LU502555B1 - In vitro culture method of thornless green prickly ash (zanthoxylum armatum dc) - Google Patents

Abstract

The present disclosure discloses an in vitro culture method of thornless green prickly ash (Z. armatum cv ‘Rongchang wucihuajiao’), belongs to the technical field of plant tissue culture. In the present disclosure, the stem segment without the axillary bud of the thornless green prickly ash is used as an explant and successfully induced to grow callus tissues on optimal callus induction medium , and then adventitious buds grow for propagation, thus the technical blank of plants regeneration via callus induction from stem segment without axillary bud explants in thornless green prickly ash is filled. In the present disclosure, an in vitro regeneration propagation system of the stem segment without axillary bud of the thornless green prickly ash (Z. armatum cv ‘Rongchang wucihuajiao’) is established for the first time, and the large-scale production is realized.

Description

BL-5531

IN VITRO CULTURE METHOD OF THORNLESS GREEN PRICKLY ASH LU502555 (ZANTHOXYLUM ARMATUM DC)

TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of plant tissue culture, specifically relates to an in vitro culture method of Thornless green prickly ash (Zanthoxylum armatum DC).

BACKGROUND ART

[0002] Zanthoxylum armatum DC is an evergreen, thorny shrub belonging to the genus Zanthoxylum. Thornless green prickly ash is a natural bud mutation variety of Z. armatum with no thorns in leaves and less thorns in stems. This new cultivar has been approved as a fine forest variety (the accession number is Yu S-SV-ZA-001-2016) in

Chongqing City and named as Z. armatum cv ’Rongchang wucihuajiao’, which is the only thornless Z. armatum variety reported at present. In terms of morphological characteristics (leaves, fruits, etc.), phenophase and distribution, the thornless green prickly ash (Z. armatum cv ’Rongchang wucihuajiao’) is significantly different from other thornless prickly ash cultivar of genus Zanthoxylum, including Z. piperitum

DC.var.Inerme Makino domesticated in Japan, and the thornless Z. bungeanum varieties cultivated in China, such as Longnan no-prickly ash, Nongcheng No. 1 no-prickly ash, and Hanyuan no-prickly ash.

[0003] Generally, seed propagation is the common propagation method for Z. armatum. The Thornless green prickly ash is derived from natural bud mutation and displays genetic instable. The trait of thornless character will disappear via seed propagation and thus thorn restored, that is an atavistic phenomenon. Therefore, vegetative propagation methods such as cuttage, grafting and in vitro culture, rather than seed propagation, are usually applied in the propagation of bud mutation varieties. For cottage, the cutting slips of the thornless green prickly ash are limited and the rooting rate is extremely low. For grafting, the number of scions is far from enough right now, while the labor cost is high and grafting seasons are limited. Therefore, compared with such traditional vegetative propagation, the in vitro propagation technology for producing seedlings of Z. armatum , has great advantages, such as annual production, industrial production, high-efficiency propagation and products with good uniformity, thus can better meet the large-scale requirement of the market.

[0004] In the prior art, a method was developed for in vitro propagation of Z. armatum by using stem segments with axillary buds as explants (reference to IN

VITRO PROPAGATION METHOD OF TISSUE CULTURE SEEDLINGS OF

ZANTHOXYLUM ARMATUM, PCT patent; Authorized country: Netherlands; Patent number: 2027681). However, there are limitations for this method in explants selection.

The stem segments with axillary buds can be used as explants, but these buds are prone to browning and died during explant disinfection process.

[0005] At present, there is no report refer to in vitro culture method of Z.armatum cv ‘Rongchang wucihuajiao’ using stem segments without axillary buds as explants. 1

BL-5531

LU502555

SUMMARY

[0006] In view of this, an object of the present disclosure is to provide an in vitro culture method of the thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’). In the present disclosure, the stem segment without the axillary bud of the thornless green prickly ash is used as an explant to induce the formation of calli and adventitious buds for rapid propagation, which fills the technical gaps in the field of plants regeneration of the thornless green prickly ash via callus induction from the stem segment without the axillary bud explants.

[0007] The present disclosure provides an in vitro culture method of thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’). The in vitro culture method comprises the following steps:

[0008] 1) sterilized stem segments without axillary buds of the thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’) were inoculated into callus induction medium for callus induction, and then calli were formed and grown on the stem segment;

[0009] 2) inoculating the stem segment with the callus into the differentiation medium, and carrying out differentiation culture to obtain cluster buds;

[0010] 3) inoculating the cluster buds into the proliferation medium, and carrying out proliferation culture to obtain bud seedlings;

[0011] 4) inoculating the bud seedlings into the rooting medium, and carrying out rooting culture to obtain rooted seedlings.

