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2022 Volume 2
Article Contents
Abstract
Introduction
Types of functional rice
High resistant starch rice
Colored rice
Giant embryo rice
Low glutelin rice
Golden rice
Fe and zn fortified rice
Breeding technology of functional rice
Traditional polymerization breeding
Transgenic polymerization breeding
Molecular marker-assisted selection polymerization breeding
Cultivation and regulation technology of functional rice
Effect of light on the accumulation of active components in rice
Effect of temperature on the accumulation of active components in rice
Effect of water management on the accumulation of active components in rice
Effect of fertilizer application on the accumulation of active components in rice
Effect of harvesting time on the accumulation of active components in rice
Prevention and control of pests and diseases for functional rice
Conclusions
Acknowledgments
Conflict of interest
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References
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REVIEW   Open Access    

Mini review: Application of the somatic embryogenesis technique in conifer species

  • 1.

    State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of National Forestry and Grassland Administration, Chinese Academy of Forestry, Haidian District, Dongxiaofu 1, Beijing 100091, PR China

  • 2.

    State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of National Forestry and Grassland Administration , Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, PR China

More Information
  • The somatic embryogenesis (SE) process is better suited to large-scale production and automation than other clonal propagation methods such as the rooting of cuttings. SE is becoming a key technique to promote the asexual industrialization of conifers. Furthermore, somatic embryos are an ideal material to study the molecular mechanism of conifer embryo development, as the processes of somatic and zygotic embryo development are very similar. This brief review introduces the culturing techniques of the SE process in conifers and outlines the progress and deficiencies in conifer SE research. Emphasis is placed on the patterning formation of conifer somatic embryos.

  • Introduction

    With the development of the world economy, people's lifestyles have changed dramatically, and long-term high-intensity work has put many people's bodies in a sub-healthy state. The increasing incidence of various chronic diseases has not only put enormous pressure on society's healthcare systems but also caused endless suffering to people[1]. Therefore, people's demands on the functionality and safety of food are increasing, and it has become the consensus of people that 'not just eating enough, but more importantly eating well'.

    Rice is the staple food for more than half of the world's population and the main economic source for a large number of rural people[2]. However, due to the rising cost of rice cultivation, farmers are gaining less and less economic benefits from growing rice, which seriously undermines their incentive to grow rice and poses a serious threat to world food security. Increasing the added value of rice not only helps to increase farmers' income but also helps to ensure world food security. The presence of a large number of functional ingredients in rice makes it possible to increase the added value of rice, and functional rice has therefore been widely noticed.

    Functional rice refers to rice containing certain specific components that play a regulatory and balancing role in human physiological functions in addition to the nutrients necessary for human growth and development in the endosperm, embryo, and rice bran. They can increase human physiological defense mechanisms, prevent certain diseases, help recovery, delay aging, and boost physical strength and energy levels[3]. Rice is a staple food for more than half of the world's population[4], and its functional components have a great potential to be exploited for human welfare. Using functional rice as a carrier to address health problems and realize 'medicine-food homology' is an excellent motivation for promoting functional rice. The current typical functional rice is introduced in this paper. It also summarizes the breeding and cultivation technologies of functional rice.

    Types of functional rice

    High resistant starch rice

    Rice has a high glycemic index. Its long-term consumption leads to obesity, diabetes, and colon disease in many people[5]. However, the consumption of rice rich in resistant starch (RS) can greatly reduce the risk of these diseases[6]. Therefore, breeding rice varieties with high RS content has attracted considerable attention from breeders in various countries. However, the variability of RS content between different rice varieties is low, and there are few germplasm resources available for selection, thus making it challenging to breed rice varieties with high RS content using traditional breeding methods. Combining traditional and modern molecular breeding techniques can greatly improve the successful production of high RS rice breeds. Nishi et al.[7] selected a high RS rice variety EM10 by treating fertilized egg cells of Kinmaze with N-methyl-N-nitrosourea. However, its yield was very low, and it was not suitable for commercial production. Wada et al.[8] crossed 'Fukei 2032' and 'EM129' as parents and selected Chikushi-kona 85, a high RS rice variety with a higher yield than EM10. Miura et al.[9] bred ultra-high RS BeI-BEIIB double mutant rice by crossing the Abe I and Abe IIB mutant strains, and the content of RS in the endosperm reached 35.1%. Wei et al.[10] found that the simultaneous inhibition of starch branching enzyme (SBE) genes SBEIIb and SBEI in Teqing by antisense RNA could increase the RS content in rice to 14.9%. Zhu et al.[11] used RNAi technology to inhibit the expression of SBEI and SBEII genes in rice, which increased the content of RS in rice endosperm from 0 to 14.6 %. Zhou et al.[6] found that rice RS formation is mainly controlled by soluble starch synthase (SSIIA). However, its regulation is dependent on the granule-bound starch synthase Waxy (Wx), and SSIIA deficiency combined with high expression of Wxa facilitates the substantial accumulation of RS in the rice. The results of Tsuiki et al.[12] showed that BEIB deficiency was the main reason for the increased accumulation of RS in rice. Itoh et al.[13] developed new mutant rice lines with significantly higher levels of RS in rice by introducing genes encoding starch synthase and granule-bound starch synthase in the rice into the BEIB-deficient mutant line be2b.

    Colored rice

    The accumulation of anthocyanins/proanthocyanidins in the seed coat of the rice grain gives brown rice a distinct color[14]. Most common rice varieties lack anthocyanins in the seed coat, and so far, no rice variety with colored endosperm in its natural state has been identified. However, Zhu et al.[15] bred rice with purple endosperm using transgenic technology. Red rice contains only proanthocyanidins, while black and purple rice contain anthocyanidins and proanthocyanidins[16]. Red seed coat of rice was found to be controlled by the complementary effects of two central effect genes Rc and Rd. The loss of function of the Rc gene prevented the synthesis of proanthocyanidins, while the Rd gene could enhance the effect of the Rc gene in promoting proanthocyanidins synthesis[17]. Purple seed coat color is controlled by two dominant complementary genes Pb and Pp. Pb determines the presence or absence of seed coat color, and Pp determines the depth of seed coat color[18]. In addition, phycocyanin synthesis is also regulated by transcription factors such as MYB, bHLH, HY5, and WD40[14], but the exact regulatory mechanism is not clear. Colored rice is rich in bioactive components, such as flavonoids, phenolic acids, vitamin E (VE), glutelin, phytosterols, and phytic acid (PA). It also contains large amounts of micronutrients such as Ca, Fe, Zn, and Se[19], and has a much higher nutritional and health value than ordinary white rice. In addition, Zhu et al.[20] successfully developed rice with enriched astaxanthin in the endosperm by introducing the genes sZmPSY1, sPaCrtI, sCrBKT, and sHpBHY. This achievement has laid a solid foundation for the further development of functional rice industry.

