NL2027681A - In vitro propagation method of tissue culture seedlings of zanthoxylum armatum - Google Patents

Abstract

The present disclosure discloses a propagation method of tissue culture seedlings of Zanthoxylum armatum cv Rongchangwucihuajiao (Z. armatum), and belongs to the technical field of vegetative propagation of cash crops. The propagation method specifically comprises the following steps: primary culture, cutting and propagation, wherein the propagation is finished until the amount of PPM in the culture medium reaches 0, and there are no less than 4 generations in the propagation. In the propagation method of tissue culture seedlings of Z. armatum ofthe present disclosure, the technical system of propagation ofthe tissue culture seedlings of Z. armatum has been established by optimizing cultivation conditions, appropriately adjusting the composition of the culture medium according to the number of the propagation, and the comprehensive study of a special cutting process and other factors, which enables the stem segments of the primary culture of Z. armatum to achieve rapid and stable propagation, so as to realize large-scale breeding in a short time. It has great practical value in the breeding and production of Z. armatum.

Description

IN VITRO PROPAGATION METHOD OF TISSUE CULTURE SEEDLINGS OF ZANTHOXYLUM ARMATUM

TECHNICAL FIELD The present disclosure belongs to the technical field of vegetative propagation of cash crops, and specifically relates to an in vitro propagation method of tissue culture seedlings of Zanthoxylum armatum cv Rongchangwucihuajiao (Z. armatum).

BACKGROUND Chinese prickly ash (also called “Huajiao”), a deciduous aromatic shrub or small tree belongs to the family Rutaceae, is one of the traditional eight major condiments in China. With the growth of economic and medicinal benefits and the increasing market demand, Huajiao has been widely introduced and cultivated. There are two species native to China: Zanthoxylum bungeanum Maxim and Zanthoxylum armatum DC, which were in color of red and green at fruit ripening stages, respectively. The major cultivars in Z. bungeanum were “Hancheng Dahongpao” in Shaanxi province and “Hanyuan Zhenglujiao” in Sichuan province. While “Jiuyeqing” was the most famous cultivars of Z. armatum, distributed in the hilly regions of Southwest China, including Chongqing, Sichuan, Guizhou and Yunnan. With its strong resistance to barren, drought, and disease as well as high economic value, Chinese prickly ash is a primary economic plant cultured in China for the characteristic agricultural industry development and targeted poverty alleviation. However, there are many prickles in the stems, branches, and leaves of the main “Huajiao” cultivars in China. This leads to the increasing of labor costs in field management and harvest process, and hence seriously affects its direct economic benefits and restricts its industrial development. Therefore, it is extremely urgent to develop new “Huajiao” varieties with little prickles or thornless. Recently, we discovered a bud sport from wild Zanthoxyium armatum, called “Zanthoxylum armatum cv Rongchangwucihuajiao”, which was thornless on the abaxial leaf surface and has few thorns on the stems. Besides, the new variety displayed excellent characteristics, including large leaves and fruits, long inflorescence and infructescence, early fruiting, high yield, and strong resistance to pests and diseases. Therefore, this variety deserves wide promotion and planting in the world. However,

we observed that plantlets derived from seed propagation exhibited prickly stems and leaves, indicating that the thornless trait cannot be inherited via sexual reproduction. Currently, the strategy for maintaining the thornless phenotype of Z. armatum cv Rongchangwucihuajiao is mediated by asexual reproduction, mainly grafting propagation. But these technologies need a long time and largely limited by environmental conditions, and thus the amounts of seedlings obtained therefrom is far from being able to meet the market demand. It is the best way to product seedling all year-round using the tissue culture technology in order to maintain the thornless traits and avoid the shortage of seedling supply. Therefore, it is urgently needed to develop a method for in vitro propagation of Z. armatum. Up to date, there were few reports focus on the propagation of Zanthoxylum by in vitro tissue culture, such as Z. piperitum DC var. inerme Makino. However, the degree of difficulty of explants regeneration and propagation in vitro is largely different among the different varieties of Z. armatum and the different species of Zanthoxyhum, which is dependent on the genotype of the plant species or varieties. Hence, the previous methods is rarely referenced for the in vitro propagation of Z. armatum cv Rongchangwucihuajiao. Here, we developed an effective protocol for rapid propagation of Z. armatum cv Rongchangwucihuajiao in vitro.

