CN104304032A - Method suitable for efficient induction and plant regeneration of multi-genotype watermelon somatic embryos - Google Patents
Method suitable for efficient induction and plant regeneration of multi-genotype watermelon somatic embryos Download PDFInfo
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Abstract
The invention discloses a method suitable for efficient induction and plant regeneration of multi-genotype watermelon somatic embryos. The method comprises the following steps: preprocessing, inducing calluses, inducing the somatic embryos, forming a complete plant, hardening seedlings, and transplanting, wherein a plant growth regulator used in the preprocessing process is TDZ (thidiazuron) with the concentration of 0.01-0.1 mg/L; growth regulators used in the callus and somatic embryo inducing processes are 6-BA (6-benzylaminopurine) with the concentration of 2-4 mg/L and IAA (indole acetic acid) with the concentration of 0.01-0.5 mg/L. The method is not only suitable for inducing the multi-genotype watermelon somatic embryos, but also has the advantages of high callus induction rate, high somatic embryo induction rate, a large amount of somatic embryos of each explant, simple implementation steps and non-severe implementation conditions.
Description
Technical field
The present invention relates to biological technical field, the watermelon somatic embryo being particularly applicable to polygene type is efficiently induced and plant regeneration method.
Background technology
Watermelon is a kind of important vegetable crop, and in the large fruit in the world 10, occupy the 5th, the economic crops as a kind of Peak output are widely cultivated all over the world.Along with the raising of living standards of the people, the requirement of people to watermelon kind and quality is more and more higher, and the breeding objective of watermelon also trends towards the development of diversification.But there is germ plasm resource scarcity in traditional genetic breeding, breeding cycle is oversize, the deficiencies such as the genetic character that Posterity phenotype goes out is unstable; And obtain the kind of high yield and high quality by the biotechnology such as cultured in vitro, genetic transformation and become one of important plant breeding means.
Not through fertilization process under cultured in vitro, but be called somatic embryo through the class embryonic knob structure that embryo occurs and embryo development procedure is formed.Somatic embryo not only can be research Cell Differentiation, cellular omnipotency mechanism and artificial seed and improves gedanken experiment system and technical foundation, also have can preserve for a long time, genetic stability is high, regeneration frequency is high advantage, also be beneficial to the selection of mutant simultaneously, also can be used as the material preparing protoplast.Concrete, for the optimization etc. of the Fast-propagation of watermelon, the preservation of germ plasm resource, the cultivation of merit and gene engineering receptor system, all there is very important theory significance and using value through somatic embryo approach regeneration plant.
But the reported success about watermelon somatic embryo inducement and plant regeneration in prior art is little, and the condition of tissue culture difference of different variety of watermelon is very remarkable, and successfully report repeatability is very low, and can only for one to two genotype.Compton and Gray watermelon Immature Cotyledons in 1993 is that explant success regenerates induction of somatic embryo, inductivity is the highest only has about 7%, and widely different between different genotype, afterwards Niu Shanshan (2006) and Song Shangwei etc. (2007) with watermelon seedling cotyledon for explant, report the somatic embryo inducement rate of the highest 25% and 27.5%, and induce the quality of resultant bulk blast and every explant number of somactic embryos also all lower or have no data available.In tissue culture procedures, the particularly important is the selection of medium and the interpolation of hormone thereof, this for the quality of aseptic seedling, the induction of callus and somatic embryo there is important impact, be determine that explant somatic embryo occurs and the key link of plant regeneration.But the performance in different genotype of different culture media and hormone has very big-difference, prior art does not find a kind of watermelon somatic embryo being applicable to polygene type efficiently to induce and plant regeneration method yet.
