Thesis: Research on Cryopreservation of 24 Seeds of the Landscape Trees (Originally written in Chinese)

• The following content is an excerpt and translation of my undergraduate thesis, aimed at personal knowledge reviewing and writing practicing.
• The original version was written in Chinese, awarded as an Outstanding Undergraduate Thesis of the Class of 2018 at Beijing Forestry University.
• The thesis was supported by the National Engineering Research Center for Floriculture, China, and was advised by Prof. Dr. Yan Liu, whose integrity, kindness, and rigorous character constantly benefit students for life.

• Abstract
• 1 Introduction
  • 1.1 Background
  • 1.2 Research status
  • 1.3 Research purpose
  • 1.4 Research approach and technical route
• 2 Materials and methods
  • 2.1 Materials
  • 2.2 Methods
  • 2.2.1 Observation and record of seed size and morphology
  • 2.2.2 Determination of seed moisture
  • 2.2.3 Determination of seed viability
  • 2.2.4 Pretreatment of seed germination
  • 2.2.5 Determination of seed germination rate
  • 2.2.6 Outdoor sowing and observation of growth status
  • 2.2.7 Preservation and thawing of seeds in liquid nitrogen
• 3 Results
  • 3.1 Basic information of the seeds
  • 3.1.1 Record of seed size and morphology
  • 3.1.2 Determination results of seed moisture
  • 3.2 Determination results and analysis of seed viability after liquid nitrogen preservation for 1h
  • 3.3 Determination results and analysis of seed germination rate after liquid nitrogen preservation
  • 3.4 Outdoor sowing status and analysis of seeds after liquid nitrogen preservation
• 4 Conclusion and Discussion
• 5 Appendix
  • 5.1 Appendix A: Part of the seeds’ germination in laboratory
  • 5.2 Appendix B: Part of the seeds’ growth status after cryopreservation and transplanting

Abstract

• Germplasm resources are the foundation of human survival and development, and germplasm preservation is of significant importance. Cryopreservation is a method for long-term preservation of germplasm, typically referring to a biotechnological approach of storing biological materials at -196°C (liquid nitrogen temperature). It is currently a hot topic in international germplasm preservation. However, its application in the preservation of seeds from landscape trees is not yet widespread.

• The present research focuses on 24 species of landscape tree seeds, determining their vitality and moisture content. Additionally, it investigates the germination and growth conditions of seeds after cryopreservation at different time intervals, as well as their performance upon field sowing. The aim is to explore the feasibility and technical methods of cryopreservation for these 24 species of landscape tree seeds. The results are as follows:
  • Among the 24 species of landscape tree seeds studied, seeds from 7 species including Syringa oblata, Lonicera maackiie, Acer truncatum, Chimonanthus praecox, Berberis thunbergii ‘Atropurpurea’, Albizia julibrissin and Forsythia suspensa retain their germination ability after 30-50 days of cryopreservation.
  • Seeds from 5 species including Syringa oblata, Acer truncatum, Chimonanthus praecox, Albizia julibrissin, and Forsythia suspensa still possess the ability to develop into healthy plants after being sown in the field following 30-50 days of cryopreservation. Their growth condition shows no significant difference compared to the control group. Cryopreservation technology is feasible for the application of these 5 species of seeds.
  • Seeds from Lonicera maackii and Acer truncatum experience significant enhancement in germination after undergoing cryopreservation for a minimum of 30 days. Additionally, there is a notable decrease in the germination time for Acer truncatum seeds.
  • Germination rate of Syringa oblata seeds significantly increases after 10 days of cryopreservation. However, after 30 days of cryopreservation, the germination rate is lower than that of the 10-day treatment but higher than the control group. This indicates that cryopreservation treatments of different durations have varying effects on the germination of Syringa oblata seeds, and further research is needed to determine the optimal treatment duration.
• Key words: Cryopreservation, germplasm preservation, seeds, landscape trees

1 Introduction

1.1 Background

• For most landscape trees, seeds are easily obtainable, and seed propagation is widely used in nurseries.

• Conventional germplasm preservation of woody plants is limited by factors such as land, manpower, high cost, low efficiency, and genetic variation is prone to occur during the preservation process. The application of cryopreservation in woody plants greatly maintains genetic stability of germplasm, which holds certain potential value for landscape trees.

1.2 Research status

• From previous research, studies on cryopreservation have primarily focused on crops, medicinal plants, and economically significant forest trees. Research specifically targeting cryopreservation of landscape trees has been relatively limited.

