CN109906939A - A kind of pepper in vitro regeneration method and used medium - Google Patents
A kind of pepper in vitro regeneration method and used medium Download PDFInfo
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Abstract
The present invention relates to a kind of suitable for capsicum in-vitro regeneration method and its culture medium used, it is using capsicum aseptic seedling Cotyledons with petiole as explant, regeneration bud induced medium hormone prescription is 3.0-9.0 mg/L ZT+5.0-9.0 mg/L 6-BA+0.05 mg/L IAA, 1-2 normal adventitious buds are retained and deleting lopsided rose petaloid thallus, the hormone prescription of regeneration bud subculture medium is 5.0-7.0 mg/L ZT+7.0 mg/L 6-BA+0.05 mg/L IAA, and the hormone in medium formula taken root is 0.5-0.7 mg/L IBA.Solid foundation has been established in the foundation for being established as further establishing pepper transformation system of the capsicum vitro Regeneration System.
Description
Technical field
The invention belongs to field of plant tissue culture, and in particular to the vitro Regeneration System of capsicum and its culture used
Base.
Background technique
Capsicum (Capsicum annuum L.) belongs to Solanaceae, is a kind of important vegetable crop and tune being favored by people
Taste product, worldwide cultivation extensively.Due to the vulnerability day of one's doom of biology and abiotic stress and capsicum autogene
The development of capsicum is made.It is currently to make up crop using Regeneration in Vitro and genetic transfoumation as crucial Plant Biotechnology
Conventional breeding deficiency and the effective means for promoting crop breeding process.
Carried out capsicum tissue culture work more than 40 year so far for the first time in 1978 from Gunay, in succession about explore capsicum from
The document report of body Regeneration System.It according to statistics, is peppery using seedling cotyledon, stem apex and plumular axis as the Regeneration in Vitro of explant
One field of green pepper Regeneration System most study, however these capsicum Regeneration in Vitro technologies predominantly stay in bud induction rank
Section, however it remains the problems such as bud is difficult to extend, difficulty of taking root.In the Induction Process of capsicum shoots derived in vitro, generally use
The hormone prescription of culture medium is the proportion of 6-BA and IAA, and regeneration induction efficiency is 90% or more.In order to explore capsicum in vitro again
The elongation of adventitious bud during life, researchers lay particular emphasis on hormone combination optimization, it is believed that GA is one of evoking adventive bud elongation
Key factor.Such as Chinese patent literature CN109258469A, adventitious bud induction culture base used are MS+3.0mg/L 6-BA
+ 0.1mg/L IAA, bud elongation medium add GA on the basis of adventitious bud induction culture base3This hormone；In Chinese patent
Bud elongation Fiber differentiation in document CN108338073A similarly uses addition GA3.However, GA induces capsicum Regeneration in Vitro
The problems such as effect of Elongation of adventitious bud does not comply with one's wishes, the elongation low efficiency of bud, and there are genotype dependences.In addition, Yang Bozhi etc.
(Molecular Plant Breeding, 2018, volume 16, the 4th phase, the 1244-1249 pages) report 6-BA dipping pretreatment Shoot Organogenesis from Cotyledon of Pepper
Explant, obtains that best adventitious bud induction culture base is MS+4.0mg/L 6-BA and best Elongation of adventitious bud culture medium is MS+
6.0mg/L 6-BA+0.1mg/L IAA, for emphasis seemingly in dipping pretreatment, hormone prescription is unsatisfactory, and bud extends efficiency
Only 23.21%.
Up to the present, capsicum is not broken through always by the technology that tissue cultures obtain regenerating system, utilizes DNA weight
The progress that group technology carries out pepper transformation is also very slow.There is researcher to attempt some genetic transformation sides in capsicum
Method, such as using embryo callus subculture, proembryo and mature somatic embryo as receptor carry out genetic transformation or using protoplast fusion,
Mediated by agriculture bacillus obtains under the methods of pollen tube and Gene Knock-out Mice, or water planting nursery+external environment for directly being improved
Take capsicum transformation seedlings, but that there are transformation efficiencies is low, regeneration frequency is low, the period is long, Elongation of adventitious bud is difficult or real for these methods
Test the problems such as at high cost, operation is relative complex.
Efficient Regeneration in Vitro and genetic conversion system are established, is that crop utilization technique for gene engineering carries out effective heredity
Improvement basis.However, the vitro Regeneration System foundation of capsicum does not obtain always important breakthrough, to limit pepper transformation
Application of the foundation and genetic engineering of system on capsicum genetic improvement, it would be highly desirable to establish efficient, stable capsicum Regeneration in Vitro body
System.