    Giant embryo rice

    Giant embryo rice refers to rice varieties whose embryo volume is more than twice that of ordinary rice[21]. Rice embryo contains more nutrients than the endosperm; therefore, the nutritional value of giant embryo rice greatly exceeds that of ordinary rice. Studies have found that the levels of γ-aminobutyric acid (GABA), essential amino acids, VE, γ-oryzanol, phenols, and trace elements in giant embryo rice are considerably higher than that in ordinary rice[21]. Satoh & Omura[22] used the chemical mutagen N-methyl-N-nitrosourea to treat the fertilized egg cells of the rice variety Kinmaze to obtain a 'giant embryo' mutant. The mutants’ embryo occupied 1/4–1/3 of the rice grain volume and was 3–4 times larger than normal rice embryo[23]. Its GABA content increased dramatically after the rice was soaked in water. Maeda et al.[24] crossed the giant embryo mutant EM40 of Kinmaze with the high-yielding variety Akenohoshi to produce the giant embryo rice variety 'Haiminori'. The embryo size of 'Haiminori' is 3–4 times that of ordinary rice, and the GABA content of its brown rice is 3–4 times higher than that of 'Nipponbare' and 'Koshihikari' after soaking for four hours in water. A few genes that can regulate the size of rice embryos have been identified, and GE is the first identified rice giant embryo gene[25]. Nagasawa et al.[26] found that the loss of GE gene function resulted in enlarged embryos and smaller endosperm in rice. Lee et al.[27] found that the inhibition of LE gene expression by RNAi technology could lead to embryo enlargement in rice, but the regulatory mechanism remains to be investigated.

    Low glutelin rice

    Protein is the second most crucial nutrient in rice, accounting for 7–10% of the grain weight, and glutenin accounts for 60%–80% of the total protein content in rice grains[28]. Compared to other proteins, glutenin is more easily digested and absorbed by the body[29]. Therefore, higher glutenin content in rice can improve its nutritional value. However, people with renal disease (a common complication of diabetes) have impaired protein metabolism, and consumption of rice with lower glutelin content can help reduce their protein intake and metabolic burden[30]. Japanese breeders treated Nihonmasari with the chemical mutagen ethyleneimine and selected the low-glutelin rice mutant NM67[31]. Iida et al.[31] developed a new rice variety LGC-1 (Low glutelin content-1) with a glutelin content of less than 4% by backcrossing the NM67 mutant with the original variety 'Nihonmasari'. According to Miyahara[32], the low glutelin trait in LGC-1 is controlled by a single dominant gene Lgc-1 located on chromosome 2. Subsequently, Nishimura et al.[33] produced two rice varieties, 'LGC Katsu' and 'LGC Jun' with lower glutelin content by crossing LGC1 with a mutant line Koshikari (γ-ray induction) lacking 26 kDa globulin (another easily digestible protein).

    Golden rice

    Vitamin A (VA) is one of the essential nutrients for the human body[34]. However, rice, a staple food, lacks VA, leading to a VA deficiency in many people. β-carotene is a precursor for VA synthesis and can be effectively converted into VA in the human body[35]. Therefore, breeding rice varieties rich in β-carotene has attracted the attention of breeders in various countries. Ye et al.[36] simultaneously transferred phytoene synthase (psy), phytoene desaturase (crt I), and lycopene β-cyclase (lcy) genes into rice using the Agrobacterium-mediated method and produced the first generation of golden rice with a β-carotene content of 1.6 µg·g−1 in the endosperm. However, due to the low content of β-carotene in rice, it is difficult to meet the human body's demand for VA. To increase β-carotene content in rice, Paine et al.[37] introduced the phytoene synthase (psy) gene from maize and the phytoene desaturase (crt I) gene from Erwinia into rice. They obtained the second generation of golden rice with 37 µg g−1 of β-carotene in the endosperm, with nearly 23-fold increase in β-carotene content compared to the first generation of golden rice.

    Fe and Zn fortified rice

    Fe and Zn are essential trace elements for human beings. The contents of Fe and Zn in common rice are about 2 μg·g−1 and 16 μg·g−1, respectively[38], which are far from meeting human needs. In 2004, to alleviate micronutrient deficiencies among underprivileged people in developing countries, the Consultative Group on International Agricultural Research launched the HarvestPlus international collaborative program for improving Fe, Zn, and β-carotene levels in staple crops, with breeding targets of 13 μg·g−1 and 28 μg·g−1 for Fe and Zn in rice, respectively. Masuda et al.[39] found that expression of the nicotianamine synthase (NAS) gene HvNAS in rice resulted in a 3-fold increase in Fe and a 2-fold increase in Zn content in polished rice. Trijatmiko et al.[38] overexpressed rice OsNAS2 gene and soybean ferritin gene SferH-1 in rice, and the Fe and Zn content in polished rice of rice variety NASFer-274 reached 15 μg·g−1 and 45.7 μg·g−1, respectively. In addition, it has been found that increasing Fe intake alone does not eliminate Fe deficiency but also decreases the amount of Fe absorption inhibitors in the diet or increases the amount of Fe absorption enhancers[40]. The negatively charged phosphate in PA strongly binds metal cations, thus reducing the bioavailability of Fe and Zn in rice[41], while the sulfhydryl group in cysteine binds Fe, thereby increasing the absorption of non-heme Fe by the body[42]. To improve the bioavailability of Fe and Zn, Lucca et al.[40] introduced a heat-tolerant phytase (phyA) gene from Aspergillus fumigatus into rice and overexpressed the cysteine-rich protein gene (rgMT), which increased the content of phytase and cysteine residues in rice by 130-fold and 7-fold, respectively[40].

    Breeding technology of functional rice

    The functional quality of rice is highly dependent on germplasm resources. Current functional rice breeding mainly adopts transgenic and mutagenic technologies, and the cultivated rice varieties are mainly enriched with only one functional substance and cannot meet the urgent demand by consumers for rice enriched with multiple active components. The diversity of rice active components determines the complexity of multifunctional rice breeding. In order to cultivate multifunctional rice, it is necessary to strengthen the application of different breeding technologies. Gene polymerization breeding is a crop breeding technology that can polymerize multiple superior traits that have emerged in recent years, mainly including traditional polymerization breeding, transgenic polymerization breeding, and molecular marker-assisted selection polymerization breeding.

    Traditional polymerization breeding

    The transfer of beneficial genes in different species during traditional polymeric breeding is largely limited by interspecific reproductive isolation, and it is challenging to utilize beneficial genes between different species effectively. Gene transfer through sexual crosses does not allow accurate manipulation and selection of a gene and is susceptible to undesirable gene linkage, and in the process of breed selection, multiple backcrosses are required[43]. Thus, the period of selecting target plants is long, the breeding cost is high, and the human resources and material resources are costly[44]. Besides, it is often difficult to continue the breakthrough after a few generations of backcrossing due to linkage drag. Thus, there are significant limitations in aggregating genes by traditional breeding methods[45].

