阅读说明：本技术 一种西番莲无菌外植体的培育方法 (Method for cultivating sterile passion flower explants ) 是由 管艳 李玲 刘世红 殷振华 桂明春 粱国平 唐敏 田海 于 2021-08-24 设计创作，主要内容包括：本发明提供了一种西番莲无菌外植体的培育方法,涉及植物组培技术领域,包括以下步骤：采集西番莲幼嫩茎段作为外植体,冲洗后采用饱和洗衣粉液浸泡,漂洗,得预处理外植体；采用利福平溶液对预处理外植体进行无菌预培养,得到预培养外植体；将所述预培养外植体剪切成茎段后进行消毒处理,得到无菌茎段；将所述无菌茎段在MS基本培养基中进行培养,获得无菌西番莲外植体。本发明所述的培育方法通过外植体的两次预处理方法,升汞杀菌方法及时间等结合,在降低污染率的同时也降低了因消毒灭菌导致的褐化率和死亡率,从而大大提高了外植体的存活率,降低了组培的生产成本。(The invention provides a method for cultivating a sterile passion flower explant, which relates to the technical field of plant tissue culture and comprises the following steps: collecting tender stem segments of passion flower as explants, soaking the tender stem segments by using saturated laundry powder liquid after washing, and rinsing to obtain pretreated explants; carrying out aseptic pre-culture on the pretreated explant by adopting a rifampicin solution to obtain a pre-cultured explant; shearing the pre-culture explant into stem sections and then carrying out disinfection treatment to obtain sterile stem sections; and culturing the sterile stem section in an MS minimal medium to obtain a sterile passion flower explant. The cultivation method of the invention combines the two pretreatment methods of the explant, the mercuric chloride sterilization method and time, and the like, reduces the pollution rate and the browning rate and the death rate caused by disinfection and sterilization, thereby greatly improving the survival rate of the explant and reducing the production cost of tissue culture.)

1. A method for cultivating a sterile passion flower explant is characterized by comprising the following steps:

collecting tender stem segments of passion flower as explants, soaking the tender stem segments by using saturated laundry powder liquid after washing, and rinsing to obtain pretreated explants;

carrying out aseptic pre-culture on the pretreated explant by adopting a rifampicin solution to obtain a pre-cultured explant;

shearing the pre-culture explant into stem sections and then carrying out disinfection treatment to obtain sterile stem sections;

and culturing the sterile stem section in an MS minimal medium to obtain a sterile passion flower explant.

2. The method for culturing the sterile passion flower explant according to claim 1, wherein the length of the explant is 12-15 cm, and 3-4 axillary buds, terminal buds and 1-2 leaves at the upper end of the terminal buds are retained.

3. The method for cultivating a sterile passion flower explant according to claim 1 or 2, wherein the time for soaking the explant is 10-30 min.

4. The method for culturing the sterile passion flower explant according to claim 3, wherein the number of rinsing is 2-4.

5. The method for cultivating a sterile passion flower explant according to claim 1 or 4, wherein the concentration of the rifampicin solution is 80-120 mg/L, and the explant is immersed in the rifampicin solution for 2-3 cm.

6. The method for culturing the sterile passion flower explant according to claim 5, wherein the pre-culture temperature is 22-30 ℃, the illumination time is 8-12 h/d, and the illumination time is 2-4 d.

7. The method for culturing the sterile passion flower explant according to claim 1 or 6, wherein the sterile stem section is 0.8-2.0 cm long with 1-2 axillary buds.

8. A method of growing a sterile passion flower explant as claimed in claim 7, wherein the sterile stem is sterilized by the method of: and (3) disinfecting for 10-60 s by using alcohol with the volume fraction of 75-90%, disinfecting for 10-12 min by using mercury bichloride with the mass concentration of 0.05-0.15%, and washing for 3-5 times.

9. The method for culturing the passion flower sterile explant according to claim 1, wherein the passion flower is cultured in an MS minimal medium for 8-12 days, the culture temperature is 25-28 ℃, the illumination intensity is 1000-2000 lux, and the illumination time is 8-12 h/d.

10. A method of growing a sterile passion flower explant according to claim 9 wherein 25 to 30ml of ms minimal medium is required for each stem inoculated.

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a cultivation method of a passion flower sterile explant.