Summary of the invention
Present invention firstly provides a kind of methods that capsicum explant induces differentiation adventitious bud, with capsicum aseptic seedling Cotyledons with petiole
For explant, regeneration bud inductive differentiation medium hormone prescription is 3.0-9.0mg/L ZT+5.0-9.0mg/L 6-BA+0.04-
0.06mg/L IAA。
The Shoot Organogenesis from Cotyledon of Pepper for being in differentiation state is returned to splitting status and starts quick Differentiation needs
Exogenous hormone, the basic element of cell division and auxin collocation have facilitation to the differentiation of bud.The present inventor research and development when unintentionally
It was found that the concentration of ZT and 6-BA is in the case where relatively high, better effect, auxin IAA and basic element of cell division 6-BA and/or
ZT collocation can 100% induction regeneration of cotyledons formation thallus.But the type and its concentration of thallophytic state and the basic element of cell division
Closely related: 6-BA mainly induces Excised cotyledons petiole end notch rose petaloid thallus to be formed, ZT regeneration induction it is lobate
Body includes rose petaloid and strip, and cut ends calli induction and 6-BA are closely related.Callus it is quick
Growth is conducive to supply thallophytic growth from the nutriment obtained in culture medium including plant hormone.Therefore deeply
On the basis of research and many experiments, in the suitable situation of IAA concentration, 3.0-9.0mg/L ZT and 5.0-9.0mg/L 6-BA
The thallus that combination can make Cotyledon Segments regeneration many, most of thallus are in rose petaloid, and small part is elongated, and
Thallus growth is fast, therefore the concentration combination is suitable for the regenerated induction of Cotyledon Segments.
In one embodiment, regeneration bud inductive differentiation medium is using MB as minimal medium, preferably the concentration of ZT
For 5.0-7.0mg/L ZT, 6-BA concentration is 6.0-8.0mg/L, IAA concentration be 0.05mg/L. thus, the present invention also provides use
In the regeneration inductive differentiation medium of above-mentioned induction differentiation.
Further, the acquisition of regeneration bud induction atomization aseptic seedling is conventional method, such as selects health without germ
Capsicum fresh seeds (current year harvest), soaked seed with pure water and carried out disinfection after 48h, such as first with 75% alcohol disinfecting 60s, then
Use 0.1%HgCl210min is sterilized, then uses aseptic water washing 4 times, 2min/ times, seed is finally inoculated in 3% sucrose of addition
With the MB culture medium (MS a great number of elements+MS microelement+MS molysite+B5 organic principle, pH=5.8) of 0.5% agar powder, illumination
It cultivates (temperature: 25 ± 2 DEG C, light intensity: 2500Lx, the photoperiod: 14h illumination/10h is dark), seed, which starts to sprout, after about 7d forms
Aseptic seedling.
Wherein explant is Cotyledons with petiole, more specifically cuts the cotyledon of capsicum aseptic seedling from petiole end and obtains, preferably
Ground has the petiole of 0.1-0.2cm long, and blade tip is cut off at nearly blade tip 1-2cm.
Comprising the concrete steps that for regeneration bud induction differentiation culture first passes through dark culture, then carries out optical culture, preferably dark training
Support 5d to 30d, more preferably dark culture 10d to 25d, most preferably dark culture 21d；Optical culture condition is temperature: 25 ± 2 DEG C, light
Strong: 2500Lx, the photoperiod: 14h illumination/10h is dark.Preferably the cotyledon explant outside of belly is inoculated with the back side downward upward when culture
In on regeneration bud inductive differentiation medium.
The elongation of adventitious bud is also one of the key link of capsicum Regeneration in Vitro seedling.Although can be using conventional method
It carries out, but in order to reach better effect, the present inventor has developed one kind and deletes regeneration bud quantity and hormone scheme is cooperated to obtain
The effect of good regeneration bud elongation, constitutes another aspect of the present invention.The rose petaloid thallus of deformity is cut off,
Staying 1-2 blade is in the normal adventitious bud to raw strip, carries out squamous subculture, and squamous subculture hormone prescription is 0.04-
0.06mg/L IAA, 3.0-7.0mg/L ZT is combined with 5.0-9.0mg/L 6-BA, and wherein IAA preferred concentration is 0.05mg/L,
ZT preferred concentration is 5.0-7.0mg/L, and more preferably 6.0mg/L, 6-BA preferred concentration is 6.0-8.0mg/L, more preferably
7.0mg/L.Further, subculture medium is using MB as minimal medium, pH5.8.
