium prescription is 3/4MS+SA 0.25mg/L or 3/4MS+ ABA2.0mg/L+SA0.05mg/L。

The genotype of described hybridized Chinese tuliptree is 243012, and culture medium prescription is 3/4MS+ABA2.0mg/L+ SA0.05mg/L。

The genotype of described hybridized Chinese tuliptree is 253010, and culture medium prescription is 3/4MS+ABA2.0mg/L+ SA0.05mg/L or 3/4MS+ABA2.0mg/L+SA0.25mg/L.

Described salicylic consumption is 0.05~0.25mg/L.

Beneficial effect：Compared with prior art, the side that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid of the invention Method, in hybridized Chinese tuliptree Somatic Embryogenesis, adds plant growth regulator SA to effectively improve raising body embryo induction Rate, effectively reduces the generation of its abnormal rate during somatocyte development, improves the breeding quality of hybridized Chinese tuliptree cell engineering. In growth course, the anti-environment stress ability of somatic embryo regeneration plant can be improved, improve hybridized Chinese tuliptree body cell engineering kind The benefit that seedling is bred, promotes the scale application of hybridized Chinese tuliptree somatic embryo regeneration plant, improves forestry large-scale production effect, drop Low final-period management abiotic stress cost.

Brief description of the drawings

Fig. 1 is different genotype inductivity otherness result figure；

Fig. 2 is the unicellular figure under microscope；In figure, a is C138, and b is that 243012, c is 253010；

Fig. 3 is c138 body embryonic development situation maps on different culture media；

Fig. 4 be 243012 on different culture media body embryonic development situation map；

Fig. 5 be 253010 on different culture media body embryonic development situation map；

Fig. 6 is C138 body embryonic development situation maps on different culture media；

Fig. 7 be 243012 on different culture media body embryonic development situation map；

Fig. 8 be 253010 on different culture media body embryonic development situation map；

Fig. 9 is different genotype body embryonic development result figure on different culture media；Figure is A.253010 in No. 1 culture medium, figure B.243012 in No. 1 culture medium, figure C.c138 in No. 1 culture medium, D.253010 in No. 7 culture mediums scheme E.243012 at No. 7 by figure Culture medium, figure F.c138 in No. 7 culture mediums, G.253010 after No. 7 culture medium illumination, scheme H.243012 in No. 7 culture mediums by figure After illumination, figure I.c138 is after No. 7 culture medium illumination；

Figure 10 is that different genotype examines under a microscope result figure；A figures are that 253010, A-1 is its enlarged drawing, and B figures are 243012, B-1 is its enlarged drawing, and C figures are C138, and C-1 is its enlarged drawing.

Specific embodiment

With reference to specific embodiment, the present invention is described further.

The material to be tested used in following examples is selected from the seminar of the applicant during 2012 and 2013 The excellent combination of hybridized Chinese tuliptree matched somebody with somebody, the Callus material induced by immature embryo, specific abductive approach is [old referring to document There is research [D] Nanjing in the intelligent hybridized Chinese tuliptrees somatic embryo of gold：Nanjing Forestry University, 2003.].Genotype is respectively 253010、c38、c138、243012。

The configuration of bigcatkin willow acid mother liquor：The salicylic acid of use is purchased from the Sigma companies pure level product of chemistry, in powdered, purity More than 95%.Take the SA absolute ethyl alcohol hydrotropies of required weight, add water constant volume be configured to 0.1mg/mL mother liquor it is standby.

In following examples, data statistics with analysis：Body embryo number on different culture media is counted after 30d, body embryo state is used EXCEL softwares carry out variance analysis.Callus state is divided into yellow tight type, brown tight type, white rough type, white round and smooth Type, the excellent and poor of culture medium is determined according to callus state.