Therefore, set up a watermelon high frequency somatic embryo being applicable to polygene type and occur and plant regeneration method, be significant, can be other related fields researchs such as the good breeding of watermelon and transgenic research and good acceptor regenerative system is provided.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, provide a kind of watermelon high frequency somatic embryo being applicable to polygene type to occur and plant regeneration method.The method comprises the following steps:
1) pre-treatment: carry out sterilizing after watermelon seed being peelled off seed coat, the MS medium be seeded in by embryo containing TDZ is cultivated again, every bottle graft kind 8, cultivates 3 ds under being first placed in dark condition after inoculation, and cultivation temperature is 23-27 DEG C, transfer to CMC model 3 d that light application time is 16 h/d again, cultivation temperature is 23-27 DEG C, and intensity of illumination is the concentration of 3000 lx, TDZ is 0-0.1 mg/L, add the sucrose of 30 g/L and the agar of 6.0 g/L in medium, pH value is adjusted to 6;
2) induction of callus: cut cotyledon explant, put into the 1/2 MS medium containing 6-BA and IAA, 7 d are cultivated under being first placed in dark condition, transfer to CMC model 14 d that light application time is 16 h/d again, cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000 lx, the concentration of 6-BA is 2-4mg/L, the concentration of IAA is 0.01-0.5mg/L, and add the sucrose of 30 g/L and the agar of 6.0 g/L in medium, pH value is adjusted to 6;
3) induction of somatic embryo: by step 2) gained callus puts into 1/2 MS medium containing 6-BA and IAA, CMC model 28 d being 16 h/d in light application time obtains somatic embryo, cultivation temperature is 23-27 DEG C, intensity of illumination is 3000 lx, the concentration of 6-BA is 2-4mg/L, the concentration of IAA is 0.01-0.5mg/L, and add the sucrose of 30 g/L and the agar of 6.0 g/L in medium, pH value is adjusted to 6;
4) formation of whole plant: the blake bottle that 1/2 MS medium is housed put into by the somatic embryo of step 3) gained without hormone is cultivated, incubation time is 28 d, cultivation temperature is 23-27 DEG C, intensity of illumination is 3000 lx, light application time is 16 h/d, add the sucrose of 30 g/L and the agar of 6.0 g/L in medium, pH value is adjusted to 6;
5) hardening: choose having 3 and the lid of blake bottle is first unscrewed placement 2 d with the healthy and strong seedling of blade and tetanic of 5-6 cm after step 4) is cultivated, more half-open 2 d, and then standard-sized sheet 2 d, need constantly to keep the skin wet during half-open and standard-sized sheet lid;
6) transplant: the plant after hardening being completed pulls up, wash root medium, be transplanted in the nutritive cube that peat soil, perlite, ash and leaf mould matrix are housed, at 23-27 DEG C, put freshness protection package and be placed in climatic cabinate moisturizing cultivation, 1-2 Zhou Houzai moves on to outdoor and cultivates.
Described sterilization process is: the seed coat peelling off watermelon seed, first with 75% alcohol-pickled sterilization 1min, then use 0.1% mercuric chloride soaking disinfection 5min, finally use aseptic water washing 5 times.
In described nutritive cube, the mass ratio of peat soil, perlite, ash and leaf mould matrix is 1:1:1:1.
Preferably, the TDZ concentration in step 1) is 0.01mg/L.When the concentration of TDZ is 0.01mg/L, the somatic embryo number that optimal somatic embryo inducement rate and every explant produce can be obtained.
Preferably, step 2) and step 3) in the concentration of 6-BA be the concentration of 3mg/L, IAA be 0.05mg/L.When the concentration of IAA is 0.05mg/L, on somatic embryo inducement rate and every explant, somatic embryo quantity reaches optimum.
The present invention has following beneficial effect:
1, the watermelon somatic embryo inducement of polygene type is applicable to;
2, callus induction rate somatic embryo number that is high, high, the every explant of somatic embryo inducement rate is many;
3, implementation step is simple, and implementation condition is not harsh.
