A method to accelerate the early morphological development of Japanese larch somatic seedlings

Abstract

The present invention relates to the field of plant tissue culture. To address the current problems of low embryoid germination rate, low seedling success rate, and an imperfect somatic embryogenesis system in Japanese larch tissue culture, the present invention provides a method for accelerating early morphological development of Japanese larch. The method comprises the steps of first germinating and culturing Japanese larch embryoids under dark conditions and then continuing germination and culturing under light conditions. The method accelerates early morphological development of Japanese larch and provides technical support for commercial seedling cultivation and genetic improvement of Japanese larch.

Description

Technical Field

[0001] The invention relates to the field of plant tissue culture, in particular to a method for accelerating the early morphological construction of Japanese larch somatic embryo seedlings. Background Art

[0002] Japanese larch (Larix kaempferi (Lamb.) Carr.) is a tree of the genus Larix in the family Pinaceae, widely distributed in temperate and cold temperate zones. It has the characteristics of rapid early growth, few pests and diseases, short rotation period, rapid forest formation, and strong stress resistance. Traditional larch breeding has problems such as difficulty in maintaining excellent traits and long growth cycle, which greatly restricts the large-scale utilization of excellent Japanese larch germplasm resources. The somatic embryogenesis technology of Japanese larch has the advantages of strong genetic stability and high reproduction coefficient, and has become the most promising asexual reproduction technology at present. However, it also has problems such as low embryoid germination rate, low seedling rate, and imperfect somatic embryogenesis system. Summary of the Invention

[0003] The present invention aims to solve the problems of low embryoid germination rate, low seedling rate and imperfect somatic embryogenesis system in the current tissue culture of Japanese larch, and provides a method for accelerating the early morphological establishment of Japanese larch somatic embryo seedlings.

[0004] The method for accelerating the early morphological formation of Japanese larch embryos of the present invention comprises the steps of first germinating and culturing the Japanese larch embryos under dark conditions and then switching to light conditions for further germination and cultivation.

[0005] In the above method, the germination culture time under dark conditions is preferably 14 days.

[0006] In the above method, the dark condition is at a temperature of 22°C.

[0007] In the above method, the illumination conditions include an illumination duration of 14 h / d, an illumination intensity of 1000 lx, and a temperature of 24±2°C.

[0008] In the above method, the embryoid is a type I embryoid, that is, an embryoid with 4-10 cotyledons and a symmetrical embryonic axis.

[0009] In the above method, the Japanese larch can be selected as Japanese larch embryonic callus.

[0010] The present invention also provides a Japanese larch propagation method, comprising the following steps: inoculating Japanese larch embryonic callus onto a proliferation culture medium, performing proliferation culture under dark conditions to obtain proliferated embryonic callus, then transferring the culture to a maturation culture medium, performing maturation culture under dark conditions to obtain embryoids, and finally inoculating the embryoids onto a germination culture medium, first performing germination culture under dark conditions, and then switching to light conditions to continue germination culture to obtain Japanese larch. Early somatic embryos .

[0011] In the above method, the Japanese larch can be selected as Japanese larch embryonic callus.

[0012] In the above method, the proliferation medium is a medium using BM medium as the basal medium, with an NAA content of 0.5 mg / L, a 6-BA content of 0.05 mg / L, a KT content of 0.05 mg / L, a glutamine content of 1000 mg / L, an acid-hydrolyzed casein content of 500 mg / L, a sucrose content of 25 g / L, and an agar content of 4 g / L.

[0013] In the above method, the maturation medium is a medium using MLV medium as the basal medium, with an ABA content of 60 μM, an IBA content of 1 μM, an activated carbon content of 10 g / L, a sucrose content of 0.2 M, and an agar content of 4 g / L.

[0014] In the above method, the germination medium is a medium using WPM medium as the basic medium, with a sucrose content of 20 g / L, an activated carbon content of 2.0 g / L, a VB1 content of 3.0 mg / L, and an agar content of 4 g / L.

[0015] In the above method, the proliferation culture is subcultured every 13 days.

[0016] In the above method, the mature culture is cultured for 55 days.

