阅读说明：本技术 一种红掌的快速培养方法 (A kind of fast culture process of the red palm ) 是由 周国权 郑彬江 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快速培养方法,包括无菌苗培养、原生质体分离、原生质体培养、原生质体植株再生,其通过在原生质体植株再生阶段,加入等量的第二MS培养基,并通过培养基的组分选择及配比,降低了渗透压和细胞密度,而且,培养板与第三MS培养基的结合使用,确保了植物来自单个细胞,提高了萌芽率及再生速度。(The present invention relates to a kind of fast culture process of red palm, including Aseptic seedling culture, protoplast electrofusion, Protoplast cuhnre, Plant Regeneration from Protoplast, it passed through in the Plant Regeneration from Protoplast stage, the 2nd MS culture medium of equivalent is added, and passes through the component selection and proportion of culture medium, reduces osmotic pressure and cell density, and, culture plate is used in combination with the 3rd MS culture medium, it is ensured that plant comes from individual cells, improves germination rate and reproduction speed.)

1. a kind of fast culture process of the red palm, which comprises the steps of:

(1) Aseptic seedling culture: using the red palm seedling of no hormone MS culture medium in vitro culture as material, illumination in 20 hours is carried out；

(2) protoplast electrofusion: selection spire is slitting, is placed in the flask equipped with enzyme solutions, enzyme solutions contain 5%(w/v) Cellulase R-10,0.5%(w/v) pectase Y-23 and 10%(w/v) mannitol, stirs and seeps enzyme solutions by vacuum pump Thoroughly, then the molten object of enzyme and spire strip are poured into culture dish, is cultivated 30 minutes under 25 °C, stirred 10s, trained again under 25 °C It supports 30 minutes；Isolated protoplast is centrifuged 10 minutes at 100 x g by the screen to filtrate, 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 50 × g After ten minutes, the intact protoplasts of interface are sucked out by suction pipe, in 10%(w/v) it washes twice in mannitol；

(3) Protoplast cuhnre: protoplast with the first MS culture medium culture, contains MS in the first MS culture medium in culture dish Salt, the thiamine hydrochloride of 0.3 mg/L, the NAA of 0.5mg/L, 0.1 mg/L BAP, 10%(w/v) glucose and 6%(w/v) sucrose, The pH of first MS culture medium is 5.5；

(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 thiamine hydrochloride of 0.3 mg/L, the NAA of 0.5mg/L, 0.1 mg/L) glucose And 3%(w/v) then group is transferred to culture plate, which includes filter paper base until group grows to 0.2-0.3mm by sucrose Plate and transfer blade, the culture plate are placed on the 3rd MS culture medium 10-20 days, and single callus is then placed on third 14-20 days on MS culture medium, to generate the callus of 2-6 mm, then callus is transferred in HSM culture medium and is regenerated, sprouted After bud, it is transferred to no hormone MS culture medium, the earth that buries then is transplanted after 7-10 days.

2. the fast culture process of the red palm as described in claim 1, which is characterized in that 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 It is 5.8.

3. the fast culture process of the red palm as described in claim 1, which is characterized in that the HSM culture medium contains MS salt, dimension Raw element 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 cultural 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.

MSCulture mediumIt is to use most common culture medium at present.Its inorganic salt concentration with higher, can guarantee tissue Needed for growthMineral nutritionIt can also accelerateCallusGrowth.Since the ion concentration in formula is high, preparation, storage and During disinfection etc., even if some ingredients are slightly different, interionic balance will not be influenced.MSSolid mediumIt can be used for luring Lead callus, it can also be used to embryo, stem section,Stem apexAnd the culture of anther,Fluid nutrient mediumEnergy when for cell suspension cultures Obtain apparent success.The quantity and ratio of the inorganic nutrients of MS culture medium are proper, it is sufficient to meet plant cell in nutrition It goes up and needs physiologically.Therefore, under normal circumstances, without adding againAmino acid、CaseinHYPERLINK "https:// baike.baidu.com/item/%E6%B0%B4%E8%A7%A3"HydrolysisObject, yeast extract and coconut milk etc. are organic to be added into Point.With it is otherCulture mediumBasis compare, in MS culture mediumNitrate, potassium andAmmoniumContent it is high, this is the significant of it Feature.

Summary of the invention

It is red that the purpose of the present invention is to provide a kind of reproduction speeds is fast, germination rate is high, sprout grows stable fast culture The method of the palm.Specifically, red palm cultural method of the invention in turn includes the following steps:

(1) Aseptic seedling culture: using the red palm seedling of no hormone MS culture medium in vitro culture as material, illumination in 20 hours is carried out；(2) Protoplast electrofusion: selection spire is slitting, is placed in the flask equipped with enzyme solutions, enzyme solutions contain 5%(w/v) cellulose Enzyme R-10,0.5%(w/v) pectase Y-23 and 10%(w/v) mannitol, stirs and permeates enzyme solutions by vacuum pump, so The molten object of enzyme and spire strip are poured into culture dish afterwards, cultivated 30 minutes under 25 °C, 10s is stirred, is further cultured for 30 under 25 °C Minute；Isolated protoplast is centrifuged 10 minutes at 100 x g by the screen to filtrate, makes protoplast in 20%(w/v) It suspends in sucrose solution, 10%(w/v) mannitol solution is lightly dripped in the top of suspension, be centrifuged 10 minutes at 50 × g Afterwards, the intact protoplasts of interface are sucked out by suction pipe, in 10%(w/v) it washes twice in mannitol；(3) protoplast Culture: protoplast in culture dish with the first MS culture medium culture, the salt containing MS salt, 0.3 mg/L in the first MS culture medium BAP, 10%(w/v of allithiamine, the NAA of 0.5mg/L, 0.1 mg/L) glucose and 6%(w/v) sucrose, the first MS culture medium PH is 5.5；(4) Plant Regeneration from Protoplast: being added the 2nd MS culture medium of equivalent for every 5 days into the culture dish of step (3), should 2nd MS culture medium contains BAP, 5%(w/v of MS salt, the thiamine hydrochloride of 0.3 mg/L, the NAA of 0.5mg/L, 0.1 mg/L) Portugal Grape sugar and 3%(w/v) then group is transferred to culture plate until group grows to 0.2-0.3mm by sucrose, and which includes filter Paper base plate and transfer blade, the culture plate are placed on the 3rd MS culture medium 10-20 days, are then placed on single callus 14-20 days on 3rd MS culture medium, to generate the callus of 2-6 mm, then callus is transferred in HSM culture medium again It is raw, after rudiment, it is transferred to no hormone MS culture medium, the earth that buries then is transplanted after 7-10 days.Preferably, the 3rd MS culture Base contains MS salt, vitamin, 0.5 mg/L BAP, 1 mg/L NAA, 3%(w/v) sucrose and 0.6%(w/v) agar, the 3rd MS training The pH for supporting base is 5.8.Preferably, the HSM culture medium contain 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 by the way that the 2nd MS training of equivalent is added in the Plant Regeneration from Protoplast stage Base is supported, and passes through the component selection and proportion of culture medium, osmotic pressure and cell density are reduced, moreover, culture plate and the 3rd MS The combined use of culture medium, it is ensured that plant comes from individual cells, improves germination rate and reproduction speed.

Specific embodiment