There is sum/observation of cell number (group) in body embryo incidence (%)=body embryo；

Body embryo maturing rate (%)=mature embryo sum/observation of cell number (group)；

Lopsided embryo formation frequency (%)=Embryos sum/observation of cell number (group)；

Embodiment 1

1st, the foundation of hybridized Chinese tuliptree suspension system

Embryo callus are transferred to concussion and cultivate on callus 250mL fluid nutrient mediums, culture environment is dark training Support, 24 DEG C, shaking speed is 96rpm.Every kind of genotype callus is inoculated with 3 bottles, according to 1：9 proportional arrangement suspension cell training Thing is supported, volume is 50mL after each triangular flask suspension cell and nutrient solution mixing.After material subculture cycle is 7 days, subculture 2 times, Carry out cell sieving.Culture medium concussion and cultivate 1d is adjusted through liquid by the way that 400 purposes are unicellular, culture medium is 3/4MS+ NAA0.2mg/L+KT0.5mg/L+BA0.2mg/L+ABA1.0mg/L+VC5.0mg/L, sugared 50g/L, caseinhydrolysate 0.5mg/ L, culture environment is light culture, and 24 DEG C, shaking speed is 96rpm.

2nd, fluid nutrient medium carries out the induction of body embryo

The liquid suspension cell of excessively culture 2 days is examined under a microscope, body is carried out according to its concentration and cell state The first one-step inducing of embryo.2mL liquid suspension cells are drawn with liquid-transfering gun, addition various concentrations SA (table 1) solid training is uniformly layered on Support on base, different embryonic stage states are observed using Stereo microscope etc., count embryo date and body embryo seedling growth potential.At each concentration 6 wares are managed, 3 experiments are repeated.

The SA of table 1 adds concentration

Culture medium number	ABA(mg/L)	SA(mg/L)
			1	0	0
2	2.0	0
			3	0	0.01
4	0	0.05
			5	0	0.25
6	0	1.25
			7	2.0	0.01
8	2.0	0.05
			9	2.0	0.25
10	2.0	1.25

Wherein agar 6.8g/L, caseinhydrolysate 0.2g/L, sucrose 30g/L, pH 5.7-5.8.

Under many circumstances, genotype is a key factor for influenceing body embryonic development, different genotype induction results In the presence of obvious difference.The present embodiment has counted genotype for c38, c138,253010,243012 totally four hybridized Chinese tuliptree embryos Property callus induction rate, asks for different average values, as a result as shown in Table 2 and Figure 1.

The SA of table 2 makes a difference to different genotype hybridized Chinese tuliptree body embryo

Found out by table 2 and Fig. 1, SA has obvious difference to different genotype hybridized Chinese tuliptree body embryo.At this 4 In genotype, body embryo inductivity genotype higher is 243012, c138, the inductivity of the two genotype reach 60% with On, and callus status are also preferable.Next to that 253010, body embryo inductivity reaches 55%, but callus state labile. Finally, the body embryo inductivity of c38 is minimum, and callus state is worst, and particle is rougher, color whiting.Therefore, in this step-by-step test It may be concluded that the two genotype of 243012 and c138 are preferable.

Otherness is carried out using method of analysis of variance to different genotype body embryo inductivity significantly to analyze, as a result as shown in table 3.

The notable analysis result of different genotype Inducement difference of table 3

Note：SS represents sum of sguares of deviation from mean, represents total variation of data；MS represents average sum of sguares of deviation from mean；F is represented The statistic that variance analysis is obtained；P is exactly P values, is obtained according to F values.Table when crit represents that the standard of F values, i.e. F values are more than crit Show that difference is statistically significant, P values are less than 0.05.

By the significantly analysis of the otherness of table 3 as can be seen that P values are less than 0.05, notable type level has been reached.Illustrate genotype pair Body embryo inductivity there are key factor.