[0017] In the above method, the germination culture time under dark conditions is preferably 14 days.

[0018] In the above method, the temperature of the dark condition is 22°C.

[0019] In the above method, the illumination conditions include an illumination duration of 14 h / d, an illumination intensity of 1000 lx, and a temperature of 24±2°C.

[0020] In the above method, the number of cotyledons of the embryoid body inoculated on the germination medium is 4-10, and the embryonic axis has a symmetrical morphology.

[0021] This study uses Japanese larch as a material to construct a somatic embryo regeneration system for Japanese larch. This system, utilizing germination and culture in darkness, accelerates the early morphological development of Japanese larch somatic embryos, providing technical support for commercial seedling cultivation and genetic improvement of Japanese larch. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The following are photos of embryonic callus cultured on each proliferation culture in the preliminary experiment 1 of the present invention. Figure 1 The culture medium used in A is 1 / 2LM proliferation medium, and the scale length is 500 μm; Figure 1 The culture medium used in B is 1 / 2MLV proliferation medium, and the scale length is 500 μm; Figure 1 The culture medium used in C is 1 / 2DCR proliferation medium, and the scale length is 500 μm; Figure 1 The culture medium used for D is BMⅡ proliferation medium, and the scale length is 750μm.

[0023] Figure 2 This is the relationship between the proliferation rate of embryogenic callus cultured on the proliferation culture screened in preliminary experiment 1 of the present invention and the culture time.

[0024] Figure 3 These are photos of mature embryoid bodies under light and dark conditions in Preliminary Experiment 2 of the present invention. Figure 3 A represents maturation on MLV maturation medium under light conditions, with a scale bar length of 750 μm; Figure 3 B was matured in the dark on MLV maturation medium. Scale bar length: 750 μm.

[0025] Figure 4 These are photos of embryoid bodies cultured in the dark on different maturation media in preliminary experiment 2 of the present invention for 55 days. Figure 4 The culture medium used in A is BMⅢ maturation medium, and the scale length is 750 μm; Figure 4 The medium used in B is MLV maturation medium, and the scale length is 750 μm; Figure 4 The culture medium used for C is 1 / 2MLV maturation medium, and the scale length is 750 μm; Figure 4 The culture medium used for D is BM IV maturation medium, and the scale length is 750 μm.

[0026] Figure 5 This is the hydroponic germination process of mature zygotic embryos of Japanese larch in Example 1 of the present invention.

[0027] Figure 6 This is the early somatic embryo seedling in Example 1 of the present invention.

[0028] Figure 7 These are photos of 20 early somatic embryo seedlings in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0030] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0031] The abbreviations and full names of the plant hormones used in the culture medium in the following examples are shown in Table 1.

[0032] Table 1 Comparison table of abbreviations and full names of plant hormones used

[0033] abbreviation Full name 6-BA 6-Benzylaminoadenine NAA Naphthaleneacetic acid 2,4-D 2,4-Dichlorophenoxyacetic acid ABA Abscisic acid KT Kinetin VB1 Vitamin B1

[0034] The BM powdered culture medium, LM powdered culture medium, MLV liquid culture medium, and WPM liquid (WPM dry powder mixture + 1000× calcium concentrate) culture medium in the following examples were all purchased from Coolaibo.

[0035] The embryonic callus tissue of Japanese larch in the following examples is recorded in the non-patent literature "Sun Haitao, Yang Ling, Qi Liwang, Li Wanfeng. Effect of desiccation treatment of Japanese larch embryoids on germination. Forestry Science Research. 2024, 37(1): 1-6, http: / / www.lykxyj.com", which can be obtained from the applicant to repeat the experiments in the examples.

[0036] Preliminary experiment 1 Screening of proliferation culture medium and proliferation culture cycle

[0037] 1. Screening of proliferation culture medium

[0038] Embryogenic callus of Japanese larch was used as material and the embryogenic callus was inoculated into 5 proliferation culture media with the formulas shown in Table 2:

[0039] Table 2 Specific configuration scheme of proliferation culture medium

[0040]

[0041] Five plates of each proliferation medium were inoculated with five clumps of Japanese larch embryogenic callus per plate, weighing 0.1 g per clump. Only white, transparent, and well-growing embryogenic callus was selected and inoculated onto each proliferation medium. Cultures were maintained in the dark at 23 ± 1°C. During the culture process, the growth of the embryogenic callus on the different media was observed and recorded. The fresh weight of the embryogenic callus after 15 days of culture was measured, and the proliferation rate of the embryogenic callus on the different media was calculated to identify the optimal proliferation medium for Japanese larch.

