Tissue culture and rapid propagation system and tissue culture and rapid propagation method of hippeastrum anephrii

Abstract

The invention provides a tissue culture and rapid propagation system and a tissue culture and rapid propagation method of hippeastrum 'Anephrite', and belongs to the technical field of tissue culture. The invention provides a group of hippeastrum 'Anephrite' tissue culture and rapid propagation culture media, and an'induction-proliferation-rooting 'integrated system is constructed based on an induction culture medium, a proliferation culture medium and a rooting culture medium. According to the method, an exclusive rapid propagation system is established for the double-petal variety 'Anephrite' for the first time, and the method has outstanding application value. When the culture medium is used for tissue culture of hippeastrum 'Anephre', double scales with bulb discs are used as explants, and after the explants are disinfected, induction culture, multiplication culture and rooting culture can be sequentially carried out. According to the method, the inductivity, the multiplication coefficient and the rooting rate are remarkably improved by regulating and controlling the content of hormones in each culture medium. Therefore, the method has the advantages of being high in propagation coefficient, good in seedling quality, stable in character and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture, and particularly relates to a tissue culture rapid propagation system and a tissue culture rapid propagation method of Hippeastrum 'Afre'. Background Art

[0002] As important vehicles for beautifying the environment and improving the quality of life, ornamental plants are attracting significant attention for their diversity and ornamental value. Among these numerous ornamental plants, Hippeastrum vittatum stands out for its unique charm. Also known as amaryllis, amaryllis, centaurium, and morning glory, this perennial bulbous plant of the genus Hippeastrum in the Amaryllidaceae family is characterized by large, vibrant flowers, distinctive shapes, rich colors, and slender leaves. Its ornamental value has generated a steady consumer demand.

[0003] Currently, compared to other horticultural ornamental plants, Hippeastrum 'Double Delight' offers both ornamental and economic benefits, making it widely used in landscape gardens, potted plants, and cut flowers. Its high ornamental value makes it highly sought after in the market, with demand increasing year by year. With the expansion of the flower market and rising quality requirements, coupled with the high potential market value of Hippeastrum, research on new varieties has also increased. 'Double Delight' (Hippeastrum 'Double Delight') is one of the best varieties of Hippeastrum. Its large, pure white flowers, thick petals, and beautiful shape create a high ornamental value. In the flower market, Hippeastrum 'Double Delight' is a popular choice among consumers, and demand is increasing. However, due to its inherent self-fertility, Hippeastrum 'Double Delight' is typically propagated through seed and bulb division, but this method has a low reproduction coefficient and cannot meet market demand. Summary of the Invention

[0004] The present invention provides a tissue culture rapid propagation system and a tissue culture rapid propagation method for Hippeastrum 'Afre', which can complete the efficient tissue culture of Hippeastrum 'Afre' and has the advantages of high propagation coefficient, good seedling quality, stable traits, etc., and can provide reliable technical support for commercial seedling production.

[0005] The present invention provides a group of tissue culture rapid propagation culture media for Hippeastrum 'Afre', comprising an induction culture medium, a proliferation culture medium and a rooting culture medium;

[0006] The induction medium is based on MS medium and further includes 1.0-2.0 mg / L 6-BA and 0.1 mg / L NAA;

[0007] The proliferation culture medium is based on MS culture medium and further includes 1.0-3.0 mg / L 6-BA and 0.2-0.6 mg / L NAA;

[0008] The rooting culture medium uses 1 / 2 MS culture medium as the basic culture medium and further comprises 0.5-1.5 mg / L IBA and 0.5-1.5 mg / L NAA.

[0009] In a preferred embodiment of the present invention, the induction medium is based on MS medium and further comprises 2.0 mg / L 6-BA, 0.1 mg / L NAA, 30.0 g / L sucrose and 7.0 g / L agar.

[0010] In a preferred embodiment of the present invention, the proliferation culture medium is based on MS culture medium and further comprises 2.0 mg / L 6-BA, 0.2 mg / L NAA, 30.0 g / L sucrose and 7.0 g / L agar.

