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Progress on medicinal plant regeneration and the road ahead

  • # Authors contributed equally: Juan Wang, Pin-Han Zhou

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  • Medicinal plants, which are valuable to human beings, play indispensable roles in various fields, such as health care, health promotion, and quality of life enhancement. They are not only pillars of traditional medicine but also valuable sources of modern pharmaceutical research and innovation. Although China has a rich variety of medicinal plants, in recent years, the drastic reduction in wild medicinal plant resources due to over-exploitation and over-utilization has affected the quality of Chinese herbal medicine. Therefore, the development of efficient in vitro regeneration culture technology for medicinal plants is particularly urgent. Here, the main regeneration pathways of medicinal plants are discussed, scientific progress of medicinal plant regeneration culture reviewed, and the main factors affecting the regeneration of medicinal plants analyzed, including the molecular mechanism of phytohormones in inducing the regeneration process, as well as the challenges faced by medicinal plant regeneration technology and directions for future development. Moreover, the challenges and future directions of medicinal plant regeneration technology are summarized, allowing us to find new ideas for the establishment of regeneration systems for rare and endangered medicinal plants, the screening of new regeneration-promoting drug molecules, and the preservation of traditional Chinese medicine (TCM) and its innovation.
  • Agricultural management practices impact soil physicochemical properties to a remarkable extent. Degradation of soil health has led to a contraction in agricultural production and soil biodiversity particularly due to conventional farming practices, indiscriminate use of inorganic fertilizers (INF) and inadequate input of residues[1]. Organic or inorganic fertilizers have been regarded as a critical component of agriculture to accomplish global food security goals[2]. The exogenous supply of fertilizers could easily alter soil properties by restoring the nutrients that have been absorbed by the plants[2]. Thus, implementing adequate nutrient management strategies could boost plant yield and sustain plant health. Tillage affects the soil, especially for crop production and consequently affects the agro-ecosystem functions. This involves the mechanical manipulation of the soil to modify soil attributes like soil water retention, evapotranspiration, and infiltration processes for better crop production. Thus, tillage practices coupled with fertilizer inputs may prove a viable strategy to improve soil health components such as nutrient status, biodiversity, and organic carbon.

    Soil serves as a major reservoir of nutrients for sustainable crop production. Intensive cultivation due to growing population burden has led to the decline of soil nutrient status that has adversely affected agricultural production. Various researchers have assessed the soil nutrient budget and the reasons behind decline of nutrient content in soil[3]. Soil management strategies have assisted in overcoming this problem to a greater extent. Tillage practices redistribute soil fertility and improve plant available nutrient content due to soil perturbations[4]. Different tillage and fertilization practices alter soil nutrient cycling over time[5]. Fertilization is an important agricultural practice which is known to increase nutrient availability in soil as well as plants[6]. A report has been compiled by Srivastava et al.[7], which assessed the effectiveness of different fertilizers on soil nutrient status in Indian soils.

    Soil biota has a vital role in the self-regulating functions of the soil to maintain soil quality which might reduce the reliance on anthropogenic activities. Soil microbial activities are sensitive to slight modifications in soil properties and could be used as an index of soil health[8]. Maintenance of microbial activity is essential for soil resilience as they influence a diverse range of parameters and activities including soil structure formation, soil SOM degradation, bio-geochemical cycling of nutrients etc.[9]. Various researchers have identified microbial parameters like microbial biomass carbon (MBC), potentially mineralizable nitrogen (PMN), soil respiration, microbial biomass nitrogen (MBN), and earthworm population as potential predictors of soil quality. Geisseler & Scow[10] have compiled a review on the affirmative influence of long-term mineral fertilization on the soil microbial community.

    Being the largest terrestrial carbon (C) reservoir, soil organic carbon (SOC) plays a significant role in agricultural productivity, soil quality, and climate change mitigation[11]. Manure addition, either solely or along with INF augments SOC content which helps in the maintenance and restoration of SOM more effectively as compared to the addition of INF alone[12]. Enhancement of recalcitrant and labile pools of SOC could be obtained through long-term manure application accentuating the necessity of continuous organic amendments for building up C and maintaining its stability[13]. Generally, compared with manure addition, INF application is relatively less capable of raising SOC and its labile fractions[14]. Alteration in SOC content because of management strategies and/or degradation or restoring processes is more prominent in the labile fraction of soil C[15]. Several fractions of soil C play vital roles in food web and nutrient cycles in soils besides influencing many biological properties of soil[16]. Thus, monitoring the response of SOC and its fractions to various management practices is of utmost importance.

    A positive impact on SOC under manure application coupled with INF in rice-wheat systems has been reported, as compared to sole applications of INFs[17]. Although ploughing and other mechanical disturbances in intensive farming cause rapid OM breakdown and SOC loss[18], additional carbon input into the soil through manure addition and rational fertilization increases carbon content[13]. Wei et al.[19] in light sandy loam soil of China found that the inclusion of crop straw together with inorganic N, P and K fertilizers showed better results for improving soil fertility over sole use of inorganic fertilizers. Zhu et al.[20] studied the influence of soil C through wheat straw, farmyard manure (FYM), green manure, and rice straw on plant growth, yield, and various soil properties and found that the recycling of SOM under intensive cultivation is completely reliant on net OM input and biomass inclusion. However, most of the studies on residue management and organically managed systems could not provide clear views regarding the relations between the quality of OM inputs and biological responses towards it. The disintegration of soil aggregates due to ploughing, use of heavy machinery, and residue removal has been reported widely under conventional tillage (CT) practices[21]. On the contrary, improvement in SOC stabilization has also been observed by some scientists[22]. Under CT, the disintegration of macro-aggregates into micro-aggregates is a prominent phenomenon, while conservation tillage has been identified as a useful practice for increasing macro-aggregates as well as carbon sequestration in agricultural soils[23]. By and large, the ploughing depth (0–20 cm) is taken into consideration for evaluating the impact of tillage and straw retention on soil aggregation[24], while degradation in deeper layers of soil is becoming a major constraint towards soil quality together with crop yield[25].

    Hence, the present review would be useful in determining how tillage practices and inorganic and organic fertilization impact nutrient availability in the soil, microbial composition and SOC fractions besides stocks under different land uses.

    Agricultural production is greatly influenced by nutrient availability and thus nutrient management is required for sustaining higher yields of crop. The term 'nutrient availability' refers to the quantity of nutrients in chemical forms accessible to plant roots or compounds likely to be converted to such forms throughout the growing season in lieu of the total amount of nutrients in the soil. For optimum growth, different crops require specifically designed nutrient ratios. Plants need macronutrients [nitrogen (N), phosphorus (P), potassium (K) in higher concentrations], secondary nutrients [calcium (Ca), magnesium (Mg), sulphur (S) in moderate amounts as compared to macronutrients] and Micronutrients [Zn (zinc), Fe (iron), Cu (copper), B (boron), Mn (manganese), Mo (molybdenum) in smaller amounts] for sustainable growth and production[26]. Fertilizers assist the monitoring of soil nutrient levels by direct addition of required nutrients into the soil through different sources and tillage practices may alter the concentration of available plant nutrients through soil perturbations. Various studies on the influence of fertilization and tillage practices on available plant nutrients have been discussed below.