[0042] The results of statistical analysis of the proliferation rates of embryonic calli on different culture media are shown in Table 3:

[0043] Table 3 Proliferation rate of Japanese larch callus tissue in 15 days on 5 kinds of proliferation medium

[0044] Basic culture medium Initial weight proliferation rate 1 / 2LM 0.1g 0.1±0.1d 1 / 2MLV 0.1g 3.64±0.76ab 1 / 2DCR 0.1g 3.22±0.4b BMⅠ 0.1g 3.2±0.19b BMⅡ 0.1g 4.3±0.88a

[0045] Note: Different letters indicate significant differences at the 0.05 level.

[0046] The results showed that all five proliferation media produced embryonic callus, but there were certain differences in the proliferation rates. The proliferation rates of embryonic callus at 15 days were as follows: BMⅡ>1 / 2MLV>1 / 2DCR>BMⅠ>1 / 2LM. The results showed that BMⅡ had the fastest proliferation rate.

[0047] During the 15-day proliferation culture process, the callus tissues in different proliferation media were observed, as shown in Tables 4 and Figure 1 :

[0048] Table 4 Morphology of Japanese larch callus in 5 proliferation media

[0049]

[0050] The results showed that the callus tissue cultured in BMⅡ proliferation medium had a granular surface with filamentous protrusions, was pure white in color, and proliferated vigorously. The callus tissue cultured in other proliferation media was mostly brown, grayish white, or white with yellowing.

[0051] Therefore, based on the callus tissue status and proliferation rate statistics, BMⅡ was selected as the proliferation medium. During the subculture process, the inventors found that the materials subcultured with BMⅡ medium were somewhat water-deficient, and the callus tissue showed a snowflake-like shape. It was speculated that this might be due to the excessive amount of agar added. Therefore, during the preparation of BM medium, the agar was reduced from 5.8g / L to 4g / L. The callus tissue did not show the above problem. Therefore, in subsequent experiments, the other ratios were kept unchanged, and only the agar addition amount was increased to 4g / L. The selected proliferation medium is as follows:

[0052] The basal medium is BM medium (BM powdered medium dissolved in water, the content of BM powdered medium is 1434 mg / L), the content of NAA is 0.5 mg / L, the content of 6-BA is 0.05 mg / L, the content of KT is 0.05 mg / L, the content of glutamine is 1000 mg / L, the content of acid hydrolyzed casein is 500 mg / L, the content of sucrose is 25 g / L, and the content of agar is 4 g / L.

[0053] 2. Screening of proliferation culture cycles

[0054] On the above-selected proliferation medium, embryonic callus began to grow rapidly after the 7th day, showing exponential growth. On the 15th day of proliferation, due to the continuous increase of callus, yellow substance appeared on the surface of the callus, and the color changed from white transparent to translucent, and the callus turned brown. As the proliferation continued, the proliferation rate of the callus began to decline after 19 days, and the browning of the callus intensified on the 23rd day. If the proliferation culture cycle is too long, the embryonic callus will lose its embryonic properties and turn brown (see Figure 2 Therefore, 13 days is a more appropriate period for proliferation and subculture, which not only maintains a high vitality of embryonic callus but also can obtain a larger proliferation amount.

[0055] Preliminary experiment 2: Screening of maturation conditions and maturation culture medium

[0056] Embryogenic calli obtained by culturing on the proliferation medium selected in Preliminary Experiment 1 for 13 days were used as materials and inoculated into MLV maturation medium with the formula shown in Table 5, and placed under light conditions and dark conditions for maturation culture respectively:

[0057] Lighting conditions: temperature 24±2℃, light duration 14h, light intensity 1000Lx.

[0058] Dark conditions: temperature 22°C.

