Somatic Embrygenesis of Symplocos paniculata

Yan YANG, Lijuan JIANG, Changzhu LI, Beilei XIE, Wenjun LI, Qiang LIU, Simeng LI

Abstract [Objectives] This study was conducted to provide a new way for the rapid reproduction of Symplocos paniculata.

[Methods]With mature embryos of S. paniculata as explants， through the study of somatic embryogenesis， the effects of different media on the induction and differentiation of S. paniculata somatic embryos were discussed， and the somatic embryogenesis system for S. paniculata was preliminarily established.

[Results] Calli suitable for somatic embryogenesis could be induced using modified MS （referred to as GMS） as the basic medium with the addition of 0.2 mg/L 6-BA and 0.1 mg/L NAA; the best medium combination for somatic embryo differentiation was GMS+0.25 mg/L 6-BA+0.15 mg/L NAA， and the differentiation rate could reach 71.99%; and plant regeneration needed to be carried out at low sugar concentrations.

[Conclusions]This study provides a technical basis for resources protection， genetic transformation， artificial seed production， and large-scale development and utilization of S. paniculata.

Key words Symplocos paniculata; Somatic embrygenesis; Plant regeneration

Received： May 5， 2021  Accepted： July 6， 2022

Supported by Changsha Science and Technology Plan Key Project （kq1801080）; State Key Laboratory of Woody Oil Resource Utilization Project （2019XK2002）.

Yan YANG （1980-）， female， P. R. China， PhD， devoted to research about tree breeding and cultivation.

*Corresponding author.

Symplocos paniculata （Thunb.） Miq.， also known as Shanguihua and Pishuangzi， belongs to Symplocos of Symplocaceae. It is a native tree species in China with strong adaptability and a wide distribution range from Liaoning in the north and Taiwan in the south. S. paniculata trees has a beautiful shape， graceful branches and leaves， white flowers and blue fruit， endowing them with a high ornamental value， so S. paniculata is one of the ideal tree species for landscaping. S. paniculata has strong adaptability and a developed root system and can effectively prevent soil erosion， and is thus a good tree species for shelter forest afforestation. S. paniculata fruit has a high oil content. The fruit oil contains a variety of unsaturated fatty acids， which can meet various nutrients required by the human body. Meanwhile， the fruit is also one of the ideal raw materials for the preparation of biodiesel. Therefore， S. paniculata is a rare woody oil plant with both edible and industrial uses[1-5]. It can be seen that S. paniculata has high comprehensive utilization value and is a biomass resource with great development potential.

S. paniculata is mostly a wild resource， and its reproduction method is mostly seed propagation， which has not been paid much attention by people. Due to severe man-made damage， the quantity of S. paniculata resources is shrinking. It can be seen that the top priority is to further protect the existing resources of S. paniculata and reduce the loss of excellent germplasm resources. As a rapid propagation technology， tissue culture can not only achieve rapid propagation， but also maintain the excellent genetic traits of plants， and is thus an ideal propagation method. So far， there is no report on the tissue culture and rapid propagation technology of S. paniculata at home and abroad. Therefore， on the basis of related research on S. paniculata， preliminary screening and discussion on suitable media for embryogenic callus induction， proliferation and somatic embryogenesis of S. paniculata was conducted， in order to provide a new way for the rapid propagation of S. paniculata and a technical basis for the resource protection， genetic transformation， artificial seed production and large-scale development and utilization of S. paniculata.

Materials and Methods

Experimental materials

The mature fruit of excellent S. paniculata plants picked from the experimental forest farm of Hunan Academy of Forestry at the end of October 2019 was selected.

The flesh was removed from the harvested fruit， and the seeds were rinsed under running water to ensure that the seed shells were free of oil. Then， on an ultra-clean workbench， the clean seeds were washed 2-3 times with sterile water， then soaked in 75% ethanol solution for 30 s， rinsed with sterile water 2-3 times， and finally soaked in 0.1% mercury bichloride for 10 min. After rinsing with sterile water for 4-5 times， the moisture of the exotesta was absorbed with sterile filter paper. Finally， the seed coat and endosperm of the seeds were removed， and the seed embryos were taken out for use.

