Tissue culture and rapid propagation method for dendrobium nobile

Abstract

The invention provides a tissue culture and rapid propagation method for dendrobium nobile, and belongs to the technical field of plant tissue culture. Comprising the following steps: taking a middle stem section of a pretreated new lateral bud as an explant, and sequentially inoculating into a bud induction culture medium for induction culture, a bud proliferation culture medium for proliferation culture and a rooting and seedling strengthening culture medium for rooting culture, the bud induction culture medium comprises the following components: plant hormones, potato powder, zeatin and activated carbon; the bud proliferation culture medium is prepared from the following components: plant hormones, potato powder, mashed apples and trehalose. The rooting and seedling strengthening culture medium comprises the following components: plant hormones, potato powder, mashed bananas and a yeast extract. According to the method disclosed by the invention, the 'Hooke summer end' is taken as a tissue culture object, the rooting rate can be obviously improved by adjusting the components of the culture medium in different stages, improving the budding rate of the explant and increasing the bud proliferation multiple, and the elongation of roots and the growth of plants are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for rapid propagation of Dendrobium officinale tissue culture. Background Art

[0002] Dendrobium officinale is a perennial epiphytic herbaceous plant of the genus Dendrobium in the Orchidaceae family, possessing high ornamental value. Based on flowering time and inflorescence location, it can be divided into spring and autumn dendrobiums. Autumn dendrobiums, which bloom in autumn, are both important cut flowers in the Orchidaceae family and a key ornamental potted plant. They are a major export cut flower for several Southeast Asian countries. In recent years, commercial production and breeding research of autumn dendrobiums have rapidly developed in my country. However, limited seedling supply has hindered large-scale production. Furthermore, the low propagation efficiency of superior hybridization plants has hindered the selection of new autumn dendrobium varieties adapted to the domestic environment. Currently, conventional tissue culture techniques for autumn dendrobiums suffer from low explant survival rates, low budding rates, and low multiplication times (<3.0). We aim to provide a tissue culture method for autumn dendrobiums to address these challenges. Summary of the Invention

[0003] The invention aims to provide a method for rapid propagation of Dendrobium officinale tissue culture, so as to solve the problems of low survival rate, low budding rate and low proliferation multiple (<3.0) in the Dendrobium officinale tissue culture process.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for rapid propagation of Dendrobium officinale tissue culture, comprising the following steps:

[0006] (1) The middle stem segment of the newly-formed lateral buds that had been pretreated was used as an explant and inoculated into a bud induction medium for induction culture to obtain clustered buds of Dendrobium officinale;

[0007] (2) dividing the clustered buds of Dendrobium officinale and inoculating them into a bud proliferation medium for proliferation culture to obtain robust clustered buds;

[0008] (3) inoculating the robust clustered buds into a rooting and seedling-strengthening medium for rooting and culture to obtain complete plants;

[0009] The bud induction culture medium comprises the following components: plant hormones, potato flour, zeatin and activated carbon;

[0010] The bud proliferation culture medium comprises the following ingredients: plant hormones, potato flour, applesauce and trehalose;

[0011] The rooting and seedling strengthening culture medium comprises the following components: plant hormones, potato flour, banana puree and yeast extract.

[0012] Preferably, the pretreatment in step (1) is as follows: after washing the middle stem segment of the newly formed lateral bud, the explant is treated with 75% ethanol for 30-40 seconds and 0.1% HgCl2 for 8-10 minutes, and then inoculated into the culture medium after surface disinfection.

[0013] Preferably, the composition of the bud induction medium in step (1) is: MS + 6-BA 2.8-3.2 mg / L + NAA 0.3-0.8 mg / L + potato flour 70-90 g / L + zeatin 0.8-1.2 mg / L + activated carbon 0.2-0.6 g / L.

[0014] Preferably, the induction culture conditions in step (1) are: culture at 23-27° C. for 25-30 days, a photoperiod of 15-16 h / d, a light intensity of 1800-2000 lx, and a relative humidity of 70%-80%.

[0015] Preferably, the composition of the bud proliferation culture medium in step (2) is: MS + 6-BA 1.8-2.2 mg / L + NAA 0.3-0.8 mg / L + potato flour 70-90 g / L + applesauce 40-50 g / L + trehalose 0.5-2 g / L.

[0016] Preferably, the conditions for the proliferation culture in step (2) are: culture at 23-27° C. for 30-35 days, a photoperiod of 15-16 h / d, a light intensity of 1800-2000 lx, and a relative humidity of 70%-80%.

[0017] Preferably, the composition of the rooting and seedling strengthening culture medium is: 1 / 2MS + NAA 0.3-0.8 mg / L + potato flour 70-90 g / L + banana puree 45-55 g / L + yeast extract 0.1-0.5 g / L.

