A culture medium for tissue culture of limonium hybridum

Abstract

The application discloses a culture medium for tissue culture of Limosella australis, which comprises a primary culture medium, a proliferation and differentiation culture medium and a rooting culture medium. The primary culture medium is MS+6BA 0.4-0.6 mg / L+NAA 0.04-0.06 mg / L+AC 0.8-1.2 mg / L+2,4-D 0.04-0.06 mg / L. The proliferation and differentiation culture medium is modified MS+6BA 0.09-0.11 mg / L+NAA 0.04-0.06 mg / L+AC 1.5-2.5 mg / L. The rooting culture medium is 1 / 2MS+IAA 0.09-0.11 mg / L+AC 2.5-3.5 mg / L. The technical scheme has the highest callus induction rate of 86.7%, the highest differentiation rate of 86.7% and the highest rooting rate of 93.3%.

Description

Technical Field

[0001] This invention relates to the field of plant culture technology, and specifically to a culture medium for tissue culture of *Limonium bicolor*. Background Technology

[0002] *Limonium bicolor* is a perennial herb belonging to the genus *Limonium* in the family Plumbaginaceae. It can grow up to 50 cm tall, with sparse, spoon-shaped or oblong-spatulate leaves that are rounded or obtuse at the apex and gradually narrowed at the base. The inflorescence axis and branches are angular, located at the lower part of the inflorescence or at the forking point. The calyx is funnel-shaped with a pale purplish-red or white lip, and the corolla is yellow. The "two colors" in *Limonium bicolor* refer to the white and yellow hues of the calyx and corolla, while "blood-tonifying" refers to its medicinal properties. Seed collection is difficult, and its genetic traits are unstable. Artificial propagation is limited by seasonality and long cycles, resulting in slow reproduction. While tissue culture has been attempted to increase its propagation speed, existing culture media have shown poor results. Therefore, a suitable culture medium for the tissue culture of *Limonium bicolor* is urgently needed. Summary of the Invention

[0003] To address the above technical issues, and considering the characteristics of wild *Limonium bicolor*, a culture medium for tissue culture of *Limonium bicolor* is provided, as detailed below:

[0004] A culture medium for tissue culture of *Limonium bicolor* includes a primary culture medium, a proliferation and differentiation culture medium, and a rooting culture medium; the primary culture medium is used to induce callus tissue in explants; the proliferation and differentiation culture medium is used to proliferate the callus tissue to form adventitious shoots; and the rooting culture medium is used to root the adventitious shoots to form sterile seedlings.

[0005] The primary culture medium was: MS + 6BA 0.4-0.6 mg / L + NAA 0.04-0.06 mg / L + AC 0.8-1.2 mg / L + 2,4-D 0.04-0.06 mg / L;

[0006] The proliferation and differentiation medium was: modified MS + 6BA 0.09-0.11 mg / L + NAA 0.04-0.06 mg / L + AC 1.5-2.5 mg / L;

[0007] The rooting medium was: 1 / 2 MS + IAA 0.09-0.11 mg / L + AC 2.5-3.5 mg / L.

[0008] Furthermore, the modified MS is as follows: the amount of macroelements ammonium nitrate and potassium nitrate in the MS medium is reduced by 1 / 3 mg / L, and the amount of microelement boric acid is reduced by 1 / 5 mg / L.

[0009] Furthermore, the primary culture medium was: MS + 6BA 0.4 mg / L + NAA 0.04 mg / L + AC 0.8 mg / L + 2,4-D 0.04 mg / L;

[0010] The proliferation and differentiation medium was: modified MS + 6BA 0.09 mg / L + NAA 0.04 mg / L + AC 1.5 mg / L;

[0011] The rooting medium was: 1 / 2 MS + IAA 0.09 mg / L + AC 2.5 mg / L.

[0012] Furthermore, the primary culture medium was: MS + 6BA 0.6 mg / L + NAA 0.06 mg / L + AC 1.2 mg / L + 2,4-D 0.06 mg / L;

[0013] The proliferation and differentiation medium was: modified MS + 6BA 0.11 mg / L + NAA 0.06 mg / L + AC 2.5 mg / L;

[0014] The rooting medium was: 1 / 2 MS + IAA 0.11 mg / L + AC 3.5 mg / L.