Accompanying drawing explanation
Fig. 1: the callus that watermelon cotyledon induces;
Fig. 2: the somatic embryo that callus produces;
Fig. 3: somatic embryo sprouts;
Fig. 4: somatic embryo is taken root;
Fig. 5: regrowth;
Fig. 6: transplanted seedling.
Embodiment
Below by embodiment, the present invention is specifically described; what be necessary to herein means out is that following examples are just for being further detailed the present invention; limiting the scope of the invention can not be interpreted as; some nonessential improvement and adjustment that the person skilled in the art in this field makes according to foregoing invention content, still belong to protection scope of the present invention.
Embodiment 1
Choose the watermelon seed that size is even, full, seed coat peelled off by superclean bench, first with 75% alcohol-pickled sterilization 1min, use 0.1% mercuric chloride soaking disinfection 6min again, then aseptic water washing is used 5 times, then be placed on the aseptic filter paper in culture dish, with sterilization after tweezers by embryo the MS medium be seeded in containing TDZ cultivate, every bottle graft kind 8.Cultivate 3 d under being first placed in dark condition after inoculation, then transfer to 16 h/d photoperiod CMC model 3 d, cultivation temperature is 23-27 DEG C, intensity of illumination is the concentration of 3000 lx, TDZ is 0-0.1 mg/L, adds sucrose 30 g/L in medium, agar 6.0 g/L, pH value is adjusted to 6.During induction, if three groups of experimental group, in first group, the concentration of 6-BA is the concentration of 2.0mg/, IAA is 0.01mg/L; In second group, the concentration of 6-BA is the concentration of 4.0mg/L, IAA is 0.5mg/L; In 3rd group, the concentration of 6-BA is the concentration of 3.0mg/L, IAA is 0.1mg/L; Experimental result gets three groups of experimental result mean values.
TDZ pre-treatment on the impact of callus and somatic embryo inducement in table 1.
As can be seen from Table 1, when carrying out pre-treatment with 0-0.1 mg/L TDZ, callus induction rate does not have significant difference.But TDZ has significant impact to the somatic embryo number that somatic embryo inducement rate and each explant produce.Somatic embryo inducement rate reaches maximum when being 0.01 mg/L with TDZ concentration, is 52.7%, and when TDZ concentration rises to 0.1 mg/L, somatic embryo inducement rate declines again.The somatic embryo number that each explant produces reaches maximum when being also 0.01 mg/L with TDZ concentration, is 7.3.To sum up, add the TDZ of 0.01 mg/L, the somatic embryo number that optimal somatic embryo inducement rate and every explant produce can be obtained.
Embodiment 2
During pre-treatment, the concentration of TDZ is 0.01mg/L, after pre-treatment, cuts cotyledon explant, and the 1/2 MS medium put into containing 6-BA and IAA is cultivated, and cultivates 7 d under being first placed in dark condition, then transfers to 16 h/d photoperiod CMC model 14 d.Then the callus of induction gained is proceeded in the fresh culture of same formula, continue cultivation 28 d in 16 h/d photoperiod conditions.Cultivation temperature is 23-27 DEG C, and the concentration of intensity of illumination to be the concentration of 3000 lx, 6-BA be 2-4mg/L, IAA is 0.01-0.5mg/L, and add sucrose 30 g/L in medium, agar 6.0 g/L, pH value is adjusted to 6.
6-BA and IAA combination on the impact of callus and somatic embryo inducement in table 2.
6-BA and the IAA callus induction incidence of variable concentrations is variant as shown in Table 2, but difference is not obvious, and entirety is all higher, at 81.7 more than %.And somatic embryo generation rate and quality because 6-BA with IAA concentration is different significant difference, from 25.9-60.7% not etc., on every explant, somatic embryo quantity is not from 3.2-8.3 etc. for inductivity.When IAA concentration one timing, along with the increase of 6-BA concentration, Callus induction rate slowly declines gradually, but somatic embryo quantity is in rising trend on somatic embryo inducement rate and every explant, when 6-BA concentration is 3.0 mg/L, somatic embryo inducement rate reaches maximum, and on every explant, somatic embryo quantity also reaches maximum.And when 6-BA concentration one timing, along with the rising of IAA concentration, somatic embryo inducement rate is on a declining curve, when the highest somatic embryo inducement rate appears at IAA 0.05 mg/L.When 6-BA concentration be 3.0 mg/L, IAA concentration is 0.05 mg/L, on somatic embryo inducement rate and every explant, somatic embryo quantity reaches optimum.