Therefore, the present invention also provides a kind of subculture methods of Elongation of adventitious bud on explant.The method is to be based on
The inventors discovered that more than the capsicum Excised cotyledons thallus quantity that regeneration induction is formed on regeneration differential medium and most of
For the rose petaloid of deformity, minority is strip, their continued growths on differential medium have no the elongation of stem.And it is existing
Research is thought to add GA in the Elongation of adventitious bud stage3It is required.The present invention shows through test result, GA3It is lobate to different conditions
The influence of the stem elongation of body has differences: GA3It is few elongated to raw thallophytic that Excised cotyledons are regenerated with the quantity to be formed
Stem elongation has apparent facilitation, unknown to rose petaloid thallus and the non-effect to the thallophytic stem elongation of raw shape
It is aobvious.Suitable GA3Concentration is 1.0-2.0mg/L, GA3Concentration easily makes thallus in water stain shape when being 3.0mg/L.In addition, GA3Make
Thallus yellow is thinning, even if the stem of regeneration induction bud extends, it is also difficult to take root.Because the regeneration of capsicum Excised cotyledons is indefinite
Taking root for bud is closely related with the state of adventitious bud.Through GA3The adventitious bud of induction elongation is fine, leaf yellow is thinning, difficulty of taking root,
And induce the adventitious bud stalwartness of stem elongation, leaf dark green by deleting thallophytic method, easily take root.
And the subculture method in the present invention is used, discovery carries out squamous subculture, and regeneration bud obviously extends after 2 weeks, leaf
Dark green piece is in oblong, and stem thickness is strong.Influence of the ZT and 6-BA that various concentration matches in subculture medium to Elongation of adventitious bud
Aspect is it can be seen that 1.0mg/L ZT combines stem elongation≤40% that can make regeneration bud with 1.0-9.0mg/L 6-BA；With
The elongation of the increase of ZT concentration, thallus stem is significantly raised, and 3.0-7.0mg/L ZT combines energy with 5.0-9.0mg/L 6-BA
Regeneration bud elongation is set to be higher than 67%.The regeneration bud elongation highest that 5.0-7.0mg/L ZT is combined with 7.0mg/L 6-BA is
88%-90% is significantly higher than other combinations, the hormone combinations being as suitable in the subculture medium of regeneration bud elongation.Through deleting
Subtract and only remains after the normal adventitious buds of 1-2 (blade is in raw strip) grow about 15d on suitable subculture medium, bud
4-5cm or so is extended, is the growth phase of 1 heart of 3-4 leaf, effect is obviously.
In turn, the present invention also provides the subculture mediums of above-mentioned Elongation of adventitious bud.
Conventional step can be taken poly- step of taking root, but preferably take following step: will be deleted thallus stretches stem
Long regeneration bud is cut from basal part of stem, is forwarded on root media and is carried out illumination cultivation, wherein preferably use culture medium for
1/2MB culture, wherein addition 0.5-0.7mg/L IBA, can further add 0.1% active carbon.Based on research discovery by deleting
Adventitious bud stalwartness, the leaf for subtracting thallophytic method induction stem elongation are dark green, easily take root, add 0.5- in 1/2MB culture
About 30d is grown in the root media of 0.7mg/L IBA+0.1% active carbon and forms flourishing root system, and seedling robust growth, through refining
After transplantation of seedlings, high survival rate is up to 100%.The present inventor also carries out the IBA of various concentration proportion to the influence that regeneration bud is taken root
Research, IBA take root there are concentration effect to regeneration bud, and low concentration and higher concentration IBA are unfavorable for taking root for regeneration bud,
Rooting rate is substantially less than the IBA of intermediate concentration, and beginning rootage duration is long, and the low and average root long of coefficient of taking root is short.As a result table
Bright to be suitable for the IBA concentration that regeneration bud is taken root as 0.5-0.7mg/L, rooting rate is up to 100%, and about 10d starts to take root, and taking root is
Number is 3.5-3.8, and average root is up to 9.8-10.5cm, and root shows great-hearted white, and lateral root is very flourishing.
Conventional steps can be used for hardening and transplant step, but preferably take following step: by culture of rootage about 30d's
The aseptic seedling of well developed root system is first gradually uncapped hardening 4-6d in illumination cultivation room, then cleans root culture medium with clear water, finally
Transplanting is to turf: vermiculite: garden mould=1: shading heat preservation 10-20d, preferably 15d in the matrix of 1: 1 (volume ratio).Experimental result
Show the high survival rate of seedling after transplanting up to 100%, and ratoon growth is healthy and strong, and leaf color is dark green glossy.
