CN101322475B - Method for obtaining regeneration plant from in-vitro culture of black seed pumpkin - Google Patents
Method for obtaining regeneration plant from in-vitro culture of black seed pumpkin Download PDFInfo
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- CN101322475B CN101322475B CN 200810138494 CN200810138494A CN101322475B CN 101322475 B CN101322475 B CN 101322475B CN 200810138494 CN200810138494 CN 200810138494 CN 200810138494 A CN200810138494 A CN 200810138494A CN 101322475 B CN101322475 B CN 101322475B
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Abstract
The invention discloses a method for obtaining regenerated plantlets by isolated culture of black-seeded pumpkin, comprising the steps of obtaining explant, inducing adventitious bud, adventitious bud extending, rooting cultivation and transplanting, wherein, the inducing culture medium of adventitious buds is MS, 3% sucrose by weight, 0.9% agaragar by weight and 0.5mg/L thidiazuron; the extending culture medium of adventitious buds is MS, 3% sucrose by weight, 0.9% agaragar by weight and 0.25mg/L kinetin; rooting culture medium is MS, 3% sucrose by weight, 0.9% agaragar by weight and 0.1mg/L indoleacetic acid. The invention overcomes the defects of low recurring frequency, long cultivation period, and the like, in the current recurring cultivation of black-seeded pumpkin plant, thereby establishing a technology of recurring system of high-frequency adventitious buds, providing an efficient practical technology for asexual propagation of black-seeded pumpkin and genetic transformation of black-seeded pumpkin induced by tumefaciens, and having certain significance.
Description
Technical field
The present invention relates to a kind of method of obtaining regeneration plant from in-vitro culture of black seed pumpkin, belong to the Plant Tissue Breeding field.
Background technology
Black seed pumpkin is a kind of annual vining plant, mainly is distributed in the high altitude localities, compares with other kinds that belong to together to differ greatly, and can not hybridize the generation offspring with them.The centre of origin is ununified as yet at present, and most people think America.Mainly be distributed in Mexico, Chile, Germany, France, Japan, Philippine and China (Andr é s 1990 at present; Meng and Zeng 2000).Black seed pumpkin is of many uses, and green fruit can be boiled and be eaten or as canned vegetable, and ripe fresh fruit can be made alcoholic beverage; The most important nutritive value of black seed pumpkin is to contain amounts of protein and essential oil (Andr é s, 1990) in its seed; Black seed pumpkin also has hypotensive and slows down effect (the Alarcon-Aguilar et al.2002 of diabetes; Roman-Ramos et al.1995; Roman-Ramos et al.1992); Chilean recently studies show that, the extraction enzyme of black seed pumpkin fresh fruit can be handled effectively and come from the waste water that fish processing enterprise discharges.
The black seed pumpkin root system is powerful, impoverishment tolerant, cold-resistant, drought-enduring, anti-withered verticillium wilt, the stock of the many melon graftings of Chang Zuowei [European and Mediterranean Plant Protection Organization (OEPP/EPPO) 2004].But black seed pumpkin also has many bad proterties, and for example therefore responsive the and root-knot nematode resistant etc. not to root rot need carry out genetic improvement research to it.
Present research and other melon crops (Colijn-Hooymansa, 1988 about the black seed pumpkin regenerating system; Trulsonand Shahin, 1986; Kathal, 1988; Moreno, 1985; Niedz, 1989) Comparatively speaking, report lessly, already present regenerating system is that stem apex expands numerous (Liu, 2004) and cotyledon knot regenerating system (Xu, 2007), but has shortcomings such as regeneration frequency is low, cultivation cycle is grown.
Plumule is made explant, its superiority is that regeneration frequency height, cultivation cycle are short, be subjected to external environment and physiological status influence little, the result more easily repeats etc.
Summary of the invention
At above-mentioned the deficiencies in the prior art, the invention provides the method for the isolated culture adventive bud evoked plant regeneration of black seed pumpkin plumule of a kind of regeneration frequency height, cultivation cycle weak point.
The present invention is achieved by the following technical solutions:
The method of obtaining regeneration plant from in-vitro culture of black seed pumpkin, comprise the inducing of acquisition, indefinite bud, the elongation of indefinite bud, culture of rootage, the transplant step of explant, it is characterized in that: the inducing culture of described indefinite bud is the agar+0.5mg/L Thidiazuron of sucrose+0.9% weight of MS+3% weight; The elongation medium of described indefinite bud is the agar+0.25mg/L kinetin of sucrose+0.9% weight of MS+3% weight; Described root media is the agar+0.1mg/L heteroauxin of sucrose+0.9% weight of MS+3% weight; The pH of described various medium is 5.8; The inducing of the acquisition of described explant, indefinite bud, the elongation of indefinite bud, the condition of culture in culture of rootage stage are 25 ℃ ± 1 ℃ of cultivation temperature.