    Transgenic polymerization breeding

    Transgenic technology is an effective means of gene polymerization breeding. Multi-gene transformation makes it possible to assemble multiple beneficial genes in transgenic rice breeding rapidly and can greatly reduce the time and workload of breeding[46]. The traditional multi-gene transformation uses a single gene transformation and hybridization polymerization method[47], in which the vector construction and transformation process is relatively simple. However, it is time-consuming, laborious, and requires extensive hybridization and screening efforts. Multi-gene-based vector transformation methods can be divided into two major categories: multi-vector co-transformation and multi-gene single vector transformation[47]. Multi-vector co-transformation is the simultaneous transfer of multiple target genes into the same recipient plant through different vectors. The efficiency of multi-vector co-transformation is uncertain, and the increase in the number of transforming vectors will increase the difficulty of genetic screening, resulting in a reduced probability of obtaining multi-gene co-transformed plants. Multi-gene single vector transformation constructs multiple genes into the T-DNA region of a vector and then transfers them into the same recipient plant as a single event. This method eliminates the tedious hybridization and backcrossing process and solves the challenges of low co-transformation frequency and complex integration patterns. It can also avoid gene loss caused by multi-gene separation and recombination in future generations[47]. The transgenic method can break through the limitations of conventional breeding, disrupt reproductive isolation, transfer beneficial genes from entirely unrelated crops to rice, and shorten the cycle of polymerizing target genes significantly. However, there are concerns that when genes are manipulated, unforeseen side effects may occur, and, therefore, there are ongoing concerns about the safety of transgenic crops[48]. Marker-free transgenic technology through which selective marker genes in transgenic plants can be removed has been developed. This improves the safety of transgenic crops, is beneficial to multiple operations of the same transgenic crop, and improves the acceptance by people[49].

    Molecular marker-assisted selection polymerization breeding

    Molecular marker-assisted selection is one of the most widely used rice breeding techniques at present. It uses the close linkage between molecular markers and target genes to select multiple genes directly and aggregates genes from different sources into one variety. This has multiple advantages, including a focused purpose, high accuracy, short breeding cycle, no interference from environmental conditions, and applicability to complex traits[50]. However, few genes have been targeted for the main effect of important agronomic traits in rice, and they are mainly focused on the regulation of rice plant type and the prevention and control of pests and diseases, and very few genes related to the synthesis of active components, which can be used for molecular marker-assisted selection are very limited. Furthermore, the current technical requirements and costs for analyzing and identifying DNA molecular markers are high, and the identification efficiency is low. This greatly limits the popularization and application of functional rice polymerization breeding. Therefore, to better apply molecular marker-assisted selection technology to breed rice varieties rich in multiple active components, it is necessary to construct a richer molecular marker linkage map to enhance the localization of genes related to functional substance synthesis in rice[51]. Additionally, it is important to explore new molecular marker technologies to improve efficiency while reducing cost.

    It is worth noting that the effects of gene aggregation are not simply additive. There are cumulative additive effects, greater than cumulative epistatic effects, and less than cumulative epistatic effects among the polymerization genes, and the effects are often smaller than the individual effect. Only with a clearer understanding of the interaction between different QTLs or genes can functional rice pyramiding breeding be carried out reasonably and efficiently. Except for RS and Se, other active components of rice mainly exist in the rice bran layer, and the content of active components in the endosperm, the main edible part, is extremely low. Therefore, cultivating rice varieties with endosperm-enriched active components have broad development prospects. In addition, because crops with high quality are more susceptible to pests and diseases[52], the improvement of rice resistance to pests and diseases should be considered during the polymerization breeding of functional rice.

    Cultivation and regulation technology of functional rice

    The biosynthesis of active components in rice is influenced by rice varieties but also depends on cultivation management practices and their growth environment.

    Effect of light on the accumulation of active components in rice

    Environmental conditions have a greater effect on protein content than genetic forces[53]. Both light intensity and light duration affect the synthesis and accumulation of active components in rice. Low light intensity in the early stage of rice growth is not conducive to the accumulation of glutelin in rice grains but favors the accumulation of amylose, while the opposite is true in the late stage of rice growth[54]. Low light intensity during the grain-filling period reduces the accumulation of total flavonoids in rice[55] and decreases Fe ions' movement in the transpiration stream and thereby the transport of Fe ions to rice grains[56]. An appropriate increase in light intensity is beneficial to the accumulation of flavonoids, anthocyanins, and Fe in rice, but the photostability of anthocyanins is poor, and too much light will cause oxidative degradation of anthocyanins[57]. Therefore, functional rice is best cultivated as mid-late rice, which would be conducive to accumulating active components in rice.

    Effect of temperature on the accumulation of active components in rice

    The temperature has a great influence on the synthesis of active components in rice. An appropriate increase in the temperature is beneficial to the accumulation of γ-oryzanol[58] and flavonoids[59] in rice. A high temperature during the grain-filling period leads to an increase in glutelin content in rice[60], but an increase in temperature decreases the total phenolic content[61]. The results regarding the effect of temperature on the content of PA in rice were inconsistent. Su et al.[62] showed that high temperatures during the filling period would increase the PA content, while Goufo & Trindade[61] reported that the increase in temperature would reduce the PA content. This may be due to the different growth periods and durations of temperature stress on rice in the two studies. The synthesis of anthocyanins/proanthocyanidins in colored rice requires a suitable temperature. Within a certain range, lower temperatures favor the accumulation of anthocyanins/proanthocyanidins in rice[63]. Higher temperatures will lead to degradation, and the thermal stability of proanthocyanidins being higher than that of anthocyanins[64]. In addition, cold or heat stress facilitates GABA accumulation in rice grains[65]. Therefore, in actual production, colored rice and low-glutelin rice are best planted as late rice, and the planting time of other functional rice should be determined according to the response of its enriched active components to temperature changes.

    Effect of water management on the accumulation of active components in rice

    Moderate water stress can significantly increase the content of glutelin[66] and GABA[67] in rice grains and promote the rapid transfer of assimilation into the grains, shorten the grain filling period, and reduce the RS content[68]. Drought stress can also induce the expression of the phytoene synthase (psy) gene and increase the carotenoid content in rice[69]. Soil moisture is an important medium in Zn diffusion to plant roots. In soil with low moisture content, rice roots have low available Zn, which is not conducive to enriching rice grains with Zn[70]. Results from studies on the effect of soil water content on Se accumulation in rice grains have been inconsistent. Li et al.[71] concluded that flooded cultivation could significantly increase the Se content in rice grains compared to dry cultivation. However, the results of Zhou et al.[72] showed that the selenium content in rice grains under aerobic and dry-wet alternative irrigation was 2.44 and 1.84 times higher than that under flood irrigation, respectively. This may be due to the forms of selenium contained in the soil and the degree of drought stress to the rice that differed between experiments[73]. In addition, it has been found that too much or too little water impacts the expression of genes related to anthocyanin synthesis in rice, which affects the accumulation of anthocyanins in rice[74]. Therefore, it is recommended to establish different irrigation systems for different functional rice during cultivation.