For above-mentioned various aspects, the present invention also provides a kind of Shoot Organogenesis from Cotyledon of Pepper in-vitro regeneration method, using Cotyledons with petiole outside
Implant, wherein the step of induction differentiation adventitious bud, squamous subculture walks poly-, step of taking root, before hardening and transfer step are taken respectively
The method to continue is given an account of to carry out.This complete regenerating system through the invention, not only bud induction is broken up and its extends effect very
It is good, and take root and transfer after survival rate it is all very high, this constitutes the method for the most preferred Shoot Organogenesis from Cotyledon of Pepper Regeneration in Vitro of the present invention.Its
In above-mentioned each step preferred parameter constitute the preferred steps of regeneration method, thus wherein most preferably: with Cotyledons with petiole explant
Body dark culture in the differential medium of 5.0mg/L ZT+7.0mg/L 6-BA+0.05mg/L IAA hormone combinations, then identical
Illumination cultivation on the differential medium of hormone combinations, then deletes regeneration bud, remains one to two pair to raw strip leaf
Shape body continues illumination training in the subculture medium that hormone combinations are 5.0mg/L ZT+7.0mg/L 6-BA+0.05mg/L IAA
It supports, then the illumination cultivation in the root media of 1/2MS+0.7mg/L IBA+0.1% active carbon, after taking root after finishing scouring seedling 5d
Transplanting shading moisturizing continued growth into matrix, finally grows under normal conditions.
Above-mentioned to be illustrated with regard to various aspects of the invention, wherein beneficial effect is correspondingly said respectively
It is bright, and have verifying in specific embodiment and embodiment.Therefore, luring the present invention provides the adventitious bud for being suitable for capsicum
It leads, squamous subculture and complete regeneration method and its used culture medium prescription, further to establish pepper transformation
Solid foundation has been established in the foundation of system.
Detailed description of the invention
Fig. 1: it is secretly trained in the differential medium of 5.0mg/L ZT+7.0mg/L 6-BA+0.05mg/L IAA hormone combinations
Support the Excised cotyledons of 21d.
Fig. 2: it is secretly trained in the differential medium of 5.0mg/L ZT+7.0mg/L 6-BA+0.05mg/L IAA hormone combinations
Support 21d after again on the differential medium of identical hormone combinations illumination cultivation 7d Excised cotyledons.
Fig. 3: it is added on the basis of hormone combinations are 5.0mg/L ZT+7.0mg/L 6-BA+0.05mg/L IAA
2.0mg/L GA3Subculture medium in thallus after illumination cultivation 1 week, stem has no elongation.
Fig. 4: it is added on the basis of hormone combinations are 5.0mg/L ZT+7.0mg/L 6-BA+0.05mg/L IAA
2.0mg/L GA3Subculture medium in thallus after illumination cultivation 1 week, stem obviously extends.
Fig. 5: in hormone combinations being 5.0mg/L ZT+7.0mg/L 6-BA+0.05mg/L IAA without the thallus deleted
Subculture medium in continue illumination cultivation 2 weeks after, stem has no elongation.
Fig. 6: being deleted only surplus a pair is 5.0mg/L ZT+7.0mg/L 6- in hormone combinations to raw strip thallus
After continuing illumination cultivation 2 weeks in the subculture medium of BA+0.05mg/L IAA, stem obviously extends.
Fig. 7: the capsicum bottle in 1/2MS+0.7mg/L IBA+0.1% active carbon root media after illumination cultivation 4 weeks
Seedling.
Fig. 8: the capsicum in 1/2MS+0.7mg/L IBA+0.1% active carbon root media after illumination cultivation 4 weeks is raw
Offspring.
Fig. 9: the capsicum of shade moisturizing continued growth 15d is transplanted into matrix after hardening of uncapping in illumination cultivation room 5d again
Raw seedling.
Specific embodiment
Below in conjunction with specific embodiment, technical solution of the present invention is clearly and completely described.Based on the present invention
In embodiment, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.
The acquisition of 1 aseptic seedling of embodiment
The aseptic capsicum fresh seeds of health (current year harvests) is selected, with pure water seed soaking 48h, is transferred to ultra-clean work
Platform.First with 75% alcohol disinfecting 60s, 0.1%HgCl is then used210min is sterilized, then with aseptic water washing 4 times, 2min/ times,
Seed is finally inoculated in the MB culture medium (MS a great number of elements+MS microelement+MS iron of addition 3% sucrose and 0.5% agar powder
Salt+B5Organic principle, similarly hereinafter, pH=5.8), illumination cultivation (temperature: 25 ± 2 DEG C, light intensity: 2500Lx, the photoperiod: 14h illumination/
10h is dark, similarly hereinafter), seed, which starts to sprout, after about 7d forms aseptic seedling.