    Yue et al.[27] reported that long-term fertilization through manure/INF improved the macronutrient content of Ultisol soil in China. Two doses of NPK (2NPK) considerably improved soil properties over a single dose (NPK). Combined application (NPK + OM) resulted in higher hydrolysable N and available P over the sole OM application. The total K content was higher under the treatments NPK, 2NPK and NPK + OM than sole OM treatment, whereas available K was higher in treatments NPK + OM and 2NPK over the sole OM and NPK. Likewise, OM, INF, and OM + INF were evaluated for their potential to regulate the soil macronutrient dynamics. Organic manure significantly improved the soil N content, whereas INF showed comparable results to that of the control treatment. Besides, all the treatments improved available P and exchangeable K concentration[28].

    Hasnain et al.[29] performed comparative studies of different ratios of INF + compost and different application times for the chemical N fertilizer on silty loamy soils of China. The available nitrogen and phosphorous content were greater in conjoint OM + INF application over the bare INF and control application irrespective of N application time. Soil quality substantially improved with increasing ratio of compost and 70:30 (INF to compost ratio) was found to be most suitable to maintain soil fertility and nutrient status. Another study by Liu et al.[30] reported the superior effects of NPK + pig manure and NPK + straw to improve soil available P and K over the control and sole NPK treatments. However, total N concentration did not exhibit any significant variation under any treatment.

    Shang et al.[31] accounted the positive impact of vermicompost and mushroom residue application on grassland soil fertility in China. The addition of organic manures improved available P and K content to a considerable extent. Under moisture-deficit winter wheat-fallow rotation, another study quantified the influence of residue management approaches and fertilizer rate on nutrient accrual. Residue burning resulted in no decline in soil macronutrient content, whereas the perpetual addition of FYM for 84 years significantly improved total N and extractable K and P concentration. Thus, residue incorporation along with FYM application may prove beneficial in reducing the temporal macronutrient decline[32].

    Ge et al.[33] examined the effects of NPK and NPK along with manure (NPKM) addition on the macronutrient status of Hapli-Udic Cambisol soil. The NPKM application resulted in the highest increase in total N, available-P and K concentration as compared to NPK and control. Likewise, mineral fertilization reduction and partial substitution with organic amendments have posed a significant influence on soil macronutrient status. Soil available P and K decreased after INF reduction[34]. Chen et al.[35] evinced that integrated application of manure and mineral fertilizers to red clay soil (typical Ultisols) improved hydrolyzed nitrogen and available P due to an increase in the decomposition of organic matter (OM) and N bio-fixation than sole mineral fertilizers and control.

    A long-term experiment was carried out out by Shiwakoti et al.[36] to ascertain the influence of N fertilization and tillage on macronutrient dynamics in soil. Nitrogen fertilization produced higher crop biomass which might have improved total N and P concentration in soil. Moreover, the reduced interaction between soil colloids and residue or greater cation exchange sites due to tillage practices could have augmented K concentration in 0−10 cm soil depth. Likewise, among tillage systems combined organic (poultry manure) and inorganic (lime and fertilizers) fertilization, no-tillage, and reduced tillage with organic fertilization resulted in higher availability of P owing to minimal disturbance of soil which decreases contact surface between phosphate ions and adsorption sites. Greater losses of K in runoff water under NT resulted in lower K availability under NT than CT[37].

    The influence of tillage systems on soil nutrient dynamics showed that minimal soil disturbances under zero tillage prohibited redistribution of soil nutrients and resulted in the highest available N, P, and K in the surface soil[38]. The influence of tillage timing on soil macronutrient status has also been assessed under tillage treatments that are fall tillage (FT), spring tillage (ST), no tillage (NT), and disk/chisel tillage (DT/CT) on mixed mesic Typic Haploxerolls soil. All the tillage systems differed in the quantity of residues generated. Thus, variation in the decomposition of crop residue and mineralization of SOM resulted in variable rates of nutrient release. The FT and ST had the highest N content over DT/CT and NT systems at corresponding depth. The N content also decreased with soil depth irrespective of tillage treatment. The available P and extractable K were highest under NT at the top 10 cm soil depth and increased over time[39]. Residue management in combination with tillage treatments (ST and CT) has been reported to affect the soil macronutrient status in Bangladesh. Tillage treatments enhanced the total N content to a considerable extent. Moreover, 3 years of residue retention led to a higher concentration of total N, available P and K in the soil.

    The combinations of N, P, and K in different ratios together with two rates of organic fertilizer (OF) applied on the aquic Inceptisol having sandy loam texture influenced the micronutrient status of the soil[40]. Soil Zn content decreased with time when no fertilizer was applied as compared to organic fertilizer (OF) application. The mineral fertilizer treatments led to a substantial increase in DTPA-extractable micronutrients in the soil. The higher micronutrient concentration due to higher OM highlights the importance of maintaining OM for soil fertility and higher crop production. Further studies revealed that long-term application of sole N fertilizers led to a significant decline in total Zn and Cu, whereas Mn and Fe status improved through atmospheric deposition. Phosphorus and OF addition along with straw incorporation markedly increased total Zn, Cu, Fe, and Mn. The DTPA-extractable Mn, Zn, Fe, and Cu were also higher in OF treatment, thus demonstrating the beneficial effects of constant OM application for maintaining the nutrient status of soil[41].

    López-Fando & Pardo[42] quantified the impact of various tillage practices including NT, CT, minimum tillage (MT), and zone-tillage (ZT) on soil micronutrient stocks. Tillage systems did exhibit a significant influence on plant available Fe stocks in the topsoil; however, diminished with depth under ZT, NT and MT. Manganese was higher in NT and ZT at all depths and increased with soil depth. Zinc was highest under NT and other results did not vary significantly as in the case of Cu. The SOC levels were also found to be responsible to affect micronutrients due to tillage practices. Likewise, in Calciortidic Haploxeralf soil the distribution of soil micronutrients (Zn, Mn, Fe, Cu) was ascertained under different tillage practices (CT, MT, and NT). The micronutrient status was highest under NT in the upper layers due to the higher SOC level[43].

    Sharma & Dhaliwal[44] determined that the combined application of nitrogen and rice residues facilitated the transformation of micronutrients (Zn, Mn, Fe, Cu). Among different fractions, the predominant fractions were crystalline Fe bound in Zn, Mn, and Cu and amorphous Fe oxide in Fe with 120 kg N ha˗1 and 7.5-ton rice residue incorporation. The higher content of occluded fractions adduced the increment in cationic micronutrient availability in soil with residue incorporation together with N fertilization due to increased biomass. Rice straw compost along with sewage sludge (SS) and INF also affected the micronutrient availability under the RW cropping system. Nitrogen fertilization through inorganic fertilizers and rice straw compost and sewage sludge (50% + 50%) improved soil micronutrient status due to an increase in SOM over sole NPK fertilizers[45]. Earlier, Dhaliwal et al.[46] in a long-term experiment determined that different combinations of NPK along with biogas slurry as an organic source modified the extractable micronutrient status of the soil.