1.3 Research purpose

• This research focuses on 24 common landscape tree species and aims to explore the feasibility and technical methods of cryopreservation of their seeds. Additionally, it discusses the impact of liquid nitrogen preservation duration on the tested seeds. The goal is to provide methods and support for the multi-level preservation of planting resources for landscape trees, thus enhancing the safety of the germplasm preservation.

1.4 Research approach and technical route

• Initially, the basic information of the seeds was determined, including observations of seed morphology, size, and measurement of seed moisture content. Subsequently, the seeds were subjected to 1-hour liquid nitrogen treatment, with room temperature storage serving as a control. The viability of the seeds was assessed using the Tetrazolium chloride (TTC) staining method to preliminarily determine if these 24 species of landscape tree seeds could maintain activity after liquid nitrogen treatment. After the assessment, the 24 species of seeds were subjected to liquid nitrogen preservation for no less than 30 days, with a subset also preserved for 10 days, again with room temperature storage as a control. Laboratory germination rates of the seeds after liquid nitrogen preservation were measured, and the outdoor sowing performance of seeds preserved for over 30 days in liquid nitrogen was evaluated. Based on the experimental results, the feasibility of cryopreservation and the establishment of technical methods for such preservation were determined for these 24 species of landscape tree seeds.

2 Materials and methods

2.1 Materials

• The study utilized seeds from 24 common landscape tree species as experimental materials.

Table.1 Catalogue of the species

Number	Species	Family
01	Acer buergerianum	Aceraceae
02	Acer palmatum	Aceraceae
03	Acer palmatum ‘Atropurpureum’	Aceraceae
04	Acer truncatum	Aceraceae
05	Cotinus coggygria	Anacardiaceae
06	Berberis thunbergii ‘Atropurpurea’	Berberidaceae
07	Betula platyphylla	Betulaceae
08	Buxus sinica	Buxaceae
09	Chimonanthus praecox	Calycanthaceae
10	Lonicera maackii	Caprifoliaceae
11	Cephalotaxus sinensis	Cephalotaxaceae
12	Cornus kousa subsp. chinensis	Cornaceae
13	Cornus officinalis	Cornaceae
14	Liquidambar formosana	Hamamelidaceae
15	Albizia julibrissin	Leguminosae
16	Cercis chinensis	Leguminosae
17	Gleditsia sinensis	Leguminosae
18	Magnolia denudata	Magnoliaceae
19	Chionanthus retusus	Oleaceae
20	Forsythia suspensa	Oleaceae
21	Syringa oblata	Oleaceae
22	Koelreuteria paniculata	Sapindaceae
23	Paulownia tomentosa	Scrophulariaceae
24	Ailanthus altissima	Simaroubaceae

2.2 Methods

2.2.1 Observation and record of seed size and morphology

• Randomly select an appropriate amount of seeds on graph paper, and take photos to record the morphology and size of each species of seed.

2.2.2 Determination of seed moisture

• Take 30 seeds of each species as a sample, evenly spread them in a sample box; Place them in the oven, and start timing when the oven reaches 103°C ± 2°C, maintaining it for 12 hours; then transfer them to a desiccator for cooling for 30 minutes. Seeds with a diameter greater than or equal to 15mm and seeds with hard seed coats are sliced and measured. After cutting the large seeds into 4-5 pieces, approximately equivalent to the amount of 5 intact seeds are randomly selected from them for measurement, ensuring that the total exposure time to air during the entire operation does not exceed 60 minutes. Each species is repeated 3 times.

• Weigh the covered sample box without the sample as \(M_{1}\) ; after adding the sample, the weight of the sample box along with the lid is \(M_{2}\) ; after drying and cooling, the weight of the sample and the sample box along with the lid is \(M_{3}\) ; Unit in grams, rounded to 3 decimal places. The final moisture content is expressed as a percentage and calculated using the following formula, with the moisture content of each seed type being the average of the three repetitions.

\[\text{Moisture content(%)} = \frac{ M_{2} - M_{3} }{ M_{2} - M_{1} } \times 100\% \quad (1)\]

2.2.3 Determination of seed viability

• The Tetrazolium chloride (TTC) staining method was employed to assess seed viability. Groups of 15 large seeds and 30 small seeds were utilized. Seeds were soaked in room temperature water for 24 hours to soften the seed coat for easier dissection. After removing the seed coat and dissecting the seeds to expose the embryos as much as possible for staining and observation, they were divided into groups and immersed in a 0.75% TTC solution for 24 hours for staining, followed by subsequent observation and analysis. Seed viability was evaluated based on the stained area, categorized as ”+”, “++”, or “+++”, respectively represented staining areas of 0-30%, 30%-60%, and 60%-100%, to indicate the degree of staining. Examples of stained seeds are illustrated in Figure 1.