[0012] The callus induction medium uses MS as basal medium and comprises the following components in concentration: 1.3-1.7 mg/L of zeatin riboside (ZR), 5.8-6.2 g/L of agar, 29.8-30.2 g/L of sucrose and 0.1% (v/v) PPM.

[0013] Preferably, the differentiation medium uses MS as basal medium and comprises the following components in concentration: 1.8-2.2 mg/L of ZR, 5.8-6.2 g/L of agar, 29.8-30.2 g/L of sucrose and 0.1% (v/v) PPM.

[0014] Preferably, the proliferation medium uses MS as basal medium and comprises the following components in concentration: 1-1.5 mg/L of ZR, 0.08-0.12 mg/L of IBA, 5.8-6.2 g/L of agar, and 29.8-30.2 g/L of sucrose.

[0015] Preferably, the rooting medium uses 1/2 MS as basal medium and comprises the following components in concentration: 0.1-0.15 mg/L of IBA, 5.8-6.2 g/L of agar, and 29.8-30.2 g/L of sucrose.

[0016] Preferably, the induction culture comprises the steps of culturing for 2-3 d under dark condition and then culturing under light condition for 20-25 d, wherein the light intensity is 800-1,000 lux.

[0017] Preferably, the differentiation culture is carried out under light condition, wherein the light conditions of the differentiation culture include that the light intensity is 1,800-2,000 lux, and the time of the differentiation culture is 25-30 d.

[0018] Preferably, the proliferation culture comprises the steps of culturing for 8-9 d under dark condition and then culturing under light condition until stem segments grow to 3-4 cm in height and 3-4 internodes grow; the light culture comprises first light culture and second light culture which are carried out sequentially; the light intensity of 2

BL-5531 the first light culture is 800-1,000 lux; the time of the first light culture is 7-8 d; the light LU502555 intensity of the second light culture is 1,800-2,000 lux.

[0019] Preferably, the rooting culture comprises the steps of culturing for 7-9 d under dark environment and then culturing under light condition for 7-21 d, wherein the light intensity of the light culture is 1,800-2,000 lux.

[0020] Preferably, after rooted seedlings are obtained, the rooted seedlings are further subjected to seedling hardening and domestication transplanting to obtain tissue culture seedlings.

[0021] Preferably, the seedling hardening is carried out in a greenhouse with the shading rate of 65-80%, the time of the seedling hardening is 8-12 d, and the temperature of the seedling hardening is 20-30°C.

[0022] The present disclosure provides the in vitro culture method of the thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’). In the present disclosure, the stem segment without the axillary bud is used as an explant. Calli are successfully induced on the optimized callus induction medium, and then adventitious buds grow for propagation, thus the technical blank of plants regeneration via calli formation from the stem segment without the axillary bud explants in thornless green prickly ash is filled.

In the present disclosure, an in vitro regeneration propagation system from the stem segment without the axillary bud in the thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’) is established for the first time, and the large-scale production is realized.

[0023] For the stem segment with the axillary bud, the axillary buds are induced to be grown and elongated, thus obtain the bud seedlings. Because without dedifferentiation and redifferentiation processes, the induction culture process is relatively simple.

However, if axillary bud primordia do not exist in the stem segment, adventitious buds can regenerate to form the bud seedlings only after a dedifferentiation stage and a redifferentiation stage in sequence, and the induction process is complex. In the present disclosure, the stem segment without the axillary bud in thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’) is taken as the explant, and the present disclosure has the following advantages: the stem segment without the axillary bud is enough as the raw material; the contamination points of stem segment culture are mainly from axillary buds, and the stem segment without the axillary bud is taken as the explant, so that the pollution rate is greatly reduced, and the obtaining rate is higher; the bud seedlings obtained from the stem segment with the axillary bud are single buds obtained from the axillary buds; and the bud seedlings obtained from the stem segment without the axillary bud are bud clusters (multiple buds) obtained from calli. With the adoption of the method, the cluster bud clusters can be obtained, and rapid propagation of the seedlings of the thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’) is realized.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows branches and leaves of thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’); 3