[0059] Three culture dishes were prepared for each treatment of each medium, with five embryogenic callus clumps (0.1 g each) placed in each dish. After 45 days of culture, the callus status and embryoid formation under different conditions were observed.

[0060] After the embryonic callus is inoculated into the maturation culture medium, some callus tissues will turn brown and die under light conditions, and most callus tissues will turn from white and transparent to grayish white. Basically, the embryoids formed are relatively weak, the cotyledons are green, and the hypocotyls are red. They cannot develop further, and die together with the callus tissue as the maturation time increases. Under dark culture conditions, the embryonic callus can maintain a proliferative state, and the callus tissue changes from white and transparent to light yellow, and the surface becomes granular. As the maturation culture continues, light yellow, smooth protrusions appear on the surface, these protrusions become elongated and cylindrical, and early embryoids appear (see Figure 3 ).

[0061] 2. Screening of mature culture medium

[0062] Embryogenic calli obtained by culturing on the proliferation medium selected in Preliminary Experiment 1 for 13 days were used as materials and inoculated onto four maturation culture media with the formulations described in Table 5:

[0063] Table 5 Specific configuration scheme of mature culture medium

[0064]

[0065] Three culture dishes were prepared for each maturation medium, with five embryonic callus clumps (0.1 g each) placed in each dish. Culture conditions were 21°C in the dark.

[0066] The embryoids after 55 days of mature culture were divided into 10 types according to the number of cotyledons and the morphology of the hypocotyl, and the embryoids of type I (occurrence frequency above 50%) were selected. The classification criteria of the embryoids are shown in Table 6:

[0067] Table 6 Morphology and classification of Japanese larch embryoids

[0068]

[0069] Embryogenic calli were transferred to different maturation media and matured in the dark at 21°C. During maturation, cotyledonary embryos gradually formed on the surface of the callus. The embryoids were short, with the radicle end not completely hardened. A long stalk-like structure at the end connected to the callus. The callus browned and died during maturation. After 35 days, the embryoids turned pale yellow, indicating the late cotyledonary embryo stage. At this point, expanded cotyledons, intact hypocotyls, and radicles can be observed in the embryoids. As maturation culture continued, the embryoids' appearance remained largely unchanged. After 70 days of maturation culture, the embryoids became overmature, with the cotyledons beginning to elongate. The number of embryoids on different media was counted.

[0070] Table 73 kinds of mature culture medium for the embryogenesis rate of Japanese larch embryoids

[0071]

[0072]

[0073] Note: Different letters indicate significant differences at the 0.05 level

[0074] From Table 7, Figure 4It can be seen that since no complete embryoids were observed on BMⅢ medium, most embryoids were in the proembryonic stage and their number could not be counted. BMⅣ produced the largest number of somatic embryos per gram of callus (21.66), followed by MLV medium. However, the proportion of type I embryoids in MLV medium was the highest, reaching 63.16%. According to previous studies, type I embryoids have complete germination characteristics, so MLV medium was selected as the maturation medium for Japanese larch. The selected maturation medium is as follows:

[0075] Maturation culture medium: MLV medium (large mother solution & iron salt 100×, MgSO4 & trace mother solution 100×, organic mother solution 100×, CaCl2 100×. Take 10 ml / L of each concentration) is used as the basic culture medium, with an ABA content of 60 μM, an IBA content of 1 μM, an activated carbon content of 10 g / L, a sucrose content of 0.2 M, and an agar content of 4 g / L.

[0076] Preliminary experiment 3: Screening of germination medium

[0077] After 55 days of maturation culture on the maturation medium selected in preliminary experiment 3, embryoids with 4-7 cotyledons and symmetrical hypocotyls were selected as plant materials and inoculated onto the four germination media with the formulations shown in Table 8 for germination culture:

[0078] Table 8 Specific configuration scheme of germination culture medium

[0079] Culture medium type sucrose agar activated carbon Other additives 1 / 2MS 20g / L 4g / L / / WPMⅠ 10g / L 6.5g / L 1.0g / L / WPMⅡ 20g / L 4g / L 2.0g / L 3.0mg / LVB1 1 / 2MLV 10g / L 3.2g / L 2.0g / L 0.5g / L NH4NO3, 0.5g / L glutamine