    Effect of fertilizer application on the accumulation of active components in rice

    Both the amount and method of nitrogen application affect the accumulation of glutelin. Numerous studies have shown that both increased and delayed application of nitrogen fertilizer can increase the accumulation of lysine-rich glutelin to improve the nutritional quality of rice (Table 1). However, this improvement is not beneficial for kidney disease patients who cannot consume high glutelin rice. Nitrogen stress can down-regulate the expression of ANDs genes related to the anthocyanins biosynthesis pathway in grains, resulting in a decrease in anthocyanins synthesis[55]. Increased nitrogen fertilizer application can also increase the Fe, Zn, and Se content in rice[75,76]. However, some studies have found that increased nitrogen fertilizer application has no significant effect on the Fe content of rice[77], while other studies have shown that increased nitrogen fertilizer application will reduce the Fe content of rice[78]. This may be influenced by soil pH and the form of the applied nitrogen fertilizer. The lower the soil pH, the more favorable the reduction of Fe3+ to Fe2+, thus promoting the uptake of Fe by rice. Otherwise, the application of ammonium fertilizer can improve the availability of soil Fe and promote the absorption and utilization of Fe by rice. In contrast, nitrate fertilizer can inhibit the reduction of Fe3+ and reduce the absorption of Fe by rice[79].

    Table 1.  Effect of nitrogen fertilizer application on glutelin content of rice.
    SampleN level
    (kg ha−1)    		Application timeGlutelin content
    (g 100 g−1)    		References
    Rough rice05.67[66]
    270Pre-transplanting : mid tillering : panicle initiation : spikelet differentiation = 2:1:1:16.92
    300Pre-transplanting : mid tillering : panicle initiation : spikelet differentiation = 5:2:2:16.88
    Brown rice05.35[83]
    90Pre-transplanting : after transplanting = 4:16.01
    Pre-transplanting : after transplanting = 1:16.60
    180Pre-transplanting : after transplanting = 4:16.53
    Pre-transplanting : after transplanting = 1:17.29
    270Pre-transplanting : after transplanting = 4:17.00
    Pre-transplanting : after transplanting = 1:17.66
    Rough rice05.59[84]
    187.5Pre-transplanting : after transplanting = 4:16.47
    Pre-transplanting : after transplanting = 1:16.64
    300Pre-transplanting : after transplanting = 4:17.02
    Pre-transplanting : after transplanting = 1:17.14
    Polished rice03.88[85]
    90Pre-transplanting : tillering : booting = 2:2:14.21
    180Pre-transplanting : tillering : booting = 2:2:14.43
    270Pre-transplanting : tillering : booting = 2:2:16.42
    360Pre-transplanting : tillering : booting = 2:2:14.87
    Brown rice09.05[86]
    120Flowering22.14
     | Show Table
    DownLoad: CSV

    Appropriate application of phosphorus fertilizer is beneficial in promoting the translocation of Fe and Zn from leaves to rice grains, thus increasing the content in rice grains[80]. However, the excessive application of phosphate fertilizer will reduce the availability of Fe and Zn in soil, resulting in less uptake by the roots and a lower content in the rice grains[81]. The content of PA in rice increased with a higher phosphorus fertilizer application rate[80]. Increasing the phosphorus fertilizer application rate would increase the values of [PA]/[Fe] and [PA]/[Zn] and reduce the effectiveness of Fe and Zn in rice[80]. Currently, there are few studies on the effect of potassium fertilization on the synthesis of active components in rice. Available studies report that increased application of nitrogen fertilizer can increase the Zn content in rice[82]. Therefore, the research in this area needs to be strengthened.

    Because the iron in soil mainly exists in the insoluble form Fe3+, the application of iron fertilizer has little effect on rice biofortification[87]. There are different opinions about the effect of Zn fertilizer application methods. Phattarakul et al.[88] believed that foliar spraying of Zn fertilizer could significantly improve the Zn content in rice grains. Jiang et al.[89] concluded that most of the Zn accumulated in rice grains were absorbed by the roots rather than from the reactivation of Zn in leaves. In contrast, Yuan et al.[90] suggested that soil application of Zn fertilizer had no significant effect on Zn content in rice grains. The different results may be affected by the form of zinc fertilizer applied and the soil conditions in the experimental sites. Studies have found that compared with the application of ZnEDTA and ZnO, zinc fertilizer in the form of ZnSO4 is most effective for increasing rice's Zn[70]. In addition, the application of zinc fertilizer reduces the concentration of PA in rice grains[70].

    The form of selenium fertilizer and the method and time of application will affect the accumulation of Se in rice grains. Regarding selenium, rice is a non-hyperaccumulative plant. A moderate application of selenium fertilizer can improve rice yield. However, the excessive application can be toxic to rice, and the difference between beneficial and harmful supply levels is slight[91]. Selenite is readily adsorbed by iron oxide or hydroxide in soil, and its effectiveness in the soil is much lower than selenite[92]. In addition, selenate can migrate to the roots and transfer to rice shoots through high-affinity sulfate transporters. In contrast, selenite is mainly assimilated into organic selenium in the roots and transferred to the shoots in smaller amounts[93]. Therefore, the biological effectiveness of Se is higher in selenate-applied soil than in selenite application[94] (Table 2). Zhang et al.[95] found that the concentration of Se in rice with soil application of 100 g Se ha-1 was only 76.8 μg·kg-1, while the concentration of Se in rice with foliar spray of 75 g Se ha-1 was as high as 410 μg·kg-1[73]. However, the level of organic selenium was lower in rough rice with foliar application of selenium fertilizer compared to soil application[96], while the bioavailability of organic selenium in humans was higher than inorganic selenium[97]. Deng et al.[73] found that the concentrations of total selenium and organic selenium in brown rice with selenium fertilizer applied at the full heading stage were 2-fold higher than those in brown rice with selenium fertilizer applied at the late tillering stage (Table 2). Although the application of exogenous selenium fertilizer can rapidly and effectively increase the Se content of rice (Table 2), it can easily lead to excessive Se content in rice and soil, which can have adverse effects on humans and the environment. Therefore, breeding Se-rich rice varieties is a safer and more reliable way to produce Se-rich rice. In summary, functional rice production should include the moderate application of nitrogen and phosphorus fertilizer and higher levels of potassium fertilizer, with consideration to the use of trace element fertilizers.