    A comparative study was carried out by Dhaliwal et al.[47] to ascertain the long-term impact of agro-forestry and rice–wheat systems on the distribution of soil micronutrients. The DTPA-extractable and total Cu, Zn, Fe, and Mn were greater in the RW system due to the reduced conditions because of rice cultivation. Under the RW system Zn removal was higher which was balanced by continuous Zn application. The higher availability of Fe under the RW system was due to reduced conditions. Contrarily, Mn was greater under the agro-forestry system owing to nutrient recycling from leaf litter.

    The long-term impact of integrated application of FYM, GM, WCS (wheat-cut straw) and INF on the soil micronutrients (Zn, Mn, Cu, and Fe) have been studied by Dhaliwal et al.[48]. The FYM application substantially improved DTPA-extractable Zn status followed by GM and WCS, whereas Cu content was maximum in the plots with OM application. The highest Fe concentration was recorded in treatment in which 50% recommended N supplied through FYM. This could be ascribed to the release of micronutrients from OM at low soil pH.

    Shiwakoti et al.[49] studied the dual effects of tillage methods (MP, DP, SW) and variable rates of N (0, 45, 90, 135 and 180 kg ha−1) on the distribution of micronutrients under a moisture-deficit winter wheat-fallow system. The soil Mn content was highest under the DP regime. Inorganic N application reduced Cu content in the soil. Comparative studies with adjacent undisturbed grass pasture indicated the loss of Zn and Cu to a significant extent. Thus, DP along with nitrogen added through inorganic fertilizers could improve micronutrient concentration in the soil. Moreover, the results implied that long-term cultivation with nitrogen fertilization and tillage results in the decline of essential plant nutrients in the soil. Thus, organic amendments along with INF may prove an effective approach to increase soil micronutrient content. In another study conducted by Lozano-García & Parras-Alcántara[50] tillage practices such as NT under apple orchard, CT with the wheat-soybean system and puddling (PD) in the rice-rice cropping system were found to affect nutrient status. Under CT, Cu content was lowest and Zn content was highest. On the contrary, puddling caused an increase in Fe and Mn concentration owing to the dispersion of soil aggregates which reduced the percolation of water and created an anaerobic environment thereby enhancing the availability of Fe and Mn.

    Tillage practices along with gypsum fertilization have been known to affect secondary nutrient concentrations in soil. In a long-term experiment, FYM application showed maximum response to increased S concentration due to the maximum addition of OM through FYM over other treatments as S is an essential component of OM and FYM[32]. Higher Mg content was recorded in FYM and pea vine treatments because the application of organic matter through organic manure or pea vines outright led to Mg accrual. The lower Mg concentration in topsoil than the lower layers was due to the competition between Mg and K for adsorbing sites and thus displacement of Mg by K. Han et al.[28] while ascertaining the impact of organic manures and mineral fertilizers (NPK) on soil chemical attributes determined that INF application reduced exchangeable calcium, whereas no significant changes were exhibited in the magnesium concentrations. The OM application significantly increased both the calcium and magnesium concentrations in the soil.

    While ascertaining the effect of different tillage treatments such as CT, NT, and MT on exchangeable and water-soluble cations, Lozano-García & Parras-Alcántara[50] recorded that NT had greater content of exchangeable Ca2+ and Mg2+ than MT and CT. The exchangeable Ca2+ decreased with depth, however, opposite results were observed for Mg2+ which might be due to the higher uptake of Mg2+ by the crop. On another note, there might be the existence of Mg2+-deficient minerals on the surface horizon. Alam et al.[51] studied the temporal effect of tillage systems on S distribution in the soil and observed that available S was 19%, 31%, and 34% higher in zero tillage than in minimum tillage, conventional tillage, and deep tillage, respectively.

    Kumar et al.[38] appraised the impact of tillage systems on surface soil nutrient dynamics under the following conditions: conventional tillage, zero till seeding with bullock drawn, conventional tillage with bullock drawn seeding, utera cropping and conservation tillage seeding with country plough and observed that tillage had a significant impact on the available S content. Compared with conventional tillage, zero and minimum tillage had higher S content as there was none or limited tillage operations which led to the accumulation of root stubble in the soil that decomposed over time and increased S concentration.

    Soil is considered a hotspot for microbial biodiversity which plays an important role in building a complex link between plants and soil. The microbial components exhibit dynamic nature and, therefore, are characterized as good indicators of soil quality[52]. These components include MBC, MBN, PMN and microbial respiration which not only assist in biological transformations like OM conversion, and biological nitrogen fixation but also increase nutrient availability for crop uptake. Management strategies such as fertilizer inputs and tillage practices may exert beneficial effects on soil biota as discussed below.

    Soil is an abode to a considerable portion of global biodiversity. This biodiversity not only plays a pivotal role in regulating soil functions but also provides a fertile ground for advancing global sustainability, especially agricultural ventures. Thus, the maintenance of soil biodiversity is of paramount importance for sustaining ecosystem services. Soil biodiversity is the diverse community of living creatures in the soil that interact not only with one another but also with plants and small animals to regulate various biological activities[53]. Additionally, it increases the fertility of soil by converting organic litter to SOM thereby enhancing SOC content. Thus, the SOM measures the number and activity of soil biota. Furthermore, the quality and amount of SOC, as well as plant diversity have a considerable impact on the soil microbial community structure[54].

    Dangi et al.[55] ascertained the impact of integrated nutrient management and biochar on soil microbial characteristics and observed that soil amended with biochar or the addition of organic manures influenced microbial community composition and biomass and crop yield. After two years, the higher rates of biochar significantly enhanced the levels of gram-positive and gram-negative bacterial phospholipid fatty acid (PLFA), total arbuscular mycorrhizal fungal (AMF) than lower rates, unfertilized and non-amended soil. Luan et al.[56] conducted a comparison study in a greenhouse to assess the effects of various rates of N fertilizer and kinds (inorganic and organic) on enzyme activities and soil microbial characteristics. Microbial growth (greater total PLFAs and microbial biomass carbon) and activity were promoted by manure substitution of mineral fertilizer, particularly at a higher replacement rate. On account of lower response in bacterial over fungal growth, manure addition led to a greater fungi/bacteria ratio. Furthermore, manure application significantly enhanced microbial communities, bacterial stress indicators and functional diversity. Lazcano et al.[57] determined the influence of different fertilization strategies on microbial community structure and function, soil biochemical properties and crop yield three months after addition of fertilizer. The integrated fertilizer regimes augmented microbial growth with improved enzyme activity as compared to sole inorganic amendments. Bacterial growth showed variable response with variation in fertilizer regime used whereas fungal growth varied with the amount of fertilizer added. Compared to mineral fertilizers, manure application led to a rapid increase in PLFA biomarkers for gram-negative bacteria. The organic amendments exhibited significant effects even at small concentration of the total quantity of nutrients applied through them; thus, confirming the viability of integrated fertilizer strategies in the short term.