SUMMARY In view of this, an objective of the present disclosure is to provide a propagation method of tissue culture seedlings of Zanthoxylum armatum cv Rongchangwucihuajiao (Z. armatum). The present disclosure can improve the survival rate and yield of Z. armatum by grafting propagation, In order to achieve the above object, the present disclosure provides the following technical solutions: A propagation method of tissue culture seedlings of Z. armatum includes the following steps of: S1. primary culture: placing a sprouted Z. armatum stem segment in a culture medium and culturing in the dark for 5-9 days, culturing the stem segment under a light intensity of 900-1100 Lux for 6-8 days, and increasing the light intensity to 1900-2100 Lux for culture for 12-16 days, until the sprouted Z. armatum stem segment grows to a height of 3-4 cm and has 3-4 internodes, and there are no less than 2 sprouted axillary buds;

S2. cutting: placing a sprouted Z. armatum stem segment following the primary culture in a sterile Petri dish on a clean bench, and cutting the base of the sprouted Z. armatum stem segment, namely one end of a tissue immersed in the culture medium, with a sterilized tool to expose a fresh tissue, and cutting the tissue into stem segments with 1- 3leaves; S3. second-generation culture: culturing the stem segment cut in step S2 in a culture medium in the dark for 5-9 days, culturing the stem segment under a light intensity of 900-1100 Lux for 6-8 days in time, and increasing the light intensity to 1900-2100 Lux for culture for 12-16 days, until the cut stem segment grows to a height of 3-4 cm and has 3-4 internodes, and there are no less than 2 sprouted axillary buds; where the culture medium during the primary culture and the second-generation culture includes: Murashige and Skoog (MS) Basal Medium, 2.0 mgeL! zeatin (ZT), 0.1 mgeL" t 1-naphthalene acetic acid (NAA), 6.0 geL"! carrageenan, 30 geL™! sucrose, and 0.1% Plant Preservative Mixture (PPM); S4. repeating steps S2 and S3 for propagation until the amount of PPM in the culture medium reaches 0, and there are no less than 4 generations in the propagation; where the culture medium during the propagation includes: MS Basal Medium, 1.0-2.0 mgeL?! ZT, 0.1 mgeL™! NAA, 6.0 geL! carrageenan, 30 geLt sucrose, and 0-0.05% PPM; the amount of the PPM is reduced by 0.01% every time 1-2 generations are increased during the propagation.

Among them, sprouted Z. armatum stem segment materials are 2-3 cm stem segments cut from Z. armatum saplings, and the sprouted stem segments grown after 30-40 d of culture in the culture medium.

During the culture process of the sprouted Z. armatum stem segments, one of the purposes of starting the culture in the dark is to avoid browning at the cuts of the newly cut stem segments, and the dark conditions are conducive to the growth of stem segment buds. The second purpose is to prevent the newly cut stem segments from growing too fast and aging. The third purpose is that the Z. armatum body is grafted from the thorny Zanthoxylum, and its cultivation in the dark can prevent the growth of thorny axillary buds from stem segments. After a period of culture in the dark, the light culture intensity is gradually increased, so that the stem segments gradually adapt to the light conditions.

After the stem segments adapt to the external conditions, they are placed under stronger light conditions to make the stem segments grow quickly and age. Preferably, in step S4, the amount of ZT in the composition of the culture medium of third-generation culture and fourth-generation culture is both 2.0 mgeL"t.

Preferably, in step S4, after there are more than 4 generations during the propagation, the amount of ZT in the composition of the culture medium needs to be adjusted according to the internode length and the number of axillary buds of multiplied axillary bud stem segments; when the axillary bud stem segments are dense and the axillary buds are clustered, the amount of ZT is 1.0-1.25 mgeL™, and when the axillary bud stem segments have long internodes and less spouted axillary buds, the amount of ZT is 1.25-