[0080] The results showed that embryoids could germinate in all four culture media, but there were significant differences in germination rates. The highest germination rate was achieved when inoculated on hormone-free WPMⅡ germination medium, so WPMⅡ was selected as the germination medium for Japanese larch. The selected germination media are as follows:

[0081] Germination medium: WPM medium (WPM dry powder mixture content 1991.34 mg / L + 1000× calcium concentrate content 1 mL / L) is used as the basic culture medium, the sucrose content is 20 g / L, the activated carbon content is 2.0 g / L, the VB1 content is 3.0 mg / L, and the agar content is 4 g / L.

[0082] Example 1 Effect of Darkness Treatment on Early Morphological Construction of Japanese Larix

[0083] The embryonic callus of Japanese larch was first inoculated on a proliferation medium for proliferation culture, the embryonic callus was proliferated, the embryonic callus was transferred to a maturation medium for maturation culture to obtain embryoids, and finally inoculated on a germination medium for germination culture to obtain Japanese larch early somatic embryo seedlings (the early somatic embryo seedlings were cultured in the dark for 14 days).

[0084] The specific configuration schemes of proliferation medium, maturation medium, and germination medium are as follows:

[0085] Proliferation medium: BM medium as the basal medium (BM powdered medium dissolved in water, the content of BM powdered medium is 1434 mg / L), NAA content of 0.5 mg / L, 6-BA content of 0.05 mg / L, KT content of 0.05 mg / L, glutamine content of 1000 mg / L, acid hydrolyzed casein content of 500 mg / L, sucrose content of 25 g / L, and agar content of 4 g / L.

[0086] Maturation culture medium: MLV medium (large mother solution & iron salt 100×, MgSO4 & trace mother solution 100×, organic mother solution 100×, CaCl2 100×. Take 10 ml / L of each concentration) is used as the basic culture medium, with an ABA content of 60 μM, an IBA content of 1 μM, an activated carbon content of 10 g / L, a sucrose content of 0.2 M, and an agar content of 4 g / L.

[0087] Germination medium: WPM medium (WPM dry powder mixture content 1991.34 mg / L + 1000× calcium concentrate content 1 mL / L) is used as the basic culture medium, the sucrose content is 20 g / L, the activated carbon content is 2.0 g / L, the VB1 content is 3.0 mg / L, and the agar content is 4 g / L.

[0088] The pH value of the above culture medium was 5.80. After high-pressure sterilization at 121°C, the culture medium was aliquoted for use under a sterile environment.

[0089] Proliferation culture and maturation culture were carried out in a dark environment (temperature 22° C.) Embryoid bodies were obtained after 13 days of proliferation culture and 55 days of maturation culture.

[0090] Germination culture was first carried out in a dark environment (temperature 22°C), and then in a light environment (temperature 24±2°C, light for 14 hours, light intensity 1000lx). Different dark germination culture durations were set for the selected type I embryoids:

[0091] Treatment 1: dark germination culture for 5 days;

[0092] Treatment 2: dark germination culture for 7 days;

[0093] Treatment 3: dark germination culture for 9 days;

[0094] Treatment 4: dark germination culture for 12 days;

[0095] Treatment 5: dark germination culture for 14 days;

[0096] Treatment 6: dark germination culture for 16 days.

[0097] A total of 103 embryoid bodies were processed.

[0098] The results are shown in Table 9, and the embryoid of germination culture in the dark, about germination culture 5 days, the embryoid axis place is slightly arched, and the embryoid axis of tender white color is grown, and germination culture about 7 days, the embryoid axis continues to elongate, with the generation of white radicle simultaneously.Along with the growth of dark germination culture time, the embryoid quantity that embryoid axis grows, radicle produces constantly increases, and the embryoid quantity with germination trend is also increasing, and when dark germination culture 7 days, the embryoid number with germination trend is arranged to obviously increase.And dark treatment 14 days, the embryoid with germination trend is arranged to reach 92, and the ratio of increase obviously descends, continues dark treatment, and the embryoid quantity with germination trend no longer increases, illustrates that dark germination culture can accelerate the early stage form of embryoid in 14 days and built up.