Embodiment 2

1) through liquid suspension culture it is excessive after, different genotype liquid cell state is examined under a microscope, such as Fig. 2 institutes Show.The hybridized Chinese tuliptree suspension cell of Select gene type C138 is cultivated.The SA that 2mL is layered on various concentrations is drawn with liquid-transfering gun (SA concentration is numbering 1 on body embryo inducing culture：0mg/L, numbering 2：0.01mg/L, numbering 3：0.05mg/L, numbering 4： 0.25mg/L, numbering 5：1.25mg/L), examine under a microscope after 28 days and count body embryonic development quantity, counted after 45 days The quantity of body embryo seedling, statistics is as shown in table 4.

The genotype of table 4 is C138 in different culture media upper body embryonal induction situation

Note：+ growth is poor, ++ growth is general；+++ grow；++++growth is preferable；+++ ++ growth is best, similarly hereinafter.

Found out by table 4, the SA of various concentrations is added in the medium, as a result shown, inductivity is above control group, it is different Medium body embryonic development situation is as shown in Figure 3.On without the minimal medium of any hormone, genotype is the hybridization of C138 Liriodendron body embryo inductivity is 0, and when SA is added in minimal medium, its body embryo inductivity substantially rises, and with concentration Rising, inductivity also improves therewith.But, when SA concentration is higher than 0.25mg/L, body embryo inductivity begins to decline.From form In as can be seen that genotype inductivity (3/4MS+SA0.25mg/L) highest in No. 4 culture mediums, reached 60.2%, it is raw Growing way is best.Maturing rate is higher by 4%-8% or so compared with other culture mediums, but abnormal rate difference is less, 8% Left and right.Therefore the optium concentration that single SA is induced C138 body embryos is 0.25mg/L.

2) the liquid suspension cell that 2mL genotype is 243012 is drawn with liquid-transfering gun, is uniformly layered on various concentrations not With on the SA body embryo inducing cultures of concentration, (SA concentration is numbering 1：0mg/L, numbering 2：0.01mg/L, numbering 3：0.05mg/L, Numbering 4：0.25mg/L, numbering 5：1.25mg/L), examine under a microscope after 28 days and count body embryonic development quantity, 45 days The quantity of body embryo seedling is counted afterwards, and different culture media body embryonic development situation is as shown in figure 4, statistics is as shown in table 5：

The genotype of table 5 is 243012 in different culture media upper body embryonal induction situation

Found out by table 5, genotype be 243012 hybridized Chinese tuliptree single SA treatment after body embryo induction situation and gene Type is similar for the hybridized Chinese tuliptree body embryo induction situation of C138, and its inductivity is above positive control, that is, be not added with the 3/ of any hormone 4MS minimal mediums.When with the rising of SA concentration, its body embryo inductivity is also improved therewith.Found in this experiment, induction Rate highest culture medium remains No. 4 culture mediums (3/4MS+SA0.25mg/L), and inductivity has reached 60%, and growth potential is higher. , it is evident that when SA concentration reaches 1.25mg/L, body embryo inductivity is decreased obviously from upper table, than No. 1 culture medium (SA It is 0.01mg/L) low by 5.4%, it is lower than No. 4 culture mediums (SA0.25mg/L) by 9%.Illustrate SA bar of the genotype in high concentration There is certain inhibitory action under part to body embryo.

3) the liquid suspension cell that 2mL genotype is 253010 is drawn with liquid-transfering gun, is uniformly layered on various concentrations different (SA concentration is numbering 1 on the SA body embryo inducing cultures of concentration：0mg/L, numbering 2：0.01mg/L, numbering 3：0.05mg/L, compiles Numbers 4：0.25mg/L, numbering 5：1.25mg/L), examine under a microscope after 28 days and count body embryonic development quantity, after 45 days The quantity of body embryo seedling is counted, as figure 5 illustrates, statistics is as shown in table 6 for different culture media body embryonic development situation.