The induction of 2 regeneration bud of embodiment
Choosing will be unfolded or the aseptic seedling cotyledon that is just unfolded is explant, with sharp sterile razor blade by cotyledon from petiole
(petiole with 0.1-0.2cm long) is cut at end, and blade tip is cut off at nearly blade tip 1-2cm.Both ends are respectively had to the cotyledon abdomen of notch
It is inoculated on differential medium downward with the back side up, first dark culture (temperature: 25 ± 2 DEG C) 21d, then illumination cultivation 7d,
Observe the differentiation and growth situation of regeneration bud, statistics regeneration inductivity.Regeneration bud inductive differentiation medium is basic training with MB
Support base, the 6-BA (0,1.0,3.0,5.0,7.0,9.0mg/L) and/or ZT of addition IAA (0.05mg/L) and various concentration proportion
(0,1.0,3.0,5.0,7.0,9.0mg/L) combines (pH=5.8), and each combination is inoculated with 10 pairs of cotyledon explants, if 3 weights
It is multiple.
Regeneration inductivity %=induces total explant number × 100 of explant number/inoculation of regeneration bud, similarly hereinafter.
Experimental result:
Excised cotyledons first carry out dark culture: the visible cotyledon of dark culture 3d switchs to yellowish green, dark culture 5d cotyledon increase by dark green
It thickens, incision initially forms white callus, and dark culture 10d cotyledon volume further increases, in cotyledon petioles end notch
The visible light yellow clear shape callus in surface, dark culture 21d cotyledon petioles end incision differentiate yellow thallus (Fig. 1).
Light-exposed culture 7d, color switch to green by light yellow after Excised cotyledons dark culture 21d, form white callus group at the cotyledon back side
It knits, the petiole end regenerated thallus of incision switchs to green, and volume significantly increases (Fig. 2).
On the basis of IAA is 0.05mg/L, the basic element of cell division 6-BA and ZT various concentration regenerate capsicum Excised cotyledons
The effect of induction is shown in Table 1.As can be seen from Table 1, Cotyledons with petiole is a kind of explant for being easy to regeneration induction, individually adds 6-
The combination of BA and ZT or various concentration BA and ZT energy 100% induce the regeneration of Cotyledons with petiole petiole end notch, regenerate shape
At thallophytic state have differences, but have no the formation for the regeneration bud that can normally extend.Individually addition 1.0-9.0mg/L
The thallus of 6-BA, the petiole end notch differentiation of Cotyledons with petiole are in rose petaloid, and quantity is more；1.0-4.0mg/L 6-BA
Under the conditions of the callus that is formed of cotyledon it is relatively smaller, thallus slow growth, and cotyledon shape under the conditions of 5.0-9.0mg/L 6-BA
At callus it is more, thallus growth is very fast.Individually the addition various concentration ZT induction regenerated lobate volume morphing of cotyledon is deposited
In notable difference: the short and small thallus formed under the conditions of low concentration ZT (1.0-3.0mg/L) is in rose petaloid, quantity
It is few；When ZT concentration increases to 5.0-9.0mg/L, the thallus that cotyledon petioles end incision is formed is larger, and most of thallus is in
Rose petaloid, small part are elongated.However, due to ZT elicitor it is leaf at callus it is few, thallus slow growth.
Various concentration 6-BA and ZT combination energy 100% induces cotyledon petioles end notch to be differentiated to form thallus, and quantity is more, but not
It is had differences with the thallus state of concentration combination regeneration induction and its growth: low concentration 6-BA and low concentration ZT combination induction
It is thallophytic small in size in rose petaloid, negligible amounts, slow growth, with the increase of 6-BA and ZT concentration, the leaf of induction
Shape body volume is larger and quantity is more, as 6-BA >=5.0mg/L, since cotyledon forms more callus, thallus growth
Comparatively fast.The thallus that 3.0-9.0mg/L ZT and 5.0-9.0mg/L 6-BA combination can make Cotyledon Segments regeneration many, big portion
Lobulated body is in rose petaloid, and small part is elongated, and thallus growth is fast, thus the concentration combination be suitable for cotyledon from
The regenerated induction of body.