    Kamaa et al.[58] assessed the long-term effect of crop manure and INF on the composition of microbial communities. The organic treatments comprised of maize (Zea mays) stover (MS) at 10 t ha−1 and FYM @ 10 t ha−1, INF treatments (120 kg N, 52.8 kg P-N2P2), integrated treatments (N2P2 + MS, N2P2 + FYM), fallow plot and control. The treatment N2P2 exhibited unfavourable effects on bacterial community structure and diversity that were more closely connected to the bacterial structure in control soils than integrated treatments or sole INF. In N2P2, fungal diversity varied differently than bacterial diversity but fungal diversity was similar in the N2P2 + FYM and N2P2 + MS-treated plots. Thus, the total diversity of fungal and bacterial communities was linked to agroecosystem management approaches which could explain some of the yield variations observed between the treatments. Furthermore, a long-term experiment was performed by Liu et al.[59] to study the efficiency of pig manure and compost as a source for N fertilization and found unique prokaryotic communities with variable abundance of Proteobacteria under compost and pig manure treatments.

    Recently, Li et al.[60] assessed the influence of different tillage practices (no-tillage, shallow tillage, deep tillage, no-tillage with straw retention, shallow tillage with straw retention and deep tillage with straw retention) on microbial communities and observed that tillage practices improved the bacterial Shannon index to a greater extent over the no-tillage plots in which the least value was recorded. Another research study by He et al.[61] reported the effect of tillage practices on enzyme activities at various growth stages. Across all the growth stages, enzyme activities of cellobiohydrolase (CBH), β-xylosidase (BXYL), alkaline phosphatase (AP), β-glucosidase (BG), β-N-acetylglucosamines (NAG) were 17%−169%, 7%−97%, 0.12%−29%, 3%−66%, 23%−137% greater after NT/ST, NT, ST, ST/PT, and PT/NT treatments as compared to plow tillage. The NT/ST treatment resulted in highest soil enzyme activities and yield, and thus was an effective and sustainable method to enhance soil quality and crop production.

    Microbes play a crucial role in controlling different soil functions and soil ecology and microbial community show significant variation across as well as within the landscape. On average, the total biomass of microbes exceeds 500 mg C kg soil−1[62]. Microbial biomass carbon is an active constituent of SOM which constitutes a fundamental soil quality parameter because SOM serves as a source of energy for microbial processes and is a measure of potential microbial activity[48,63]. Soil systems that have higher amounts of OM indicate higher levels of MBC. Microbial biomass carbon is influenced by many parameters like OM content in the soil, land use, and management strategies[64]. The MBC and soil aggregate stability are strongly related because MBC integrates soil physical and chemical properties responds to anthropogenic activities.

    Microbial biomass is regarded as a determinative criterion to assess the functional state of soil. Soils having high functional diversity of microbes which, by and large, occurs under organic agricultural practices, acquire disease and insect-suppressive characteristics that could assist in inducing resistance in plants[65]. Dou et al.[66] determined that soil microbial biomass C (SMBC) was 5% to 8% under wheat-based cropping systems and zero tillage significantly enhanced SMBC in the 0−30 cm depth, particularly in the upper 0 to 5 cm. According to Liang et al.[67], SMBC and soil microbial biomass N (SMBN) in the 0−10 cm surface layer were greater in the fertilized plots in comparison to the unfertilized plots on all sampling dates whereas microbial biomass C and N were highest at the grain filling stage. Mandal et al.[68] demonstrated that MBC also varied significantly with soil depth. Surface soil possessed a maximum MBC value than lower soil layers due to addition of crop residues and root biomass on the surface soil. The MBC content was highest with combined application of INF along with farmyard manure and GM, whereas untreated plots showed minimum MBC values. The incorporation of CR slows down the rate of mineralization processes; therefore, microbes require more time to decompose the residues and utilize the nutrients released[69]. On the other hand, incorporation of GR having a narrow C:N ratio enhances microbial activity and consequently accelerates mineralization in the soil. Malviya[70] also recorded that the SMBC contents were significantly greater under RT than CT, regardless of soil depth which was also assigned to residue incorporation which increases microbial biomass on account of higher carbon substrate in RT.

    Naresh et al.[71] studied the vertical distribution of MBC under no-tillage (NT), shallow (reduced) tillage and normal cultivated fields. A shallow tillage system significantly altered the tillage induced distribution of MBC. In a field experiment, Nakhro & Dkhar[72] examined the microbial populations and MBC in paddy fields under organic and inorganic farming approaches. The organic source used was a combination of rock phosphate, FYM and neem cake, whereas a mixture of urea, muriate of potash and single super phosphate was used as an inorganic source. The organically treated plots exhibited the highest MBC compared to inorganically treated plots and control. Organic carbon exhibited a direct and significant correlation with bacterial and fungal populations. The addition of organic fertilizers enhanced the content of SOC and consequently resulted in higher microbial count and MBC. Ramdas et al.[73] investigated the influence of inorganic and organic sources of nutrients (as minerals or INF) applied over a five-year period on SOC, MBC and other variables. It was observed that the addition of FYM and conjoint application of paddy straw (dry) and water hyacinth (PsWh) (fresh) significantly increased the SOC content than vermicompost, Chromolaena adenophorum (fresh) and Glyricidia aculeate (fresh), and Sesbania rostrata (fresh).

    Xu et al.[74] evaluated the influence of long-term fertilization strategies on the SOC content, soil MBN, soil MBC, and soil microbial quotient (SMQ) in a continuous rice system and observed that MBC at the main growth stages of early and late rice under 30% organic matter and 70% mineral fertilizer and 60% organic matter and 40% mineral fertilizer treatments was greater as compared to mineral fertilizer alone (MF), rice straw residues and mineral fertilizer (RF), and no fertilizer (CK) treatments. However, SMBC levels at late growth stages were greater in comparison to early growth stages. A recent study by Xiao et al.[75] demonstrated that increasing tillage frequency (no-tillage, semi-annual tillage, and tillage after every four months, two months, and one month) decreased soil MBC. Microbial biomass carbon content was significantly greater in no-till treatment (597 g kg−1) than in tillage every four months (421 g kg−1), two months (342 g kg−1) and one month (222 g kg−1). The decrease in the content of MBC in association with tillage practices is due to soil perturbations which enhanced soil temperature, diminished soil moisture content, and resulted in the destruction of microbial habitat and fungal hyphae. Therefore, the MBC content eventually affected the N cycle.

    Li et al.[76] reported that in comparison to CT, NT and RT resulted in increased MBC content and NT significantly increased MBC by 33.1% over CT. Furthermore, MBC concentration was 34.1% greater in NT than RT. The increase in MBC concentration was correlated with the results of increase in SOC concentration. Site-specific factors including soil depth and mean annual temperature significantly affected the response ratio of MBC under NT as compared to the duration of NT.

    Microbial biomass nitrogen (MBN) is a prominent indicator of soil fertility as it quantifies the biological status of soil. Soil MBN is strongly associated with organic matter of the soil. The nitrogen in MBN has a rapid turnover rate thereby reflecting the changes in management strategies way before the transformations in total N are discernable[77].

