CN111543325A - Induction culture method and induction culture medium for slash pine embryonic callus - Google Patents
Induction culture method and induction culture medium for slash pine embryonic callus Download PDFInfo
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- CN111543325A CN111543325A CN202010502795.1A CN202010502795A CN111543325A CN 111543325 A CN111543325 A CN 111543325A CN 202010502795 A CN202010502795 A CN 202010502795A CN 111543325 A CN111543325 A CN 111543325A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
- A01H4/006—Encapsulated embryos for plant reproduction, e.g. artificial seeds
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Abstract
The invention belongs to the field of plant tissue culture, and discloses an induction culture method and an induction culture medium for slash pine embryonic callus. The induction culture method of the slash pine embryonic callus comprises the following steps: (1) collecting strobilus Pini in the first ten days of 7 months, completely immersing strobilus Pini in anhydrous ethanol for 10-20min, washing with sterile water for 2-4 times, removing seed shell, soaking in 70-80% ethanol for 2-4min, washing with sterile water for 5-8 times, placing the seed in a culture dish with filter paper, sealing, and refrigerating; (2) cutting along the side of the seed at the distance from the tail under aseptic condition, taking out the complete immature zygotic embryo, placing on solid induction culture medium, inoculating, sealing membrane, and placing in dark culture at 23 + -3 deg.C for 46-52 days to obtain embryogenic callus. The method lays a foundation for establishing a slash pine somatic embryogenesis technical system, and provides an efficient and stable method for large-scale seedling culture of improved slash pine seeds.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to an induction culture method and an induction culture medium for slash pine embryonic callus.
Background
The slash pine (Pinus elliottii Engelm.) is originally produced in the southeast United states, and has become an exotic resin-producing tree species with the largest artificial cultivation area in China due to the advantages of strong adaptability, quick growth, good rosin quality, difficult crystallization, difficult solidification, less impurities and the like after being introduced into China in 1933. However, the traditional and extensive seedling propagation is mainly used for producing the slash pine, the production facilities are simple and crude, the scientific and technological investment is less, and a uniform propagation and cultivation technology is lacked, so that the quality of finished nursery stocks is unstable, and the quantity of the finished nursery stocks is far from the actual requirement; however, the existing cutting propagation technology has low rooting rate, high requirements on grafting technology and is limited by seasons, so that excellent families and clone lines cannot be produced in a large scale.
Somatic embryogenesis (Somatic embryogenesis) refers to a phenomenon that through artificial control, Somatic cells of plants are not subjected to cell fusion in a fertilization process, but new individuals are generated through a process similar to a zygotic embryo development path, and the Somatic embryogenesis has the advantages of strong genetic stability, high regeneration rate, no limitation of natural conditions and the like. Conifer somatic embryogenesis is generally divided into four stages, embryogenic callus (embryogenic suspensor mass) induction, embryogenic callus proliferation, somatic embryo differentiation and maturation, somatic embryo germination and plant regeneration. Embryogenic callus induction plays an important role in the pinus elliottii somatic embryogenesis system as the first step in somatic embryogenesis.
At present, although the induction of embryogenic callus of slash pine has been reported, the problem of low induction rate of embryogenic callus still exists. The induction rate is low due to many reasons, such as the genotype of the explant, the sampling time and the culture conditions. Therefore, a stable embryogenic callus induction system is established by selecting proper genotypes, sampling time, culture conditions and the like, a foundation can be laid for establishing a somatic embryogenesis technical system of the slash pine, and an efficient and stable method is provided for large-scale seedling culture of improved varieties of the slash pine.
Disclosure of Invention
The invention aims to overcome the defects of the background technology, provides an induction culture method of the slash pine embryonic callus and a special induction culture medium thereof, establishes a stable embryonic callus induction system, lays a foundation for establishing a slash pine somatic embryogenesis technical system, and provides an efficient and stable method for large-scale seedling culture of slash pine fine varieties.
In order to achieve the purpose of the invention, the induction culture method of the slash pine embryonic callus comprises the following steps:
(1) collecting strobilus Pini in the first ten days of 7 months, completely immersing strobilus Pini in anhydrous ethanol for 10-20min, washing with sterile water for 2-4 times, removing seed shell, soaking in 70-80% ethanol for 2-4min, washing with sterile water for 5-8 times, placing the seed in a culture dish with filter paper, sealing, and refrigerating;
(2) cutting along the side of the seed at the distance from the tail under aseptic condition, taking out the complete immature zygotic embryo, placing on solid induction culture medium, inoculating, sealing membrane, and placing in dark culture at 23 + -3 deg.C for 46-52 days to obtain embryogenic callus;
wherein the solid induction culture medium is a DCR culture medium added with 2,4-D, KT and 6-BA.