[0011] In a preferred embodiment of the present invention, the rooting medium is based on 1 / 2 MS medium and further comprises 0.5 mg / L IBA, 1.5 mg / L NAA, 30.0 g / L sucrose and 7.0 g / L agar.

[0012] The present invention also provides a method for tissue culture of Hippeastrum 'Afre' using the tissue culture rapid propagation medium, comprising the following steps: processing the sterilized bulb into double scales with bulb discs as explants, and placing them on an induction medium to induce bulblets;

[0013] The induced bulblets are broken down into several small bud pieces with bulb discs and inoculated onto proliferation medium for proliferation culture;

[0014] The proliferated bulblets were separated and inoculated onto rooting medium for rooting culture to obtain complete tissue culture seedlings of Hippeastrum 'Afre'.

[0015] In a preferred embodiment of the present invention, the disinfection comprises sequentially soaking in a 75% by volume ethanol aqueous solution for 30 seconds, and then transferring to a 1.0-2% by volume sodium hypochlorite aqueous solution and soaking for 15-18 minutes.

[0016] In a preferred embodiment of the present invention, the temperature of the induction culture is 25° C., the photoperiod is 14 hours of light per day, and the light intensity is 1500 to 2000 lux.

[0017] In a preferred embodiment of the present invention, after the leaves on the induced small bulbs are removed, each bulb is decomposed into 2 to 4 small bud blocks with bulb disks.

[0018] In a preferred embodiment of the present invention, the temperature of the proliferation culture is 25° C., the photoperiod is 14 hours of light per day, and the light intensity is 1500 to 2000 lux.

[0019] In a preferred embodiment of the present invention, the rooting culture temperature is 25° C., the photoperiod is 14 hours of light per day, and the light intensity is 1500 to 2000 lux.

[0020] Beneficial Effects: The present invention provides a set of tissue culture rapid propagation culture media for Amaryllis 'Afre' and constructs an integrated "induction-proliferation-rooting" system based on induction, proliferation, and rooting media. When configuring each culture medium, the present invention adjusts the culture medium type according to the growth stage, using a fully nutrient MS culture medium for the initial induction and proliferation stages and a 1 / 2 MS culture medium for the rooting stage. This meets the physiological needs of the plant and can effectively promote growth and development at all stages. This invention establishes a dedicated rapid propagation system for the double-flowered variety 'Afre' for the first time, with outstanding application value.

[0021] When the culture medium is used to carry out tissue culture of Amaryllis 'Afre', double scales with bulb discs are used as explants. The selection of the explants focuses more on the activity of the thin-walled cells at the base of the bulb, which avoids the problem of unstable receptacle induction rate, while reducing the damage to the mother bulb, and the operation is simpler and more efficient. After the explants are disinfected, induction culture, proliferation culture and rooting culture can be carried out in sequence. In the embodiments of the present invention, the reproduction efficiency is significantly improved by regulating the content of hormones in each culture medium. According to statistics, the induction rate can be as high as 63.3%, the proliferation coefficient reaches 5.02 times, and the rooting rate is as high as 100%. Therefore, the method of the present invention has the advantages of high reproduction coefficient, good seedling quality, stable traits, etc., and can provide reliable technical support for commercial seedling production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The field photos of the adventitious buds induced by double scales under different treatments, A: T0 treatment, B: T1 treatment, C: T2 treatment, D: T3 treatment, E: T4 treatment;

[0023] Figure 2 This is a statistical chart showing the effects of different treatment combinations on bulb induction of Amaryllis ‘Afre’;

[0024] Figure 3 This is a field photo of the proliferation of small bulbs of Amaryllis 'Afre'. In the figure, A: T1 treatment, B: T2 treatment, C: T3 treatment, D: T4 treatment, E: T5 treatment, F: T6 treatment, G: T7 treatment, H: T8 treatment, I: T9 treatment;