BL-5531

[0025] FIG. 2 shows the underside of the leaves inthornless green prickly ash LU502555 (Z.armatum cv ‘Rongchang wucihuajiao’);

[0026] FIG. 3 shows that stem segments without axillary bud are taken as explants;

[0027] FIG. 4 shows that callus is induced from stem segments without axillary buds and adventitious buds are differentiated;

[0028] FIG. 5 shows that adventitious buds form bud clusters;

[0029] FIG. 6 shows subculture of cluster buds;

[0030] FIG. 7 shows that cluster buds growing and elongating;

[0031] FIG. 8 shows rooting induction of cluster buds;

[0032] FIG. 9 shows greenhouse substrate seedlings;

[0033] FIG. 10 shows commercial seedlings.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The present disclosure provides an in vitro culture method of thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’). The method comprises the following steps:

[0035] 1) inoculating a stem segment without an axillary bud of the thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’) into callus induction medium, and carrying out induction culture until callus grows from the stem segment;

[0036] 2) inoculating the stem segment with the callus into differentiation medium, and carrying out differentiation culture to obtain cluster buds;

[0037] 3) inoculating the cluster buds into proliferation medium, and carrying out proliferation culture to obtain bud seedlings;

[0038] 4) inoculating the bud seedlings into rooting medium, and carrying out rooting culture to obtain rooted seedlings.

[0039] The callus induction medium takes MS as basic medium and comprises the following components in concentration: 1.3-1.7 mg/L of ZR, 5.8-6.2 g/L. of agar, 29.8-30.2 g/L of sucrose and 0.1% (v/v) PPM.

[0040] In the present disclosure, a stem segment without an axillary bud of the thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’) was inoculated into callus induction medium for induction culture until callus grows from the stem segment; the callus induction medium takes MS as basic medium and comprises the following components in concentration: 1.3-1.7 mg/L of ZR, 5.8-6.2 g/L. of agar, 29.8-30.2 g/L of sucrose and 0.1% (v/v) PPM. In the present disclosure, the thornless green prickly ash is preferably Z.armatum cv ‘Rongchang wucihuajiao’.

[0041] In the present disclosure, the callus induction medium preferably comprises the following components in concentration: 1.5 mg/L of ZR, 6 g/L of agar, 30 g/L of sucrose and 0.1% (v/v) PPM.

[0042] In the present disclosure, a stem segment without an axillary bud of thornless green prickly ash is preferably selected from a current-year branch of Z.armatum cv ‘Rongchang wucihuajiao’; the stem segment is preferably a semi-lignified stem segment; the stem segment is preferably free of leaves; the length of the stem segment is preferably 0.5-1 cm. In the present disclosure, the stem segment without the axillary bud of the thornless green prickly ash (Z.armatum cv ‘Rongchang wucihuajiao’) is 4

BL-5531 inoculated in front of a callus induction medium, preferably the stem segment 1s LU502555 disinfected. The present disclosure had no particular limitation on the method of disinfection, and conventional explant disinfection methods in the art could be used.

[0043] In the present disclosure, the induction culture preferably comprises the steps of culturing for 2-3 d under dark condition and then culturing under light condition for 20-25 d, wherein the light intensity of the light culture is 800-1,000 lux. In the present disclosure, callus grows from the stem segment through induction culture, and adventitious buds further grow from a small amount of callus in this stage. The adventitious buds can continuously elongate in differentiation medium, develop into subsequent cluster buds like adventitious buds obtained by callus differentiation, and are cut into rooted seedlings.

[0044] After the callus grows from the stem segment, the stem segment with the callus is inoculated into the differentiation medium, and differentiation culture 1s performed, so that the cluster buds are obtained.

[0045] In the present disclosure, the differentiation medium uses MS as a basal medium and comprises the following components in concentration: 1.8-2.2 mg/L of ZR, 5.8-6.2 g/L. of agar, 29.8-30.2 g/L. of sucrose and 0.1% (v/v) PPM; more preferably comprises the following components in concentration: 2 mg/L of ZR, 6 g/L of agar, 30 g/L of sucrose and 0.1% (v/v) PPM.

[0046] In the present disclosure, the differentiation culture is preferably carried out under light condition, wherein the light conditions of the differentiation culture preferably include that the light intensity is 1,800-2,000 lux, and the time of the differentiation culture is preferably 25-30 d.