[0018] Preferably, the conditions for rooting and seedling cultivation are: photoperiod 15-16h / d, light intensity 1800-2000lx, relative humidity 70%-80%, until the taproot of the intact plant reaches 3-5cm in length.

[0019] The present invention provides a method for rapid propagation of Dendrobium officinale tissue culture, wherein a bud induction culture medium in an induction culture stage uses a hormone combination with an optimal ratio, adds components such as potato flour and zeatin that can promote cell division to facilitate budding, and further adds activated carbon as a browning inhibitor to reduce browning and improve the survival rate; the bud proliferation culture medium provided by the application uses a hormone combination with an optimal ratio, adds apple puree and potato flour to promote proliferation and bud growth, and further adds trehalose to reduce oxidative damage to buds, which has a better effect than other combinations and can significantly increase the bud proliferation multiple; the addition of potato flour, banana puree and yeast extract to the rooting and seedling strengthening culture medium provided by the application can provide trace elements such as potassium and vitamins required for bud growth, supplement natural growth factors, significantly improve the rooting rate, and maintain the growth of seedlings while promoting rooting.

[0020] In summary, the present invention takes the autumn Dendrobium variety 'Reko Xiamo' as the object and provides a method for rapid propagation of autumn Dendrobium by tissue culture. By adjusting the components of the culture medium at different stages, the germination rate of the explant is improved, and the bud proliferation multiples are increased, the rooting rate can be significantly improved, and it is conducive to the elongation of the roots and the growth of the plant. DETAILED DESCRIPTION

[0021] The variety of Dendrobium used in this application for Mid-Autumn Festival is 'Reke Xiamo'.

[0022] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1

[0024] (1) A 10 cm section of the middle stem of a newly formed lateral bud was treated with 75% ethanol for 30 seconds, then rinsed with tap water for 30 minutes, and then treated with 0.1% HgCl2 for 8 minutes. After rinsing and surface disinfection, the stem was cut into 2-3 cm long segments with nodes and placed in a bud induction medium for induction. The buds were cultured at 25°C for 30 days with 14 hours of light per day at a light intensity of 2000 lx and a relative humidity of 80% to obtain clustered buds.

[0025] The bud induction medium is composed of: MS+6-BA3mg / L+NAA 0.5mg / L+potato flour 80g / L+zeatin 1.0mg / L+0.4g / L activated carbon;

[0026] (2) Cut the induced clustered buds into buds with two small buds, inoculate them into bud proliferation medium for proliferation culture, and culture them at 25°C for 30 days with 14 hours of light per day, a light intensity of 2000 lx, and a relative humidity of 80% to obtain robust clustered buds;

[0027] The composition of the bud proliferation medium is: MS+6-BA2 mg / L+NAA 0.5 mg / L+potato flour 80 g / L+apple paste 55 g / L+trehalose 1 g / L;

[0028] (3) cutting the robust clustered buds obtained in step (2) into single buds and inoculating them on a rooting and seedling-strengthening medium for cultivation at 25° C., with 14 hours of light per day, a light intensity of 2000 lx, and a relative humidity of 80%, until the taproot of the intact plant reaches a length of 3 cm and the plant height is greater than 4 cm;

[0029] The composition of the rooting and seedling strengthening culture medium is: 1 / 2MS+NAA0.5mg / L+potato flour 80g / L+banana puree 50g / L+yeast extract 0.3g / L.

[0030] Comparative Example 1

[0031] Different from Example 1, the formula of the bud induction medium in this comparative example is as follows:

[0032] A.MS + 6-BA 3mg / L + NAA 0.5mg / L + potato flour 80g / L + coconut water 10% (v / w) + 0.4g / L activated carbon;

[0033] B.MS + 6-BA 3mg / L + NAA 0.5mg / L + potato flour 80g / L + zeatin 1.0mg / L + polyvinylpyrrolidone (PVP) 50mg / L;

[0034] C.MS+6-BA3mg / L+NAA0.5 mg / L+potato flour80g / L+coconut water10% (v / w)+polyvinylpyrrolidone (PVP)50mg / L.

[0035] Comparative Example 2

[0036] Different from Example 1, the formula of the bud proliferation culture medium in this comparative example is as follows:

[0037] A.MS + 6-BA 2mg / L + NAA 0.5mg / L + potato flour 80g / L + banana puree 55g / L + trehalose 1g / L;

[0038] B.MS + 6-BA 2mg / L + NAA 0.5mg / L + potato flour 80g / L + applesauce 55g / L + zeatin 1.0mg / L;

[0039] C.MS+6-BA 2mg / L+NAA 0.5mg / L+potato flour 80g / L+banana puree 55g / L+zeatin 1.0mg / L.

