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CN-121986717-A - Litsea cubeba tissue culture method and culture medium thereof

CN121986717ACN 121986717 ACN121986717 ACN 121986717ACN-121986717-A

Abstract

The application relates to a litsea cubeba tissue culture method and a culture medium thereof. The culture medium for litsea cubeba tissue culture comprises a specific induction culture medium, a proliferation culture medium and a rooting culture medium. The method takes a new semi-lignified branch with axillary buds after pruning as an explant, and sequentially carries out induction culture, proliferation culture and rooting culture after disinfection by PPM at the tail end, and tissue culture seedlings can be obtained through further domestication and transplantation, wherein the induction culture medium is based on WPM and contains sodium citrate and PPM for cooperatively controlling bacteria and controlling brown, the proliferation culture medium is based on WPM and contains sodium citrate and Meta-Topolin for improving proliferation multiple and inhibiting basal brown, and the rooting culture medium is based on 1 ⁄ WPM and contains active carbon for controlling brown and promoting root. The application provides a reproducible and cost-controllable technical path for the preservation of litsea cubeba good germplasm resources and the industrialized seedling.

Inventors

  • XUE LEI
  • CHEN YING
  • LIU YANG

Assignees

  • 广东省林业科学研究院

Dates

Publication Date
20260508
Application Date
20260320

Claims (10)

  1. 1. A culture medium for litsea cubeba tissue culture, which is characterized by comprising an induction culture medium, a proliferation culture medium and a rooting culture medium; The induction culture medium comprises a first basic culture medium, and further comprises 0.5 g/L-1.5 g/L sodium citrate and 0.5 ml/L-2.0 ml/L plant tissue culture antibacterial agent based on the first basic culture medium, wherein the first basic culture medium is selected from WPM; The proliferation culture medium comprises a second basal culture medium, and further comprises 0.2 mg/L-1.0 mg/L Meta-Topolin and 0.5 g/L-1.5 g/L sodium citrate based on the second basal culture medium, wherein the second basal culture medium is selected from WPM; the rooting culture medium comprises a third basic culture medium, and further comprises 1.0 g/L-2.0 g/L of activated carbon based on the third basic culture medium, wherein the third basic culture medium is selected from 1 ⁄ WPM.
  2. 2. The culture medium for litsea cubeba tissue culture according to claim 1, wherein the induction culture medium comprises 0.5 g/L-1.5 g/L sodium citrate, 0.5 ml/L-2.0 ml/L plant tissue culture antibacterial agent, 0.5 mg/L-1.0 mg/L6-BA, 0.1 mg/L-0.5 mg/L NAA, 15 g/L-30 g/L sucrose and 5 g/L-10 g/L coagulant; And/or the proliferation medium comprises 0.2 mg/L-1.0 mg/L Meta-Topolin, 0.5 g/L-1.5 g/L sodium citrate, 0.2 mg/L-0.8 mg/L IBA, 15 g/L-30 g/L sucrose, and 5 g/L-10 g/L coagulant; and/or the rooting medium comprises 1.0 g/L-2.0 g/L active carbon, 0.5 mg/L-2.0 mg/L IBA, 15 g/L-30 g/L sucrose and 5 g/L-10 g/L coagulant.
  3. 3. A tissue culture method of litsea cubeba, characterized in that the litsea cubeba is subjected to tissue culture by using the culture medium for the tissue culture of litsea cubeba according to any one of claims 1-2.
  4. 4. The method for tissue culture of litsea cubeba as claimed in claim 3, wherein the tissue culture comprises induction culture, multiplication culture and rooting culture, and culture mediums adopted by the induction culture, the multiplication culture and the rooting culture are respectively the induction culture medium, the multiplication culture medium and the rooting culture medium.
  5. 5. The tissue culture method of litsea cubeba as claimed in claim 4, wherein the tissue culture method comprises the following steps: selecting newly germinated semi-lignified litsea cubeba twigs after trimming as explants; Inoculating the explant to the induction culture medium after disinfection, and performing induction culture to obtain axillary buds; placing the axillary buds into a proliferation culture medium for proliferation culture to obtain proliferation buds; transferring the proliferation buds into a rooting culture medium for rooting culture to obtain tissue culture seedlings, and transplanting the tissue culture seedlings to a substrate after domestication.
  6. 6. The tissue culture method of litsea cubeba according to claim 5, wherein the explant comprises young shoots with axillary buds collected in 2-7 months after pruning parent branches in 12-4 months of the next year, and the diameter of the young shoots is 0.1-0.3 cm.
  7. 7. The tissue culture method of litsea cubeba as claimed in any one of claims 5 to 6, wherein the step of sterilizing comprises: (a) Treating the explant with alcohol for 15-30 s; (b) After washing for 2-4 times with sterile water, soaking for 15-20 min by using a mixed solution containing 15-25% of sodium hypochlorite and 0.1-0.5% of Tween-20; (c) Washing with sterile water for 3-5 times; (d) And soaking the explant for 5-10 min by using a plant tissue culture antibacterial agent.
  8. 8. The tissue culture method of litsea cubeba according to any one of claims 5 to 6, wherein the culture conditions of the induced culture comprise a temperature of 22-28 ℃, an illumination intensity 1000 lux~2000 lux, an illumination time of 12-h/d-16 h/d and a culture period of 8-20 d; and/or the culture conditions of proliferation culture comprise the temperature of 22-28 ℃, the illumination intensity 1000 lux~2000 lux, the illumination time of 12-h/d-16 h/d and the culture time of 30-45 d; and/or culturing conditions of rooting culture comprise the temperature of 22-28 ℃, the illumination intensity 1000 lux~2000 lux, the illumination time of 12-h/d-16 h/d and the culturing time of 20-40 d.
  9. 9. The method for tissue culture of litsea cubeba according to any one of claims 5 to 6, wherein the domestication step comprises the steps of moving the tissue culture seedlings to an environment with shading degree of 70% -80% and daytime illumination of 2000 lux~3000 lux for 20 d to 30 d, and transplanting after the seedlings are 3 cm to 8 cm in height and have 5 to 10 true leaves.
  10. 10. The method for tissue culture of litsea cubeba according to any one of claims 5 to 6, wherein the substrate comprises peat soil and perlite, and the volume ratio of peat soil to perlite is (2 to 4): 1.