[0047] In the present disclosure, the cluster buds are obtained through differentiation culture, the method can improve the plant tissue culture efficiency. In the present disclosure, the cluster buds grow from the stem segment, and the method further comprises the step of cutting off the cluster buds from the stem segment, wherein this step is carried out in sterile environment through a sterile tool.

[0048] After the cluster buds are obtained, the cluster buds are inoculated to proliferation medium for proliferation culture to obtain bud seedlings.

[0049] In the present disclosure, the proliferation medium uses MS as a basal medium and preferably comprises the following components in concentration: 1-1.5 mg/L of ZR, 0.08-0.12 mg/L of IBA, 5.8-6.2 g/L of agar, and 29.8-30.2 g/L of sucrose, more preferably comprises the following components in concentration: 1-1.5 mg/L of ZR, 0.1 mg/L of IBA, 6 g/L of agar, and 30 g/L of sucrose.

[0050] In the present disclosure, the proliferation culture comprises the steps of culturing for 8-9 d under the dark condition and then culturing under a light condition until stem segments grow to 3-4 cm in height and 3-4 internodes grow; the light culture comprises first light culture and second light culture which are sequentially carried out; the light intensity of the first light culture is 800-1,000 lux; the time of the first light culture is 7-8 d; the light intensity of the second light culture is 1,800-2,000 lux. In the present disclosure, the time of the second light culture is preferably 13-14 d. The present disclosure is beneficial to the smooth transition of the stem segments from the dark environment to the light environment by gradually increasing the light intensity, so as to

BL-5531 facilitate the growth of stem segments. LU502555

[0051] After the bud seedlings are obtained, the bud seedlings are inoculated to rooting medium for rooting culture to obtain rooted seedlings.

[0052] In the present disclosure, the height of the bud seedlings is preferably larger than or equal to 2.0 cm, and the stem diameter of the bud seedlings is preferably larger than or equal to 2.0 mm; the depth of the bud seedlings inserted into the rooting medium is preferably 1.5-2 cm.

[0053] In the present disclosure, the rooting medium uses MS as a basal medium and preferably comprises the following components in concentration: 0.1-0.15 mg/L of IBA, 5.8-6.2 g/L of agar and 29.8-30.2 g/L of sucrose, and more preferably comprises the following components in concentration: 0.1-0.15 mg/L of IBA, 6 g/L. of agar and 30 g/L. of sucrose.

[0054] In the present disclosure, the rooting culture comprises the steps of culturing for 7-9 d under dark environment and then culturing under light condition for 7-21 d, wherein the light intensity of the light culture is preferably 1,800-2,000 lux. The light culture time is preferably determined according to the conditions that the number of root systems of the rooted seedlings is larger than or equal to 2, and the root length of the rooted seedlings is larger than or equal to 2.0 cm.

[0055] In the present disclosure, the culture in dark environment is preferably carried out under the conditions that lamplight and curtains are closed in a culture room and a culture bottle is covered by a black film.

[0056] In the present disclosure, from the induction culture stage to the rooting culture stage, if light culture is needed, the light culture time per day is preferably 10 h; and from the induction culture stage to the rooting culture stage, the culture temperature is preferably 23-25°C.

[0057] Preferably, after rooted seedlings are obtained, the rooted seedlings are further subjected to seedling hardening and domestication transplanting to obtain tissue culture seedlings.

[0058] In the present disclosure, the seedling hardening is preferably carried out in a greenhouse with the shading rate of 65-80%. In the seedling hardening process, the seedlings of the thornless green prickly ash (Z. armatum cv ‘Rongchang wucihuajiao’)are placed in a greenhouse, and a culture bottle cover is loosened; the seedling hardening time is preferably 8-12 d, and more preferably 10 d; the seedling hardening temperature is preferably 20-30°C; and the seedling hardening is to enable rooted seedling leaves to be thickened.

[0059] After the seedling hardening, the method of the present disclosure further comprises the steps of washing the rooting medium on the rooted seedlings, and soaking the whole plant in a sterilizing solution, wherein a reagent adopted for washing is preferably clear water, the sterilizing solution is preferably a 1000-time carbendazim solution, and the soaking time is preferably 1-2 min.

[0060] After soaking, the rooted seedlings soaked in the sterilizing solution are transplanted into a matrix hole tray for domestication, wherein the matrix comprised the following raw materials in parts by volume: 7 parts of peat and 3 parts of perlite.