Background

Passion flower (Passifloracaerulea L.) is a typical tropical and subtropical berry type fruit tree, belongs to the Passifloraceae (Passifloraceae) Passiflora genus (Passiflora L.), and is a perennial evergreen vine. The passion fruit has unique and intense fragrance, is sour, sweet and delicious, is a famous tropical fruit for fruit juice and has the reputation of 'the king of fruit juice'. The tissue culture technology of passion flower is difficult to popularize and apply to seedling breeding up to now, wherein one of the main reasons is that the explant is difficult to disinfect, and particularly, the explant collected from the field carries a certain amount of bacteria and fungi on the surface and inside, particularly, endophyte. The currently developed research on the passion flower tissue culture technology takes passion flower stems as explants, two disinfectants, namely 2% sodium hypochlorite and 0.1% -0.2% mercury bichloride, are adopted for disinfection, and the disinfection time is 10-20 min. The result is that the disinfection effect of mercury bichloride is obviously better than that of sodium hypochlorite. Therefore, in the existing passion flower explant disinfection and sterilization technology, 0.1% -0.2% of mercuric chloride is generally selected for disinfection and sterilization for 10-20 min. However, it still has the following disadvantages: 1. with increasing concentration of disinfectant or disinfection time, the contamination rate is in a downward trend, while the mortality rate and browning rate are in an upward trend, and the survival rate is in a downward trend. 2. Endophytes are difficult to eradicate, and with the increase of the number of subcultures, the pollution of the endophytes is serious.

Disclosure of Invention

The invention aims to provide a cultivation method of a passion flower sterile explant, which reduces the pollution rate, the browning rate and the death rate caused by disinfection and sterilization, greatly improves the survival rate of the explant and reduces the production cost of tissue culture.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for cultivating a passion flower sterile explant, which comprises the following steps:

collecting tender stem segments of passion flower as explants, soaking the tender stem segments by using saturated laundry powder liquid after washing, and rinsing to obtain pretreated explants;

carrying out aseptic pre-culture on the pretreated explant by adopting a rifampicin solution to obtain a pre-cultured explant;

shearing the pre-culture explant into stem sections and then carrying out disinfection treatment to obtain sterile stem sections;

and culturing the sterile stem section in an MS minimal medium to obtain a sterile passion flower explant.

Preferably, the length of the explant is 12-15 cm, and 3-4 axillary buds, terminal buds and 1-2 leaves at the upper ends of the terminal buds are reserved.

Preferably, the time for soaking the explants is 10-30 min.

Preferably, the rinsing is performed for 2-4 times.

Preferably, the concentration of the rifampicin solution is 80-120 mg/L, and the explant is immersed in the rifampicin solution for 2-3 cm.

Preferably, the pre-culture temperature is 22-30 ℃, the illumination time is 8-12 h/d, and the time is 2-4 d.

Preferably, the sterile stem section is 0.8-2.0 cm long and has 1-2 axillary buds.

Preferably, the specific mode of the sterile stem section disinfection treatment is as follows: and (3) disinfecting for 10-60 s by using alcohol with the volume fraction of 75-90%, disinfecting for 10-12 min by using mercury bichloride with the mass concentration of 0.05-0.15%, and washing for 3-5 times.

Preferably, the culture is carried out in an MS minimal medium for 8-12 days, the culture temperature is 25-28 ℃, the illumination intensity is 1000-2000 lux, and the illumination time is 8-12 h/d.

Preferably, 25-30 ml of MS minimal medium is needed for each inoculated stem segment.

The invention provides a method for cultivating a sterile passion flower explant, which greatly improves the disinfection and sterilization effects, reduces the pollution of surface fungi and bacteria to below 8 percent, reduces the pollution of endophytes to about 30 percent, and does not aggravate the severity of the endophytes with the increase of the times of subculture proliferation. And through the combination of the two pretreatment methods of the explant, the mercuric chloride sterilization method, the time and the like, the pollution rate is reduced, and simultaneously the browning rate and the death rate caused by disinfection and sterilization are reduced, so that the survival rate of the explant is greatly improved, and the production cost of tissue culture is reduced.

Detailed Description

The invention provides a method for cultivating a passion flower sterile explant, which comprises the following steps:

collecting tender stem segments of passion flower as explants, soaking the tender stem segments by using saturated laundry powder liquid after washing, and rinsing to obtain pretreated explants;

carrying out aseptic pre-culture on the pretreated explant by adopting a rifampicin solution to obtain a pre-cultured explant;

shearing the pre-culture explant into stem sections and then carrying out disinfection treatment to obtain sterile stem sections;

and culturing the sterile stem section in an MS minimal medium to obtain a sterile passion flower explant.

In the invention, the length of the explant is preferably 12-15 cm, more preferably 13-14 cm, and still more preferably 13.5 cm; the number of the axillary buds to be reserved is preferably 3-4, and more preferably 3; the number of terminal buds and the upper leaves of the terminal buds is preferably 1 to 2, and more preferably 1.