Fig.1 Examples of stained seeds

2.2.4 Pretreatment of seed germination

• For seeds subjected to liquid nitrogen storage for 10 days and the control group, different pre-treatment methods were employed to break dormancy in various seed species.

2.2.5 Determination of seed germination rate

• Random samples, each containing approximately 30 seeds, were taken and replicated three times. Following liquid nitrogen storage or germination pre-treatment, the seeds were placed into prepared culture plates for germination experiments. Two layers of filter paper or a suitable thickness of defatted cotton were spread as the germination bed in each culture plate. The culture plates were then placed in a constant temperature incubator set at 25°C for germination experiments, ensuring a minimum of 8 hours of light exposure every 24 hours. The germination beds were kept moist and clean throughout the experiment. Germination progress was continuously monitored until cessation, and germination rates were calculated accordingly.

2.2.6 Outdoor sowing and observation of growth status

• Random samples, each containing approximately 30 seeds, were taken and replicated three times. Seeds were sown using the furrow seeding method, and after sowing, a layer of mulch was applied to maintain soil moisture. Several days later, the germination progress of the seeds was continuously observed and recorded, including the time of emergence of the first true leaves after seed germination. Germination rates were calculated accordingly. Following the appearance of the first pair of true leaves, plant height was recorded every two days. Throughout this period, plants were watered daily to maintain soil moisture, and timely weed removal was conducted to ensure optimal growth conditions.

2.2.7 Preservation and thawing of seeds in liquid nitrogen

• For each of the 24 different seed varieties, approximately 30 seeds were randomly selected for each group. Seeds within each group were sorted based on size and placed into cryogenic tubes of varying capacities. These tubes were then subjected to liquid nitrogen storage for durations of 1 hour, 10 days, or 30 days and above, with room temperature storage serving as the control. Upon reaching the desired storage durations, the cryogenic tubes were retrieved and the seeds were rapidly thawed by rinsing them with room temperature water for approximately 5 minutes. After thawing, the seeds were subjected to either TTC staining, germination tests following pre-treatment, or direct sowing in the field.

3 Results

3.1 Basic information of the seeds

3.1.1 Record of seed size and morphology

• The size and morphology of the tested seeds are depicted in Figure 2.

Fig.2 The size and morphology of the seeds

3.1.2 Determination results of seed moisture

• For the cryopreservation of seeds, the moisture content of the preservation material is commonly regarded as having a significant impact on the success of preservation. It is generally believed that the lower the moisture content of the material, the less likely it is to suffer from cryo-injuries. However, each preservation material has a specific safe moisture content range, as excessive dehydration can also harm the material. The moisture content of the tested seeds was determined under normal room temperature conditions. Specific results can be found in Table 2.

Table.2 Moisture content of the seeds

Number	Species	Moisture Content / %
01	Acer buergerianum	6.7±0.058
02	Acer palmatum	8.4±0.071
03	Acer palmatum ‘Atropurpureum’	5.1±0.032
04	Acer truncatum	7.2±0.108
05	Cotinus coggygria	6.5±0.235
06	Berberis thunbergii ‘Atropurpurea’	7.2±0.057
07	Betula platyphylla	6.3±0.211
08	Buxus sinica	7.3±0.152
09	Chimonanthus praecox	7.8±0.073
10	Lonicera maackii	7.6±0.059
11	Cephalotaxus sinensis	6.9±0.195
12	Cornus kousa subsp. chinensis	6.6±0.373
13	Cornus officinalis	7.5±0.088
14	Liquidambar formosana	6.8±0.061
15	Albizia julibrissin	3.9±0.055
16	Cercis chinensis	8.3±0.247
17	Gleditsia sinensis	6.7±0.147
18	Magnolia denudata	5.5±0.318
19	Chionanthus retusus	4.5±0.067
20	Forsythia suspensa	5.6±0.074
21	Syringa oblata	4.2±0.033
22	Koelreuteria paniculata	10.1±0.415
23	Paulownia tomentosa	4.4±0.079
24	Ailanthus altissima	6.3±0.028

3.2 Determination results and analysis of seed viability after liquid nitrogen preservation for 1h

• The criterion for assessing seed viability is based on the degree of seed staining, where a larger stained area indicates a higher degree of staining and thus stronger seed viability.

• The seeds of the 24 tested species of landscape trees exhibited varying degrees of staining after 1 hour of liquid nitrogen storage, as did the control group. This indicates that the seeds under test possessed varying levels of viability both before and after 1 hour of liquid nitrogen storage.