[0099] Table 9 Number of embryoids in dark germination culture at different times

[0100]

[0101] Germination culture was performed according to the method of treatment 5, that is, germination culture was carried out in the dark for 14 days (temperature 22°C), and then in a light environment (temperature 24±2°C, light intensity 14h / d, light intensity 1000lx). There were three different morphological characteristics of embryoids after 14 days of dark germination culture, namely:

[0102] (1) Hypocotyl elongation and rooting;

[0103] (2) only the embryonic axis elongates;

[0104] (3) No roots are formed and the hypocotyl does not elongate.

[0105] The proportions of different morphological characteristics were statistically analyzed, and the results are shown in Table 10.

[0106] Table 10 Proportions of different morphological characteristics

[0107] Somatic embryo seedling type Number of occurrences Frequency Hypocotyl elongation and rooting 144 90％ Only hypocotyl elongation 11 6.875％ No roots, no hypocotyl elongation 5 3.125％

[0108] The results showed that the morphological characteristics of hypocotyl elongation and rooting had the highest occurrence frequency, reaching 90%, while the other morphological characteristics had lower occurrence frequencies.

[0109] According to the process of Japanese larch seed germination and culture (see Figure 5 ) and found that when the zygotic embryo germinates, it will first swell due to water absorption, and the hypocotyl breaks through the seed coat. As the hydroponic germination culture continues, the hypocotyl continues to elongate and the radicle is produced. According to the results of hydroponic culture of zygotic embryos, it was found that the germination of embryoids is accompanied by the elongation of the hypocotyl and the production of radicles. Therefore, the inventors only selected embryoids with elongated hypocotyls and rooting for subsequent experiments, and called them early somatic embryo seedlings (see Figure 6 ).

[0110] Thirty early somatic embryo seedlings (hypocotyl elongation and rooting) were randomly selected and their hypocotyl lengths were measured. The results are shown in Table 11. 20 of them were randomly selected and photographed. Figure 7 .

[0111] Table 1 Statistics of hypocotyl length of 1130 early somatic embryo seedlings

[0112]

[0113]

[0114] The results showed that after dark germination, the hypocotyls of early somatic embryos showed significant growth, with the minimum elongation being 0.55 cm and the maximum elongation being 1.22 cm. This suggests that dark germination is beneficial for the morphological development of early somatic embryos.

[0115] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, contents, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope disclosed herein, in accordance with the principles of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A Japanese larch breeding method, characterized in that, The method comprises the following steps: inoculating Japanese larch embryonic callus onto a proliferation medium, performing proliferation culture under dark conditions to obtain proliferated embryonic callus, then transferring the callus onto a maturation medium, performing maturation culture under dark conditions to obtain embryoids, wherein the embryoids have 4-10 cotyledons and a symmetrical hypocotyl; and finally inoculating the embryoids onto a germination medium, first performing germination culture under dark conditions, and then switching to light conditions to continue germination culture, thereby obtaining early somatic embryo seedlings of Japanese larch. The proliferation medium is a medium using BM medium as a basal medium, with NAA content of 0.5 mg / L, 6-BA content of 0.05 mg / L, KT content of 0.05 mg / L, glutamine content of 1000 mg / L, acid hydrolyzed casein content of 500 mg / L, sucrose content of 25 g / L, and agar content of 4 g / L; The maturation medium is a medium using MLV medium as a basal medium, with an ABA content of 60 μM, an IBA content of 1 μM, an activated carbon content of 10 g / L, a sucrose content of 0.2 M, and an agar content of 4 g / L; The germination medium is a medium using WPM medium as a basic medium, with a sucrose content of 20 g / L, an activated carbon content of 2.0 g / L, a VB1 content of 3.0 mg / L, and an agar content of 4 g / L; The proliferation culture is subcultured every 13 days; The mature culture was cultured for 55 days; The germination culture time under the dark conditions is 14 days; The dark condition is 22°C. The illumination conditions are as follows: illumination duration of 14 h / d, illumination intensity of 1000 lx, and temperature of 24±2.

Images