In the invention, the soaking time of the explant is preferably 10-30 min, more preferably 15-25 min, and still more preferably 20 min.

In the present invention, the number of rinsing is preferably 2 to 4, more preferably 2 to 3, and still more preferably 3.

In the invention, the concentration of the rifampicin solution is preferably 80-120 mg/L, more preferably 90-110 mg/L, and still more preferably 100 mg/L; the length of the explant immersed in the rifampicin solution is preferably 2-3 cm, and more preferably 2.5 cm.

In the invention, the pre-culture temperature is preferably 22-30 ℃, more preferably 24-28 ℃, even more preferably 25-27 ℃, even more preferably 26 ℃; the illumination time is preferably 8-12 h/d, more preferably 9-11 h/d, and still more preferably 10 h/d; the pre-culture time is preferably 2-4 d, more preferably 3-4 d, and still more preferably 3 d.

In the invention, the length of the sterile stem is preferably 0.8-2.0 cm, more preferably 1-1.8 cm, and even more preferably 1.5 cm; the number of axillary buds is preferably 1-2, and more preferably 1.

In the step of sterilizing the sterile stem segments, the volume fraction of the alcohol is preferably 75-90%, and is further preferably 75%; the alcohol disinfection treatment time is preferably 10-60 s, more preferably 20-50 s, and even more preferably 30 s; the mass concentration of the mercuric chloride is preferably 0.05-0.15%, and more preferably 0.1%; the surface disinfection time is preferably 10-12 min, more preferably 10-11 min, and still more preferably 11 min; the number of washing is preferably 3 to 5, more preferably 4 to 5, and still more preferably 4.

In the invention, the culture time in the MS culture medium is preferably 8-12 d, more preferably 9-11 d, and even more preferably 10 d; the culture temperature is preferably 25-28 ℃, more preferably 26-27 ℃, and even more preferably 26 ℃; the illumination intensity is preferably 1000-2000 lux, more preferably 1250-1750 lux, and even more preferably 1500 lux.

In the invention, the MS minimal medium is preferably 25-30 ml, more preferably 26-29 ml, and even more preferably 27ml for each stem segment to be inoculated.

The method for culturing sterile passion flower explants provided by the invention is described in detail below with reference to the following examples, which should not be construed as limiting the scope of the invention.

Example 1

The explant is taken at 10 am, terminal buds and 3 axillary buds are selected, tender stem segments of passion flower newly-extracted 1 leaf at the upper end of the terminal buds, which are about 13.5cm in length, are taken as the explant, the explant is soaked in saturated laundry powder liquid for 20min, rinsed clean in tap water, and rinsed for 3 times by using sterile water. Adopting a rifampicin solution with the concentration of 100mg/L, immersing the explant in the rifampicin solution for 2.5cm, and pre-culturing for 3d under the conditions that the temperature is 26 ℃ and the illumination time is 10h/d to obtain the pre-cultured explant. Precultured explants were cut to 1.5cm long stem segments with 1 axillary bud. Disinfecting the stem section with 75% alcohol for 30s, disinfecting the surface with 0.1% mercury bichloride for 11min, and washing for 4 times to obtain sterile stem section. And inoculating each sterile stem segment into 27ml MS minimal medium without adding any hormone, and culturing for 10d under the conditions of the culture temperature of 26 ℃, the illumination intensity of 1500lux and the illumination time of 10h/d to obtain a sterile passion flower explant.

Example 2

The explant is collected at 12 am, a tender stem section newly extracted from passion flower with terminal bud and 3 axillary buds, the length of the tender stem section is about 12cm, 1 leaf at the upper end of the terminal bud is selected as the explant, the explant is soaked in saturated laundry powder liquid for 10min, then the explant is rinsed clean in tap water, and then the explant is rinsed for 2 times by using sterile water. The explant is immersed in rifampicin solution with the concentration of 80mg/L for 2cm, and is pre-cultured for 2 days under the conditions that the temperature is 22 ℃ and the illumination time is 8h/d, so that the pre-cultured explant is obtained. Precultured explants were cut to 0.8cm stem segments with 1 axillary bud. Disinfecting the stem section with 75% alcohol by volume for 10s, then disinfecting the surface with 0.05% mercury bichloride for 10min, and washing for 3 times to obtain the sterile stem section. And inoculating each sterile stem segment into 25ml MS minimal medium without adding any hormone, and culturing for 8d under the conditions of culture temperature of 25 ℃, illumination intensity of 1000lux and illumination time of 8h/d to obtain a sterile passion flower explant.