Described explant obtains by the following method: after the black seed pumpkin seed is shelled, 70% ethanol (volume parts) surface sterilization 30 seconds, sterile water wash once, the mercury chloride of 0.1% (w/v) sterilization 5 minutes is used sterile water wash 5 times then; The seed of sterilization after the soaked overnight, is inoculated into vernalization on the 1/2MS medium in the dark, and the photoperiod is 8h dark/16h illumination; Separate two cotyledons after 3 days, the protrusion position of picking two slice, thin piece leaf bases obtains plumule, is explant, can peel off plumule under anatomical lens in case of necessity.
The step of described transplanting is: culture of rootage is after 30 days, select the good stand seedling of taking root, in blake bottle, inject the water of 1/3 volume, open wide to cultivate bottle cap after placing 2 days on the culturing rack, water flush away agar (agar in the former medium) gently, be transplanted to the sand that has sterilization soil: native volume ratio is in 1: 1 the polypots, be used to preserve moisture with transplanted seedling on the plastic pocket, watered the MS liquid nutrient medium one time every 3 days, remove plastic sack after 2 weeks, support 2 weeks of adaptation in the environment at earth culture.
Compared with prior art, the present invention has following beneficial effect:
1, the present invention, draws materials conveniently as explant with the black seed pumpkin plumule, and it is little influenced by external environment and physiological status, as a result good reproducibility;
2, on the inducing culture of MS+0.5mg/L TDZ, inducing culture regeneration frequency height reaches 100%; The indefinite bud quantity that each explant produces is many, and cluster takes place;
3, can continue a large amount of new indefinite buds that generate behind the excision elongation bud, indefinite bud have certain continuation.
The present invention has overcome shortcomings such as the regeneration frequency in the black seed pumpkin plant regeneration is cultivated is low at present, cultivation cycle is long, set up the technology of high frequency adventitious shoot regeneration system, for the vegetative propagation of black seed pumpkin and agriculture bacillus mediated black seed pumpkin genetic transformation provide effective practical technique, has certain meaning.
Description of drawings
Fig. 1 is plumule and the plumule tender tissue schematic diagram on every side after peeling off;
Fig. 2 is the schematic diagram of inducing of indefinite bud;
The sprouting schematic diagram of Fig. 3 indefinite bud;
Fig. 4 is the elongation schematic diagram of indefinite bud;
Fig. 5 is the plant schematic diagram after taking root;
Fig. 6 is the regeneration plant schematic diagram after transplanting.
Embodiment
The present invention is further illustrated below in conjunction with embodiment:
Embodiment 1: the isolated culture adventive bud evoked plant regeneration of black seed pumpkin plumule:
1. materials and methods
1.1 vegetable material: black seed pumpkin is produced in Yunnan.
1.2 plant hormone and medium: this tests used plant hormone all available from sigma company, is respectively Thidiazuron (TDZ), kinetin (KT), heteroauxin (IAA).Minimal medium is MS medium (Murashige T., Skoog F.A revisedmedium for rapid growth and bio-assays with tobacco tissue cultures.Physiol Plant, 1962,15:473-497), in incubation, added different hormones according to different developmental stages in medium, wherein, the inducing culture of indefinite bud is the agar+0.5mg/L Thidiazuron of sucrose+0.9% weight of MS+3% weight; The elongation medium of indefinite bud is the agar+0.25mg/L kinetin of sucrose+0.9% weight of MS+3% weight; Root media is the agar+0.1mg/L heteroauxin of sucrose+0.9% weight of MS+3% weight; Each medium is adjusted pH value to 5.8 with sodium hydroxide and hydrochloric acid before autoclaving.
1.3 method
1.3.1 the acquisition of explant: after the black seed pumpkin seed shells, 30 seconds of 70% ethanol surface sterilization, sterile water wash once, 0.1% mercury chloride sterilization 5 minutes, use sterile water wash then 5 times, the seed of sterilization after the soaked overnight, is inoculated into vernalization on the 1/2MS medium in the dark, and the photoperiod is 8h dark/16h illumination.Separate two cotyledons with tweezers and blade after 3 days, the protrusion position of dissecting needle picking two slice, thin piece leaf bases obtains plumule (can carry out) under anatomical lens.
1.3.2 inducing of indefinite bud: the plumule explant of peeling off is transferred on the inducing culture of indefinite bud, and as shown in Figure 1, a situation arises for the statistics indefinite bud after 30 days.
1.3.3 the elongation of indefinite bud and taking root: at inducing culture on the inducing culture of indefinite bud after 30 days, will have from the explant of the original hase of sprouting and be transferred on the elongation medium of indefinite bud, and be used for the elongation of bud.Shen Chang indefinite bud is cut to forward to and carries out culture of rootage on the root media subsequently.Cultivation temperature is 25 ± 1 ℃.
1.3.4 transplant: culture of rootage is after 30 days, select the good stand seedling of taking root, in blake bottle, inject the water of 1/3 volume, open wide to cultivate bottle cap after placing 2 days on the culturing rack, with running water flush away agar gently, the good plant seedling of will taking root is transplanted to the sand that has sterilization soil: the volume ratio of soil is in 1: 1 the polypots, be used to preserve moisture with transplanted seedling on the plastic pocket, watered the MS liquid nutrient medium one time every 3 days, remove plastic sack after 2 weeks, support 2 weeks of adaptation in the environment at earth culture.