Description

Litsea cubeba tissue culture method and culture medium thereof Technical Field The application relates to the technical field of plant tissue culture, in particular to a litsea cubeba tissue culture method and a culture medium thereof. Background Litsea cubeba (Litsea cubeba) is a plant of litsea (Litsea) belonging to Lauraceae (Lauraceae), is an economic woods tree species which integrates natural spice, medical use, edible use, material use and ornamental value, and has the content of citral in essential oil extracted from fruits as high as 60% -90%. However, litsea cubeba is a hermaphrodite plant, the offspring population generated by sexual propagation has extremely large variation, and the excellent properties consistent with the parent plant cannot be completely maintained, so that the yield and quality of the essential oil extracted from the litsea cubeba seedlings obtained by sexual propagation are difficult to be ensured. In asexual propagation, tissue culture is higher than that of cutting and grafting, and seedlings are more orderly and consistent, so that the tissue culture of litsea cubeba is one of important means for maintaining the excellent characters of the litsea cubeba female plant. In the prior art, a tissue culture scheme aiming at litsea cubeba cluster buds induction, proliferation or rooting is reported, wherein the disclosed method for inducing litsea cubeba cluster buds needs 21 d, the proliferation coefficient is about 4 times, but no rooting and transplanting method is mentioned, the other traditional technology discloses a litsea cubeba asexual fast propagation seedling method, the litsea cubeba tissue culture proliferation buds are used for in-vitro rooting, the survival rate can reach 80% -95%, the technology is complicated in operation and high in management cost after cutting, the other technology discloses a method for remarkably improving the rooting rate of litsea cubeba cluster buds, the method sequentially comprises the steps of soaking the cluster buds with an anti-browning agent, inoculating the cluster buds to a strong seedling culture medium, an induction rooting culture medium and a proliferation rooting culture medium, adding root tip tissue extracting solution into the proliferation culture medium, so that cluster buds death caused by tissue is greatly reduced, the rooting rate and root growth speed are remarkably improved, but the preparation method of an anti-rooting agent and the root tip extracting solution is tedious, the time consuming and high in cost is consumed, and the browning of the brown tissue is even the brown tissue is affected by the browning and the browning of the base. Therefore, a litsea cubeba tissue culture rapid propagation method which is simple and convenient to operate, has controllable cost, and can cooperatively control bacteria and brown in each culture stage and improve proliferation and rooting quality is still needed. Thus, the conventional technology has yet to be improved. Disclosure of Invention Based on the above, the application provides a litsea cubeba tissue culture rapid propagation method and a culture medium used for the method. In one aspect, the application provides a culture medium for litsea cubeba tissue culture, which comprises an induction culture medium, a proliferation culture medium and a rooting culture medium; The induction culture medium comprises a first basic culture medium, and further comprises 0.5 g/L-1.5 g/L sodium citrate and 0.5 ml/L-2.0 ml/L plant tissue culture antibacterial agent based on the first basic culture medium, wherein the first basic culture medium is selected from WPM; The proliferation culture medium comprises a second basal culture medium, and further comprises 0.2 mg/L-1.0 mg/L Meta-Topolin and 0.5 g/L-1.5 g/L sodium citrate based on the second basal culture medium, wherein the second basal culture medium is selected from WPM; the rooting culture medium comprises a third basic culture medium, and further comprises 1.0 g/L-2.0 g/L of activated carbon based on the third basic culture medium, wherein the third basic culture medium is selected from 1 ⁄ WPM. In some embodiments, the induction medium comprises 0.5 g/L-1.5 g/L sodium citrate, 0.5 ml/L-2.0 ml/L plant tissue culture antibacterial agent, 0.5 mg/L-1.0 mg/L6-BA, 0.1 mg/L-0.5 mg/L NAA, 15 g/L-30 g/L sucrose, and 5 g/L-10 g/L coagulant; And/or the proliferation medium comprises 0.2 mg/L-1.0 mg/L Meta-Topolin, 0.5 g/L-1.5 g/L sodium citrate, 0.2 mg/L-0.8 mg/L IBA, 15 g/L-30 g/L sucrose, and 5 g/L-10 g/L coagulant; and/or the rooting medium comprises 1.0 g/L-2.0 g/L active carbon, 0.5 mg/L-2.0 mg/L IBA, 15 g/L-30 g/L sucrose and 5 g/L-10 g/L coagulant. The application also provides a litsea cubeba tissue culture method, which uses the culture medium for litsea cubeba tissue culture to perform tissue culture on litsea cubeba. In some embodiments, the tissue culture comprises induction culture, proliferation culture and rooting cultur