1.5 mgeL"!. Preferably, in step S4, during the propagation, the amount of PPM is reduced by 0.01% each time as propagation time increases. Where, after there are 3-4 generations during the propagation, the multiplied axillary bud stem segments gradually adapt to in vitro environment, and endophytic fungi in the body have been inhibited, and the hormone level in the body has gradually accumulated. Herein, the composition of the culture medium in the propagation needs to be adjusted appropriately according to the situation. As the number of the propagation continues to increase, the cell division number and the ratio of auxin are gradually reduced, and the amount of PPM is gradually reduced until it reaches 0 on the original MS Basal Medium basis. Preferably, in step S2, apical buds are cut into 1-2 cm long stem segments with 2-4 leaves when cutting the seedlings of Z. armatum. Preferably, in step S2, all the other leaves need to be preserved except that water-stained and yellow old leaves adjacent to the culture medium need to be cut when cutting the seedlings of Z. armatum. The cutting process is one of the key factors affecting the propagation of Z. armatum. The subsequent multiplication varies greatly with the same material using different cutting methods. Therefore, the key points of the cutting process for the propagation of Z armatum include the following four points: 1) placing material to be cut in a sterile Petri dish on a clean bench, and cutting the old tissue of the base of the stem segments, namely one end of a tissue immersed in the culture medium, with a sterilized tool first to expose a fresh tissue; 2) cutting it according to the micro-multiplication form of the axillary buds. That is, 3-4 cm stem segments are cut into ones with 1-3 leaves, depending on the spouting of stem segment axillary buds. If the internodes of the stem segments are longer and the axillary buds spout better, the stem segments can be cut into ones with 1 leaf and 1 bud; if the internodes of the stem segments are tighter and 5 the axillary buds do not spout long enough, the stem segments can be cut into ones with 2-3 leaves, 3) cutting the apical buds of the stem segment generally into 1-2 cm long stem segments with 2-4 leaves; 4) preserving all the other leaves completely except for water-stained and yellow old leaves adjacent to the culture medium.

During the cutting process, if the base of the sprouted Z. armatum stem segments and the water-stained and yellow old leaves adjacent to the culture medium are not cut, the base and the old leaves will absorb nutrients during the propagation process of the stem segments, which will slow down the multiplication of new stem segments, and also cause some weak stem segments to wither.

Preferably, throughout the propagation, daily lighting culture is conducted for 10 h at a temperature of 23-27°C.

The present disclosure has the following beneficial effects: 1) in the propagation method of tissue culture seedlings of Z. armatum of the present disclosure, culturing them in the dark first and then gradually increasing the light intensity can make hormones in the tissue culture seedlings gradually stabilize, and a special cutting process can greatly reduce the number of the generations of the propagation to achieve the purpose of rapid propagation of Z. armatum saplings; 2) in the propagation method of tissue culture seedlings of Z. armatum of the present disclosure, the technical system of propagation of the tissue culture seedlings of Z. armatum has been established by optimizing cultivation conditions, appropriately adjusting the composition of the culture medium according to the number of the propagation, and the comprehensive study of a special cutting process and other factors, which enables the stem segments of the primary culture of Z. armatum to achieve rapid and stable propagation, so as to realize large-scale breeding in a short time. It has great practical value in the breeding and production of Z. armatum, 3) the propagation method of the tissue culture seedlings of Z. armatum of the present disclosure is not affected by external diseases and insect pests, and the breeding method is simple and fast. At the same time, it can also save the cost of manpower and material resources caused by grafting of Z. armatum. The quantity of the seedlings can be effectively guaranteed, and the yield of Zanthoxvium can be greatly increased in the subsequent planting and fruiting process.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a picture of the seedlings of Zanthoxylum armatum cv Rongchangwucihuajiao (Z. armatum) after primary culture in example 1; FIG. 2 shows a picture of the cut stem segments in example 1.