Example 3

The explant is collected at 16 pm, the tender stem segment of passion flower with 2 leaves, which has a top bud length of about 15cm and a length of 4 axillary buds and is newly extracted from the top end of the top bud, is selected as the explant, the explant is soaked in a saturated laundry powder solution for 30min, rinsed clean in tap water, and rinsed 4 times with sterile water. The explant is immersed in rifampicin solution with the concentration of 120mg/L for 3cm, and is pre-cultured for 4 days under the conditions that the temperature is 30 ℃ and the illumination time is 12h/d, so that the pre-cultured explant is obtained. Precultured explants were cut to 2.0cm stem segments with 2 axillary buds. Sterilizing the stem segment with 90% alcohol by volume for 60s, sterilizing the surface with 0.15% mercuric chloride by mass concentration for 12min, and washing for 5 times to obtain sterile stem segment. And inoculating each sterile stem segment into 30ml of MS minimal medium without adding any hormone, and culturing for 12 days under the conditions that the culture temperature is 28 ℃, the illumination intensity is 2000lux and the illumination time is 12h/d to obtain a sterile passion flower explant.

Example 4

The explant is collected at 14 pm, the tender stem segment of passion flower newly extracted from 1 leaf of terminal bud and 4 axillary buds with the length of about 13cm and the upper end of the terminal bud is selected as the explant, the explant is soaked in a saturated laundry powder solution for 15min, rinsed clean in tap water, and rinsed 3 times with sterile water. Adopting a rifampicin solution with the concentration of 90mg/L, immersing the explant in the rifampicin solution for 2.2cm, and pre-culturing for 3d under the conditions that the temperature is 24 ℃ and the illumination time is 9h/d to obtain the pre-cultured explant. Precultured explants were cut to 1.0cm stem segments with 1 axillary bud. Disinfecting the stem section with 75% alcohol for 20s, disinfecting the surface with 0.1% mercury bichloride for 11min, and washing for 4 times to obtain sterile stem section. And inoculating each sterile stem segment into 26ml of MS minimal medium without adding any hormone, and culturing for 9d under the conditions that the culture dimension is 26 ℃, the illumination intensity is 1250lux and the illumination time is 9h/d to obtain a sterile passion flower explant.

Example 5

The explant is collected at 14 pm, the tender stem segment of passion flower newly extracted from 1 leaf of terminal bud and 3 axillary buds with the length of about 14cm and the upper end of the terminal bud is selected as the explant, the explant is soaked in a saturated laundry powder solution for 25min, rinsed clean in tap water, and rinsed 3 times with sterile water. Adopting a rifampicin solution with the concentration of 110mg/L, immersing the explant in the rifampicin solution for 2.8cm, and pre-culturing for 3d under the conditions that the temperature is 28 ℃ and the illumination time is 11h/d to obtain the pre-cultured explant. Precultured explants were cut to 1.8cm stem segments with 1 axillary bud. Disinfecting the stem section with 75% alcohol by volume for 50s, disinfecting the surface with 0.1% mercury bichloride for 11min, and washing for 4 times to obtain sterile stem section. And inoculating each sterile stem segment into 27ml of MS minimal medium without adding any hormone, and culturing for 11d under the conditions that the culture temperature is 27 ℃, the illumination intensity is 1750lux and the illumination time is 11h/d to obtain a sterile passion flower explant.

Comparative example 1

Adopts the conventional passion flower explant disinfection mode in the prior art and adopts 0.1 percent mercuric chloride disinfection treatment for 11 min.

Comparative example 2

Adopts the conventional passion flower explant disinfection mode in the prior art and adopts 2 percent sodium hypochlorite disinfection treatment for 11 min.

TABLE 1 comparative examples 1, 2 compare the effect of example 1 on the disinfection of passion flower explants

Experimental group	Contamination ratio (%)	Endogenous contamination ratio (%)	Mortality (%)	Survival rate (%)
					Comparative example 1	33.33	37.50	0	29.17
Comparative example 2	38.45	53.85	7.7	0
					Example 1	8.33	29.17	0	62.5

According to the embodiment, compared with the conventional mode of 2% sodium hypochlorite disinfection treatment in the comparative example 2, the method for cultivating the sterile passion flower explants reduces the pollution rate of nearly 1/3, reduces the endogenous pollution rate by half, can control the death rate to be zero, and improves the survival rate to 62.5%; compared with the 0.1% mercuric chloride disinfection treatment mode described in comparative example 1, the method also achieves the technical effects of reducing the pollution rate, reducing the endogenous pollution rate and doubling the survival rate.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.