Embodiment 3
The formation of whole plant: the 1/2 MS medium put into by somatic embryo without hormone is cultivated, and incubation time is 28 days, and cultivation temperature is 23-27 DEG C, intensity of illumination is 3000 lx, and light application time is 16 h/ days, adds sucrose 15 g/L in medium, agar 6.0 g/L, pH value is adjusted to 6.As shown in Figure of description 3 and accompanying drawing 4, method provided by the invention can make somatic embryo form complete plant.
Embodiment 4
Hardening and transplanting: after the forming step of pre-treatment, callus induction, somatic embryo inducement and whole plant, be equipped with in the blake bottle of 1/2 MS medium without hormone, choose in formed whole plant and there are 3 with the healthy and strong seedling of blade and tetanic of 5-6 cm, the lid of blake bottle is first unscrewed placement 2 d, half-open 2 d again, and then standard-sized sheet 2 d, need constantly to keep the skin wet during half-open and standard-sized sheet lid.Then, the plant after hardening being completed pulls up, and washes root medium, be transplanted in the nutritive cube that peat soil, perlite, ash and leaf mould matrix are housed, at 23-27 DEG C, put freshness protection package and be placed in climatic cabinate moisturizing cultivation, 1-2 Zhou Houzai moves on to outdoor and cultivates.The mass ratio of peat soil, perlite, ash and leaf mould matrix is 1:1:1:1.The effect of hardening and transplanting is shown in Figure of description 5 and accompanying drawing 6 respectively.
Embodiment 5
Get early good, special little phoenix, Jing Xin and carbuncle in early spring four genotype watermelon seeds respectively and carry out the induction of somatic embryo.In pre-treatment, process is: carry out sterilizing after watermelon seed being peelled off seed coat, the MS medium be seeded in by embryo containing TDZ is cultivated again, every bottle graft kind 8,3 d are cultivated under being first placed in dark condition after inoculation, cultivation temperature is 23-27 DEG C, transfer to CMC model 3 d that light application time is 16 h/d again, cultivation temperature is 23-27 DEG C, intensity of illumination is 3000 lx, the concentration of TDZ is 0.01 mg/L, add the sucrose of 30 g/L and the agar of 6.0 g/L in medium, the concentration that pH value is adjusted to 6TDZ is 3mg/L; When callus and somatic embryo inducement, cut cotyledon explant, the 1/2 MS medium put into containing 6-BA and IAA is cultivated, and cultivates 7 d under being first placed in dark condition, then transfers to 16 h/d photoperiod CMC model 14 d; Then the callus of induction gained is proceeded in the fresh culture of same formula, continue cultivation 28 d in 16 h/d photoperiod conditions.Cultivation temperature is 23-27 DEG C, and the concentration of intensity of illumination to be the concentration of 3000 lx, 6-BA be 3mg/L, IAA is 0.05mg/L, and add sucrose 30 g/L in medium, agar 6.0 g/L, pH value is adjusted to 6.
Genotype on the impact of callus and somatic embryo inducement in table 3.
As can be seen from Table 3, method provided by the invention successfully can induce somatic embryo to different genotype, and body embryonal induction rate is more than 45%, and on every explant, the body embryo number of induced synthesis is between 7.6-8.5.In table 3, various genotype utilizes this technical system can as genetically modified acceptor regenerative system.