[0025] Figure 4 This is a statistical chart showing the effects of different treatment combinations on the proliferation of Hippeastrum 'Afre' bulbs;

[0026] Figure 5The color change of the root system of Amaryllis 'Afre' bulblets, in the figure A: 7 days of T4 treatment, B: 14 days of T4 treatment, C: 21 days of T4 treatment, D: 28 days of T4 treatment;

[0027] Figure 6 This is the result diagram of the effect of different treatment combinations on the rooting of Hippeastrum 'Afre' bulbs;

[0028] Figure 7 This is a diagram of the growth of Hippeastrum 'Afre' small bulblets in rooting medium. In the figure, A and a: T1 treatment, B and b: T2 treatment, C and c: T3 treatment, D and d: T4 treatment, E and e: T5 treatment, F and f: T6 treatment, G and g: T7 treatment, H and h: T8 treatment, I and i: T9 treatment. DETAILED DESCRIPTION

[0029] The present invention provides a group of tissue culture rapid propagation culture media for Hippeastrum 'Afre', comprising an induction culture medium, a proliferation culture medium and a rooting culture medium;

[0030] The induction medium is based on MS medium and further includes 1.0-2.0 mg / L 6-BA and 0.1 mg / L NAA;

[0031] The proliferation culture medium is based on MS culture medium and further includes 1.0-3.0 mg / L 6-BA and 0.2-0.6 mg / L NAA;

[0032] The rooting culture medium uses 1 / 2 MS culture medium as the basic culture medium and further comprises 0.5-1.5 mg / L IBA and 0.5-1.5 mg / L NAA.

[0033] The induction culture medium of the present invention is based on MS culture medium, which contains 6-BA, NAA, sucrose and agar, wherein the concentration of 6-BA is 1.0-2.0 mg / L, such as 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L or 2.0 mg / L; the concentration of NAA is 0.1 mg / L; the concentration of sucrose is 30.0 g / L, and the concentration of agar is 7.0 g / L. In the embodiment of the present invention, it was found that when 2.0 mg / L 6-BA and 0.1 mg / L NAA were combined, the efficiency of inducing small bulbs was the highest, with an induction rate of 63.3%. When inducing using the induction culture medium of the present invention, adventitious buds can be directly induced, skipping the callus stage, and significantly shortening the culture cycle.

[0034] The proliferation culture medium of the present invention is based on MS culture medium, which contains 6-BA, NAA, sucrose and agar, wherein the concentration of 6-BA is 1.0 to 3.0 mg / L, such as 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2.0 mg / L, 2.1 mg / L , 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, 2.5 mg / L, 2.6 mg / L, 2.7 mg / L, 2.8 mg / L, 2.9 mg / L or 3.0 mg / L; the concentration of NAA is 0.2-0.6 mg / L, such as 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L or 0.6 mg / L; the concentration of sucrose is 30.0 g / L, and the concentration of agar is 7.0 g / L. In the examples of the present invention, it was found that when 2.0 mg / L 6-BA and 0.2 mg / L NAA were combined in the proliferation medium, the proliferation efficiency could reach 5.02 times.

[0035] The rooting medium of the present invention uses 1 / 2 MS medium as a basic medium, which contains IBA, NAA, sucrose and agar, wherein the concentration of IBA is 0.5-1.5 mg / L, such as 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L or 1.5 mg / L; the concentration of NAA is 0.5-1.5 mg / L, such as 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L or 1.5 mg / L; the concentration of sucrose is 30.0 g / L, and the concentration of agar is 7.0 g / L. In the examples of the present invention, a combination of 0.5 mg / L IBA and 1.5 mg / L NAA in the rooting medium achieved a 100% rooting rate, with an average root count of 5.2 roots per plant, demonstrating a robust, well-developed root system. Furthermore, the combination of high NAA concentration (1.5 mg / L) and low IBA concentration (0.5 mg / L) exhibited a significant synergistic effect, reversing the conventional wisdom that high NAA concentrations can easily lead to root deformities.