3.3 Determination results and analysis of seed germination rate after liquid nitrogen preservation

• After liquid nitrogen storage, seeds from seven species of landscape trees, including lilac, Ginkgo biloba, maple, purple leaf barberry, mimosa, wintersweet, and forsythia, retained their ability to germinate. The data of Germination rate is analized in SPSS, using the Least Significant Difference (LSD) multiple comparison to determine which groups have significant differences, as shown in Table 3.

Table.3 Germination rate of the seeds

Treatment	Germination rate / %
	Albizia julibrissin	Lonicera maackii	Chimonanthus praecox	Forsythia suspensa	Acer truncatum	Syringa oblata	Berberis thunbergii 'Atropurpurea'
Liquid nitrogen storage for no less than 30 days	5.6a	5.6a	2.2a	20.0a	20.0a	47.8b	4.4a
Liquid nitrogen storage for 10 days	14.4a	2.2ab	1.1a	11.1a	5.6b	74.4a	3.3a
Room temperature storage (control group)	16.7a	0.0b	1.1a	10.0a	1.1b	32.2b	3.3a

Note: In the table, “a” and “b” represent the LSD multiple comparison results (P=0.05).

• There was a significant difference between the Albizia julibrissin subjected to liquid nitrogen storage for no less than 30 days and the control treatment, indicating a significant promotion of germination for Albizia julibrissin after liquid nitrogen storage for no less than 30 days.

• For the Acer truncatum, after being stored in liquid nitrogen for no less than 30 days, there was a significant difference compared to the storage for 10 days in liquid nitrogen and the control treatment, indicating a significant promotion of germination for Acer truncatum after liquid nitrogen storage for no less than 30 days, while the storage for 10 days in liquid nitrogen had no significant effect on the germination of Acer truncatum.

• Apart from the above two species, the treatment with liquid nitrogen had no significant effect on the germination rate of the other 5 species of seeds. To further investigate the effect of liquid nitrogen storage duration on seed germination, the time required for the first seed to germinate was determined, and the results are presented in Table 4.

Table.4 Days required for seeds’ germination

Treatment	The time required for the first seed to germinate / d
	Albizia julibrissin	Chimonanthus praecox	Forsythia suspensa	Acer truncatum	Syringa oblata	Berberis thunbergii 'Atropurpurea'
Liquid nitrogen storage for no less than 30 days	4a	9a	6.3a	2.7b	3a	15.5a
Liquid nitrogen storage for 10 days	1b	9a	6a	3.7b	1.7b	15.5a
Room temperature storage(control group)	1b	6a	6a	10a	3a	17a

Note: In the table, “a” and “b” represent the LSD multiple comparison results (P=0.05).

• From the perspective of the time required for the first seed to germinate, there was a significant difference in the Albizia julibrissin after liquid nitrogen storage for no less than 30 days compared to the storage for 10 days in liquid nitrogen and the control treatment. This indicates that the treatment of liquid nitrogen storage for no less than 30 days prolonged the time required for Albizia julibrissin to germinate to a certain extent, while the treatment of liquid nitrogen storage for 10 days had no significant effect on the time required for Albizia julibrissin to germinate.

• For the Acer truncatum, after liquid nitrogen storage for no less than 30 days and for 10 days, there was a significant difference compared to the control treatment, indicating that both treatments of liquid nitrogen storage for no less than 30 days and for 10 days could shorten the time required for Acer truncatum to germinate to some extent.

• For the Syringa oblata, after liquid nitrogen storage for 10 days, there was a significant difference compared to the storage for no less than 30 days in liquid nitrogen and the control treatment, indicating that the treatment of liquid nitrogen storage for 10 days could shorten the time required for Syringa oblata to germinate to a certain extent, while the treatment of liquid nitrogen storage for no less than 30 days had no significant effect on shortening the time required for Syringa oblata to germinate.

3.4 Outdoor sowing status and analysis of seeds after liquid nitrogen preservation

• After liquid nitrogen storage, the seeds of Syringa oblata, Acer truncatum, Chimonanthus praecox, Albizia julibrissin, and Forsythia suspensa, totaling 5 species, still possessed the capability to grow into healthy seedlings when sown in the field. Additionally, the control group of Gleditsia sinensis exhibited a certain germination rate. Specific data can be found in Table 5.