The genotype of table 6 is 253010 in different culture media upper body embryonal induction situation

As can be seen from Table 6, genotype is lured for 253010 hybridized Chinese tuliptrees body embryo on No. 4 culture mediums (SA0.25mg/L) Conductance highest, is 51.9%.On the SA inducing cultures of various concentrations, body embryo incidence is carried the genotype with SA concentration It is high and rise, but compared with preceding two groups of genotype, the inductivity of the genotype is relatively low, and 50% or so, body embryo maturing rate exists 40% or so.And less, maturing rate amplitude of variation is between 2%-3%, and growth potential is more uniform for the otherness between various concentrations, But compared with control group, its inductivity is higher.Compared with first 2 groups, when SA concentration is more than 0.25mg/L, now body embryo is sent out Raw rate does not significantly decrease, and is reduced only by 0.5%.

4) with liquid-transfering gun draw 2mL genotype be C138 liquid suspension cell, be uniformly layered on various concentrations SA and (hormone combination concentration is numbering 1 on the body embryo inducing culture of ABA：ABA2.0mg/L+SA0.0mg/L, numbering 2： ABA2.0mg/L+SA0.01mg/L, numbering 3：ABA2.0mg/L+SA0.05mg/L, numbering 4：ABA2.0mg/L+SA0.25mg/ L, numbering 5：ABA2.0mg/L+SA1.25mg/L), examine under a microscope after 28 days and count body embryonic development quantity, 45 days The quantity of body embryo seedling is counted afterwards, and body embryonic development situation is as shown in fig. 6, statistics is as shown in table 7.

The genotype of table 7 is C138 in different culture media upper body embryonal induction situation

As can be seen from Table 7, selection concentration is lured for ABA2.0mg/L carries out somatic embryo with the SA of various concentrations respectively Lead, as a result show, in No. 3 culture medium (SA0.05mg/L), inductivity highest is 57.8% to the genotype, and maturing rate is 54.3%, abnormal rate is 3.4%.Secondly it is No. 4 culture mediums (SA0.25mg/L), inductivity is differed only by with No. 3 culture mediums 0.2%.According to data display in table, coordinate on culture medium in SA and ABA, body embryo inductivity with the rising of SA concentration on Rise.When SA is 0.01mg/L, body embryo inductivity is higher by control group 5.9%, illustrates that SA has obvious effect to body embryo induction. It is lower than control group by 5% or so but when concentration reaches 1.25mg/L, body embryo inductivity is decreased obviously, and less than control.From Abnormal rate finds that control group abnormal rate is 7.2%, and after SA is added on the culture medium of ABA, abnormal rate is remarkably decreased, and exists 3%-4% or so, it is seen that after ABA is combined with SA, is conducive to suppressing the sprouting of Embryos.

5) the liquid suspension cell that 2mL genotype is 243012 is drawn with liquid-transfering gun, is uniformly layered on the SA of various concentrations (hormone combination concentration is numbering 1 with the body embryo inducing culture of ABA：ABA2.0mg/L+SA0.0mg/L, numbering 2： ABA2.0mg/L+SA0.01mg/L, numbering 3：ABA2.0mg/L+SA0.05mg/L, numbering 4：ABA2.0mg/L+SA0.25mg/ L, numbering 5：ABA2.0mg/L+SA1.25mg/L), examine under a microscope after 28 days and count body embryonic development quantity, 45 days The quantity of body embryo seedling is counted afterwards, and body embryonic development situation is as shown in fig. 7, statistics is as shown in table 8.