The analytically pure and biochemical reagents used in the test were purchased from Sinopharm Group. The ultra-clean workbench used was purchased from Suzhou Yida Purification Laboratory Equipment Co.， Ltd.， and the HT-JZ-1 type inoculation equipment sterilizer was purchased from Jinan Haotian Science and Technology Development Co.， Ltd.

Culture initiation

The starting media used MS， GMS and WPM as the basic mediums. According to the research in the related field， different concentrations of cytokinin 6-BA and auxin NAA were added， as shown in Table 1. Each bottle was inoculated with 10 embryos. Each treatment was assigned with 20 bottles and   repeated 3 times， and cultured for 20 d. The number of calli and the induction of calli were investigated， and the callus induction rate was calculated.

Callus induction rate=（Number of callus-producing explants/Total number of uncontaminated explants）×100%

Differentiation and proliferation culture of embryogenic calli

By starting the culture， the basic medium suitable for the induction of embryogenic calli of S. paniculata was screened out， and then this medium was used as the basic medium for proliferation culture， which was added with 0， 0.2， 0.25 mg/L 6-BA and 0， 0.1， 0.15， 0.2 NAA， a total of seven hormone levels. Effective calli induced by the culture initiation was inoculated on GMS1-GMS7 medium， and two embryogenic callus groups were inoculated in each bottle， and each treatment was inoculated with 20 bottles in three repetitions. The calli were cultured for 30 d， and the differentiation of embryogenic calli was investigated to preliminarily select an optimal proliferation combination.

The medium combination were as follows：

GMS1： basic medium+0.2 mg/L 6-BA+0.1 mg/L NAA; GMS2： basic medium+0.2 mg/L 6-BA+0.15 mg/L NAA; GMS3： basic medium+0.2 mg/L 6-BA+0.2 mg/L NAA; GMS4： basic medium+0.25 mg/L 6-BA+0.1 mg/L NAA; GMS5： basic medium+0.25 mg/L 6-BA+0.15 mg/L NAA; GMS6： basic medium+0.25 mg/L 6-BA+0.2 mg/L NAA; GMS7： basic medium+0 mg/L 6-BA+0 mg/L NAA.

Somatic embryo maturation and plant regeneration

The plant regeneration culture adopted the best basic medium selected above as the medium， without adding any hormones， and the somatic embryos induced and differentiated were transferred into the medium， and then cultured in the dark to obtain mature somatic embryos. After that， they were transferred to the medium with halved elements for dark cultivation， and when the root system was fully germinated， they were transferred to light cultivation.

Culture conditions

The media for embryogenic callus induction and proliferation were all added with 3% sucrose and 6.5 g/L agar， and the pH value was adjusted to 5.8; and 1.5% sucrose was added to the plant regeneration medium， and other conditions were the same as above.

Somatic embryos were cultured in dark during the induction and proliferation process， and the culture temperature was about 25 ℃. The early stage of the culture process of somatic embryo maturation and plant regeneration was dark culture， and after the roots were fully germinated， they were transferred to light culture under 12 h/d and a light intensity of 2 000 Lx.

Data processing

SPSS 13.0 statistical software was used for relevant data analysis.