[0015] Furthermore, the primary culture medium was: MS + 6BA 0.5 mg / L + NAA 0.05 mg / L + AC 1 mg / L + 2,4-D 0.05 mg / L;

[0016] The proliferation and differentiation medium was: modified MS + 6BA 0.1 mg / L + NAA 0.05 mg / L + AC 2 mg / L;

[0017] The rooting medium was: 1 / 2 MS + IAA 0.1 mg / L + AC 3 mg / L.

[0018] Furthermore, the primary culture medium was: MS + 6BA 0.5 mg / L + NAA 0.05 mg / L + AC 1 mg / L + 2,4-D 0.05 mg / L;

[0019] The proliferation and differentiation medium was: modified MS + 6BA 0.09-0.11 mg / L + NAA 0.04-0.06 mg / L + AC 1.5-2.5 mg / L;

[0020] The rooting medium was: 1 / 2 MS + IAA 0.09-0.11 mg / L + AC 2.5-3.5 mg / L.

[0021] Furthermore, the primary culture medium is: MS + 6BA 0.4-0.6 mg / L + NAA 0.04-0.06 mg / L + AC 0.8-1.2 mg / L + 2,4-D 0.04-0.06 mg / L;

[0022] The proliferation and differentiation medium was: modified MS + 6BA 0.1 mg / L + NAA 0.05 mg / L + AC 2 mg / L;

[0023] The rooting medium was: 1 / 2 MS + IAA 0.09-0.11 mg / L + AC 2.5-3.5 mg / L.

[0024] Furthermore, the primary culture medium is: MS + 6BA 0.4-0.6 mg / L + NAA 0.04-0.06 mg / L + AC 0.8-1.2 mg / L + 2,4-D 0.04-0.06 mg / L;

[0025] The proliferation and differentiation medium was: modified MS + 6BA 0.09-0.11 mg / L + NAA 0.04-0.06 mg / L + AC 1.5-2.5 mg / L;

[0026] The rooting medium was: 1 / 2 MS + IAA 0.1 mg / L + AC 3 mg / L.

[0027] Moreover, the culture medium is a sterile culture medium.

[0028] Furthermore, the culture medium is sterilized after being placed into a culture container.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] This invention, through screening commonly used tissue culture agents in the prior art and repeated experiments, selected suitable primary culture medium, proliferation and differentiation medium, and rooting medium for the tissue culture of *Limonium bicolor*. The primary culture medium is: MS + 6BA 0.4-0.6 mg / L + NAA 0.04-0.06 mg / L + AC 0.8-1.2 mg / L + 2,4-D 0.04-0.06 mg / L; the proliferation and differentiation medium is: modified MS + 6BA 0.09-0.11 mg / L + NAA 0.04-0.06 mg / L + AC 1.5-2.5 mg / L; the rooting medium is: 1 / 2 MS + IAA 0.09-0.11 mg / L + AC 2.5-3.5 mg / L. Using the technical solution of this invention, the highest callus induction rate is 86.7%, the highest differentiation rate is 86.7%, and the highest rooting rate is 93.3%. Attached Figure Description

[0031] Figure 1 The proliferation effect of the proliferation and differentiation medium in Experiment 2;

[0032] Figure 2 The rooting effect of the rooting medium in Experiment 3 is shown. Detailed Implementation Example 1

[0033] A culture medium for tissue culture of *Limonium bicolor* includes a primary culture medium, a proliferation and differentiation culture medium, and a rooting culture medium; the primary culture medium is used to induce callus tissue in explants; the proliferation and differentiation culture medium is used to proliferate the callus tissue to form adventitious shoots; and the rooting culture medium is used to root the adventitious shoots to form sterile seedlings.