Table 1 various concentration ZT and/or 6-BA are with the comparison regenerated influence of capsicum Excised cotyledons
Note: wherein each processing is added to 0.05mg/L IAA
The elongation of 3 regeneration bud of embodiment
The elongation experiment of regeneration bud is carried out on the basis of embodiment 2.
1.GA3Influence to regeneration bud elongation
Thallus regeneration bud is placed in various concentration GA3Illumination cultivation in the elongation medium of proportion, 10d subculture is primary,
Regeneration bud is observed after 20d and extends situation, and counts the elongation frequency of regeneration bud.The culture medium of regeneration induction bud elongation is using MB as base
Basal culture medium adds the GA of various concentration proportion on the basis of the hormone combinations of the suitable differential medium of above-mentioned acquisition3
(1.0,2.0,3.0mg/L), each combination inoculation 10 have the cotyledon of thallus regeneration bud, 1 cotyledon/bottle, if 3 weights
It is multiple.
Regeneration bud elongation %=induces cotyledon of the cotyledon number/inoculation with regeneration bud with regeneration bud of stem elongation
Sum × 100, similarly hereinafter.
Experimental result:
Capsicum Excised cotyledons can induce the thallus that regeneration forms different conditions, about 98% cotyledon on differential medium
More and most of thallus quantity of formation is in rose petaloid, and the thallus quantity that only about 2% cotyledon is formed is few and is in
Strip.GA3There are concentration differences for influence to the elongation of different conditions thallophytic stem: when the regenerated thallus number of Excised cotyledons
When measuring mostly and being in roseleaf state, in 1.0-2.0mg/L GA3Under the conditions of grow 1 week after thallus in yellow, Ye Mingxian
It extends and thinning, but the not formed adventitious bud (Fig. 3) normally extended, the Excised cotyledons callus after continued growth 1 week are brown
Change, thallus falls off；3.0mg/L GA3Under the conditions of grow 1 week after thallus obviously extend, but easily form water stain shape, also not
Form the regeneration bud of stem elongation.When the regenerated thallus of Excised cotyledons is elongated and to raw state, and thallophytic quantity is few
(for 1-3 to) when, 1.0-2.0mg/L GA3Under the conditions of grow 1 week after, stem obviously extends, and petiole and blade also extend, Ye Huanghua
Thinning (Fig. 4)；3.0mg/L GA3Under the conditions of thallus stem also can obviously extend, but Miao Yi forms water stain shape.Therefore,
1.0-2.0mg/L GA3Excised cotyledons are regenerated with few elongated extend to raw thallophytic stem of the quantity to be formed has obviously
Facilitation, it is unobvious to the effect of rose petaloid thallus and the non-elongation to raw shape thallus stem.
2. deleting the influence that regeneration bud quantity extends regeneration bud
More than the capsicum Excised cotyledons thallus quantity that regeneration induction is formed on suitable differential medium and major part is
The rose petaloid of deformity, minority are strip, their continued growths on differential medium have no the elongation (Fig. 5) of stem.It will
The thallus regeneration bud rose petaloid thallus of sharp sterile razor blade excision deformity, only stays 1-2 normal adventitious buds
(blade is in raw strip) carries out illumination cultivation in subculture medium, and 7d subculture is primary, and regeneration bud elongation is observed after 20d
Situation, and count the elongation frequency of regeneration bud.Squamous subculture based formulas: MB is minimal medium, addition IAA (0.05mg/L) with
The 6-BA (1.0,3.0,5.0,7.0,9.0mg/L) and/or ZT (1.0,3.0,5.0,7.0,9.0mg/L) group of various concentration proportion
It closes (pH=5.8).
Experimental result:
Behind regeneration bud squamous subculture 2 weeks through deleting, stem obviously extends, and dark green blade is in oblong, the strong (figure of stem thickness
6).Influence of the ZT and BA that various concentration matches in subculture medium to Elongation of adventitious bud is shown in Table 2.From Table 2, it can be seen that
On the basis of addition IAA (0.05mg/L): 1.0mg/L ZT is combined with 1.0-9.0mg/L 6-BA can be such that the stem of regeneration bud extends
Rate≤40%；With the increase of ZT concentration, the elongation of thallus stem is significantly raised, 3.0-7.0mg/L ZT and 5.0-9.0mg/
L 6-BA combination can make regeneration bud elongation be higher than 67%.5.0-7.0mg/L ZT is stretched with the 7.0mg/L 6-BA regeneration bud combined
Long rate highest is 88%-90%, is significantly higher than other combinations, is as suitable for swashing in the subculture medium of regeneration bud elongation
Element combination.Deleted only surplus 1-2 normal adventitious buds (blade is in raw strip) lifes on suitable subculture medium
After being about 15d, bud extends about 4-5cm, is the growth phase of 1 heart of 3-4 leaf.