Results and Analysis

Induction of calli

After the mature embryos of S. paniculata were inoculated on 12 media of MS， GMS and WPM supplemented with different concentrations of hormones， they began to expand at 5 d， and calli began to appear at the hypocotyl and radicle at 10 d， and covered whole embryos at 15 d. With the increase of culture days， the callus mass also increased. On the 20th d， before the transfer， the callus induction rate was counted， and the shape and color of calli on different media were also observed and recorded， as shown in Table 2. It can be seen from Table 2 that different basic media showed great differences in the callus culture of S. paniculata， which was consistent with the results of variance analysis. The specific results are shown in Table 3. The calli cultured by MS and WPM media had a common feature： sticky， hygrophanous， soft， poor color. It is difficult for this type of calli to differentiate into embryogenic calli in the later culture process， and they will gradually brown and die with the increase of the number of passages. The calli induced by GMS medium were generally loose， obviously filamentous in structure， mostly milky white in color， and had strong proliferation and differentiation ability. Comparing the callus induction rate on each medium， it could be seen that different hormone ratios also had a great effect on the induction of S. paniculata calli， and the medium with the highest callus induction rate was GMS+0.2 mg/L 6-BA+0.1 mg/L NAA， and the callus induction rate was as high as 92.52%.

Differentiation and proliferation of embryogenic calli

After 2-3 times of subsulture， the calli with loose texture， milky white color and granular protrusions were selected and transferred to GMS1-GMS7. The callus began to differentiate into staghorn-like tissue in about 10 d. At 18 d， milky white granular tissue began to differentiate on the antler-like tissue， which was the so-called embryogenic callus. When observing and recording the test results， it was found that the differentiation and proliferation of embryogenic calli on different mediums were quite different. The specific results are shown in Table 4. As can be seen from Table 4， in the medium without adding any hormones， there were no signs of differentiation and proliferation; with the increase of 6-BA concentration， the differentiation rate also increased gradually， and the days of differentiation were shortened， but the effect on proliferation rate was not significant; and according to the change of auxin NAA concentration and the proliferation of embryogenic calli， the proliferation rate of embryogenic calli showed an increasing trend with the increase of NAA concentration， but when its concentration was greater than 0.15 mg/L， the proliferation rate of embryogenic calli showed a downward trend， indicating that NAA had a great effect on the proliferation of S. paniculata embryogenic calli and presented a parabolic shape. In addition， due to the different induction and differentiation ability of embryogenic calli in different media， some calli could simultaneously appear four different developmental stages： spherical embryo， heart-shaped embryo， torpedo embryo and cotyledon embryo during the process of differentiation. Based on the above， it could be seen that in terms of the current seven media， the best culture medium combination was GMS5， that is， GMS+0.25 mg/L 6-BA+0.15 mg/L NAA. The differentiation rate and proliferation rate of embryogenic calli cultured by GMS5 were higher at 71.11% and 76.56%， respectively.

Maturation of somatic embryos and plant regeneration

Since the previously induced somatic embryos had different developmental stages at the same time， in order to obtain sterile seedlings with a consistent growth laws， the induced somatic embryos must be inoculated into the modified MS medium without any hormones and cultured in the dark for a period of time， so that the somatic embryos at various stages develop into mature somatic embryos， that is， to the stage of cotyledon embryos （as shown in Fig. 1）. Finally， through cultivation， when the cotyledons opened into a trumpet shape， the cotyledon embryos were transferred into 1/2GMS+1.5% sucrose medium， and after a period of rooting culture， regenerated plants were obtained， but the root system was not developed enough. The specific reasons remain to be further studied.

Conclusions and Discussion

① For the process of callus induction， this study initially selected three basic media， MS， GMS and WPM. By setting different hormone ratios， the occurrence of calli was regularly observed， and it was found that the induction stage of calli had no high requirements on the types of medium， and each medium could induce S. paniculata calli， but the only medium that could achieve the culture target was the modified MS medium. Through multiple comparisons， it was concluded that the suitable medium combination for callus induction was GMS+0.2 mg/L 6-BA+0.1 mg/L NAA. The calli induced by the medium was not only loose in texture and had the same color as the embryos of the same species， but also partially showed obvious granular differentiation， which provided excellent materials for further somatic embryogenesis.