[0034] The primary culture medium was: MS + 6BA 0.4 mg / L + NAA 0.04 mg / L + AC 0.8 mg / L + 2,4-D 0.04 mg / L;

[0035] The proliferation and differentiation medium was: modified MS + 6BA 0.09 mg / L + NAA 0.04 mg / L + AC 1.5 mg / L;

[0036] The rooting medium was: 1 / 2 MS + IAA 0.09 mg / L + AC 2.5 mg / L.

[0037] Furthermore, the modified MS is as follows: the amount of macroelements ammonium nitrate and potassium nitrate in the MS medium is reduced by 1 / 3 mg / L, and the amount of microelement boric acid is reduced by 1 / 5 mg / L;

[0038] Furthermore, the culture medium is a sterile culture medium.

[0039] Furthermore, the culture medium is placed into a culture container and then sterilized. Example 2

[0040] A culture medium for tissue culture of *Limonium bicolor* includes a primary culture medium, a proliferation and differentiation culture medium, and a rooting culture medium; the primary culture medium is used to induce callus tissue in explants; the proliferation and differentiation culture medium is used to proliferate the callus tissue to form adventitious shoots; and the rooting culture medium is used to root the adventitious shoots to form sterile seedlings.

[0041] The primary culture medium was: MS + 6BA 0.6 mg / L + NAA 0.06 mg / L + AC 1.2 mg / L + 2,4-D 0.06 mg / L;

[0042] The proliferation and differentiation medium was: modified MS + 6BA 0.11 mg / L + NAA 0.06 mg / L + AC 2.5 mg / L;

[0043] The rooting medium was: 1 / 2 MS + IAA 0.11 mg / L + AC 3.5 mg / L.

[0044] Furthermore, the modified MS is as follows: the amount of macroelements ammonium nitrate and potassium nitrate in the MS medium is reduced by 1 / 3 mg / L, and the amount of microelement boric acid is reduced by 1 / 5 mg / L;

[0045] Furthermore, the culture medium is a sterile culture medium.

[0046] Furthermore, the culture medium is placed into a culture container and then sterilized. Example 3

[0047] A culture medium for tissue culture of *Limonium bicolor* includes a primary culture medium, a proliferation and differentiation culture medium, and a rooting culture medium; the primary culture medium is used to induce callus tissue in explants; the proliferation and differentiation culture medium is used to proliferate the callus tissue to form adventitious shoots; and the rooting culture medium is used to root the adventitious shoots to form sterile seedlings.

[0048] The primary culture medium was: MS + 6BA 0.5 mg / L + NAA 0.05 mg / L + AC 1 mg / L + 2,4-D 0.05 mg / L;

[0049] The proliferation and differentiation medium was: modified MS + 6BA 0.1 mg / L + NAA 0.05 mg / L + AC 2 mg / L;

[0050] The rooting medium was: 1 / 2 MS + IAA 0.1 mg / L + AC 3 mg / L.

[0051] Furthermore, the modified MS is as follows: the amount of macroelements ammonium nitrate and potassium nitrate in the MS medium is reduced by 1 / 3 mg / L, and the amount of microelement boric acid is reduced by 1 / 5 mg / L;

[0052] Furthermore, the culture medium is a sterile culture medium.

[0053] Furthermore, the culture medium is placed into a culture container and then sterilized.

[0054] Experimental Section

[0055] Experiment 1: Test of the effect of different primary culture media on induction rate

[0056] Unopened inflorescences of *Limonium bicolor* were randomly selected and inoculated onto different primary culture media (Table 1). Fifteen inflorescences were inoculated into each group, for a total of 11 groups. The culture temperature was 25℃, the light intensity was 1800 lx, and the light duration was 14 h / d. The callus induction rate was calculated after 18 days, as shown in Table 1.