The influence that the ZT and BA of 2 various concentration of table proportion extend regeneration bud
Note: same row difference lowercase indicates that above there were significant differences in 0.05 level；Wherein each processing is added to
IAA 0.05mg/L。
4 regeneration bud of embodiment is taken root
The regeneration bud (1 heart of 3-4 leaf) for choosing the robust growth height up to the 4.0-5.0cm that obtain in embodiment 3 is cut from base portion
Under (without callus), be forwarded in root media and carry out illumination cultivation, after 30d calculate rooting rate, averagely take root number and
Average root long.Root media adds the IBA of active carbon (0.1%) and various concentration proportion using 1/2MB as minimal medium
(0.1,0.3,0.5,0.7,0.9mg/L) (pH=5.8), each processing are inoculated with 10 regeneration buds, and 1 regeneration bud/bottle repeats 3
It is secondary.
Regeneration bud number/inoculation regeneration bud sum × 100 for rooting rate (%)=take root
It averagely takes root the sum/inoculation regeneration bud sum of number=root
The sum of average root long (cm)=root long overall length/root
Experimental result:
It respectively will be through GA3The regeneration bud that inducing and delete thallus extends stem is cut from basal part of stem, is forwarded to training of taking root
It supports and carries out illumination cultivation on base, there are notable differences for the efficiency of taking root of the two.Through GA3Induce the adventitious bud rooting of elongation difficult,
Leaf color jaundice is difficult to turn green.
And deleting thallus makes the regeneration bud of stem elongation easily take root, about 10d or so starts to take root in root media,
Healthy and strong rooted seedling is formed after 30d, well developed root system, stem thickness is strong, and leaf is dark green (Fig. 7,8).To various concentration proportion IBA to regeneration
The influence that bud is taken root is tested, and the results are shown in Table 3.Table 3 the result shows that, IBA takes root there are concentration effect to regeneration bud, low
Concentration and higher concentration IBA are unfavorable for taking root for regeneration bud, and rooting rate is substantially less than the IBA of intermediate concentration, and starts to give birth to
The root time is long, and the low and average root long of coefficient of taking root is short.Being suitable for the IBA concentration that regeneration bud is taken root is 0.5-0.7mg/L, is taken root
Rate is up to 100%, and about 10 days start to take root, and coefficient of taking root is 3.5-3.8, and average root is up to 9.8-10.5cm, and root, which shows, to be had
The white of vigor, lateral root are very flourishing.
The influence that 3. various concentration IBA of table takes root to regeneration bud
Note: same row difference lowercase indicates that above there were significant differences in 0.05 level
5 hardening of embodiment and transplanting
Acclimatization and transplants are carried out to the regrowth after culture of rootage in embodiment 4.By the well developed root system of culture of rootage about 30d
Aseptic seedling is first gradually uncapped hardening 5d in illumination cultivation room, is then cleaned root culture medium with clear water, is finally transplanted to turf: leech
Stone: garden mould=1: shading heat preservation 15d in 1: 1 (volume ratio) matrix counts survival rate after transplanting 15d.
The seedling number for survival rate (%)=survive/hardening sum × 100
Experimental result:
The high survival rate of 15d seedling is up to 100% after transplanting, and ratoon growth is healthy and strong, and leaf color is dark green glossy (Fig. 9).With
After can grow under normal conditions.
Claims (10)
1. a kind of Shoot Organogenesis from Cotyledon of Pepper explant method of inducing differentiation, which is characterized in that using capsicum aseptic seedling Cotyledons with petiole as explant
Body, the hormone prescription of regeneration bud inductive differentiation medium are 3.0-9.0 mg/L ZT+5.0-9.0 mg/L 6-BA+0.04-
0.06 mg/L IAA, it is preferred to use MB is minimal medium；Dark culture is further preferably first passed through, then carries out optical culture, it is excellent
Selection of land is dark culture 5d to 30 days, more preferably 10 d of dark culture to 25 d, most preferably 21 d of dark culture；Optical culture optimum condition
For temperature: 25 ± 2 DEG C, light intensity: 2500 Lx, the photoperiod: 14 h illumination/10 h are dark.
2. Shoot Organogenesis from Cotyledon of Pepper explant method of inducing differentiation as described in claim 1, which is characterized in that the concentration of ZT is 5.0-
7.0 mg/L ZT, 6-BA concentration are 6.0-8.0 mg/L, and IAA concentration is 0.05 mg/L.