[0061] After transplanting, the method of the present disclosure preferably comprises 6

BL-5531 the steps of irrigating the transplanted seedlings with rooting water, and covering the LU502555 rooted seedlings with a film for heat preservation and moisture preservation.

[0062] In the present disclosure, the domestication is preferably performed in the greenhouse; ventilation is performed in the morning and evening in the domestication process; a compound fertilizer is sprayed from the 11” day of the domestication; the ratio of N to P to K in the compound fertilizer is 15: 15: 15; the mass concentration of the compound fertilizer is preferably 0.5%; the application frequency of the compound fertilizer is preferably once every 7 d; the film is removed from the 15“ day of the domestication, and the seedlings are continuously cultured and can be taken out of the nursery after 30-45 d.

[0063] Unless otherwise specified, the present disclosure has no special requirements on the source of the used raw materials, and commercially available commodities well known to those skilled in the art can be used.

[0064] The technical solutions of the present disclosure will be clearly and completely described below with reference to the embodiments of the present disclosure.

[0065] Example 1

[0066] S1, Explant disinfection: a current-year branch of thornless green prickly ash (Z. armatum cv ‘Rongchang wucihuajiao’)was taken as an explants (FIGS. 1 to 2), a semi-lignified stem segment without an axillary bud was disinfected and then placed in a sterile inoculation disc on a clean bench, and the stem segment was cut into 0.5-1.0 cm segments (without leaves and axillary buds), as shown in FIG 3.

[0067] S2, Callus induction: the stem segments cut in the step S1 were placed into the callus induction medium, cultured for 2-3 d in dark environment, and then cultured for 20-25 d under the light intensity of 1,000 lux to obtain calli and a small amount of adventitious buds; and the calli grew and adventitious buds further grew from the small amount of calluses in this stage, as shown in FIG 4.

[0068] The callus induction medium comprised MS, 1.5 mg L* of ZR, 6.0 g L*! of agar, 30 g L* of sucrose and 0.1% (v/v) PPM.

[0069] S3, Differentiation culture: the stem segments with the calluses were flatly put into a differentiation medium and continuously cultured for 25-30 d under the light intensity of 2,000 lux until cluster buds were obtained, as shown in FIG. 5.

[0070] Tthe differentiation medium comprised MS, 2.0 mg L* 1 of ZR, 6.0 g L*! of agar, 30 g L* of sucrose and 0.1% (v/v) PPM.

[0071] S4, Cutting subculture: the stem segments with the cluster buds were placed into the sterile inoculation disc on the clean bench, and the tissues with the cluster buds were cut by a sterile tool, as shown in FIG. 6.

[0072] SS, Subculture proliferation: the cluster buds cut in the step S4 were placed into a proliferation medium, and cultured for 8-9 d in a dark environment and then cultured for 7-8 d under the condition that the light intensity was 1,000 lux, then the light intensity was increased to 1,000 lux for culturing for 13-14 d until stems grew to 3-4 cm in height and 3-4 internodes grew, as shown in FIG. 7.

[0073] The proliferation medium comprised MS, 1.0 mg L* of MS+ZR, 0.1 mg:L*! of

IBA, 6.0 g L of agar and 30 g L* of sucrose. 7

BL-5531

[0074] In the stages S1-S5, in the light culture process, the light culture time was 10 h LU502555 every day, and the temperature was 23-25°C.

[0075] S6, Rooting culture: the bud seedlings obtained by subculture proliferation were cut and inoculated into a rooting medium, wherein the height of the bud seedlings was greater than or equal to 2.0 cm, the stem diameter was greater than or equal to 2.0 mm, and the depth of the bud seedlings inserted into the medium was 1.5-2.0 cm; after inoculation, the bud seedlings were cultured in dark environment for 7-9 d, and then transferred into a light environment for continuous culture for 7-14 d, wherein the light culture time was 10 h/d, the temperature in the whole rooting culture process was 23-25°C, and the light intensity was 2,000 lux; after culturing for 14-21 d, rooted seedlings with the root system number being greater than or equal to 2 and the root length being greater than or equal to 2.0 cm were selected and transplanted, as shown in

FIG. 8.

[0076] Wherein, the medium in the rooting culture process comprised the following components: a 1/2 MS basic medium, 0.1 mg L* of IBA, 6.0 g L! of agar and 30 g L*! of sucrose.