The method for collecting the strobilus pini in the first ten days of 7 months refers to the division standard of the development stages of the zygotic embryos of Pullan loblolly pine, namely, immature zygotic embryos in the II-III stage are taken as materials. In the last ten days of 7 months, the days of 7 months are preferably 2-14.
Further, the concentration of 2,4-D in the solid induction medium is 1.8-3.6mg/L, KT, the concentration is 0.8-3.6mg/L and the concentration of 6-BA is 0.8-3.6 mg/L.
Preferably, the concentration of 2,4-D in the solid induction medium is 1.8-2.5mg/L, KT, the concentration is 1.6-2.7mg/L and the concentration of 6-BA is 1.6-2.7 mg/L.
More preferably, the concentration of 2,4-D in the solid induction medium is 2.2mg/L for 2.2mg/L, KT and the concentration of 6-BA is 2.2 mg/L.
Further preferably, the solid induction medium is further added with one or more of melezitose 1.4-1.6g/L, VC 8-15g/L, gellan gum 2.7-3.3g/L, inositol 90-110mg/L, enzymatic hydrolysis casein 0.8-1.2g/L or glutamine 480-520 mg/L.
Still more preferably, the solid induction medium further comprises one or more of melezitose 1.5g/L, VC 10g/L, gellan gum 3.0g/L, inositol 100mg/L, casein enzyme hydrolysis 1g/L, and glutamine 500 mg/L.
Further, the pH of the solid induction medium is 5.5-6.1, preferably 5.8.
In another aspect, the present invention also provides an induction medium for embryogenic callus of slash pine, wherein the concentration of 2,4-D in the solid induction medium is 1.8-3.6mg/L, KT, the concentration is 0.8-3.6mg/L, and the concentration of 6-BA is 0.8-3.6 mg/L.
Preferably, the concentration of 2,4-D in the solid induction medium is 1.8-2.5mg/L, KT, the concentration is 1.6-2.7mg/L and the concentration of 6-BA is 1.6-2.7 mg/L.
More preferably, the concentration of 2,4-D in the solid induction medium is 2.2mg/L for 2.2mg/L, KT and the concentration of 6-BA is 2.2 mg/L.
Further preferably, the solid induction medium is further added with one or more of melezitose 1.4-1.6g/L, VC 8-15g/L, gellan gum 2.7-3.3g/L, inositol 90-110mg/L, enzymatic hydrolysis casein 0.8-1.2g/L or glutamine 480-520 mg/L.
Still more preferably, the solid induction medium further comprises one or more of melezitose 1.5g/L, VC 10g/L, gellan gum 3.0g/L, inositol 100mg/L, casein enzyme hydrolysis 1g/L, and glutamine 500 mg/L.
Further, the pH of the solid induction medium is 5.5-6.1, preferably 5.8.
Compared with the prior art, the invention has the following remarkable advantages and beneficial effects:
the induction culture method of the embryogenic callus of the slash pine provided by the invention takes the immature zygotic embryo of the slash pine as a material, takes DCR as a basic culture medium, optimizes the induction culture medium by adding a proper amount of hormone, silver nitrate, VC, gellan gum and the like, enables the induction rate of the embryogenic callus to reach 40 percent at most, lays a foundation for establishing a somatic embryo regeneration system of the slash pine, and also provides a theoretical basis for fine variety breeding of the slash pine and the next genetic transformation.
Drawings
FIG. 1 is the developmental stage of zygotic embryos; wherein A, immature zygotic embryo; B. a cut immature zygotic embryo; C. stage 1; D. stage 2; E. stage 3; F. stage 4; G. stage 5; H. stage 6; I. stage 7; J. stage 8;
FIG. 2 is a process for producing the callus induced by the immature embryo of slash pine according to the present invention; wherein, from left to right are respectively: 1. at the initial stage of induction; 2. embryogenic callus; 3. non-embryogenic callus; 4. embryogenic embryonal suspensor cell masses;
FIG. 3 is the effect of different carbon sources on the induction rate of slash pine embryogenic callus in example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention. It is to be understood that the following description is only illustrative of the present invention and is not to be construed as limiting the present invention.
The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like.
The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.
The induction culture method of the slash pine embryonic callus in the embodiment of the invention comprises the following steps:
(1) collecting strobilus Pini in the first ten days of 7 months, soaking strobilus Pini in anhydrous ethanol for 15min, washing with sterile water for 2-4 times, removing seed shell, soaking seed in 75% ethanol for 3min, washing with sterile water for 5-8 times, placing seed in culture dish with filter paper, sealing, and refrigerating;
(2) cutting along the side of the seed at a position 1/3 away from the tail part under aseptic condition, taking out the complete immature zygotic embryo, placing on a solid induction culture medium, inoculating, sealing membrane, and placing in the condition of 23 + -1 deg.C for dark culture for 7 weeks to obtain embryogenic callus;
wherein the solid induction culture medium is a DCR culture medium added with 2,4-D, KT and 6-BA.