    In an experiment on continuous silage maize cultivation with crop rotation, Cerny et al.[78] observed that organic fertilizers exerted an affirmative influence on the soil MBN. During the application of organic manure MBN decreased, but there was higher MBN content as compared to control. However, addition of mineral nitrogenous fertilizers exerted an adverse effect on MBN content in experiments with maize. El-Sharkawi[79] recorded that organic matter-treated pots resulted in maximum MBN content than urea-treated pots. The sludge application enhanced total MBN and, therefore, could implicitly benefit crop production particularly in poor soils[18]. Sugihara et al.[80] observed that during the grain-filling stage in maize, residue and/or fertilizer addition exerted a pronounced influence on soil microbial dynamics; however, a clear effect of residue and ⁄or fertilizer addition was not observed. Microbial biomass nitrogen reduced dramatically from 63–71 to 18–33 kg N ha˗1 and C:N ratio at the same time increased more than ten-fold in all plots.

    Malik et al.[81] apprised that the organic amendments significantly enhanced MBN concentrations up to 50% more than the unamended soil. Wang et al.[82] evaluated the influence of organic materials on MBN content in an incubation and pot experiment with acidic and calcareous soils. The results revealed that MBN content which was affected by the different forms of organic amendments, increased by 23.37%−150.08% and 35.02%−160.02% in acidic and calcareous soils, respectively. The MBN content of both soils decreased with the increase in the C/N ratio of the organic materials, though a higher C/N ratio was effective for sustaining a greater MBN content for a very long time.

    Dhaliwal & Bijay-Singh[52] observed higher MBN levels in NT soils (116 kg ha−1) than in cultivated soils (80 kg ha−1). Kumar et al.[83] ascertained that in surface layer, MBN content was 11.8 mg kg−1 in CT which increased to 14.1 and 14.4 mg kg−1 in ZT and RT without residue retention and 20.2, 19.1 and 18.2 mg kg−1 in ZT, RT and CT with residue incorporation, respectively (Table 1). In the subsurface layer, the increased tendency on account of tillage and crop residue retention was identical to those of 0−15 cm layer but the magnitude was comparatively meagre (Table 1). In comparison to control, the persistent retention of crop residues led to significant accrual of MBN in the surface layer.

    Table 1.  Effect of different treatments on contents of various fractions of soil organic carbon[38].
    TreatmentsPMN (mg kg−1)MBC (mg kg−1)MBN (mg kg−1)DOC (mg kg−1)
    Depths (cm)
    0−1515−300−1515−300−1515−300−1515−30
    Tillage practices
    ZTR12.411.2562.5471.120.218.9198.6183.6
    ZTWR8.57.6350.4302.114.112.6167.1159.2
    RTR10.69.9490.2399.319.117.2186.4171.6
    RTWR7.66.6318.1299.814.413.7159.5148.7
    CTR9.38.5402.9354.418.216.6175.9168.9
    CT6.75.6307.9289.511.89.7142.5134.6
    Nitrogen management
    Control3.62.8218.3202.910.810.4103.792.3
    80 kg N ha−15.34.4241.1199.414.912.2128.3116.9
    120 kg N ha−18.97.6282.7220.916.516.1136.8123.6
    160 kg N ha−19.88.4343.9262.919.418.1164.8148.9
    200 kg N ha−110.49.7346.3269.622.721.7155.7136.4
    ZTR = Zero tillage with residue retention, ZTWR = Zero tillage without residue retention; RTR = Reduced tillage with residue retention, RTWR = Reduced tillage without residue retention, CTR = Conventional tillage with residue incorporation; CT = Conventional tillage without residue incorporation.
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    Xiao et al.[75] determined that the MBN content decreased with tillage treatment having highest value in no tillage treatment, however, the difference among the treatments was negligible. Soil perturbations decreased the aggregate size and thus lower the soil aeration and exposure of fresh organic matter which restricted the growth of microorganisms. The results also concluded that MBN content is highly sensitive to tillage. Ginakes et al.[84] assessed the impact of zone tillage intensity on MBN in a corn-kura clover cropping sequence. Microbial biomass nitrogen was influenced by time and type of tillage treatment. Temporal studies revealed that MBN was higher after tillage treatment than the values possessed before tillage. Under different tillage treatments, higher values were recorded in ST (shank-till) and DT (double-till) over NT and RZT (zone-till) treatments.

    Another biological parameter, PMN, is a crucial parameter of soil fertility due to its association with soil N supply for crop growth. Also, PMN indicates the status of soil microbial community associated with PMN, whether it is improving or degrading. Forest soils are characterized by greater levels of PMN than CT receiving conventional chemical fertilizers which could be assignable to improved microbial activity in the former soils than the latter[48,77]. Aulakh et al.[85] assessed the effect of various combinations of fertilizer N, P, FYM and wheat residue (WR) applied to soybean and soybean residues added to wheat under CT and CA. The added fertilizers of N and P, FYM, and crop residue enhanced the mean weight diameter and water-stable aggregates thus favoured the development of macro-aggregates. The treatment INF + FYM + crop residue performed better among all the treatments. The net flux of mineral nitrogen from the mineralizable fraction is used to measure potentially mineralizable N which indicates the balance between mineralization and immobilization by soil microbes[77]. Nitrogen mineralization is widely used to assess the ability of SOM to supply inorganic nitrogen in the form of nitrate which is the most common form of plant-available nitrogen. Kumar et al.[83] observed an increase in PMN which was higher in surface soil than sub-surface soil thereby implying that high OC accumulation on account of crop residue retention was the most probable cause.

    Verma & Goyal[86] assessed the effect of INM and organic manuring on PMN and observed that PMN was substantially affected by different organic amendments. Potentially mineralizable nitrogen varied between 19.6−41.5 mg kg−1 soil with greater quantity (2.5%) in vermicompost applied plots than FYM treated plots. The INF treatments resulted in lower PMN content which could be due to nutrient immobilization by microbes. Mahal et al.[87] reported that no-till resulted in higher PMN content than conventional tillage treatments. This trend was due to the maintenance of SOM due to the residue cover and reduction of soil erosion under no-tillage system[88]. On the contrary, tillage practices led to the loss of SOC owing to loosened surface soil and higher mineralization of SOM.

    Soil respiration is referred as the sum of CO2 evolution from intact soils because of the respiration by soil organisms, mycorrhizae and roots[89]. Various researchers have proposed soil respiration as a potential indicator of soil microbial activity[52,77]. Gilani & Bahmanyar[90] observed that addition of organic amendments enhanced soil respiration more than the control and synthetic fertilizer treatments. Moreover, among organic amendment treatments, highest soil respiration was observed in sewage-sludge treated soils. Under controlled conditions in saline-sodic soil, Celis et al.[91] reported that sewage sludge resulted in a higher soil respiration rate than mined gypsum and synthetic gypsum. The application of gypsum because of minimal organic matter intake had little effect on soil respiration. The addition of organic matter especially during early spring led to higher microbial biomass and soil respiration albeit diminished levels of nitrate-N. Moreover, SOM hinders the leaching of nitrate ions thereby resulting in a better soil chemical environment[71].