[0077] S7, Domesticating and transplanting: firstly, the bottled seedlings of the thornless green prickly ash (Z. armatum cv ‘Rongchang wucihuajiao’) were placed into a greenhouse with the shading rate of 65-80%, a culture bottle cover was loosened, and seedling hardening was performed for about 10 d at normal temperature until the rooted seedling leaves were thickened; then, the rooted seedling medium that was subjected to seedling hardening was washed with clean water and soaked in 1000-fold carbendazim solution for 1-2 min; the rooted seedling medium was planted into a prepared matrix hole tray, wherein the transplanting matrix was prepared from peat and perlite (V: V =7: 3); after planting, rooting water was irrigated, the rooted seedling medium was covered with a film to preserve heat and moisture and then cultured in the greenhouse, wherein ventilation was performed in the morning and evening in the transplanting and culturing process; after 10 d, 0.5% compound fertilizer (N: P: K =15:15:15) was sprayed, once every 7 d; the film was removed after about 15 d; and the seedlings could be taken out of the nursery after 30-45 d, as shown in FIGS. 9 and 10.

[0078] Example 2

[0079] S1, Explant disinfection: a current-year branch of thornless green prickly ash (Z. armatum cv ‘Rongchang wucihuajiao’) was taken as an explant, a semi-lignified stem segment without an axillary bud was disinfected and then placed in a sterile inoculation disc on a clean bench, and the stem segment was cut into 0.5-1.0 cm segments (without leaves and axillary buds).

[0080] S2, Callus induction: the stem segments cut in the step S1 were placed into the callus induction medium, cultured for 2-3 d in a dark environment, and then cultured for 20-25 d under the light intensity of 1,000 lux to obtain calluses and a small number of adventitious buds; and the calli grew and adventitious buds further grew from the small amount of calli in this stage.

[0081] The callus induction medium comprised MS, 1.5 mg L* of ZR, 6.0 g L*! of agar, 30 g L* of sucrose and 0.1% (v/v) PPM.

[0082] S3, Differentiation culture: the stem segments with the calluses were flatly put 8

BL-5531 into a differentiation medium and continuously cultured for 25-30 d under the light LU502555 intensity of 2,000 lux until cluster buds were obtained.

[0083] The differentiation medium comprised MS, 2.0 mg L* 1 of ZR, 6.0 g L* of agar, 30 g L* of sucrose and 0.1% (v/v) PPM.

[0084] S4, Cutting subculture: the stem segments with the cluster buds were placed into the sterile inoculation disc on the clean bench, and the tissues with the cluster buds were cut by a sterile tool.

[0085] SS, Subculture proliferation: the cluster buds cut in the step S4 were placed into a proliferation medium, and cultured for 8-9 d in a dark environment and then cultured for 7-8 d under the condition that the light intensity was 1,000 lux, then the light intensity was increased to 1,000 lux for culturing for 13-14 d until stems grew to 3-4 cm in height and 3-4 internodes grew, as shown in FIG. 7.

[0086] The proliferation medium comprised MS, 1.5 mg L* of ZR, 0.1 mg-L* of IBA, 6.0 g L! of agar and 30 g L* of sucrose.

[0087] In the stages S1-S5, in the light culture process, the light culture time was 10 h every day, and the temperature was 23-25°C.

[0088] SO, Rooting culture: the bud seedlings obtained by subculture proliferation were cut and inoculated into a rooting medium, wherein the height of the bud seedlings was greater than or equal to 2.0 cm, the stem diameter was greater than or equal to 2.0 mm, and the depth of the bud seedlings inserted into the medium was 1.5-2.0 cm; after inoculation, the bud seedlings were cultured in a dark environment for 7-9 d, and then transferred into a light environment for continuous culture, wherein the light culture time was 10 h/d, the temperature in the whole rooting culture process was 23-25°C, and the light intensity was 2,000 lux; after culturing for 14-21 d, rooted seedlings with the root system number being greater than or equal to 2 and the root length being greater than or equal to 2.0 cm were selected and transplanted.

[0089] Wherein, the medium in the rooting culture process comprised the following components: a 1/2 MS basic medium, 0.15 mg L* of IBA, 6.0 g L*! of agar and 30 g L! of sucrose.