[0036] When preparing the induction medium, proliferation medium and rooting medium of the present invention, a basic medium, sucrose as a carbon source, agar as a solidifying agent and plant hormones of various concentrations are mixed, the pH is adjusted to 5.8, and then high-pressure steam sterilization is performed at 121° C. for 20 minutes. The culture medium is obtained after cooling and solidification.

[0037] The present invention also provides a method for tissue culture of Hippeastrum 'Afre' using the tissue culture rapid propagation medium, comprising the following steps: processing the sterilized bulb into double scales with bulb discs as explants, and placing them on an induction medium to induce bulblets;

[0038] The induced bulblets are broken down into several small bud pieces with bulb discs and inoculated onto proliferation medium for proliferation culture;

[0039] The proliferated bulblets were separated and inoculated onto rooting medium for rooting culture to obtain complete tissue culture seedlings of Hippeastrum 'Afre'.

[0040] The present invention selects the lower base of the bulb of Amaryllis 'Afre', which is healthy and pest-free, with a bulb disk as experimental material. First, the surface sand is removed and the outer layer of dry and brown scales is peeled off. Then, a scalpel is used to cut off the top of the bulb and the root system of the bulb, and the bulb is divided into four equal parts longitudinally. Then, a detergent solution is used to clean the surface, and after rinsing with running water for 1 hour, excess water is absorbed with filter paper. Finally, the processed bulb is placed on a clean bench as an explant.

[0041] The explant is disinfected under sterile conditions, and the disinfection includes: disinfection with a 75% by volume ethanol aqueous solution for 30 seconds, followed by disinfection with a 2% by volume NaClO aqueous solution for 18 minutes, and after the ethanol disinfection, shaking and washing with sterile water for 3 to 4 times before subsequent NaClO disinfection. The present invention is to shake and wash with sterile water 5 times after the NaClO disinfection. After the disinfection is completed, the moisture on the surface of the explant is absorbed with sterile filter paper, and then the upper 2 / 3 of the mother bulb is completely removed with a scalpel by a cross-cutting method, retaining the base scales with the bulb disk, and cutting the scales into small squares of about 1.0 cm × 1.0 cm in layers to obtain double scales with bulb disks, ready for inoculation.

[0042] The invention inoculates the explants onto an induction culture medium for induction culture at a temperature of 25° C., a photoperiod of 14 hours per day, and a light intensity of 1500 to 2000 lux. After the induction, adventitious buds can be directly obtained from the explants.

[0043] The method removes leaves from the induced small bulbs, decomposes each bulb into 2 to 4 small bud blocks with bulb disks, and then inoculates the small bud blocks into a proliferation culture medium for proliferation culture. The proliferation culture temperature is 25°C, the photoperiod is 14 hours of light / day, and the light intensity is 1500 to 2000 lux.

[0044] The present invention separates the bulblets grown from the proliferation culture and inoculates them into a rooting medium, wherein the diameter of the separated bulblets is required to be ≥0.8 cm. The rooting culture temperature of the present invention is 25°C, the photoperiod is 14 hours of light per day, and the light intensity is 1500-2000 lux.

[0045] To further illustrate the present invention, the tissue culture and rapid propagation system and method of Hippeastrum 'Afrère' provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0046] The bulbs of Amaryllis 'Afre' used in the examples of this invention were purchased from Heidi's Garden, the official flagship store on Taobao. The bulbs had a circumference ranging from 18 cm to 22 cm. MS and 1 / 2 MS were used as the basal culture medium. 30 g / L sucrose was added as a carbon source, and 7 g / L agar was used as a solidifying agent. After adjusting the pH to 5.8, the medium was autoclaved at 121°C for 20 min. The culture environment was maintained at a constant temperature of 25°C, with a 14-hour photoperiod and a light intensity of 1500–2000 lux.