Table.5 Germination rate of the seeds after being sown in the field

Treatment	Germination rate / %
	Albizia julibrissin	Chimonanthus praecox	Forsythia suspensa	Acer truncatum	Syringa oblata	Gleditsia sinensis
Liquid nitrogen storage for no less than 30 days	24.4	12.2	14.4	15.6	47.8	0
Room temperature storage(control group)	36.7	7.8	8.9	7.8	57.8	3.3

• For seeds that retained the capability to grow into healthy seedlings after liquid nitrogen storage, the observation included selecting the first plant to develop true leaves and recording the date of the appearance of the first true leaf during its growth process. After the emergence of the first true leaf, the height of the plant was measured every two days.

• After liquid nitrogen storage, the seeds of Syringa oblata, Acer truncatum, Chimonanthus praecox, Albizia julibrissin, and Forsythia suspensa, totaling 5 species, retained the ability to grow into healthy seedlings when sown in the field.

• Among them, the Albizia julibrissin exhibited the earliest appearance of the first pair of true leaves on the 11th day after sowing, while the Chimonanthus praecox showed the latest appearance of the first pair of true leaves on the 15th day after sowing. For these 5 species, there was no significant difference between the seedlings grown after liquid nitrogen storage for no less than 30 days and those grown from seeds stored at room temperature.

4 Conclusion and Discussion

• Among the 24 species of landscape trees selected, the seeds of Syringa oblata, Lonicera maackiie, Acer truncatum, Chimonanthus praecox, Berberis thunbergii ‘Atropurpurea’, Albizia julibrissin and Forsythia suspensa, totaling 7 species, retained their germination ability after long-term cryopreservation in liquid nitrogen. Furthermore, the seeds of Syringa oblata, Acer truncatum, Chimonanthus praecox, Albizia julibrissin, and Forsythia suspensa, totaling 5 species, retained the capability to develop into healthy plants after being cryopreserved in liquid nitrogen for no less than 30 days. The growth status of these plants showed no significant difference compared to the control group. Therefore, the application of cryopreservation technology for these 5 species of plant seeds is feasible.

• The cryopreservation method for the aforementioned 5 species of seeds is as follows:
  • Seeds are sorted based on their size and morphology and then placed into cryogenic tubes of varying capacities under their natural moisture content conditions.
  • These tubes are directly submerged in liquid nitrogen to achieve cryopreservation.
  • When retrieving the seeds, the cryogenic tubes must be quickly removed from the liquid nitrogen and rinsed under running room temperature tap water for 5 minutes.
  • After rinsing, the seeds can be used for sowing, pre-treatment before sowing, or other applications.

• Among the successfully preserved 5 species of seeds mentioned, Lonicera maackiie and Acer truncatum, totaling 2 species, exhibited a significant increase in germination rate after being stored in liquid nitrogen for no less than 30 days. Additionally, the time required for germination of Acer truncatum significantly shortened after being stored in liquid nitrogen for no less than 30 days. These phenomena indicate that cryopreservation has a certain promoting effect on the germination of certain seeds.

• After 10 days of liquid nitrogen storage, Syringa oblata showed a shortened germination time and reached peak germination rates. However, after liquid nitrogen storage for no less than 30 days, the germination rate decreased but still remained higher than that of seeds stored at room temperature. This phenomenon is similar to the results obtained by Liu et al. (2019)¹ in their experiments on the pollen of Syringa oblata stored in liquid nitrogen for 30 and 180 days. The specific reasons may be related to the physiological characteristics of Syringa oblata itself and require further research.

• After being stored in liquid nitrogen for no less than 30 days, Albizia julibrissin showed an increase in the time required for germination. The same phenomenon was observed in the sowing experiments. Additionally, some seeds experienced bursting during thawing. The reasons for these phenomena may be related to the moisture content of the Albizia julibrissin seeds ².

• During the germination and sowing experiments, some seeds showed mold growth in the warm and humid incubator environment. Due to the limited duration of the experiments, some seeds were not subjected to prolonged stratification pre-treatment, which may cause the failure of germination for some seeds. In the sowing experiments, it was challenging to provide the optimal sowing treatment for all seeds based on their individual growth characteristics. For example, factors such as the thickness of the covering soil after sowing could only be adjusted to meet the general requirements for suitable growth of all seeds as much as possible. Additionally, the high winds in Beijing during the spring and the rainfall in May led to the rapid germination and growth of some weed seeds in the experimental site, which may cause the failure of some seeds to germinate properly.

5 Appendix

5.1 Appendix A: Part of the seeds’ germination in laboratory

Fig.A1 Germination of Lonicera maackii seed in laboratoryFig.A2 Germination of Syringa oblata seed in laboratory

5.2 Appendix B: Part of the seeds’ growth status after cryopreservation and transplanting

Fig.B Part of the seeds' growth status after cryopreservation and transplanting