    Faust et al.[92] observed that microbial respiration was associated with volumetric water content. The respiration declined with less availability of water, thus the lesser the tillage intensity, the more the volumetric water content which consequently resulted in higher microbial respiration. Another study by Bongiorno et al.[93] reflected the influence of soil management intensity on soil respiration. Reduced tillage practices resulted in 51% higher basal respiration than CT. Furthermore, this investigation suggested that microbial catabolic profile could be used as a useful biological soil quality indicator. Recently, Kalkhajeh et al.[94] ascertained the impact of simultaneous addition of N fertilizer and straw-decomposing microbial inoculant (SDMI) on soil respiration. The SDMI application boosted the soil microbial respiration which accelerated the decomposition of straw due to N fertilization. The C/N ratio did not affect the microbial respiration at elongation and heading stages, whereas N fertilization enhanced the microbial respiration to a greater extent than the unfertilized control. Additionally, the interaction between sampling time and basal N application significantly affected microbial respiration.

    Gong et al.[95] apprised the effect of conventional rotary tillage and deep ploughing on soil respiration in winter wheat and observed that deep ploughing resulted in a higher soil respiration rate than conventional rotary tillage. Soil moisture content and temperature are the dominating agents influencing soil respiration which is restricted by the soil porosity.

    Soil organic carbon plays a vital role in regulating various soil functions and ecosystem services. It is influenced by numerous factors like tillage practices and fertilization. Moreover, modified management practices may prove beneficial to avoid SOC loss by increasing its content. An exogenous supply of fertilizers may alter the chemical conditions of soil and thus result in transformation of SOC. Tillage practices lead to frequent soil disturbances which reduce the size of soil aggregates and accelerate the oxidation of SOC thereby reducing its content. The literature on the influence of fertilization and tillage practices on the transformation of SOC is discussed below.

    Soil organic carbon is a major part of the global carbon cycle which is associated not only with the soil but also takes part in the C cycling through vegetation, oceans and the atmosphere (Figs 1 & 2). Soil acts as a sink of approximately 1,500 Pg of C up to 1 m depth, which is greater than its storage in the atmosphere (approximately 800 Pg C) and terrestrial vegetation (500 Pg C) combined[96]. This dynamic carbon reservoir is continuously cycling in diverse molecular forms between the different carbon pools[97]. Fertilization (both organic and mineral) is one of the crucial factors that impart a notable influence on OC accretion in the soil. Many researchers have studied the soil C dynamics under different fertilizer treatments. Though inorganic fertilizers possess the advantage of easy handling, application and storage, they do not contribute to soil organic carbon. On the contrary, regardless of management method, plant residues are known to increase organic carbon content.

    Figure 1.  Impact of different fertilization regimes on abundance of the microbial biomarker groups . Error bars represent the standard error of the means and different letters indicate significant differences at p < 0.05 among treatments. Source: Li et al.[60].
    Figure 2.  Soil organic carbon (SOC) dynamics in the global carbon cycle.

    Katkar et al.[98] reported a higher soil quality index under conjunctive nutrient management strategies comprising addition of compost and green leaves along with mineral nutrients. Mazumdar et al.[99] investigated the impact of crop residue (CR), FYM, and leguminous green manure (GM) on SOC in continuous rice-wheat cropping sequence over a 25-year period. At the surface layer, the maximum SOC content was recorded under NPK + FYM than NPK + CR and NPK + GM treatments. SOC was significantly lower under sole application of INFs (NPK) than the mixed application of organic and inorganic treatments. A higher range of SOC content was recorded at a depth of 0.6 m in the rice-wheat system (1.8–6.2 g kg−1) in farmyard manure (FYM)-treated plots than 1.7–5.3 g kg−1 under NPK, and 0.9–3.0 g kg−1 in case of unfertilized plots[100]. In a research study Dutta et al.[101] reported that rice residue had a higher decomposition rate (k¼ 0.121 and 0.076 day−1) followed by wheat (0.073 and 0.042 day−1) and maize residues (0.041 day−1) when their respective residues placed on soil surface than incorporated in the soils. Naresh et al.[102] found FYM and dhaincha as GM/ sulphitation press mud (SPM) treatments are potent enough to enhance the SOC. Maximum SOC content was noted in 0–5 cm depth that reduced gradually along the profile. In surface soil, the total organic content (TOC) under different treatments varied with source used to supply a recommended dose of nitrogen (RDN) along with conventional fertilizer (CF).

    Cai et al.[103] ascertained that long-term manure application significantly improved SOC content in different size fractions which followed the sequence: 2,000–250 μm > 250–53 μm > 53 μm fraction. Naresh et al.[22] determined that mean SOC content increased from 0.54% in control to 0.65% in RDF and 0.82% in RDF + FYM treatment and improved enzyme activity; thus, ultimately influenced nutrient dynamics under field conditions. The treatments RDF + FYM and NPK resulted in 0.28 Mg C ha−1 yr−1 and 0.13 Mg C ha−1 yr−1, respectively and thus higher sequestration than control. Zhao et al.[104] determined that in the surface layer, significant increase in SOC content in each soil aggregate was noticed under straw incorporation treatments over no straw incorporated treatments (Fig. 3). Moreover, the aggregate associated OC was significantly higher in the surface layer than the sub-surface layer. The highest increment in aggregate-associated OC was noted in both maize and wheat straw (MR-WR) added plots followed by MR and least in WR. Besides, all of the three straw-incorporated treatments exhibited notable increase in SOC stock in each aggregate fraction in the surface layer of the soil. In the subsurface (20−40 cm) layer under MR-WR, significant rise in SOC stock of small macro-aggregates was observed, whereas there was a reduction in SOC stock in the silt + clay fraction than other treatments. The straw-incorporated treatments increased the quantity of mineral-associated organic matter (mSOM) and intra-aggregate particulate organic matter, (iPOM) within small macro-aggregates and micro-aggregates especially in the topmost layer of the soil.

    Figure 3.  Distribution of OC in coarse iPOM (intra-aggregate particulate organic matter) fine iPOM, mSOM (mineral-associated matter), and free LF (free light fraction) of small macro-aggregates and micro-aggregates in the 0–20 cm and 20–40 cm soil layers under MR-WR (return of both maize and wheat straw), MR (maize straw return), WR (wheat straw return). Different lowercase and uppercase letters indicate significant differences at p < 0.05 among treatments and depths respectively[104].

    Srinivasarao et al.[105] reported that SOC content was reduced with the addition of INFs (100% RDN) alone as compared to the conjunctive application of inorganic and organic or sole FYM treatments. Earlier, Srinivasarao et al.[106] reported that FYM treated plots exhibited greater per cent increase in SOC stock than mineral fertilized plots and control. Tong et al.[107] ascertained that the application of NP and NPK significantly improved SOC stocks. On the contrary, fertilized soils could also exhibit decrease in carbon content than control. Naresh et al.[108] determined that higher biomass C input significantly resulted in greater particulate organic carbon (POC) content. Zhang et al.[109] ascertained that long-term addition of NPK and animal manures significantly improved SOC stocks by a magnitude of 32%−87% whereas NPK and wheat/ and or maize straw incorporation enhanced the C stocks by 26%−38% than control. Kamp et al.[110] determined that continuous cultivation without fertilization decreased SOC content by 14% than uncultivated soil. However, super optimum dose of NPK, balanced NPK fertilization and integration of NPK with FYM not only improved SOC content but also SOC stocks over the first year. In conventionally tilled cotton-growing soils of southern USA, Franzluebbers et al.[111] estimated that carbon sequestration averaged 0.31 ± 0.19 Mg C ha−1 yr−1. Mandal et al.[112] reported maximum SOC stock in the surface layer of the soil (0–15 cm) which progressively diminished with depth in each land use system. A significant decrease in SOC stock along the profile depth was also observed by Dhaliwal et al.[47] in both croplands and agroforestry. In the topmost soil layer, highest SOC stock was recorded in rice–fallow system while the lowest was in the guava orchard[112].