[0047] In the embodiment of the present invention, the data statistical analysis method used is as follows:

[0048] Induction rate = number of induced double-scale adventitious bud explants / number of inoculated double-scale explants * 100%;

[0049] Contamination rate = number of contaminated double-scale explants after inoculation / number of inoculated double-scale explants * 100%;

[0050] Rooting rate = number of rooted plants / number of cultured bulblets;

[0051] Increase coefficient = number of bulblets after increase / number of inoculated bulblets;

[0052] The basic data in the example results were processed and counted by Excel software, and then variance analysis (ANOVA) was completed with the help of SPSS statistical software. The differences between the groups were verified by Duncan's multiple comparison method, and the test levels were set at α=0.05 and α=0.01.

[0053] Example 1

[0054] 1.1 Explant processing

[0055] The lower base of the healthy and pest-free bulbs of Zephyranthes 'Afreya' with the bulb disc was selected as the experimental material. After strict screening, the following pretreatment procedures were adopted: first, remove the surface sand, peel off the outer layer of dry and brown scales; then use a scalpel to remove the bulb tip and the bulb clinging root system, and divide the bulb longitudinally into four equal parts; then use a dishwashing liquid solution for surface cleaning, rinse with running water for 1 h, and then use filter paper to absorb excess water; finally, place the treated bulbs on the clean bench for standby.

[0056] 1.2 Disinfection of explants

[0057] During the disinfection treatment of explants under sterile conditions, 75% ethanol disinfection for 30 s + 2% NaClO disinfection for 18 min were selected, and then the explants were washed repeatedly with sterile water before being inoculated into the induction medium. After disinfection, the water on the surface of the explants was absorbed with sterile filter paper, and then the upper 2 / 3 of the mother bulb scales were removed with a scalpel, leaving the base scales with the bulb disc. The scales were cut into small squares of about 1.0 cm x 1.0 cm, and the double scales with the bulb disc were obtained for inoculation.

[0058] 1.3 Induction culture of small bulbs

[0059] The induction medium was MS basic medium with the addition of sucrose 30 g / L, agar 7 g / L, and pH 5.8. Five treatments were set up with different concentrations of 6-BA (1.0, 2.0, 3.0, 4.0 mg / L) and a fixed concentration of NAA 0.1 mg / L, and MS medium without any hormone was used as a control. Ten bottles were inoculated for each treatment (one explant per bottle), and the experiment was repeated three times. After 30 days, the induction rate was calculated and the growth of the induced small bulbs was observed.

[0060] Table 1 Design table for induction culture of Zephyranthes 'Afreya' bulbs

[0061]

[0062] After inoculation, the explants of Zephyranthes 'Afreya' were cultured and observed in the tissue culture room. It was observed that the explants turned red after 3-5 days of inoculation, and according to the different treatment groups, a small part showed signs of contamination, while some explants turned green and the double scales began to open. After 14 days of inoculation, one or more small yellow bumps appeared between the double scales, and after 21 days, the small yellow bumps formed new small bulbs, while some double scales formed callus, and the time for bud induction from callus was longer (about 30 days), which may be related to the process of dedifferentiation and redifferentiation of explant cells. Figure 1

[0063] From Table 2 and Figure 2 ​It can be seen that the concentration of 6-BA has a significant effect on the induction of bulblets in Amaryllis 'Afre'. The synergistic effect of low concentrations of 6-BA (1.0-2.0 mg / L) and NAA (0.1 mg / L) may activate the cytokinin signaling pathway, promoting the transformation of parenchyma cells at the base of the bulb disc into meristematic cell clusters, thereby inducing adventitious bud formation. This results in an increase in the induction rate of bulblets with increasing concentration, and bulblets form faster and grow better. However, when the 6-BA concentration is increased to 3.0-4.0 mg / L, the excessive cytokinin may disrupt the endogenous hormone balance and inhibit the polar transport of auxin, leading to callus formation rather than direct organogenesis. This results in a decrease in the induction rate of bulblets with increasing concentration, as well as slower bulblet formation and moderate growth. Therefore, treatment T2 (MS + 2.0 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 6.0 g / L agar) was ultimately selected as the optimal induction medium, achieving an induction rate of 63.3% and excellent growth. This suggests that the synergistic effect of moderate cytokinin and low auxin concentrations can both promote cell proliferation and maintain orderly organ differentiation.