The genotype of table 8 is 243012 in different culture media upper body embryonal induction situation

As shown in Table 8, in the combination culture medium of various concentrations SA and 2.0mg/LABA, data result shows, body embryo hair Raw rate is improved therewith the rising of SA concentration.When SA concentration is 0.05mg/L, body embryo inductivity highest, growth potential is preferable, this When body embryo incidence be 62.6%, maturing rate is 59.5%, and abnormal rate is 3.1%.With C138 genotype similarly, concentration is worked as When bringing up to 0.25mg/L, inductivity has declined, but whether it is obvious that being only below No. 3 culture mediums 1.6%.But when dense Degree continues to improve during to 1.25mg/L, and body embryo inductivity straight line declines, and 12% is have dropped than No. 4 culture mediums, meanwhile, compare control group Experiment is low by 3%.The SA of high concentration is guessed after ABA is combined, and is occurred to body embryo inhibited.From abnormal rate, can be with Find out, control group abnormal rate is higher, be 6.2%, after the SA of various concentrations is added, abnormal rate is decreased obviously, and abnormal rate exists 3% or so.It can be seen that when SA and ABA is applied in combination, can effectively suppress the generation of Embryos.

5) the liquid suspension cell that 2mL genotype is 253010 is drawn with liquid-transfering gun, is uniformly layered on the SA of various concentrations (hormone combination concentration is numbering 1 with the body embryo inducing culture of ABA：ABA2.0mg/L+SA0.0mg/L, numbering 2： ABA2.0mg/L+SA0.01mg/L, numbering 3：ABA2.0mg/L+SA0.05mg/L, numbering 4：ABA2.0mg/L+SA0.25mg/ L, numbering 5：ABA2.0mg/L+SA1.25mg/L), examine under a microscope after 28 days and count body embryonic development quantity, 45 days The quantity of body embryo seedling is counted afterwards, and body embryonic development situation is as shown in figure 8, statistics is as shown in table 9.

The genotype of table 9 is 253010 in different culture media upper body embryonal induction situation

As shown in Table 9, compared with preceding 2 groups of genotype, inductivity is relatively low, in 45%-53% or so for the genotype.With it is preceding Similarly body embryo inductivity is improved with the rising of SA concentration for two groups of experiments, but when concentration reaches 1.25mg/L, body Embryonal induction rate is decreased obviously.In the result, it is respectively No. 3 cultures it can be found that there is 2 groups of culture medium prescriptions to reach 52.6% And No. 4 culture mediums (SA0.25mg/L) (SA0.05mg/L).But the body embryo maturing rate when SA concentration is 0.05mg/L compares SA Concentration is higher by 1.4% when being 0.25mg/L；And on abnormal rate, the abnormal rate as SA0.05mg/L is than SA0.25mg/L's Abnormal rate is low by 1.4%.Therefore, in order to be able to efficiently cultivate excellent body embryo seedling, it is optimal culture that should select No. 3 culture mediums Base, i.e. 3/4MS+ABA2.0mg/L+SA0.05mg/L.The result of abnormal rate shows, on the culture medium without SA, abnormal rate It is higher, 9.1% has been reached, after SA is added, abnormal rate begins to decline, in 5%-7% or so, than the deformity of the first two genotype Rate will height.

Embodiment 3

Hybridized Chinese tuliptree callus is directly transferred on the SA solid mediums of addition various concentrations, 5 are connect per ware Cell block, each concentration is inoculated with 6 wares.Initial culture environment is light culture, after callus grows embryonic type structure, adjusts illumination Environment, is changed to optical culture 16h, light culture 8h, and temperature is 24 DEG C.Specific addition concentration is as shown in table 10.

The SA of table 10 adds concentration

Wherein agar 8.0g/L, lactoalbumin hydrolysate 0.5g/L, activated carbon 4.0g/L, sucrose 40g/L, pH5.7-5.8.

Respectively by genotype for the hybridized Chinese tuliptree callus of C138,243012,253010 is transferred in addition various concentrations SA solid mediums on (table 10), after culture 28 days, there are no obvious body embryo and occur, but subculture twice after, callus Tissue starts embryonal connective tissue occur, and statistics body embryo quantity and developmental state are observed under stereomicroscope.Result such as table 11,12 With shown in 13.