[0040] Comparative Example 3

[0041] Different from Example 1, the formula of the rooting and seedling strengthening culture medium in this comparative example is as follows:

[0042] A.1 / 2MS + NAA 0.5mg / L + potato flour 80g / L + applesauce 50g / L + yeast extract 0.3g / L;

[0043] B. 1 / 2MS + NAA 0.5mg / L + potato flour 80g / L + banana puree 50g / L + coconut water 10% (v / w);

[0044] C. 1 / 2MS + NAA 0.5 mg / L + potato flour 80 g / L + applesauce 50 g / L + coconut water 10% (v / w).

[0045] Experimental Example 1: Verification of the effects of different culture media on the cultivation process of Dendrobium officinale

[0046] (1) Bud induction medium:

[0047] Hormone ratio screening: Using MS basal medium, a two-factor experiment was conducted using 6-BA (0, 1, 2, 3, 4, and 5 mg / L) and NAA (0, 0.5, 1, and 1.5 mg / L). Six bottles of 3 explants were used per treatment, replicated three times. The germination rate and germination multiple were recorded.

[0048] Table 1 Effects of different hormone ratios on adventitious bud induction

[0049]

[0050]

[0051] It can be seen that the optimal hormone ratio is 6-BA 3.0mg / L+NAA 0.5mg / L.

[0052] Optimization of potato flour concentration: Based on MS basal medium and the medium with the best hormone ratio screened out, 0, 20, 40, 60, 80, 100, and 120 g / L potato flour were added, and the germination rate and proliferation multiple were recorded.

[0053] Table 2 Effect of potato flour on proliferation

[0054]

[0055]

[0056] As shown in Table 2, adding 80g / L potato flour has the best effect.

[0057] Based on the method for rapid propagation of Dendrobium officinale tissue culture given in Example 1, bud induction culture was carried out using the bud induction medium formulated in Example 1 and Comparative Example 1. A total of four treatments were set up, with three groups of culture medium inoculated in each treatment and three explants inoculated in each group. The explant survival rate and budding rate were calculated 30 days after inoculation.

[0058] Table 3 Effects of other ingredients on proliferation

[0059] budding multiple Example 1 6.92 Comparative Example 1-A 6.84 Comparative Example 1-B 6.83 Comparative Example 1-C 6.88

[0060] As can be seen, the optimal hormone composition of the bud induction medium provided in this application is: 6-BA 3.0mg / L + NAA 0.5mg / L (Table 1), and the best effect is achieved when potato flour is added at 80g / L (Table 2). At the same time, the technical effects of bud induction medium with different components were verified (Table 3). The bud induction medium formula provided in this application is more effective than other component combinations, and can improve explant survival rate and budding rate.

[0061] (2) Bud proliferation medium:

[0062] Hormone ratio screening: MS basal medium was used, and a two-factor experiment of 6-BA (0, 1, 2, 3, 4, 5 mg / L) and NAA (0, 0.5, 1, 1.5 mg / L) was set up. 6 bottles × 3 explants were treated for 3 times, and the proliferation coefficient was calculated.

[0063] Table 4 Effects of different hormone ratios on adventitious bud proliferation

[0064]

[0065]

[0066] As can be seen from Table 4, the optimal hormone ratio is 6-BA2.0mg / L+NAA0.5mg / L.

[0067] Optimization of potato powder concentration: Based on MS basal medium and the medium with the best hormone ratio screened out, 0, 20, 40, 60, 80, 100, and 120 g / L potato powder were added, and the proliferation coefficient was recorded.

[0068] Table 5 The synergistic effect of potato flour on sprout proliferation

[0069] serial number Potato flour dosage g / L Average proliferation coefficient variance Analysis results 1 0 5.63 0.99 5.63±0.99 2 20 6.20 0.81 6.20±0.81 3 40 6.37 1.43 6.37±1.43 4 60 6.57 1.53 6.57±1.53 5 80 6.83 1.26 6.83±1.26 6 100 6.78 0.78 6.78±0.78 7 120 6.30 0.86 6.30±0.86

[0070] As shown in Table 5, adding 80g / L potato flour has the best effect.

[0071] Based on the method for rapid propagation of Dendrobium officinale tissue culture given in Example 1, proliferation culture was carried out using the bud proliferation culture medium formulated in Example 1 and Comparative Example 2. A total of four treatments were set up, with three groups of culture medium inoculated in each treatment, and three clustered buds in each group. The bud proliferation coefficient was calculated 35 days after inoculation.