[0057] Table 1 Callus induction status

[0058] Serial Number Number of vaccinations (individuals) Number of successful inducements (units) Induction rate (%) Primary culture medium 1 15 12 80 MS+6BA 0.4mg / L+NAA 0.04mg / L+AC 0.8mg / L+2,4-D 0.04mg / L 2 15 12 80 MS+6BA 0.6mg / L+NAA 0.06mg / L+AC 1.2mg / L+2,4-D 0.06mg / L 3 15 13 86.7 MS+6BA 0.5mg / L+NAA 0.05mg / L+AC 1mg / L+2,4-D 0.05mg / L 4 15 9 60 MS+6BA 0.5mg / L+NAA 0.05mg / L+AC 1mg / L+2,4-D 0.07mg / L 5 15 9 60 MS+6BA 0.5mg / L+NAA 0.05mg / L+AC 1mg / L+2,4-D 0.03mg / L 6 15 8 53.3 MS+6BA 0.3mg / L+NAA 0.05mg / L+AC 1mg / L+2,4-D 0.05mg / L 7 15 9 60 MS+6BA 0.7mg / L+NAA 0.05mg / L+AC 1mg / L+2,4-D 0.05mg / L 8 15 8 53.3 MS+6BA 0.5mg / L+NAA 0.03mg / L+AC 1mg / L+2,4-D 0.05mg / L 9 15 9 60 MS+6BA 0.5mg / L+NAA 0.07mg / L+AC 1mg / L+2,4-D 0.05mg / L 10 15 8 53.3 MS+6BA 0.5mg / L+NAA 0.05mg / L+AC 0.7mg / L+2,4-D 0.05mg / L 11 15 8 53.3 MS+6BA 0.5mg / L+NAA 0.05mg / L+AC 1.3mg / L+2,4-D 0.05mg / L

[0059] As shown in Table 1, groups 1-3 used the primary culture medium of Examples 1-3 of this invention, with 15 inoculated cells in each group, and all successfully induced 12-13 callus tissues, with the highest induction rate of 86.7%. Groups 4 and 5 increased or decreased the amount of 2,4-D compared to this invention, and only 9 callus tissues were successfully induced in each group, with an induction rate of 60%. Groups 6 and 7 decreased or increased the amount of 6BA compared to this invention, groups 8 and 9 decreased or increased the amount of NAA compared to this invention, and groups 10 and 11 decreased or increased the amount of AC compared to this invention. Each group successfully induced 8-9 callus tissues, with induction rates of 53.3% and 60%, respectively. It can be seen that the induction rate of groups 4-11 was significantly lower than that of this invention, proving that the primary culture medium used in this invention has the best effect on callus induction.

[0060] Experiment 2: Test of the effect of different proliferation and differentiation media on differentiation rate

[0061] After successfully inducing callus tissue using the scheme of Group 3 in Experiment 1, the callus tissue was then inoculated into proliferation and differentiation medium. 15 callus tissues were inoculated into each group, for a total of 10 groups. The proliferation and differentiation medium for each group is shown in Table 2. The culture temperature was 25℃, the light intensity was 1800 lx, and the light duration was 14 h / d. The callus induction rate was calculated after 20 days.

[0062] Table 2. Callus differentiation

[0063] Serial Number Total number of callus tissues (units) Seedlings (plants) that differentiate into an indeterminate number of buds. Differentiation rate (%) Proliferation and differentiation culture medium 1 15 13 86.7 Modified MS + 6BA 0.09 mg / L + NAA 0.04 mg / L + AC 1.5 mg / L 2 15 12 80 Improved MS+6BA 0.11mg / L+NAA 0.06mg / L+AC 2.5mg / L 3 15 13 86.7 Improved MS+6BA 0.1mg / L+NAA 0.05mg / L+AC 2mg / L 4 15 10 66.7 MS+6BA 0.1mg / L+NAA 0.05mg / L+AC 2mg / L 5 15 9 60 Improved MS+6BA 0.08mg / L+NAA 0.05mg / L+AC 2mg / L 6 15 8 53.3 Modified MS + 6BA 0.12 mg / L + NAA 0.05 mg / L + AC 2 mg / L 7 15 8 53.3 Modified MS + 6BA 0.1 mg / L + NAA 0.03 mg / L + AC 2 mg / L 8 15 9 60 Modified MS + 6BA 0.1 mg / L + NAA 0.07 mg / L + AC 2 mg / L 9 15 8 53.3 Improved MS+6BA 0.1mg / L+NAA 0.05mg / L+AC 1mg / L 10 15 9 60 Improved MS+6BA 0.1mg / L+NAA 0.05mg / L+AC 3mg / L