The genotype of table 11 is C138 body embryonic development situations on different culture media

The genotype of table 12 be 243012 on different culture media body embryonic development situation

The genotype of table 13 be 253010 on different culture media body embryonic development situation

As shown in table 11,12,13, the hybridized Chinese tuliptree of these three genotype goes out embryo most on these different culture medias Many culture mediums are No. 7 culture mediums, i.e. 3/4MS+SA0.05mg/L.Although can successfully be induced on the culture medium of addition SA Body embryo, but the SA inductivities of various concentrations have obvious difference, and with the raising of the concentration of SA, germ extraction rate also rises therewith. As the concentration of SA is from 0.01mg/L to 0.05mg/L, body embryo inductivity has brought up to 30% to C138 from 11%；243012 with The concentration of SA from 0.01mg/L to 0.05mg/L, body embryo inductivity from 15% brought up to 47.5% from；253010 with SA's From 0.01mg/L to 0.05mg/L, body embryo inductivity has brought up to 95% to concentration from 5%.Gene is can be seen that from whole result Type for C138 and 243012 hybridized Chinese tuliptree germ extraction rate it is relatively low, and genotype be 253010 germ extraction rate it is higher, peak reaches To 95%, meanwhile, on without the culture medium of any hormone, the genotype can also go out embryo.

From the point of view of callus status, 253010 state is optimal, is yellow, and particle is smaller.In the SA bars of high concentration Under part, though there is brown calli to occur, germ extraction rate is not influenceed.And other two genotype callus status have Coarse, the cell water content more situation such as Fig. 9, shown in 10 of grain.

From the point of view of the embryo time is gone out, on the inducing culture without SA, regardless of genotype, it is general that it goes out the embryo time All over later, 80d or so is arrived, best genotype 253010 will also arrive 40d or so can just go out embryo, but add different in the later stage After the SA of concentration, going out the embryo time starts to shift to an earlier date.When SA concentration is 0.01mg/L, C138 and 243012 can just go out embryo after 57d, And 253010 to go out the embryo time more early, embryo can be just gone out at 30 days or so.Data result shows, with the raising of SA concentration, different bases Also shift to an earlier date therewith because type goes out the embryo time.

Claims

The foundation of a kind of 1. method that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid, including hybridized Chinese tuliptree suspension system, The single celled acquisition of liquid and transition, the generation step of inducement crossbreeding Liriodendron body embryo, it is characterised in that in the inducement crossbreeding goose palm During the generation of Chinese catalpa body embryo, salicylic acid and ABA are added simultaneously in the medium, or individually add salicylic acid.
2. the method that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid according to claim 1, it is characterised in that institute The salicylic consumption stated is 0.01 ~ 1.25mg/L, and the consumption of ABA is 2mg/L.
3. the method that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid according to claim 1, it is characterised in that institute The culture medium stated is fluid nutrient medium or solid medium.
4. the method that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid according to claim any one of 1-3, it is special Levy and be, the genotype of described hybridized Chinese tuliptree is C38, C138,243012,253010.
5. the method that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid according to claim 4, it is characterised in that institute The genotype of the hybridized Chinese tuliptree stated is C138, and culture medium prescription is 3/4MS+ SA 0.25mg/L or 3/4MS+ABA2.0mg/L +SA0.05mg/L。
6. the method that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid according to claim 4, it is characterised in that institute The genotype of the hybridized Chinese tuliptree stated is 243012, and culture medium prescription is 3/4MS+ABA2.0mg/L+SA0.05mg/L.
7. the method that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid according to claim 4, it is characterised in that institute The genotype of the hybridized Chinese tuliptree stated is 253010, and culture medium prescription is 3/4MS+ABA2.0mg/L+SA0.05mg/L or 3/4MS +ABA2.0mg/L+SA0.25mg/L。
8. the method that hybridized Chinese tuliptree body embryo generation is carried out using salicylic acid according to claim 1, it is characterised in that institute The salicylic consumption stated is 0.05 ~ 0.25mg/L.