Example 1
Research on influence of different collection dates and explant maturity on induction rate of slash pine embryogenic callus: selecting cones of a single plant with the number of 16 from a Xianhuang forest fine variety farm in Jian city in 2018, wherein the collection time is 7 months and 4 days, 7 months and 11 days, 7 months and 18 days, 7 months and 25 days, and 8 months and 1 day, randomly extracting 3 cones in each collection period, randomly selecting 10 immature seeds from each cone for sterilization, removing episperms, and obtaining an explant containing zygotic embryos and endosperm.
The development stage of the slash pine zygotic embryo was observed under 20 Xstereoscope (Leica MZ16) with reference to the division standard of the development stage of Pullan loblolly zygotic embryo, and the development stage of the slash pine zygotic embryo observed was divided into 8 stages. As shown in FIG. 1, stage 1 is the early development stage of zygotic embryo, stage 2 is the multiple embryo head stage, stage 3 is the stage of schizogenesis multiple embryo, stage 4 is the globular embryo stage, stage 5 is the rod embryo stage, stage 6 is the bullet head stage, and the zygotic embryo gradually develops and matures at stages 7 and 8.
As shown in Table 1, the induction rates of the calli and the embryogenic calli of cones at different collection times as explants are significantly different, the induction rates of the calli at 7-month and 11-month days and 7-month and 4-day (stages II-III) are the highest, and the induction rate of the embryogenic calli is gradually reduced along with the development of zygotic embryos until the embryos are completely matured, and the induction rate of the embryogenic calli is 0%.
TABLE 1 Effect of different Collection dates on the Induction of embryogenic callus of slash pine
Example 2
Study of the effects of different hormones on induction of slash pine callus: explants were inoculated onto DCR minimal medium for induced proliferation according to the procedure of example 1 (cone harvest time 7 months and 11 days), and the results of induced callus derived from slash pine embryogenic tissue by adding KT, 2,4-D and 6-BA at different concentrations are shown in Table 2.
TABLE 2 Effect of different hormone concentration combinations on the callus induction rate of P.elliottii
Table 2 shows that the hormone combinations of KT, 2,4-D and 6-BA with different concentrations have obvious influence on the callus induction of the slash pine. The induction rate of the callus of No. 5 treatment is the highest and reaches 30%, and then No. 4 treatment and No. 8 treatment are carried out, the induction rate of the embryogenic callus of the slash pine is 20%, and the callus can not be induced by No. 2 treatment, so that the induction rate of the embryogenic callus of the slash pine is the most suitable culture medium for induction of the embryogenic callus of the slash pine in No. 5 treatment of DCR + KT 2.2mg/L +2,4-D2.2mg/L +6-BA 2.2 mg/L.
Example 3
Study on the influence of different carbon sources on the induction rate of embryogenic callus of slash pine: sucrose, maltose, and melezitose were added to the induction medium (No. 5) obtained in example 2 as carbon sources, and the influence of different carbon sources on the induction rate of slash pine embryogenic callus was examined, and the results are shown in FIG. 3.
FIG. 3 shows that the induction rate is the highest at 20% at a sucrose concentration of 30 g/L. When the maltose concentration is 30g/L, the induction rate reaches the highest and is 30 percent. When melezitose is used as a carbon source of an induction culture medium, the change rule is not obvious. When the concentration of melezitose is 1.0g/L, the induction rate of the callus of the slash pine is only 2.5 percent, and when the concentration of melezitose is 1.5g/L, the induction rate reaches the highest and reaches 40 percent.
Example 4
Research on influence of silver nitrate and VC on induction rate of embryogenic callus of slash pine: silver nitrate and VC were added to the induction medium (No. 5) obtained in example 2, and the effect of different concentrations of silver nitrate and VC on the induction rate of embryogenic callus of slash pine was examined, and the results are shown in tables 3 and 4.
TABLE 3 Effect of different concentrations of silver nitrate on the embryogenic callus induction rate of Pinus elliottii
As is clear from Table 3, the highest induction rate was 42.5% when silver nitrate was not added. The induction rate gradually decreases with increasing silver nitrate concentration. When 8g/L of silver nitrate is added, the induction rate is the lowest and is only 7.5%. From this, it is known that the addition of silver nitrate decreases the induction rate of embryogenic callus of slash pine.
TABLE 4 Effect of VC at different concentrations on the embryogenic callus induction rate of Pinus elliottii
As is clear from Table 4, the induction rate as a whole tends to increase with the increase in VC concentration. When the concentration of VC is 2.5g/L, the lowest inductivity is 12.5 percent; when the VC concentration is 10g/L, the embryogenic callus induction rate is the highest and reaches 37.5 percent.
