阅读说明：本技术 一种红掌的培育方法 (A kind of breeding method of the red palm ) 是由 郑彬江 周国权 郑彬旭 罗志超 于 2019-06-18 设计创作，主要内容包括：本发明涉及一种红掌的培育方法,包括红掌苗培养、原生质体分离、原生质体培养和原生质体再生,与用切片叶片制成的样本相比,研磨技术对叶组织的损伤较小,因此原生质体的产量较高,另外,本发明采用原研的水解酶材料,相比其他细胞壁水解酶,如小豆粕纤维素酶RS和纤维素酶R-10等,当以相同浓度使用时,发现释放原始细胞的效果更优,且通过荧光灯和白炽灯组合供光,室温下光照18小时后,将培养室的温度调整为22℃,然后在黑暗条件下培养10小时,且以特定的光通量密度,改善了育苗效果。(The present invention relates to a kind of breeding methods of red palm, including red palm seedling culture, protoplast electrofusion, Protoplast cuhnre and protoplast regeneration, compared with the sample made of slice blade, grinding technique is smaller to the damage of leaf texture, therefore the yield of protoplast is higher, in addition, the present invention is using the former hydrolase material ground, compared to other cytohydrolists, such as small dregs of beans cellulase RS and cellulase R-10, when being used with same concentrations, it was found that the effect of release initial cell is more excellent, and by fluorescent lamp and incandescent lamp combination for light, at room temperature after illumination 18 hours, the temperature of culturing room is adjusted to 22 DEG C, then it is cultivated 10 hours under dark condition, and with specific pharosage, improve nursery effect.)

1. a kind of breeding method of the red palm, which is characterized in that in turn include the following steps:

(1) red palm seedling culture: red palm seed to be cultivated in culturing room, condition of culture are as follows: fluorescent lamp and incandescent lamp are combined for light, At room temperature after illumination 18 hours, the temperature of culturing room is adjusted to 22 DEG C, is then cultivated 10 hours under dark condition, light is restored According to, later daily with diluted nutrient solution pour, the acrospire being fully deployed is used for protoplast electrofusion after 20 days；

(2) protoplast electrofusion: the acrospire being fully deployed is detached from from plant, and rear suck dry moisture, puts wash with distilled water It is lightly ground in the distilled water for being mixed with diamond dust using medical cotton stick, rinses the leaf of abrasion again then to remove Buddha's warrior attendant The leaf of abrasion, is cut into 1 × 1 μm of square by sand, then suck dry moisture, and is swum on culture medium, and leaf surfaces and training are made Support base contact, which contains 2.5%(w/v) cellulase, 0.5%(w/v) pectase Y-23,9%(w/v) mannitol, Calcium chloride, the 2%(w/v of 0.1 mg/L) BSA and 5mM MES, the pH of the culture medium is 5.8；It is logical in the light of 120 μ E/m2s Metric density is irradiated at 28 DEG C 4-5 hours, then by the screen to filtrate, and with containing 9%(w/v) mannitol, 0.05 mg/L's Calcium chloride, 0.1%(w/v) buffer of BSA washes twice, and filtrate is centrifuged 3 minutes at 100x g, makes protoplast 20% (w/v) it suspends in sucrose solution, 10%(w/v) mannitol solution is lightly dripped in the top of suspension, is centrifuged at 100 × g After five minutes, the intact protoplasts of interface are sucked out by suction pipe, in 9%(w/v) it washes twice in mannitol；

(3) Protoplast cuhnre: protoplast in culture dish with the BAP of NAA, 0.3mg/L containing MS salt, 0.2mg/L, 10%(w/v) glucose and 6%(w/v) sucrose, and pH be 5.8 culture medium in cultivate；

(4) Plant Regeneration from Protoplast: the 2nd MS culture medium of equivalent is added within every 5 days into the culture dish of step (3), this second MS culture medium contains BAP, 5%(w/v of MS salt, the NAA of 0.5mg/L, 0.1 mg/L) glucose and 3%(w/v) sucrose, Zhi Daoqun It falls and grows to 0.2-0.3mm, group is then transferred to culture plate, and culture plate is placed on the 3rd MS culture medium 14-21 days, Then single callus is placed on the 3rd MS culture medium 21-28 days, to generate the callus of 2-4 mm, then by callus Tissue, which is transferred in HSM culture medium, to be regenerated, and after rudiment, is transferred to no hormone MS culture medium, is then transplanted and bury after 7-10 days Earth.

2. the breeding method of the red palm according to claim 1, which is characterized in that fluorescent lamp and incandescent lamp energy in step (1) It is enough that the pharosage of 420 μ E/m2s is provided at integral shroud height.

3. the breeding method of the red palm according to claim 1, which is characterized in that the 3rd MS culture medium contain MS salt, Vitamin, 0.5 mg/L BAP, 1 mg/L NAA, 3%(w/v) sucrose and 0.6%(w/v) agar, the pH of the 3rd MS culture medium is 5.8。

4. the breeding method of the red palm according to claim 1, which is characterized in that the HSM culture medium contains MS salt, dimension life Plain B5,1 mg/L BAP, 0.1 mg/L NAA and 3%(w/v) sucrose, the pH of HSM culture medium is 5.8.

Technical field

The application belongs to technical field of plant culture, and in particular to a kind of breeding method of the red palm.