2 results and analysis
2.1 inducing of adventitious shoot regeneration: the plumule explant is on the inducing culture of indefinite bud, and inductivity is 100%, and promptly each explant all can effectively produce a large amount of indefinite buds, and cluster takes place usually, as Fig. 2, shown in Figure 3.
2.2 the elongation of bud: only have the indefinite bud of minority to extend on the inducing culture of indefinite bud, the common chap of most of indefinite bud, vitrifying, yellow show as abnormal bud on form, and produce some callus around bud.When the indefinite bud of inducing was transferred on the elongation medium of indefinite bud, the indefinite bud of inducing after three weeks can extend and be normal bud, as shown in Figure 4.After the bud excision switching, remaining explant can continue to produce a large amount of indefinite buds, also can continue elongation in the time of on the bud elongation medium of these indefinite bud switchings, develops into normal bud.This phenomenon is numerous for the expansion of black seed pumpkin to be very favourable.
2.3 the taking root and transplant of indefinite bud: the indefinite bud of lengthen by 2 cm is transferred on the medium of taking root, and occurs the projection of root after 7 days.The incidence of cultivating 20 days roots is 100%, and each seedling is taken root 4～5, as shown in Figure 5.We find that the bud of black seed pumpkin very easily takes root under study for action, and this may be relevant with the genotype of black seed pumpkin itself.Good seedling take root after 4 weeks according to the description of materials and methods, tame cultivation, transplanting survival rate reaches 85.0%, as shown in Figure 6.
3 conclusions: the present invention as explant material, on the inducing culture of indefinite bud, carries out inducing culture with 3 days black seed pumpkin plumule of vernalization, induce 30 days after, the inductivity of indefinite bud is 100%, and the indefinite bud cluster takes place; Forwarding the elongation of carrying out bud on the elongation medium of indefinite bud to cultivates, most of induced bud can normally extend, indefinite bud with lengthen by 2 cm after 30 downcuts, be transferred on the root media, rooting rate is 100%, and on average each seedling is taken root 4-5, and culture of rootage is after 30 days, tame transplanting, survival rate is 85.0%.
Claims (2)
1. the method for obtaining regeneration plant from in-vitro culture of black seed pumpkin, the inducing of acquisition, indefinite bud, the elongation of indefinite bud, culture of rootage, the transplant step that comprise explant, it is characterized in that: described explant obtains by the following method: after the black seed pumpkin seed is shelled, 70% ethanol surface sterilization 30 seconds, sterile water wash once, 0.1% mercury chloride sterilization 5 minutes is used sterile water wash 5 times then; The seed of sterilization after the soaked overnight, is inoculated into vernalization on the 1/2MS medium in the dark, and the photoperiod is 8h dark/16h illumination; Separate two cotyledons after 3 days, the protrusion position of picking two slice, thin piece leaf bases obtains plumule, and be explant: the inducing culture of described indefinite bud is the agar+0.5mg/L Thidiazuron of sucrose+0.9% weight of MS+3% weight; The elongation medium of described indefinite bud is the agar+0.25mg/L kinetin of sucrose+0.9% weight of MS+3% weight; Described root media is the agar+0.1mg/L heteroauxin of sucrose+0.9% weight of MS+3% weight; The pH of described various medium is 5.8; The inducing of the acquisition of described explant, indefinite bud, the elongation of indefinite bud, the condition of culture in culture of rootage stage are 25 ℃ ± 1 ℃ of cultivation temperature.
2. the method for obtaining regeneration plant from in-vitro culture of black seed pumpkin according to claim 1, it is characterized in that: the step of described transplanting is: culture of rootage is after 30 days, select the good stand seedling of taking root, in blake bottle, inject the water of 1/3 volume, open wide and cultivate bottle cap after placing 2 days on the culturing rack, water flush away agar gently, be transplanted to the sand that has sterilization soil: native volume ratio is in 1: 1 the polypots, be used to preserve moisture with transplanted seedling on the plastic pocket, watered one time the MS liquid nutrient medium every 3 days, remove plastic sack after 2 weeks, support 2 weeks of adaptation in the environment at earth culture.
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| CN 200810138494 CN101322475B (en) | 2008-08-07 | 2008-08-07 | Method for obtaining regeneration plant from in-vitro culture of black seed pumpkin |
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| CN107372123B (en) * | 2017-09-13 | 2019-06-11 | 界首市家丰家庭农场 | A method of improving tulip flourish |
| CN110278868A (en) * | 2019-05-17 | 2019-09-27 | 云南农业大学 | It is a kind of based on stem with bud be explant black seed pumpkin tissue culture propagation method |
| CN111218471A (en) * | 2020-02-23 | 2020-06-02 | 华中农业大学 | Agrobacterium rhizogenes-mediated pumpkin root system transformation method and gene editing method |
| CN113826552B (en) * | 2021-09-24 | 2023-03-28 | 内蒙古农业大学 | In-vitro rapid propagation method for seed cucurbita pepo |
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