    Table 2.  Effect of selenium fertilizer application on the selenium content of rice.
    SampleSe level (g Se ha−1)Selenium fertilizer formsApplication methodSe content (μg·g−1)References
    Rough rice00.002[98]
    18SeleniteFoliar spray at full heading0.411
    Polished rice00.071[99]
    20SeleniteFoliar spray at full heading0.471
    20SelenateFoliar spray at full heading0.640
    Rough rice75SeleniteFoliar spray at late tillering0.440[73]
    75SeleniteFoliar spray at full heading1.290
    75SelenateFoliar spray at late tillering0.780
    75SelenateFoliar spray at full heading2.710
    Polished rice00.027[100]
    15SeleniteFoliar spray at full heading0.435
    45SeleniteFoliar spray at full heading0.890
    60SeleniteFoliar spray at full heading1.275
     | Show Table
    DownLoad: CSV

    Effect of harvesting time on the accumulation of active components in rice

    The content of many active components in rough rice is constantly changing during the development of rice. It was found that the content of total flavonoids in brown rice increased continuously from flowering stage to dough stage and then decreased gradually[101]. The γ-oryzanol content in rice decreased by 13% from milk stage to dough stage, and then gradually increased to 60% higher than milk stage at full maturity[101]. The results of Shao et al.[102] showed that the anthocyanin content in rice reached its highest level at two weeks after flowering and then gradually decreased. At full ripeness, and the anthocyanins content in brown rice was only about 50% of the maximum level. The content of total phenolics in rice decreased with maturity from one week after flowering to the fully ripe stage, and the loss of total phenolics reached more than 47% by the fully ripe stage. In contrast, the content of total phenolics in black rice increased with maturity[102]. Moreover, RS content in rough rice decreases during rice maturation[68]. Therefore, the production process of functional rice should be timely and early harvested to obtain higher economic value.

    Prevention and control of pests and diseases for functional rice

    Pests and diseases seriously impact the yield and quality of rice[103]. At present, the two most effective methods to control pests and diseases are the use of chemical pesticides and the planting of pest and disease-resistant rice varieties. The use of chemical pesticides has greatly reduced the yield loss of rice. However, excessive use of chemical pesticides decreases soil quality, pollutes the environment, reduces soil biodiversity[104], increases pest resistance, and aggravates the adverse effects of pests and diseases on rice production[105]. It also increases residual pesticide levels in rice, reduces rice quality, and poses a severe threat to human health[106].

    Breeding pest and disease-resistant rice varieties are among the safest and effective ways to control rice pests and diseases[107]. In recent years, many pest and disease resistance genes from rice and microorganisms have been cloned[47]. Researchers have used these genes to breed rice varieties resistant to multiple pests and diseases through gene polymerization breeding techniques. Application in production practices delivered good ecological and economic benefits[108].

    Green pest and disease control technologies must consider the synergies between rice and water, fertilizer, and pest and disease management. In this regard, the rice-frog, rice-duck, and other comprehensive rice production models that have been widely used in recent years are the most representative. These rice production models significantly reduced chemical pesticide usage and effectively controlled rice pests and diseases[109]. The nutritional imbalance will reduce the resistance of rice to pests and diseases[110]. Excessive application of nitrogen fertilizer stimulates rice overgrowth, protein synthesis, and the release of hormones, increasing its attractiveness to pests[111]. Increased soluble protein content in rice leaves is more conducive to virus replication and increases the risk of viral infection[112]. Increasing the available phosphorus content in the soil will increase crop damage by pests[113], while insufficient potassium supply will reduce crop resistance to pests and diseases[114]. The application of silica fertilizer can boost the defense against pests and diseases by increasing silicon deposition in rice tissue, inducing the expression of genes associated with rice defense mechanisms[115] and the accumulation of antifungal compounds in rice tissue[116]. The application of silica fertilizer increases the release of rice volatiles, thereby attracting natural enemies of pests and reducing pest damage[117]. Organic farming increases the resistance of rice to pests and diseases[118]. In addition, rice intercropping with different genotypes can reduce pests and diseases through dilution and allelopathy and changing field microclimate[119].

    In conclusion, the prevention and control of rice pests and diseases should be based on chemical and biological control and supplemented by fertilizer management methods such as low nitrogen, less phosphorus, high potassium and more silicon, as well as agronomic measures such as rice-aquaculture integrated cultivation, organic cultivation and intercropping of different rice varieties, etc. The combined use of multiple prevention and control measures can improve the yield and quality of functional rice.

    Conclusions

    Functional rice contains many active components which are beneficial to maintaining human health and have high economic and social value with broad market prospects. However, the current development level of the functional rice industry is low. The development of the functional rice requires extensive use of traditional and modern polymerization breeding techniques to cultivate new functional rice varieties with endosperm that can be enriched with multiple active components and have broad-spectrum resistance to pests and diseases. It is also important to select suitable planting locations and times according to the response characteristics of different functional rice active components to environmental conditions.

    Acknowledgments

    This work is supported by the National Natural Science Foundation of China (Project No. 32060430 and 31971840), and Research Initiation Fund of Hainan University (Project No. KYQD(ZR)19104).

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Zhu T, Wang J, Hu J, Ling J. 2022. Mini review: Application of the somatic embryogenesis technique in conifer species. Forestry Research 2:18 doi: 10.48130/FR-2022-0018
    Zhu T, Wang J, Hu J, Ling J. 2022. Mini review: Application of the somatic embryogenesis technique in conifer species. Forestry Research 2:18 doi: 10.48130/FR-2022-0018
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Mini review: Application of the somatic embryogenesis technique in conifer species

  • 1.

    State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of National Forestry and Grassland Administration, Chinese Academy of Forestry, Haidian District, Dongxiaofu 1, Beijing 100091, PR China

  • 2.

    State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of National Forestry and Grassland Administration , Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, PR China

  • Received: 21 September 2022
  • Accepted: 21 November 2022
  • Published online: 07 December 2022
Forestry Research  2,  Article number: 18  (2022)  |  Cite this article

Abstract: The somatic embryogenesis (SE) process is better suited to large-scale production and automation than other clonal propagation methods such as the rooting of cuttings. SE is becoming a key technique to promote the asexual industrialization of conifers. Furthermore, somatic embryos are an ideal material to study the molecular mechanism of conifer embryo development, as the processes of somatic and zygotic embryo development are very similar. This brief review introduces the culturing techniques of the SE process in conifers and outlines the progress and deficiencies in conifer SE research. Emphasis is placed on the patterning formation of conifer somatic embryos.