Example 5
Study on the influence of different gellan gum concentrations on the induction rate of embryogenic callus of slash pine: the effect of different concentrations of gellan gum on the induction rate of slash pine embryogenic callus was tested by adding gellan gum to the induction medium (No. 5) obtained in example 2, and the results are shown in Table 5.
As shown in Table 5, the average induction rate of the slash pine embryogenic callus added with 3.0g/L of gellan gum is significantly higher than that of the slash pine embryogenic callus added with 6.0g/L of gellan gum, and is 42.5% and 5%, respectively.
TABLE 5 Effect of different Gellan gum concentrations on the embryogenic callus induction rate of Pinus elliottii
It will be understood by those skilled in the art that the foregoing is only exemplary of the present invention, and is not intended to limit the invention, which is intended to cover any variations, equivalents, or improvements therein, which fall within the spirit and scope of the invention.
Claims (10)
1. An induction culture method of slash pine embryogenic callus is characterized by comprising the following steps:
(1) collecting strobilus Pini in the first ten days of 7 months, completely immersing strobilus Pini in anhydrous ethanol for 10-20min, washing with sterile water for 2-4 times, removing seed shell, soaking in 70-80% ethanol for 2-4min, washing with sterile water for 5-8 times, placing the seed in a culture dish with filter paper, sealing, and refrigerating;
(2) cutting along the side of the seed at the distance from the tail under aseptic condition, taking out the complete immature zygotic embryo, placing on solid induction culture medium, inoculating, sealing membrane, and placing in dark culture at 23 + -3 deg.C for 46-52 days to obtain embryogenic callus;
wherein the solid induction culture medium is a DCR culture medium added with 2,4-D, KT and 6-BA.
2. The method for induction-culturing of embryogenic callus of slash pine according to claim 1, wherein the last 7 th month is 7 months 2-14 days.
3. The induction culture method of slash pine embryogenic callus according to claim 1, wherein the concentration of 2,4-D in the solid induction medium is 1.8-3.6mg/L, KT, the concentration is 0.8-3.6mg/L and the concentration of 6-BA is 0.8-3.6 mg/L; preferably, the concentration of 2,4-D in the solid induction medium is 1.8-2.5mg/L, KT, the concentration is 1.6-2.7mg/L and the concentration of 6-BA is 1.6-2.7 mg/L; more preferably, the concentration of 2,4-D in the solid induction medium is 2.2mg/L for 2.2mg/L, KT and the concentration of 6-BA is 2.2 mg/L; further preferably, the solid induction medium is added with one or more of melezitose of 1.4-1.6g/L, VC of 8-15g/L, gellan gum of 2.7-3.3g/L, inositol of 90-110mg/L, enzyme hydrolyzed casein of 0.8-1.2g/L or glutamine of 480-520 mg/L; still more preferably, the solid induction medium further comprises one or more of melezitose 1.5g/L, VC 10g/L, gellan gum 3.0g/L, inositol 100mg/L, casein enzyme hydrolysis 1g/L, and glutamine 500 mg/L.
4. The method for induction culture of embryogenic callus of slash pine according to claim 1, wherein pH of the solid induction medium is 5.5-6.1, preferably 5.8.
5. An induction culture medium of slash pine embryogenic callus, characterized in that the concentration of 2,4-D in the solid induction culture medium is 1.8-3.6mg/L, KT, the concentration is 0.8-3.6mg/L and the concentration of 6-BA is 0.8-3.6 mg/L.
6. The induction medium of slash pine embryogenic callus according to claim 5, wherein the concentration of 2,4-D in the solid induction medium is 1.8-2.5mg/L, KT, the concentration is 1.6-2.7mg/L and the concentration of 6-BA is 1.6-2.7 mg/L.
7. The induction medium of slash pine embryogenic callus according to claim 5, wherein the concentration of 2,4-D in the solid induction medium is 2.2mg/L, KT, the concentration is 2.2mg/L and the concentration of 6-BA is 2.2 mg/L.
8. The induction medium of the embryogenic callus of wet land of any one of claims 5 to 7, wherein the solid induction medium further comprises one or more of melezitose 1.4-1.6g/L, VC 8-15g/L, gellan gum 2.7-3.3g/L, inositol 90-110mg/L, casein hydrolysate 0.8-1.2g/L, or glutamine 480-520 mg/L.
9. The induction medium of slash pine embryogenic callus according to any one of claims 5-7, wherein the solid induction medium further comprises one or more of melezitose 1.5g/L, VC 10g/L, gellan gum 3.0g/L, inositol 100mg/L, casein hydrolysate 1g/L, and glutamine 500 mg/L.
10. The induction medium of slash pine embryogenic callus according to claim 5, wherein the pH of the solid induction medium is 5.5-6.1, preferably 5.8.
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Application publication date: 20200818 |