DETAILED DESCRIPTION The present disclosure will be further described in combination with specific examples so as to enable those skilled in the art to better understand and practice the present disclosure, but the illustrated examples do not constitute any limitation to the present disclosure. Example 1 A propagation method of tissue culture seedlings of Z. armatum included the following steps of: S1. primary culture: placing a sprouted Z. armatum stem segment in a culture medium and culturing in the dark for 8-9 days, culturing the stem segment under a light intensity of 1000 Lux for 7-8 days, and increasing the light intensity to 2000 Lux for culture for 13-14 days, until the sprouted Z. armatum stem segment grows to a height of 3-4 cm and has 3-4 internodes, and there are no less than 2 sprouted axillary buds, the cultured seedlings of the sprouted Z. armatum stem segment as shown in FIG. 1; S2. cutting: placing a sprouted Z. armatum stem segment following the primary culture in a sterile Petri dish on a clean bench, and cutting the base of the sprouted Z. armatum stem segment, namely one end of a tissue immersed in the culture medium, with a sterilized tool to expose a fresh tissue, and then cutting apical buds into 1-2 cm long stem segments with 2-4 leaves, and cutting the rest into stem segments with 1-3 leaves, as shown in FIG. 2; S3. second-generation culture: culturing the stem segment cut in step S2 in a culture medium in the dark for 8-9 days, culturing the stem segment under a light intensity of 1000 Lux for 7-8 days in time, and increasing the light intensity to 2000 Lux for culture for 13-14 days, until the cut stem segment grows to a height of 3-4 cm and has 3-4 internodes, and there are no less than 2 sprouted axillary buds;

where the culture medium during the primary culture and the second-generation culture includes: MS Basal Medium, 2.0 mgsL™ ZT, 0.1 mgeL"! NAA, 6.0 gel"! carrageenan, 30 geL! sucrose, and 0.1% PPM; S4. repeating steps S2 and S3 for propagation. The composition of the culture medium in the propagation consisting of: MS Basic Medium, ZT in the third and fourth generation medium 2.0 mgeL™!, in the fifth to seventh generations 1.20 mgeL!, NAA

0.1 mgeL!, carrageenan 6.0 geL"t, sucrose 30 geL"! and PPM 0-0.05%, PPM in the third- generation culture medium in the propagation 0.05%, reducing the amount of the PPM by 0.01% for each subsequent increase, and there are seven generations in the propagation finally; throughout the propagation, daily lighting culture is conducted for 10 h at a temperature of 23-25°C.

Through the breeding method of this example, the seedlings of Z. armatum were finally obtained by culture. The whole process took 30-40 days, and the survival rate after transplanting reached more than 95%.

Rooting culture: (1) cutting the seedlings obtained by propagation and inoculating them into rooting culture medium. The height of the seedlings was >2.0 cm, the thickness of the stems was >2.0 mm, and the depth of the seedlings inserted into the culture medium was 3.0- 40mm; (2) the formula of the culture medium: 1/2MSHBA0.5 mg Lt +AC (activated carbon) 50 mg L+carrageenan 6.0 g:L"!+sucrose 20 g Lt: (3) cultivation conditions: placing them in the dark after inoculation and cultivating them for 7 days, then transferring them to a light environment to continue to culture for 7-14 days. The light culture time was 10 h/d. During the whole rooting culture process, the temperature was 23-25°C, and the light intensity was 2000 lux; (4) after culture for 14-21 days, selecting rooting seedlings with root number >2, root length >2.0 cm, and normal leaves for transplantation. Domestication and transplantation: (1) domestication: placing the seedlings of Z. armatum in a greenhouse with a shading rate of 65%-80% before transplanting, loosening the cap of the culture bottle, and acclimatizating at room temperature for about 10 d to thicken the leaves of the rooting seedlings.

(2) transplantation: rinsing the culture medium of the rooting seedlings after acclimatizating with clean water, then placing it in 2000 times carbendazim solution and soaking it for 1-2 minutes, and culturing it in a prepared substrate tray. The transplanting substrate ratio was peat: perlite (V:V=7:3). After culturing, irrigating rooting water, covering it with film for heat preservation and moisturizing, placing it in a greenhouse for culturing, paying attention to ventilation in the morning and evening during the transplanting and culturing process, using 0.5% compound fertilizer (N:P:K=15:15:15) for spraying once every 7 d after 10 d, removing the film after about 15 d, and surviving from nursery after 30-45 d.

To sum up, in the propagation method of the tissue culture seedlings of Z. armatum of the present disclosure, the technical system of propagation of the tissue culture seedlings of Z. armatum has been established by optimizing cultivation conditions, appropriately adjusting the composition of the culture medium according to the number of the propagation, and the comprehensive study of a special cutting process and other factors, which enables the stem segments of the primary culture of Z. armatum to achieve rapid and stable propagation, so as to realize large-scale breeding in a short time. It has great practical value in the breeding and production of Z. armatum.