[0090] S7, Domesticating and transplanting: firstly, the bottled seedlings of the thornless green prickly ash (Z. armatum cv ‘Rongchang wucihuajiao’) were placed into a greenhouse with the shading rate of 65-80%, a culture bottle cover was loosened, and seedling hardening was performed for about 10 d at normal temperature until the rooted seedling leaves were thickened; then, the rooted seedling medium that was subjected to seedling hardening was washed with clean water and soaked in 1000-fold carbendazim solution for 2 min; the rooted seedling medium was planted into a prepared matrix hole tray, wherein the transplanting matrix was prepared from peat and perlite (V: V =7: 3); after planting, rooting water was irrigated, the rooted seedling medium was covered with a film to preserve heat and moisture and then cultured in the greenhouse, wherein ventilation was performed in the morning and evening in the transplanting and culturing process; after 10 d, 0.5% compound fertilizer (N: P: K =15:15:15) was sprayed, once every 7 d; the film was removed after about 15 d; and the seedlings could be taken out of the nursery after 30-45 d.

[0091] Although the present disclosure is described in detail with the foregoing 9

BL-5531 . . . LU502555 embodiments, these embodiments are only a part of rather than all of the embodiments.

Other embodiments that are obtained based on these embodiments without creative work shall also fall within the scope of protection of the present disclosure.

Claims (10)

BL-5531 CLAIMS: LU502555

1. An in vitro culture method of thornless green prickly ash (Z. armatum cv ‘Rongchang wucihuajiao’), comprising the following steps: 1) inoculating a stem segment without an axillary bud of the thornless green prickly ash into a callus induction medium, and carrying out induction culture until callus grows from the stem segment; 2) inoculating the stem segment with the callus into a differentiation medium, and carrying out differentiation culture to obtain cluster buds; 3) inoculating the cluster buds into a proliferation medium, and carrying out proliferation culture to obtain bud seedlings; 4) inoculating the bud seedlings into a rooting medium, and carrying out rooting culture to obtain rooted seedlings; the callus induction medium takes MS as a basic medium and comprises the following components in concentration: 1.3-1.7 mg/L of ZR, 5.8-6.2 g/L. of agar,

29.8-30.2 g/L of sucrose and 0.1% (v/v) PPM.

2. The in vitro culture method according to claim 1, wherein the differentiation medium uses MS as a basal medium and comprises the following components in concentration: 1.8-2.2 mg/L of ZR, 5.8-6.2 g/L of agar, 29.8-30.2 g/L of sucrose and 0.1% (v/v) PPM.

3. The in vitro culture method according to claim 1, wherein the proliferation medium uses MS as a basal medium and comprises the following components in concentration: 1-1.5 mg/L of ZR, 0.08-0.12 mg/L of IBA, 5.8-6.2 g/L. of agar, and

29.8-30.2 g/L of sucrose.

4. The in vitro culture method according to claim 1, wherein the rooting medium uses 1/2 MS as a basal medium and comprises the following components in concentration: 0.1-0.15 mg/L of IBA, 5.8-6.2 g/L of agar, and 29.8-30.2 g/L of sucrose.

5. The in vitro culture method according to claim 1, wherein the induction culture comprises the steps of culturing for 2-3 d under a dark condition and then culturing under a light condition for 20-25 d, the light intensity of the light culture is 800-1,000 lux.

6. The in vitro culture method according to claim 1, wherein the differentiation culture is carried out under a light condition, the light conditions of the differentiation culture include that the light intensity is 1,800-2,000 lux, and the time of the differentiation culture is 25-30 d.

7. The in vitro culture method according to claim 1, wherein the proliferation culture comprises the steps of culturing for 8-9 d under the dark condition and then culturing under a light condition until stem segments grow to 3-4 cm in height and 3-4 internodes grow; the light culture comprises first light culture and second light culture which are sequentially carried out; the light intensity of the first light culture is 800-1,000 lux; the time of the first light culture is 7-8 d; the light intensity of the second light culture is 1,800-2,000 lux.

8. The in vitro culture method according to claim 1, wherein the rooting culture 11

BL-5531 comprises the steps of culturing for 7-9 d under a dark environment and then culturing LU502555 under a light condition for 7-21 d, wherein the light intensity of the light culture is 1,800-2,000 lux.

9. The in vitro culture method according to claim 1, wherein after rooted seedlings are obtained, the rooted seedlings are further subjected to seedling hardening and domestication transplanting to obtain tissue culture seedlings.

10. The in vitro culture method according to claim 9, wherein the seedling hardening 1s carried out in a greenhouse with the shading rate of 65-80%, the time of the seedling hardening 1s 8-12 d, and the temperature of the seedling hardening 1s 20-30°C. 12