3. Shoot Organogenesis from Cotyledon of Pepper explant method of inducing differentiation as described in claim 1, which is characterized in that outside the Cotyledons with petiole
Implant has the petiole of 0.1-0.2 cm long, is cut off the cotyledon explant outside of belly court when blade tip is cultivated at nearly blade tip 1-2 cm
The upper and back side is inoculated in downward on regeneration bud inductive differentiation medium.
4. a kind of method of the Elongation of adventitious bud of Shoot Organogenesis from Cotyledon of Pepper explant induction differentiation, which is characterized in that each of differentiated
It is in the adventitious bud to raw strip that explant, which deletes its regeneration bud quantity to 1-2 blade, carries out squamous subculture, the subculture
The hormone prescription of culture medium is that 0.04-0.06 mg/L IAA, 3.0-7.0 mg/L ZT is combined with 5.0-9.0 mg/L 6-BA,
Wherein IAA preferred concentration is 0.05 mg/L, and ZT preferred concentration is 5.0-7.0 mg/L, and more preferably 6.0 mg/L, 6-BA's is excellent
Selecting concentration is 6.0-8.0mg/L, most preferably 7.0 mg/L；Further, subculture medium is using MB as minimal medium,
pH5.8。
5. a kind of method of hot pepper seed Regeneration in Vitro, using Cotyledons with petiole as explant, which is characterized in that induction differentiation adventitious bud
The step of use step as described in any one of claims 1 to 3.
6. method as claimed in claim 5, which is characterized in that generate adventitious bud in explant and then wanted using such as right
Method described in asking 4 carries out squamous subculture.
7. method as claimed in claim 6, which is characterized in that by the regeneration bud on the explant after by squamous subculture from
Basal part of stem is cut, and is forwarded on root media and is carried out illumination cultivation, wherein culture medium is preferably used to cultivate for 1/2 MB,
Middle addition 0.5-0.7 mg/L IBA further adds 0.1% active carbon.
8. such as the described in any item methods of claim 5 to 7, which is characterized in that further by the regrowth after taking root through refining
It is transplanted in growth substrate and grows after seedling, preferred regrowth is first gradually uncapped hardening 4-6 d in illumination cultivation room, then with clear
Water cleans root culture medium, transplants to turf: vermiculite: garden mould=1: 1: 1(volume ratio) shading heat preservation 10-20d in matrix, finally
It grows under normal conditions.
9. a kind of culture medium for the induction differentiation of Shoot Organogenesis from Cotyledon of Pepper explant, which is characterized in that bud inductive differentiation medium swashs
Element formula is 3.0-9.0 mg/L ZT+5.0-9.0 mg/L 6-BA+0.04-0.06 mg/L IAA, it is preferred to use MB is base
Basal culture medium, it is highly preferred that it is that 6.0-8.0 mg/L, IAA concentration is that the concentration of ZT, which is 5.0-7.0 mg/L ZT, 6-BA concentration,
0.05 mg/L。
10. a kind of subculture medium for the Elongation of adventitious bud for inducing differentiation to generate for capsicum explant, which is characterized in that institute
The hormone prescription for stating subculture medium is 0.04-0.06 mg/L IAA, 3.0-7.0 mg/L ZT and 5.0-9.0 mg/L 6-BA
Combination, preferably IAA concentration are 0.05 mg/L, and ZT concentration is 5.0-7.0 mg/L, and more preferably 6.0 mg/L, 6-BA's is dense
Degree is 6.0-8.0 mg/L, most preferably 7.0 mg/L；Further preferably, the subculture medium is basic culture with MB
Base.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115486372A (en) * | 2022-11-08 | 2022-12-20 | 海南大学三亚南繁研究院 | Method for constructing in-vitro regeneration system of drooping hot pepper |
| CN116218902A (en) * | 2023-03-28 | 2023-06-06 | 湖北伯远合成生物科技有限公司 | Efficient genetic transformation method for capsicum |
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| CN108338073A (en) * | 2018-03-06 | 2018-07-31 | 北京市农林科学院 | Tissue culture method of hot pepper |
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| CN115486372A (en) * | 2022-11-08 | 2022-12-20 | 海南大学三亚南繁研究院 | Method for constructing in-vitro regeneration system of drooping hot pepper |
| CN116218902A (en) * | 2023-03-28 | 2023-06-06 | 湖北伯远合成生物科技有限公司 | Efficient genetic transformation method for capsicum |
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