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  • Somatic embryogenesis (SE) is a universal phenomenon unique to the plant kingdom. The SE technique has considerable application significance in valuable varieties for which it is difficult to obtain seeds and for forests with long growth cycles. The SE technique is considered to be one of the most important asexual propagation techniques for conifers[1] and is conducive to the rapid reproduction of new varieties of valuable conifers. In 1985, Hakman et al. were the first to achieve SE in coniferous species[2]. They induced somatic proembryogenic masses (PEMs) using immature Picea abies zygotes as explants and obtained regenerated plants. To date, most conifer species can only use immature/mature embryos as explants for PEM induction, although there are a few exceptional genotypes in which PEM can be induced from primordial shoots[3,4]. The cell morphology and physiological changes associated with somatic embryo induction, maturation and germination of spruce species have been systematically studied in Picea glauca[5]. SE has been achieved in more than 50 tree species and hybrids in six coniferous genera, including Abies, Larix, Picea, Pinus, Pseudotsuga and Sequol[2,5−8].

    Somatic embryo development in conifers

    • Conifer SE techniques involve four phases: proliferation of the PEM, induction of SE, formation of the meristematic centers, and development of the somatic embryo[9]. The PEM proliferates on a proliferation medium that contains plant growth factors (PGRs), including auxin and cytokinin. The cultures must be subcultured every 10 to 21 d (depending on the species) onto fresh medium. Long-term subculture leads to a decrease or even complete loss of embryo production capacity in the PEM lines. For long-term storage, the cultures can be cryopreserved. One week of withdrawal of PGRs effectively induces the differentiation of early somatic embryos (EEs). After the SE induction phase, the cultures are transferred to a maturation medium that contains abscisic acid (ABA) and a high sucrose content. The establishment of meristematic centers characterizes the formation of late somatic embryos (LEs). In the last phase, somatic embryos achieve both morphological and physiological maturity. It takes 4 to 6 weeks for development into mature somatic embryos (MEs) on maturation medium (Fig. 1a). The MEs must be desiccated before germination, and they are finally planted in the field.

      Figure 1. 

      Conifer embryo development represented by Picea abies. (a) Schematic representation of the developmental stages of somatic embryo development. (b)−(f) Somatic embryo development process: (b) proembryogenic mass (PEM); (c) cultures after one week on maturation medium, insert presents an early somatic embryo (EE); (d) culture contains late embryos (LEs); (e) culture contains maturing and (f) matured somatic embryo (ME). (g), (h) Dissected seeds to show (g) the early zygotic embryo, indicated by the black arrow, and (h) the maturing zygotic embryo. Bar = 500 μm.

    Induction of somatic embryogenesis in conifers

    • The induction and differentiation rates of the PEMs are related to genotype. Currently, some desirable varieties are difficult to propagate with the SE technique. Somatic dedifferentiation and redifferentiation require the regulation of a multitude of genes and chromosome reprogramming, which are controlled by DNA methylation and histone modification. Related research has been widely conducted in angiosperms. However, this knowledge is very limited in gymnosperms. Some genes have been verified as major regulators of SE or plant embryo patterning in a variety of plant species. Table 1 lists some of these genes that have been reported in conifers. Homologs of BBM, LEC1, WOX2 and SERK1 have been identified in Larix decidua[10]. LEC1, WOX2 and SERK1 are presumed to conserve their function in the induction of SE based on their expression pattern[11].

      Table 1.  List of some of the major regulatory genes in somatic embryogenesis.

      Gene familyGeneDescriptionReferences
      LRR-RLKsSOMATIC EMBRYOGENESIS RECEPTOR-
      LIKE KINASE 1-5 (SERKs)      		Transmembrane proteins; involved in signal transduction and have been strongly associated with somatic embryogenesis and apomixis in a number of plant species.[23]
      AP2/ERFBABYBOOM (BBM)Tanscription factor; activates LEC1-ABI3-FUS3-LEC2 network to induce somatic embryogenesis.[24]
      EMBRYOMAKER (EMK/AIL5 )Tanscription factors; promote the formation of somatic embryo on cotyledons.[25]
      WOUND INDUCED DEDIFFERENTIATION1 (WIND1)Tanscription factor; controls cell dedifferentiation in Arabidopsis and functions as a key molecular switch for plant cell dedifferentiation.[26,27]
      B3-AFLLEAFY COTYLEDON 1 (LEC1)Tanscription factor; promote somatic embryo development in vegetative organs.[28]
      LEC1-LIKE (L1L)Tanscription factor; promote somatic embryo development in vegetative organs.[29]
      LEAFY COTYLEDON 2 (LEC2)Tanscription factor; activates the expression of embryonic traits in vegetative tissues.[28]
      ABSCISIC ACID INSENSITIVE 3 (ABI3)/VIVI PAROUS (VP1)Transcript factor; regulates embryo-specific ABA-induced genes.[30]
      FUSCA3 (FUS3)Transcription factor; promotes embryogenesis by regulating synthesis of storage proteins and lipids.[31]
      VP1/ABI3-LIKE (VAL)Transcription factor; repress plant embryo development.[32]
      WOXWUSCHEL (WUS)Transcription factor; a central player in stem cell maintenance in the SAM.[33]
      WUSCHEL-related homeobox (WOX) 2Transcription factor; promotes apical embryonic cell division.[34]
      WOX 5Transcription factor; a central player in stem cell maintenance in the SAM.[35]
      WOX 8 and WOX9WOX8 and WOX9 functionally overlap in promoting basal embryonic cell division.[34]
      NACCUP SHAPED COTYLEDONS 1-3 (CUCs)CUP SHAPED COTYLEDONS 1-3 act redundantly to specify the cotyledon boundary.[36−38]
      HD-GL2Arabidopsis thaliana meristem L1 layer (ATML1)An early molecular marker for the establishment of both apical-basal and radial patterns during plant embryogenesis.[39]
      ANTHOCYANINLESS2 (ANL2)anl2 mutant shows aberrant cellular organization.[40]
      Class I KNOX geneSHOOTMERISTEMLESS (STM)Tanscription factors regulate the architecture of the SAM by maintaining a balance between cell division and differentiation.[41]
      GRASSCARECROW (SCR)Regulates the radial organization of the root.[42]
      AGO proteinsARGONAUTE (AGO)Participate in post-transcriptional gene silencing and influence stem cell fate specification in both plants and animals.[43]
      PcG proteinsPOLYCOMB REPRESSIVE COMPLEX
      subunit genes      		Epigenetic effector proteins; stem cell self-renewal, pluripotency, gene silencing; repressive effect on dedifferentiation ability of cells.[15,16]

      Low levels of global DNA methylation have been found in the embryogenic cultures of several plants. It was found that de novo DNA methylation and its maintenance are required for the regulation of SE in Picea abies[12]. Klimaszewska et al. detected no significant differences in DNA methylation between embryogenic and nonembryogenic Pinus pinaster cultures[13]. Histone posttranslational modifications such as histone deacetylation and methylation have been implicated in the formation of somatic embryos. Trichostatin A (TSA) treatment, which inhibits histone deacetylases, interferes with somatic embryogenesis induction in conifers[14]. H3K27me3, which is written and read by polycomb repressive complex 2 (PRC2), controls cell differentiation by directing widespread transcriptional repression[15,16]. Nakamura et al.[17] reported that the H3K27me3 level was low in the productive PEM but markedly increased upon embryo induction in P. abies.