Background technique

The red palm isMonocotyledonaeAraeceaeAnthuriumPerennial evergreen herbaceous plant.Stipes is short；Leaf is raw from base portion Out, green, keratin, full edge, long round shape is heart-shaped or ovum is heart-shaped.Petiole is elongated, and spathe is cleared, and keratin simultaneously has wax gloss, orange Red or scarlet；Spadix yellow can bloom constantly throughout the year.Fancy candles lit in the bridal chamber at wedding originates in the torrid zones such as Costa Rica, Colombia Rainforest area.It often grows nonparasitically upon another plant in the tree, grown nonparasitically upon another plant on rock sometimes or is grown directly upon on the ground, property likes the ring of warm, moist half yin Direct sunlight is avoided in border.The fancy candles lit in the bridal chamber at wedding flower peculiar U.S. of appearance is beautiful.Florescence is lasting, group planting beautification at suitable potting, cut-flower or flower garden concealment.

Protoplast, which refers to, has sloughed plant cell wall, exposed, viable primary matter group with specific process. There is no cell wall, but all features with living cells.Its is cell-free, and obstacle hinders, and carries out related genetic manipulation with can be convenient, And it can be rightFilm,OrganelleDeng progress basic research；With totipotency, and it can be carried out artificial culture and develop into completelyPlant；It is former Raw plastid is appropriate for induced fusion and forms hybrid cell.

Summary of the invention

The purpose of the present invention is to provide a kind of methods of the good red palm of culture of yield height, cultivate effect.Specifically, this hair Bright red palm breeding method in turn includes the following steps:

(1) red palm seedling culture: red palm seed to be cultivated in culturing room, condition of culture are as follows: fluorescent lamp and incandescent lamp are combined for light, At room temperature after illumination 18 hours, the temperature of culturing room is adjusted to 22 DEG C, is then cultivated 10 hours under dark condition, light is restored According to, later daily with diluted nutrient solution pour, the acrospire being fully deployed is used for protoplast electrofusion after 20 days；

(2) protoplast electrofusion: the acrospire being fully deployed is detached from from plant, and rear suck dry moisture, puts wash with distilled water It is lightly ground in the distilled water for being mixed with diamond dust using medical cotton stick, rinses the leaf of abrasion again then to remove Buddha's warrior attendant The leaf of abrasion, is cut into 1 × 1 μm of square by sand, then suck dry moisture, and is swum on hydrolase, and leaf surfaces and water are made Solve enzyme contact, which contains 2.5%(w/v) cellulase, 0.5%(w/v) pectase Y-23,9%(w/v) mannitol, Calcium chloride, the 2%(w/v of 0.1 mg/L) BSA and 5mM MES, the pH of the hydrolase is 5.8；It is logical in the light of 120 μ E/m2s Metric density irradiates 4-5 hours at 28 DEG C, and keeps shaking, then by the screen to filtrate, and with containing 9%(w/v) mannitol, The calcium chloride of 0.05 mg/L, 0.1%(w/v) buffer of BSA washes twice, and filtrate is centrifuged 3 minutes at 100x g, makes original Raw plastid is in 20%(w/v) it suspends in sucrose solution, 10%(w/v) mannitol solution is lightly dripped in the top of suspension, In It is centrifuged after five minutes, the intact protoplasts of interface is sucked out by suction pipe, in 9%(w/v under 100 × g) it washs in mannitol Twice；

(3) Protoplast cuhnre: protoplast in culture dish with the BAP of NAA, 0.3mg/L containing MS salt, 0.2mg/L, 10%(w/v) glucose and 6%(w/v) sucrose, and pH be 5.8 culture medium in cultivate；

(4) Plant Regeneration from Protoplast: the 2nd MS culture medium of equivalent is added within every 5 days into the culture dish of step (3), this second MS culture medium contains BAP, 5%(w/v of MS salt, the NAA of 0.5mg/L, 0.1 mg/L) glucose and 3%(w/v) sucrose, Zhi Daoqun It falls and grows to 0.2-0.3mm, group is then transferred to culture plate, and culture plate is placed on the 3rd MS culture medium 14-21 days, Then single callus is placed on the 3rd MS culture medium 21-28 days, to generate the callus of 2-4 mm, then by callus Tissue, which is transferred in HSM culture medium, to be regenerated, and after rudiment, is transferred to no hormone MS culture medium, is then transplanted and bury after 7-10 days Earth.

Preferably, fluorescent lamp and incandescent lamp can provide the luminous flux of 420 μ E/m2s at integral shroud height in step (1) Density.

Preferably, the 3rd MS culture medium contains MS salt, vitamin, 0.5 mg/L BAP, 1 mg/L NAA, 3%(w/ V) sucrose and 0.6%(w/v) agar, the pH of the 3rd MS culture medium is 5.8.

Preferably, the HSM culture medium contains MS salt, vitamin B5,1 mg/L BAP, 0.1 mg/L NAA and 3%(w/ V) sucrose, the pH of HSM culture medium are 5.8.

Beneficial effect obtained by the present invention is compared with the sample made of slice blade, and grinding technique is to leaf texture Damage is smaller, therefore the yield of protoplast is higher, in addition, the present invention compares other cells using the former hydrolase material ground Wall hydrolase, such as small dregs of beans cellulase RS and cellulase R-10, when with same concentrations in use, discovery release it is original The effect of cell is more excellent, and by fluorescent lamp and incandescent lamp combination for light, at room temperature after illumination 18 hours, by the temperature of culturing room 22 DEG C are adjusted to, is then cultivated 10 hours under dark condition, and with specific pharosage, improves nursery effect.

Specific embodiment