[0064] Table 2 Effects of different treatments on bulb induction of Amaryllis 'Afre'

[0065] deal with Number of explants Number of buds Induction rate / % Growth potential <![CDATA[T0]]> 30 15 50.0 Bulblets form slowly and grow moderately [T1] 30 17 56.7 Small bulbs form quickly and grow well <![CDATA[T2]]> 30 19 63.3 The bulblets form quickly and grow very well <![CDATA[T3]]> 30 14 46.6 Small bulbs form slowly and grow well <![CDATA[T4]]> 30 9 42.8 Small bulbs form slowly and grow moderately

[0066] 1.4 Proliferation and culture of small bulbs

[0067] Bulblets of the Hippeastrum 'Afre' cultivar obtained through primary induction culture were used as experimental material. The bulblets were pretreated, young leaves removed, and then divided into 2-4 small buds with bulb discs according to individual size. The culture medium was based on MS supplemented with 30 g / L sucrose and 7.0 g / L agar, and the pH was adjusted to 5.8. Nine treatment groups were set up, each containing 15 bottles of culture medium, each inoculated with three small bulb discs. After 30 days of culture, the culture medium formulation was optimized by measuring the proliferation coefficient and observing the growth of the bulblets.

[0068] Table 3 Design table for the proliferation and culture of Hippeastrum 'Afre' bulbs

[0069]

[0070] The bulbs induced in the first generation were cut into 2-4 pieces according to their size and inoculated into 9 different proliferation media. After 7 days, some of the treated small bulbs showed a tendency to grow taller, while some of the treated scales just began to sprout and the scales near the bulb disk showed white. Around 21 days, the inoculated small bulbs basically formed small plants ( Figure 3 ).

[0071] The experiment adopted a completely randomized block design, setting 3 6-BA concentration gradients (1.0, 2.0, 3.0 mg / L) and 3 NAA concentration gradients (0.2, 0.4, 0.6 mg / L) for a total of 9 treatment combinations, and each treatment was repeated 3 times. The experimental results show (Table 4, Figure 4 ), with the increase of 6-BA concentration, the proliferation coefficient showed a trend of first increasing and then decreasing. The proliferation coefficient was the best when 6-BA was 2 mg / L, followed by 1 mg / L and 3 mg / L. The effect of NAA concentration was also significant. The best effect was achieved when the NAA concentration was 0.2 mg / L, followed by 0.4 mg / L and 0.6 mg / L. The proliferation coefficient showed a downward trend. Comprehensive analysis showed that the combination of 6-BA 2 mg / L and NAA 0.2 mg / L (T4 treatment) performed best, with a proliferation coefficient of 5.02±0.33, which was significantly higher than other combinations, namely MS+6-BA 2.0 mg / L+NAA 0.2 mg / L+sucrose 30 g / L+agar 7.0 g / L. This optimal ratio may be because it regulates the balance of endogenous hormones, which not only promotes cell division and bud primordium formation, but also avoids the occurrence of deformed buds caused by hormone imbalance.

[0072] The range analysis (Table 5) showed that the regulatory effect of NAA concentration (R = 13.0) on the proliferation effect was slightly higher than that of 6-BA (R = 12.2). The results of variance analysis (Table 6) further confirmed that the effects of 6-BA (F = 12.37, P < 0.01) and NAA (F = 14.05, P < 0.01) on the proliferation coefficient reached an extremely significant level.