[0064] As shown in Table 2, groups 1-3 used the proliferation and differentiation media of Examples 1-3 of this invention, with an inoculation number of 15, and 12-13 successfully differentiated, with a differentiation rate of 86.7%; group 4 replaced the modified MS medium with ordinary MS medium, and only 10 differentiated successfully, with a differentiation rate of 66.7%; groups 5 and 6 reduced or increased the amount of 6BA compared to this invention, groups 7 and 8 reduced or increased the amount of NAA compared to this invention, and groups 9 and 10 reduced or increased the amount of AC compared to this invention, with 8-9 successfully differentiated in each group, and differentiation rates of 53.3% and 60%, respectively; thus, the differentiation rates of groups 4-10 were all lower than those of this invention, proving that the proliferation and differentiation media used in this invention has the best effect on promoting callus differentiation.

[0065] Experiment 3: Test of the effect of different rooting media on rooting rate

[0066] Adventitious shoots were differentiated from callus tissue using the scheme of Group 3 in Experiment 2. Finally, the adventitious shoots were inoculated on rooting medium, with 15 shoots in each group, for a total of 8 groups. The rooting medium for each group is shown in Table 3. The culture temperature was 25℃, the light intensity was 1800lx, and the light duration was 14h / d. The rooting rate was counted on the 6th day, and the results are shown in Table 3.

[0067] Table 3. Rooting status of adventitious buds

[0068] Serial Number Total number of seedlings inoculated (plants) Number of rooted seedlings (plants) Rooting rate (%) Rooting medium is 1 15 13 86.7 1 / 2MS + IAA 0.09 mg / L + AC 2.5 mg / L 2 15 14 93.3 1 / 2MS + IAA 0.11 mg / L + AC 3.5 mg / L 3 15 14 93.3 1 / 2MS + IAA 0.1 mg / L + AC 3 mg / L 4 15 10 66.7 MS + IAA 0.1 mg / L + AC 3 mg / L 5 15 9 60 1 / 2MS + IAA 0.08 mg / L + AC 3 mg / L 6 15 8 53.3 1 / 2MS + IAA 0.12 mg / L + AC 3 mg / L 7 15 9 60 1 / 2MS + IAA 0.1 mg / L + AC 2 mg / L 8 15 9 60 1 / 2MS + IAA 0.1 mg / L + AC 4 mg / L

[0069] As shown in Table 3, groups 1-3 used the rooting culture media of Examples 1-3 of this invention, with 15 seedlings inoculated. Each group had 13-14 successfully rooted seedlings, achieving a rooting rate of 93.3%. Group 4 replaced 1 / 2 MS culture medium with MS culture medium, resulting in only 10 successfully rooted seedlings, with a rooting rate of 66.7%. Groups 5 and 6 reduced or increased the amount of IAA compared to this invention, resulting in 8-9 successfully rooted seedlings, with rooting rates of 60% and 53.3%, respectively. Groups 7 and 8 reduced or increased the amount of AC compared to this invention, with 9 successfully rooted seedlings in each group, achieving a rooting rate of 60%. Therefore, the rooting rates of groups 4-8 were lower than those of this invention, demonstrating that the rooting culture media used in this invention has the best effect on promoting seedling rooting.