Claims (3)
1. the watermelon somatic embryo being applicable to polygene type is efficiently induced and plant regeneration method, it is characterized in that: comprise the following steps:
Pre-treatment: carry out sterilizing after watermelon seed being peelled off seed coat, the MS medium be seeded in by embryo containing TDZ is cultivated again, every bottle graft kind 8, cultivates 3 ds under being first placed in dark condition after inoculation, and cultivation temperature is 23-27 DEG C, transfer to CMC model 3 d that light application time is 16 h/d again, cultivation temperature is 23-27 DEG C, and intensity of illumination is the concentration of 3000 lx, TDZ is 0-0.1 mg/L, add the sucrose of 30 g/L and the agar of 6.0 g/L in medium, pH value is adjusted to 6;
The induction of callus: cut cotyledon explant, put into the 1/2 MS medium containing 6-BA and IAA, 7 d are cultivated under being first placed in dark condition, transfer to CMC model 14 d that light application time is 16 h/d again, cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000 lx, the concentration of 6-BA is 2-4mg/L, the concentration of IAA is 0.01-0.5mg/L, and add the sucrose of 30 g/L and the agar of 6.0 g/L in medium, pH value is adjusted to 6;
The induction of somatic embryo: by step 2) gained callus puts into 1/2 MS medium containing 6-BA and IAA, CMC model 28 d being 16 h/d in light application time obtains somatic embryo, cultivation temperature is 23-27 DEG C, intensity of illumination is 3000 lx, the concentration of 6-BA is 2-4mg/L, the concentration of IAA is 0.01-0.5mg/L, and add the sucrose of 30 g/L and the agar of 6.0 g/L in medium, pH value is adjusted to 6;
The formation of whole plant: the somatic embryo of step 3) gained is put into be equipped with and do not cultivate containing the blake bottle of 1/2 MS medium of hormone, incubation time is 28 d, cultivation temperature is 23-27 DEG C, intensity of illumination is 3000 lx, light application time is 16 h/d, add the sucrose of 30 g/L and the agar of 6.0 g/L in medium, pH value is adjusted to 6;
5) hardening: choose having 3 and the lid of blake bottle is first unscrewed placement 2 d with the healthy and strong seedling of blade and tetanic of 5-6 cm after step 4) is cultivated, more half-open 2 d, and then standard-sized sheet 2 d, need constantly to keep the skin wet during half-open and standard-sized sheet lid;
6) transplant: the plant after hardening being completed pulls up, wash root medium, be transplanted in the nutritive cube that peat soil, perlite, ash and leaf mould matrix are housed, at 23-27 DEG C, put freshness protection package and be placed in climatic cabinate moisturizing cultivation, 1-2 Zhou Houzai moves on to outdoor and cultivates.
2. method according to claim 1, is characterized in that: the sterilization process described in step 1) is: the seed coat peelling off watermelon seed, first with 75% alcohol-pickled sterilization 1min, then use 0.1% mercuric chloride soaking disinfection 5min, finally use aseptic water washing 5 times.
3. method according to claim 1, is characterized in that: in nutritive cube described in step 6), the mass ratio of peat soil, perlite, ash and leaf mould matrix is 1:1:1:1.
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| CN109136259A (en) * | 2018-10-09 | 2019-01-04 | 北京市农林科学院 | A kind of watermelon High-efficient Genetic Transformation and transgenic plant identification method |
| CN106688888B (en) * | 2016-12-20 | 2019-02-01 | 湖南省西瓜甜瓜研究所 | A kind of raising watermelon Unpollinated ovules embryoid induction rate method |
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| CN106688888B (en) * | 2016-12-20 | 2019-02-01 | 湖南省西瓜甜瓜研究所 | A kind of raising watermelon Unpollinated ovules embryoid induction rate method |
| CN109136259A (en) * | 2018-10-09 | 2019-01-04 | 北京市农林科学院 | A kind of watermelon High-efficient Genetic Transformation and transgenic plant identification method |
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