② Studies have shown that the characteristics of calli have a great effect on the somatic embryogenesis of S. paniculata， and only suitable calli can differentiate into normal somatic embryos， which is the reason for the selection of callus induction medium. Due to the differences in nutrients and culture conditions at different developmental stages， during the differentiation process of somatic embryos， we screened different hormone ratios on the basis of GMS medium according to our own culture purpose， and found that the blank medium could not induce somatic embryos， which indicated that plant growth regulators played a very important role in the process of somatic embryogenesis of S. paniculata. It could be seen from the results of various hormone treatments that high concentrations of 6-BA could promote the differentiation of somatic embryos， and the combination with appropriate concentrations of NAA could well improve the differentiation rate and proliferation rate of somatic embryos. The best hormone combination was 0.25 mg/L 6-BA+0.15 mg/L NAA， which could make the differentiation rate and proliferation rate of somatic embryos reach 71.99% and 76.56%， respectively.

③ During the experiment， it was also found that somatic embryos of different developmental stages could be differentiated from the same callus， which is consistent with the research results of related scholars[6-9]. It is precisely with this mechanism that the continuous proliferation and renewal of embryogenic calli can be ensured， so as to ensure the smooth progress of plant regeneration， which lays a foundation for further research on the mechanism of somatic embryogenesis of S. paniculata and the genetic transformation of excellent characters from the molecular level.

④ The somatic embryo maturation and rooting process adopted blank GMS medium. Since the osmotic pressure required for the rooting process of seedlings is different from tissue induction and differentiation， the amount of sucrose in the rooting medium was halved， but the final result was not ideal， and the specific reasons still need further research and exploration.

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All in all， the study on somatic embryogenesis of S. paniculata not only provides a new way for the rapid reproduction of S. paniculata， but also provides a wider range of materials for related research in the field of S. paniculata， which is beneficial to the genetic improvement of the tree species in the future， and provides a technical basis for the preparation of artificial seeds.

References

[1] MA Q， JIANG LJ， LI CZ. Serial Lectures on energy plants 12 Bio-diesel source plants： Symplocos paniculata[J]. Solar Energy， 2009（12）： 27. （in Chinese）.

[2] YANG Y， JIANG LJ， LI CZ， et al. Investigation and analysis of wild Symplocos paniculata resources in Dawei Mountain[J]. Hunan Forestry Science & Technology， 2011， 38（6）： 36-38. （in Chinese）.

[3] Institute of Botany， the Chinese Academy of Sciences. Iconographia cormophytorum sinicorum （Tomus 3）[M]. Beijing： Science Press， 1980： 331. （in Chinese）.

[4] GUAN ZX， ZHU TP， QIU TQ. Analysis and utilization evaluation of oils and amino acids of Symplocos paniculata seeds[J]. Chinese Wild Plant Resources，1991（2）： 11-14.（in Chinese）.

[5] ZUO CQ， ZHU TP， DU JR. Study on value of exploition and utilization of Symplocos paniculata[J]. Jiangxi hydraulic Science & Technology， 1994， 20（1）： 65-72. （in Chinese）.

[6] PULLMAN GS， CHOPRA R， CHASE KM. Loblolly pine （Pinus taeda L.） somatic embryogenesis： Improvements in embryogenic tissue initiation by supplementation of medium with organic acids，Vitamins B12 and E[J]. Plant Science， 2006（170）： 648-658.

[7] BELMONTE MF， MACEY J， YEUNG CE. et al. The effect of osmoticum on ascorbate and glutathione metabolism during white spruce （Picea glauca） somatic embryo development[J]. Plant Physiology and Biochemistry， 2005（43）： 337-346.

[8] JIANG RC， PENG FR， TAN PP， et al. Study on somatic embryogenesis of Catalpa bungei[J]. Journal of Nanjing Forestry University： Natural Science Edition， 2010， 34（2）： 15-18. （in Chinese）.

[9] FELIPE A， PATRIEIO AJ. Identification of genes expressed during early somatic embryogenesis in Pinus radiate[J]. Plant Physiology and Biochemistry， 2008（46）： 559-568.