Claims

1. A culture medium for tissue culture of *Limonium bicolor*, characterized in that, It includes a primary culture medium, a proliferation and differentiation culture medium, and a rooting culture medium; the primary culture medium is used to induce callus tissue in explants; the proliferation and differentiation culture medium is used to proliferate the callus tissue to form adventitious shoots; and the rooting culture medium is used to root the adventitious shoots to form sterile seedlings. The primary culture medium was: MS + 6BA 0.4-0.6 mg / L + NAA 0.04-0.06 mg / L + AC 0.8-1.2 mg / L + 2,4-D 0.04-0.06 mg / L; The proliferation and differentiation medium was: modified MS + 6BA 0.09-0.11 mg / L + NAA 0.04-0.06 mg / L + AC 1.5-2.5 mg / L; The rooting medium was: 1 / 2 MS + IAA 0.09-0.11 mg / L + AC 2.5-3.5 mg / L; The modified MS is achieved by reducing the levels of macroelements ammonium nitrate and potassium nitrate in the MS medium by 1 / 3 mg / L and the levels of microelements boric acid by 1 / 5 mg / L.

2. The culture medium for tissue culture of *Limonium bicolor* as described in claim 1, characterized in that, The primary culture medium was: MS + 6BA 0.4 mg / L + NAA 0.04 mg / L + AC 0.8 mg / L + 2,4-D 0.04 mg / L; The proliferation and differentiation medium was: modified MS + 6BA 0.09 mg / L + NAA 0.04 mg / L + AC 1.5 mg / L; The rooting medium was: 1 / 2 MS + IAA 0.09 mg / L + AC 2.5 mg / L.

3. The culture medium for tissue culture of *Limonium bicolor* as described in claim 1, characterized in that, The primary culture medium was: MS + 6BA 0.6 mg / L + NAA 0.06 mg / L + AC 1.2 mg / L + 2,4-D 0.06 mg / L; The proliferation and differentiation medium was: modified MS + 6BA 0.11 mg / L + NAA 0.06 mg / L + AC 2.5 mg / L; The rooting medium was: 1 / 2 MS + IAA 0.11 mg / L + AC 3.5 mg / L.

4. The culture medium for tissue culture of *Limonium bicolor* as described in claim 1, characterized in that, The primary culture medium was: MS + 6BA 0.5 mg / L + NAA 0.05 mg / L + AC 1 mg / L + 2,4-D 0.05 mg / L; The proliferation and differentiation medium was: modified MS + 6BA 0.1 mg / L + NAA 0.05 mg / L + AC 2 mg / L; The rooting medium was: 1 / 2 MS + IAA 0.1 mg / L + AC 3 mg / L.

5. The culture medium for tissue culture of *Limonium bicolor* as described in claim 1, characterized in that, The primary culture medium was: MS + 6BA 0.5 mg / L + NAA 0.05 mg / L + AC 1 mg / L + 2,4-D 0.05 mg / L; The proliferation and differentiation medium was: modified MS + 6BA 0.09-0.11 mg / L + NAA 0.04-0.06 mg / L + AC 1.5-2.5 mg / L; The rooting medium was: 1 / 2 MS + IAA 0.09-0.11 mg / L + AC 2.5-3.5 mg / L.

6. The culture medium for tissue culture of *Limonium bicolor* as described in claim 1, characterized in that, The primary culture medium was: MS + 6BA 0.4-0.6 mg / L + NAA 0.04-0.06 mg / L + AC 0.8-1.2 mg / L + 2,4-D 0.04-0.06 mg / L; The proliferation and differentiation medium was: modified MS + 6BA 0.1 mg / L + NAA 0.05 mg / L + AC 2 mg / L; The rooting medium was: 1 / 2 MS + IAA 0.09-0.11 mg / L + AC 2.5-3.5 mg / L.

7. The culture medium for tissue culture of *Limonium bicolor* as described in claim 1, characterized in that, The primary culture medium was: MS + 6BA 0.4-0.6 mg / L + NAA 0.04-0.06 mg / L + AC 0.8-1.2 mg / L + 2,4-D 0.04-0.06 mg / L; The proliferation and differentiation medium was: modified MS + 6BA 0.09-0.11 mg / L + NAA 0.04-0.06 mg / L + AC 1.5-2.5 mg / L; The rooting medium was: 1 / 2 MS + IAA 0.1 mg / L + AC 3 mg / L.

8. A culture medium for tissue culture of *Limonium bicolor* as described in any one of claims 1-7, characterized in that, The culture medium is a sterile culture medium.

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