      Zygotic embryos are nourished via the phloem tissue, whereas somatic embryos use an exogenous supply of carbohydrates. It is assumed that the existence of ‘nurse cells’, which can provide an endosperm-like environment to facilitate the initial development of somatic embryos, is critical for the proliferation of PEMs. Conditioned medium (spent medium harvested from cultured cells) from embryogenic cultures can promote embryogenesis. Elhiti et al. reported 51 proteins that function in early somatic embryogenesis[18]. A glycosylated acidic endochitinase, which is involved in the cleavage of compounds such as lipo-chitooligosaccharides (LCOs)[19] and arabinogalactan proteins (AGPs)[20], can stimulate embryo development and growth. In P. abies, the chitinase 4-encoding gene Chia4-Pa is expressed in the single cell-layered zone surrounding the corrosion cavity of the megagametophyte and surrounding the early somatic embryo[21]. Furthermore, LCOs and AGPs have been isolated from P. abies conditioned medium and have been demonstrated to be effective stimulators of somatic embryogenesis[19]. Vanillyl benzyl ether has been confirmed to be an inhibitory compound that leads to the development of new somatic embryos[22]. This compound could inhibit the differentiation of suspensors.

    Comparison of zygotic and somatic embryo development in conifers

    • In angiosperms, asymmetric cell division of the zygote produces one cell that gives rise to the suspensor and another that give rise to the embryo proper. This phenomenon is not observed in SE. In conifers, zygotes undergo several rounds of nuclear duplication without cytokinesis to enter a free nuclear phase after fertilization, which is followed by cellularization to form an eight-celled proembryo. The cells of the apical portion multiply to form the embryo proper, whereas cells of the basal part elongate and undergo limited cell division to form the embryonic suspensor[44]. The free nuclear phase is absent in SE. The development of somatic embryos (Fig. 1b−f) is morphologically similar to that of zygotic embryos (Fig. 1g, h) in the later phases. After the induction of SE in the somatic embryo, the embryonal mass and the suspensor are separated by a layer of conifer-specific cells called embryonal tube cells. These cells produce apical meristem cells and basal suspensor cells through asymmetric anticlinal division. The primary body plan is established during embryogenesis. Somatic embryos are not hidden behind ovules and can be obtained throughout the year. These characteristics make somatic embryos an ideal material to study the physiological characteristics and molecular mechanism of conifer embryo development. Combining the relevant knowledge of angiosperms and reverse genetics, a general understanding of the molecular regulatory mechanism of conifer embryo development has been acquired.

    Patterning formation of conifer somatic embryos

    • Apical-basal differentiation is the foundation of plant embryonic development. Polar auxin transport is essential for the correct patterning of both the apical and basal parts of conifer embryos throughout the entire developmental process. Auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA) treatment of early embryos leads to Indole-3-acetic acid (IAA) accumulation in the suspensor, which inhibits programmed cell death (PCD) of the suspensor, thereby resulting in aberrant suspensor development. NPA treatment of late embryos leads to fused cotyledons, the absence of shoot apical meristem (SAM) and aberrant root apical meristem (RAM)[45]. The aberrant morphologies of NPA-treated spruce embryos are comparable to several auxin response and transport mutants in Arabidopsis.

      PCD eliminates unwanted cells during embryogenesis, which is necessary for correct embryonic pattern formation[46]. Two successive waves of PCD were observed during SE of P. abies[47]: the first wave was responsible for the degradation of ECs when they develop into somatic embryos; the second wave eliminated terminally differentiated embryo-suspensor cells during early embryogenesis. A reverse genetics study demonstrated that autophagic PCD is regulated by the type II metacaspase mcII-Pa. RNAi inhibition of mcII-Pa both inhibits autophagy in the suspensor cells and induces necrosis of the differentiated cells caused by autophagy disorder[48,49].

      WUSCHEL-related homeobox (WOXs) are a family of plant-specific transcription factors that play important roles in cell fate determination during plant development. There are 15 WOX genes in Arabidopsis. AtWUS, AtWOX2, AtWOX5, AtWOX8 and AtWOX9 are most relevant to embryo patterning. AtWUS and AtWOX5 are crucial regulators of SAM and RAM, respectively[35,50]; they are necessary for meristem maintenance but are not required for their initiation. AtWOX2 is specifically expressed in apical embryonic cells, whereas AtWOX8 and AtWOX9 are specifically expressed in suspensor cells after the first zygotic cell division[34]. Eleven and 14 WOX genes have been identified in P. abies and P. pinaster, respectively[51,52]. Notably, only one homolog of WUS/WOX5 has been be detected in gymnosperms[51,52]. P. pinaster WOX5 shows maximum expression at the mature embryo stage with transcripts preferentially located at the root tip of seedlings[52]. The WUS and WOX5 genes are the result of angiosperm-specific gene duplication[53]. PaWOX2 mRNA has been detected in the embryonal mass and upper suspensor during early embryogenesis. Functional studies show that PaWOX2 conserves a function in protoderm formation during early embryo development and may exert a unique function in suspensor expansion in gymnosperms[54]. PaWOX8/9, a P. abies homolog of AtWOX8 and AtWOX9, is highly expressed in PEMs and EEs. With the degradation of the suspensor, the expression level of PaWOX8/9 decreases gradually. Knockdown of PaWOX8/9 by RNAi leads to aberrant cell division orientation in tube cells[55]. In addition, the transcript levels of some cell cycle-regulating genes such as PaE2Fs and PaCYCBLs are directly or indirectly regulated by PaWOX8/9. Cell cycle-regulating genes have a significant role in the regulation of asymmetric cell division[56].

      The outermost protoderm differentiates into the epidermis during embryogenesis[57]. Unlike angiosperms, conifer protoderm cells not only divide periclinally but also anticlinally. This makes it difficult to identify the epidermal layer of conifers. Several genes related to epidermal development in P. abies have been identified, including PaWOX2, P. abies Homeobox1 (PaHB1), PaHB2 and Pa18, a lipid transport protein (LTP) coding gene[58]. The expression patterns of PaHB1 and PaHB2 are similar to their Arabidopsis homologs, AtML1 and AtANL2[59]. AtML1 is a master regulator of epidermal cell fate. The expression of AtML1 becomes restricted to the protoderm at the dermatogen stage[39,60]. Ectopic expression of PaHB1 leads to early developmental arrest caused by a lack of protoderm[59]. AtANL2 is involved in maintaining the subepidermal-layer identity[40]. PaHB2 is uniformly expressed in PEMs and EEs. In MEs, PaHB2 expression was mainly detected in the outermost layer of the cortex and the root cap[59]. However, it is still unclear if PaHB2 is involved in the development of the cortex. LTPs are crucial for the formation of the cuticle layer[61]. Pa18 is expressed in all cells in PEMs and its expression is restricted to the protoderm in developing embryos[58].