[0073] Table 4 Effects of different treatments on the proliferation of Hippeastrum 'Afre' bulbs

[0074] deal with 6-BA (mg / L) NAA (mg / L) Total number of explants / (pieces) Total number of bulblets proliferated (number) Value-added coefficient <![CDATA[T1]]> 1.0 0.2 45 170 <![CDATA[3.77±0.21 b ]]> [T2] 1.0 0.4 45 150 <![CDATA[3.34±0.18 bc ]]> [T3] 1.0 0.6 45 124 <![CDATA[2.75±0.15 cd ]]> <![CDATA[T4]]> 2.0 0.2 45 226 <![CDATA[5.02±0.33 a ]]> <![CDATA[T5]]> 2.0 0.4 45 154 <![CDATA[3.42±0.20 bc ]]> <![CDATA[T6]]> 2.0 0.6 45 124 <![CDATA[2.75±0.16 cd ]]> <![CDATA[T7]]> 3.0 0.2 45 165 <![CDATA[3.66±0.25 bc ]]> <![CDATA[T8]]> 3.0 0.4 45 132 2.93 ± 0.17 bcd ]] <![CDATA[T9]]> 3.0 0.6 45 100 <![CDATA[2.22±0.12 d ]]>

[0075] Note: Different lowercase letters indicate significant differences among treatments (P<0.05, Duncan method).

[0076] Table 5 Main effect / range analysis of the proliferation culture of Amaryllis 'Afre' bulbs

[0077]

[0078] Note: X is the average value of each concentration level; R is the range.

[0079] Table 6 Variance analysis of the proliferation coefficient of Hippeastrum 'Afre' bulblets under different treatments

[0080] Source of variation sum of squares degrees of freedom mean square F ratio P critical value 6-BA concentration 298.44 2 149.22 12.37 ** NAA concentration 338.89 2 169.45 14.05 ** error 216.34 18 12.02 total 853.67 26

[0081] Note: ** indicates extremely significant differences.

[0082] 1.5 Rooting culture of bulblets

[0083] Bulblets (≥0.8 cm in diameter) of subcultured Amaryllis 'Afre' were isolated and inoculated into different rooting media. The basal medium used was 1 / 2 MS, containing 30 g / L sucrose, 7.0 g / L agar, and a pH of 5.8. Nine treatments were set up, with varying concentrations of IBA (0.5, 1.0, and 1.5 mg / L) and NAA (0.5, 1.0, and 1.5 mg / L) added as shown in Table 7. Each treatment group was inoculated with 15 bottles, with three plants in each. After 30 days, root growth was observed and rooting rates were calculated to identify the optimal rooting medium.

[0084] Table 7 Design table for rooting culture of Amaryllis 'Afre' bulbs

[0085]

[0086] The proliferated bulblets of Amaryllis ‘Afre’ were inoculated into rooting medium with 9 different hormone ratios. Around 14 days, cylindrical roots gradually grew from the bottom of the bulblets. Around 21 days, the roots of the bulblets basically turned from milky white to green, reflecting the transformation process from storage organs to functional organs, as shown in the attached figure. Figure 5 .

[0087] During the rooting culture stage, experimental data showed that the ratio of different concentrations of IBA and NAA had a significant effect on the rooting effect of Amaryllis 'Afre' ( Figure 6 and Figure 7). Table 8 shows that treatment T3 (1 / 2MS+IBA 0.5mg / L+NAA 1.5mg / L) performed the best, with a rooting rate of 100%, and the largest number of roots (141), significantly better than other treatment groups. Analysis found that the treatment group with low concentration of IBA (0.5mg / L) had the highest average rooting rate (66.7%), while as the IBA concentration increased to 1.0mg / L and 1.5mg / L, the rooting rate decreased to 47.4% and 52.6%, respectively. In terms of NAA concentration, the treatment group with 1.5mg / L had the highest average rooting rate (63.7%), but it needed to be combined with low concentration of IBA (0.5mg / L) to achieve the best effect. It is worth noting that the combination of high concentration of IBA (1.5mg / L) and high concentration of NAA (1.5mg / L) (T9) had a rooting rate of only 53.3%, indicating that excessive auxin will have an inhibitory effect. The range analysis (Table 9) showed that the range of IBA (R = 19.3) was slightly higher than that of NAA (R = 18.5), indicating that IBA played a dominant role in the rooting process. The results of the variance analysis (Table 10) showed that the effects of IBA (F = 15.83, P < 0.01) and NAA (F = 14.86, P < 0.01) on the rooting rate were both extremely significant.