The above examples are merely preferred examples provided to more fully illustrate the present invention, and the scope of the present invention is not limited thereto. Any equivalent replacement or modification made by those skilled in the art based on the present invention should be included in the protection scope of the invention. The protection scope of the present invention is subject to the protection scope defined by the claims.

Claims (7)

Claims L Propagation method of tissue culture seedlings of Zanthoxylum armatum cv Rongchangwucihuajiao (Z. armatum), wherein the propagation method comprises the following steps: Sl. primary culture: placing a germinated stem segment of Z. armatum in a culture medium and culturing it in the dark for 5-9 days, culturing the stem segment under a light intensity of 900-1100 Lux for 6-8 days, and increasing from the light intensity to 1900-2100 Lux for cultivation for 12-16 days, until the germinated stem segment of Z. armatum grows to a height of 3 - 4 cm and has 3 - 4 stem members, and there are not less than two germinated axillary buds; S2. cutting: placing a germinated stem segment of Z. armatum followed by the primary culture in a sterile petri dish on a clean bench, and cutting the base of the germinated stem segment of Z. armatum, i.e. one end of a tissue submerged in the culture medium, using a sterilized tool to expose a fresh tissue, and cutting the tissue into 1 - 3 leaf stem segments, S3. second generation culture: culturing the stem segment cut in step S2 in a culture medium in the dark for 5 - 9 days, culturing the stem segment under a light intensity of 900 - 1100 Lux for 6 - 8 days, and increasing the light intensity up to 1900 - 2100 Lux for cultivation for 12 - 16 days, until the cut stem segment grows to a height of 3 - 4 cm and has 3 - 4 stem members, and there are not less than 2 germinated axillary buds; wherein the culture medium during the primary culture and the second generation culture comprises the following: Murashige and Skoog (MS) basal medium, 2.0 mg-L™! zeatin (ZT), 0.1 mg Lt I-naphthaleneacetic acid (NAA), 6.0 g-L™! carrageenan, 30 µl-t sucrose and 0.1% plant preservation mixture (PPM); S4. repeating steps S2 and S3 for propagation until the amount of PPM in the culture medium reaches 0 and there are not less than 4 generations in propagation; wherein the culture medium comprises the following during propagation: MS basal medium, 10-2.0 mgL+ZT, 0.1 mg Lt NAA, 6.0 g-1 L! carrageenan, 30 g of L° sucrose and 0-0.05% PPM; -10- where the amount of the PPM is reduced by 0.01% each time 1 - 2 generations are increased during propagation. The propagation method of tissue culture seedlings of Z. armatum according to claim 1, wherein in step S4, the amount of ZT in the composition of the culture medium for both third-generation culture and fourth-generation culture is 2.0 mg-L. The propagation method of tissue culture seedlings of Z. armatum according to claim 1, wherein in step S4, after there are more than 4 generations during the propagation, the amount of ZT in the composition of the culture medium is to be adjusted according to the length of the stem members and the number axillary buds of multiplied axillary bud stem segments; where, when the axillary bud stem segments are closed and the axillary buds are clustered, the amount of ZT is 1.0 — 1.25 mg-L! and when the axillary bud stem segments have long stem members and fewer germinated axillary buds, the amount of ZT is 1.25 — 1.5 mg-L. The propagation method of Z. armatum tissue culture seedlings according to claim 1, wherein in step S4, during the propagation, the amount of PPM is reduced by 0.01% each time the propagation time increases. The propagation method of Z. armatum tissue culture seedlings according to claim 1, wherein in step S2, terminal buds are cut into 1 - 2 cm long stem segments with 2 - 4 leaflets when cutting the Z. armatum seedlings. The propagation method of Z. armatum tissue culture seedlings according to claim 1, wherein in step S2 all other leaves are to be preserved except that water-stained and yellow old leaves are to be cut adjacent to the culture medium when cutting the Z. armatum seedlings. The propagation method of tissue culture seedlings of Z. armatum according to claim 1, wherein through the propagation, daylight culture is carried out for 10 hours at a temperature of 23-27°C.