    Nath et al.[113] determined that there was accrual of higher TOC in surface layers as compared to lower layers of soil under paddy cultivation. This accrual could be adduced to left-over crop residues and remnant root biomass which exhibited a decreasing trend with soil depth. Das et al.[114] determined that integrated use of fertilizers and organic sources resulted in greater TOC as compared to control or sole fertilizer application. Fang et al.[115] observed that the cumulative carbon mineralization differed with aggregate size in top soils of broad-leaved forests (BF) and coniferous forests (CF). However, in deep soil it was greater in macro-aggregates as compared to micro-aggregates in BF but not in CF (Fig. 4). By and large, the percent SOC mineralized was greater in macro-aggregates as compared to micro-aggregates. Dhaliwal et al.[100] ascertained that SOC accrual was considerably influenced by residue levels and tillage in surface soil (0−20 cm); albeit no variation was observed at lower depth (20−40 cm). The SOC content was greater in zero-tilled and permanently raised beds incorporated with residues as compared to puddled transplanted rice and conventionally planted wheat. Pandey et al.[116] reported that no-tillage prior to sowing of rice and wheat increased soil organic carbon by 0.6 Mg C ha–1 yr–1. The carbon sequestration rate on account of no-tillage or reduced tillage ranged between 0−2,114 kg ha–1 yr–1 in the predominant cropping system of South Asia, Xue et al.[117] observed that the long-term conventional tillage, by and large, exhibited a significant decline in SOC owing to degradation of soil structure, exposing protected soil organic matter (intra-soil aggregates) to microbes. Therefore, the adoption of no-tillage could hamper the loss of SOC thereby resulting in a greater or equivalent quantity of carbon in comparison to CT (Fig. 5).

    Figure 4.  (a) Soil aggregate fractions of two depths in two restored plantations of subtropical China, (b) organic carbon and (c) its mineralization from various soil aggregates within 71 d at various soil depths in two restored plantations of subtropical China. Error bars show the standard error of the mean. The different letters represent significant differences among the different soil aggregate fractions within a depth at p < 0.05[115].
    Figure 5.  The concentrations of (a) SOC, (b) total nitrogen (TN), and (c) soil C:N ratio for 0–50 cm profile under different tillage treatments in 2012 and 2013. NT = no-till with residue retention; RT = rotary tillage with residue incorporation; PT = plow tillage with residue incorporation; and PT0 = plow tillage with residue removed. The lowercase letters indicate statistical difference among treatments at p < 0.05[117].

    Singh et al.[118] determined that carbon stock in the 0-40 cm layer increased by 39, 35 and 19% in zero-tilled clay loam, loam, and sandy loam soils, respectively as compared to conventional tilled soils over a period of 15 years. Kuhn et al.[119] also apprised about the advantages of NT over CT vis-a-vis SOC stocks across soil depths. In the surface layer (0−20 cm) NT, by and large, resulted in higher SOC stocks as compared to CT; however, SOC stocks exhibited a declining trend with soil depth, in fact, became negative at depths lower than 20 cm. Sapkota et al.[120] observed that over a period of seven years, direct dry-seeded rice proceeded by wheat cultivation with residue retention enhanced SOC at 0-60 cm depth by a magnitude of 4.7 and 3.0 t C ha−1 in zero-tillage (ZTDSR-ZTW + R) and without tillage (PBDSR-PBW + R), respectively. On the contrary, the conventional tillage rice-wheat cropping system (CTR-CTW) decreased the SOC up to 0.9 t C ha−1 (Table 2).

    Table 2.  Influence of tillage and crop establishment methods on SOC stock and its temporal variation under rice–wheat system[120].
    Tillage and crop establishment methodsDepths (m)
    0–0.050.05–0.150.15–0.30.3–0.60–0.6
    Total SOC (t/ha)
    CTR-CTW3.5e7.1c8.77.026.2c
    CTR-ZTW3.9d7.6bc8.86.526.7c
    ZTDSR-CTW4.2d7.5bc9.26.327.3c
    ZTDSR-ZTW4.9c8.9ab8.26.228.2bc
    ZTDSR-ZTW+R6.1a9.0ab9.86.831.8a
    PBDSR-PBW+R5.5b9.3a9.36.030.1ab
    MSD0.41.72.01.42.49
    Treatment effect
    (p value)
    < 0.0010.040.1580.267< 0.001
    Initial SOC content3.6 ±
    0.15
    8.1 ±
    1.39
    8.78 ±
    1.07
    6.7 ±
    0.73
    27.1 ±
    1.21
    Change in SOC over seven years (t/ha)
    CTR-CTW−0.16−0.99−0.040.28−0.90
    CTR-ZTW0.28−0.500.01−0.20−0.41
    ZTDSR-CTW0.62−0.570.45−0.340.16
    ZTDSR-ZTW1.340.84−0.62−0.461.09
    ZTDSR-ZTW+R2.490.961.040.164.66
    PBDSR-PBW+R1.891.220.51−0.642.98
    CTR-CTW = Conventionally tilled puddled transplanted rice followed by conventionally tilled wheat, CTR-ZTW = Conventionally tilled puddled transplanted rice followed by zero-tilled wheat, ZTDSR-CTW = Zero-tilled direct dry-seeded rice followed by conventionally tilled wheat, ZTDSR-ZTW = Zero-tilled direct dry-seeded rice followed by zero-tilled wheat, ZTDSR-ZTW+R = Zero-tilled direct dry-seeded rice followed by zero-tilled wheat with residue retention, PBDSR-PBW+R = Direct dry-seeded rice followed by direct drilling of wheat both on permanent beds with residue retention, MSD, minimum significant difference. Significant different letters indicate significant differences at p < 0.05.
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    Labile organic carbon (LC) is that fraction of SOC that is rapidly degraded by soil microbes, therefore, having the highest turnover rate. This fraction can turn over quickly on account of the change in land use and management strategies. From the crop production perspective, this fraction is crucial as it sustains the soil food cycle and, hence, considerably impacts nutrient cycling thereby altering soil quality and productivity. Short-term management could influence the labile fraction of carbon[121]. However, some site-specific problems and regional factors may influence their distribution in soil layers[102].