      The establishment of meristem centers are major patterning events during embryogenesis. STM, one of the four KNOX1 family genes in Arabidopsis, is crucial for the establishment of the embryonal SAM[41]. Four KNOX1 genes, HBK1, HBK2, HBK3 and HBK4, have been identified in P. abies[62]. HBK2 and HBK4 are expressed specifically in the SAM and are regulated by polar auxin transport[63]. HBK1 and HBK3 show more general expression patterns within the embryos[64]. Ectopic expression of the four HBK genes in transgenic Arabidopsis plants supports functions similar to those of HBK2 and HBK4 in SAM development[63]. Overexpression of HBK3 in Arabidopsis leads to enlarged meristems and an expanded expression pattern of STM[64]. In addition, HBK1 and HBK3 are expressed in all tested PEM lines, whereas HBK2 and HBK4 are only expressed in cell lines that are competent to form mature embryos[63]. The CUC genes function in the formation of cotyledon boundaries and the establishment of the embryonal SAM[36−38]. PaNAC01 and PaNAC02 belong to the NAC gene family. Based on phylogenetic analysis, PaNAC01 is more similar to CUC1 and CUC2 and can substitute for CUC2 in the Arabidopsis cuc1cuc2 mutant[65].

      Several genes that are important regulators of root meristem in angiosperms have been shown to be expressed during somatic embryogenesis in conifers. P. glauca AGO (PgAGO) is expressed at the future site of RAM[66]. The highest expression level of conifer AGO homologs is detected at the late/mature transition stage of embryogenesis[67]. Furthermore, knockdown of PgAGO leads to abnormal root meristems. The expression patterns of SCR, SHORT-ROOT (SHR) and several SCR-likes (SCLs) show expression patterns similar to their Arabidopsis homologs in several conifer species. SCR from the GRAS transcription factor family is important for the early delineation of radial patterning in the embryonic root[68].

      A genomics study based on conifer expressed sequence tag (EST) collections shows that the conifer embryo differs markedly from other gymnosperm tissues studied in terms of the range of genes transcribed. Approximately 72% of conifer embryo-expressed genes are found in Arabidopsis and have sequences similar to key genes that regulate seed development in Arabidopsis. However, approximately 11% of Pinus taeda embryo ESTs are novel[44]. The first conifer genome, the P. abies genome, was published in 2013[69]. The genomes of other conifers such as P. glauca[70] and Pinus tabuliformis[71] have been subsequently published. The sizes of the conifer genomes range from approximately 6,500 to 37,000 Mb and are highly repetitive. These factors make the genomes difficult to fully assemble. With the improvement of the genome and other biological information, a better understanding of the molecular mechanism of embryogenesis in conifers will definitely benefit the development of SE techniques.

    Conifer somatic embryogenesis techniques and the forest industry

    • The SE technique could intervene at two stages of the forest breeding strategy. First, the SE technique could be used to achieve faster offspring determination by providing an accurate assessment of genotype stability. Second, after the candidate genotype is identified, the SE technique could be used to mass produce valuable genotype copies and eventually achieve large-scale production. Forestry breeding via SE has several advantages compared with traditional forest breeding: 1) SE is not affected by the flowering and seed production cycle of forest trees, which provides greater flexibility for the deployment of forest renewable resources; 2) the intensity of genetic selection is greatly improved by SE breeding, as it is possible to achieve rapid reproduction of a small number of genotypes, which have a larger selection differential; 3) the SE technique combined with early selection on molecular labels could be used in the early development stage to better evaluate phenotypic type and plasticity and eventually shorten the breeding cycle; and 4) evaluating the traits of SE seedlings provides stronger evidence for genetic assays than traditional progeny tests. Using SE seedling evaluation, the environmental interactions with genotypes can be estimated more accurately, which improves the efficiency of clone determinations[6].

      Information on the application of the SE technique in industrial production is still limited at present. Tree species such as Abies nordmanniana, P. abies, P. glauca, P. sitchensis, Pseudotsuga menziesii, Pinus radiata and P. taeda are being researched. P. abies, P. sitchensis, P. menziesii, P. radiata and P. taeda are of interest for the commercial production of coniferous SE plants by transnational corporations. According to reports, the Arborgen (USA) and Weyerhaeuser (USA) companies, which are among the world's largest wood producers, have the largest application capacity for the SE technique. Arborgen could produce one million P. taeda SE seedlings annually. Weyerhaeuser plans to produce ten million synthetic seeds per year via the SE technique.

      It is still a long process to achieve industrialization of conifer somatic embryo production. Establishing a cryopreserved PEM library would be conducive to applying the SE technique to industrial production. In addition, plant biologists strive to expand the explant types that can be used for PEM induction. Finally, determining how to reduce costs in the SE process without affecting the quality of embryos is a problem that concerns many companies and institutions. In addition to improving the methods of PEM induction, somatic embryo differentiation, germination and planting, it is also important to connect each step effectively to maximize the production efficiency while minimizing the economic cost. Several strategies have been investigated including strict control of liquid proliferative media conditions and embryonic tissue density for different genotypes to maintain consistency in callus growth and proliferation cycle duration and manual control of the environmental conditions of germination to reduce the germination time in vitro without affecting the germination percentage. Egertsdotter et al. have summarized these studies and the progress of the SE technique in the field of conifer breeding[72]. Establishing a database and developing an automated system to monitor the status of cryopreservation and production materials, mechanizing the production process of SE, applying bioreactors and automation systems in the production of somatic embryos and studying the impact of light on the development of somatic seedlings would also be essential tasks to achieve the breeding goal of using the SE technique to achieve efficient, high-quality and economical production of tens of millions of seedlings.

      Conflict of interest

      • The authors declare that they have no conflict of interest.

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      • Copyright: © 2022 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (1)  Table (1) References (72)
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    Zhu T, Wang J, Hu J, Ling J. 2022. Mini review: Application of the somatic embryogenesis technique in conifer species. Forestry Research 2:18 doi: 10.48130/FR-2022-0018
    Zhu T, Wang J, Hu J, Ling J. 2022. Mini review: Application of the somatic embryogenesis technique in conifer species. Forestry Research 2:18 doi: 10.48130/FR-2022-0018
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  • Somatic embryo development in conifers
  • Induction of somatic embryogenesis in conifers
  • Comparison of zygotic and somatic embryo development in conifers
  • Patterning formation of conifer somatic embryos
  • Conifer somatic embryogenesis techniques and the forest industry
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