[0072] Table 6 Effects of other components on adventitious bud proliferation

[0073] deal with proliferation coefficient Example 1 6.98±0.75 Comparative Example 2-A 6.87±0.81 Comparative Example 2-B 6.84±1.46 Comparative Example 2-C 6.84±0.64

[0074] It can be seen that in the bud proliferation culture medium given in the present application, the optimal hormone composition is: 6-BA 2.0 mg / L+NAA 0.5 mg / L. At the same time, the present invention also verified the technical effects of bud proliferation culture media with different components. The results showed that (Table 6) the bud proliferation culture medium formula provided by the present application is better than other combinations and can significantly improve the bud proliferation coefficient.

[0075] (3) Rooting and seedling strengthening plan:

[0076] Based on 1 / 2MS, a two-factor experiment was conducted to compare NAA (0, 0.1, 0.5, 1 mg / L) and IBA (0, 0.1, 0.5, 1 mg / L). Each treatment consisted of 6 bottles × 3 explants and was repeated 5 times. The rooting rate, plant height, number of leaves, stem diameter and root coefficient were calculated.

[0077] Table 7 Effects of different hormone ratios on rooting effect

[0078]

[0079] Based on the method for rapid propagation of Dendrobium officinale tissue culture given in Example 1, rooting culture was performed using the rooting and seedling strengthening culture medium formulated in Example 1 and Comparative Example 3. A total of four treatments were set up, with three groups of culture medium inoculated in each treatment, and three healthy single buds in each group. Growth indicators such as the number of roots, root length, and plant height of the Dendrobium officinale seedlings were counted 55 days after inoculation.

[0080] Table 8 Effects of other ingredients on rooting effect

[0081]

[0082] As shown in Table 7, the optimal hormone composition of the rooting and seedling strengthening medium provided by this application is: NAA 0.5 mg / L. In addition, the present invention also verified the effects of different rooting and seedling strengthening medium components on the growth indicators of Dendrobium seedlings. The results showed (Table 8) that the rooting and seedling strengthening medium formula provided by this application was more effective than other combinations, and promoted root elongation and plant growth.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of Dendrobium officinale tissue culture, characterized in that: The following steps are involved: (1) The middle stem segment of the pretreated new lateral bud was used as an explant and inoculated into a bud induction medium for induction culture to obtain clustered buds of Dendrobium officinale; (2) dividing the clustered buds of Dendrobium officinale and inoculating them into a bud proliferation medium for proliferation culture to obtain robust clustered buds; (3) inoculating the robust clustered buds into a rooting and seedling-strengthening medium for rooting and culture to obtain complete plants; The bud induction medium comprises the following components: plant hormones, potato flour, zeatin and activated carbon; The bud proliferation culture medium comprises the following ingredients: plant hormones, potato flour, applesauce and trehalose; The rooting and seedling strengthening culture medium comprises the following components: plant hormones, potato flour, banana puree and yeast extract.

2. The method according to claim 1, wherein: The pretreatment in step (1) is as follows: the middle stem segment of the newly formed lateral bud is washed, treated with 75% ethanol for 30 to 40 seconds and 0.1% HgCl2 for 8 to 10 minutes, and then inoculated into the culture medium after surface disinfection.

3. The method according to claim 2, wherein: The composition of the bud induction medium in step (1) is: MS+6-BA2.8-3.2 mg / L+NAA 0.3-0.8 mg / L+potato flour 70-90 g / L+zeatin 1.0 mg / L+activated carbon 0.2-0.6 g / L.

4. The method according to claim 3, wherein: The induction culture conditions in step (1) are: culture at 23-27° C. for 25-30 days, a photoperiod of 15-16 h / d, a light intensity of 1800-2000 lx, and a relative humidity of 70%-80%.

5. The method according to claim 4, wherein: The composition of the bud proliferation culture medium in step (2) is: MS+6-BA 1.8-2.2 mg / L+NAA 0.3-0.8 mg / L+potato flour 70-90 g / L+apple puree 40-50 g / L+trehalose 0.5-2 g / L.

6. The method according to claim 5, wherein: The conditions for the proliferation culture in step (2) are: culture at 23-27° C. for 30-35 days, light intensity of 15-16 h / d, light intensity of 1800-2000 lx, and relative humidity of 70%-80%.

7. The method according to claim 6, wherein: The composition of the rooting and seedling strengthening culture medium is: 1 / 2MS+NAA 0.3-0.8 mg / L+potato flour 70-90 g / L+banana puree 45-55 g / L+yeast extract 0.1-0.5 g / L.

8. The method according to claim 7, wherein: The conditions for rooting and seedling cultivation are: 23-27° C., 15-16 h / d of light, 1800-2000 lx of light intensity, and 70%-80% relative humidity, until the main root of the complete plant reaches a length of 3-5 cm.

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