[0088] Table 8 Effects of different treatments on the rooting of Amaryllis 'Afre' bulbs

[0089]

[0090]

[0091] Note: Different lowercase letters indicate significant differences among treatments (P<0.05, Duncan method).

[0092] Table 9 Main effect / range analysis of rooting culture of Amaryllis 'Afre' bulbs

[0093]

[0094] Note: X is the average value of each concentration level; R is the range.

[0095] Table 10 Variance analysis of different plant growth regulators on rooting rate

[0096] Source of variation sum of squares degrees of freedom mean square F ratio P critical value IBA concentration 412.56 2 206.28 15.83 ** NAA concentration 387.22 2 193.61 14.86 ** error 234.78 18 13.04 total 1034.56 26

[0097] Note: ** indicates extremely significant differences.

[0098] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A tissue culture rapid propagation medium for Hippeastrum 'Afre', characterized in that: Including induction medium, proliferation medium and rooting medium; The induction medium is based on MS medium and further includes 1.0-2.0 mg / L 6-BA and 0.1 mg / L NAA; The proliferation culture medium is based on MS culture medium and further includes 1.0-3.0 mg / L 6-BA and 0.2-0.6 mg / L NAA; The rooting culture medium uses 1 / 2 MS culture medium as the basic culture medium and further comprises 0.5-1.5 mg / L IBA and 0.5-1.5 mg / L NAA.

2. The tissue culture rapid propagation medium according to claim 1, wherein The induction culture medium is based on MS culture medium and further comprises 2.0 mg / L 6-BA, 0.1 mg / L NAA, 30.0 g / L sucrose and 7.0 g / L agar.

3. The tissue culture rapid propagation medium according to claim 1, wherein The proliferation culture medium is based on MS culture medium and further comprises 2.0 mg / L 6-BA, 0.2 mg / L NAA, 30.0 g / L sucrose and 7.0 g / L agar.

4. The tissue culture rapid propagation medium according to claim 1, wherein The rooting medium is based on 1 / 2 MS medium and further comprises 0.5 mg / L IBA, 1.5 mg / L NAA, 30.0 g / L sucrose and 7.0 g / L agar.

5. A method for tissue culture of Hippeastrum 'Afrère' using the tissue culture rapid propagation medium according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: processing the sterilized bulb into double scales with bulb discs as explants, and placing the double scales on an induction culture medium to induce small bulbs; The induced bulblets are broken down into several small bud pieces with bulb discs and inoculated onto proliferation medium for proliferation culture; The proliferated bulblets were separated and inoculated onto rooting medium for rooting culture to obtain complete tissue culture seedlings of Hippeastrum 'Afre'.

6. The method according to claim 5, characterized in that The disinfection comprises soaking in an ethanol aqueous solution with a volume percentage of 75% for 30 seconds, and then transferring to an aqueous sodium hypochlorite solution with a volume percentage of 1.0-2% and soaking for 15-18 minutes.

7. The method according to claim 5, characterized in that The induction culture temperature is 25° C., the light cycle is 14 hours per day, and the light intensity is 1500 to 2000 lux.

8. The method according to claim 5, characterized in that After removing the leaves from the induced bulblets, each bulb was broken down into 2 to 4 small bud blocks with bulb disks.

9. The method according to claim 8, characterized in that The temperature of the proliferation culture is 25° C., the light cycle is 14 hours per day, and the light intensity is 1500 to 2000 lux.

10. The method according to claim 5, characterized in that: The rooting culture temperature is 25° C., the photoperiod is 14 hours of light per day, and the light intensity is 1500 to 2000 lux.

Images