    Banger et al.[122] observed significant alteration in labile pools of C, for instance, particulate organic matter (POM), water-soluble C (WSC) and light fraction of C (LFC) because of the addition of fertilizers and/or FYM over a 16-year period. Particulate organic matter, LFC and WSC contributed 24%–35%, 12%–14% and 0.6%–0.8%, respectively, towards SOC. The increase in concentration of SOC including its pools like POC and the sequestration rate due to integrated nutrient management was also reported by Nayak et al.[123]. Gu et al.[124] observed that mulch-treated soils (straw and grass mulch) had significantly greater levels of LOC, POC, DOC and EOC as compared to no mulch-treated soils which could be adduced to the addition of straw, root and its sections into the soil. The content of labile C fractions across all treatments exhibited a decreasing trend with soil depth[23, 102, 125].

    In a long-term experiment, Anantha et al.[126] observed that the total organic carbon apportioned into labile carbon, non-labile, less labile, and very labile carbon constituted around 18.7%, 19.3%, 20.6% and 41.4% of the TOC, respectively (Table 3). Zhu et al.[20] determined that straw incorporation had a substantial impact on TOC and labile C fractions of the soil which were greater in straw incorporated treatments as compared to non-straw treatments across all the depths. Wang et al.[127] reported that the light fraction organic carbon (LFOC) and DOC were significantly greater in the straw-applied treatments than the control by a magnitude of 7%–129% for both the early and late season rice. The treatments NPK + FYM or NPK + GR + FYM resulted in greater content of very labile and labile C fractions whereas non-labile and less labile fractions were greater in control and NPK + CR treatment. There was 40.5% and 16.2% higher C build-up in sole FYM treated plots and 100% NPK + FYM, respectively over control. On the other hand, a net depletion of 1.2 and 1.8 Mg ha−1 in carbon stock was recorded under 50% NPK and control treatments, respectively. Out of the total C added through FYM, only 28.9% was stabilized as SOC, though an external supply of OM is a significant source of soil organic carbon[69]. Hence, to sustain the optimum SOC level at least an input of 2.3 Mg C ha−1 y−1 is required. A comparatively greater quantity of soil C in passive pools was observed in 100% NPK + FYM treatment. The increase in allocation of C into the passive pool was about 33%, 35%, 41% and 39% of TOC in control, suboptimal dose, optimal dose and super optimal dose of NPK which indicates that the concentration of passive pools increased with an increase in fertilization doses. Water-soluble carbon (WSC) was 5.48% greater in the upper soil layer as compared to lower layer of soil. In surface soil (0−15 cm), the values of light fraction carbon (LFC) were 81.3, 107.8, 155.2, 95.7, 128.8, 177.8 and 52.7 mg kg−1 in ZT without residue retention, ZT with 4 t ha−1 residue retention, ZT with 6 t ha−1 residue retention, FIRB without residue addition and FIRB with 4 and 6 t ha−1 residue addition and CT, respectively (Table 4). Tiwari et al.[128] determined that the decrease in POC was due to reduction in fine particulate organic matter in topsoil whereas decrement in dissolved organic carbon was observed largely in subsoil. Therefore, in surface soils fine POC and LFOC might be regarded as preliminary evidence of organic C alteration more precisely, while DOC could be considered as a useful indicator for subsoil. Reduction in allocations of fine POC, LFOC and DOC to SOC caused by tillage and straw management strategies indicated the decline in quality of SOC. A higher SOC concentration was recorded in the conjoint application of INF + FYM (0.82%) and sole application of INF (0.65%) than control (0.54%). Kumar et al.[83] reported that the CT without residue retention had significantly lower labile carbon fractions (27%–48%) than zero-tillage with 6-ton residue retention. Moreover, residue-retained fertilized treatments had significantly greater labile fractions of C than sole fertilized treatments[125]. Kumar et al.[83] reported highest change in DOC in zero-till with residue retention (28.2%) in comparison to conventional tillage practices. In ZT, absence of soil perturbations resulted in sustained supply of organic substrata for soil microbes which increases their activity. On the contrary, CT practices resulted in higher losses of C as CO2 due to frequent disturbances.

    Table 3.  Oxidisable organic carbon fractions in soils (g kg−1) at different layers[126].
    TreatmentDepths (cm)
    0−1515−3030−45Total
    Very Labile C
    Control3.6 ± 0.5c1.4 ± 0.3b1.3 ± 0.2a6.3 ± 0.4b
    50% NPK4.6 ± 0.3bc2.1 ± 0.7ab1.5 ± 0.1a8.1 ± 0.9a
    100% NPK4.4 ± 0.3bc2.3 ± 0.2a1.4 ± 0.5a8.0 ± 0.7a
    150% NPK5.0 ± 0.2ab2.6 ± 0.2a1.5 ± 0.1a9.0 ± 0.3a
    100% NPK + FYM4.8 ± 0.2ab2.0 ± 0.2ab1.3 ± 0.3a8.1 ± 0.2a
    FYM5.9 ± 1.3a2.2 ± 0.2a1.4 ± 0.3a9.5 ± 1.6a
    Fallow4.2 ± 0.7bc1.5 ± 0.5b0.7 ± 0.3b6.3 ± 0.8b
    Lbile C
    Control2.4 ± 0.3a1.0 ± 0.2a0.8 ± 0.4a4.2 ± 0.6a
    50% NPK1.7 ± 0.4ab0.9 ± 0.5a0.7 ± 0.2a3.3 ± 0.7a
    100% NPK1.8 ± 0.4ab0.8 ± 0.5a0.6 ± 0.3a3.2 ± 0.8a
    150% NPK1.2 ± 0.3b0.7 ± 0.2a0.9 ± 0.2a2.8 ± 0.4a
    100% NPK + FYM1.9 ± 0.3ab0.7 ± 0.2a0.7 ± 0.3a3.4 ± 0.2a
    FYM2.5 ± 0.9a0.7 ± 0.3a0.7 ± 0.2a3.9 ± 0.9a
    Fallow2.2 ± 1.0ab1.0 ± 0.3a1.0 ± 0.4a4.1 ± 1.1a
    Less labile C
    Control1.5 ± 0.3c0.6 ± 0.4c0.4 ± 0.0c2.6 ± 0.7d
    50% NPK1.8 ± 0.1c0.4 ± 0.1c0.5 ± 0.2c2.7 ± 0.1cd
    100% NPK2.5 ± 0.3ab0.8 ± 0.1bc1.1 ± 0.2ab4.4 ± 0.1b
    150% NPK2.6 ± 0.2a0.9 ± 0.1bc0.4 ± 0.2c3.9 ± 0.1b
    100% NPK + FYM2.7 ± 0.6a1.5 ± 0.2a1.4 ± 0.1a5.6 ± 0.7a
    FYM1.9 ± 0.7bc1.7 ± 0.2a1.0 ± 0.2b4.5 ± 0.7ab
    Fallow1.5 ± 0.3c1.3 ± 0.7ab0.9 ± 0.4b3.8 ± 1.2bc
    Non labile C
    Control1.2 ± 0.5b1.2 ± 0.3a0.2 ± 0.2b2.6 ± 0.5b
    50% NPK1.2 ± 0.9b1.7 ± 0.8a0.7 ± 0.4ab3.5 ± 1.8ab
    100% NPK1.3 ± 0.6b1.5 ± 0.6a0.5 ± 0.2ab3.3 ± 1.0ab
    150% NPK1.4 ± 0.3b1.5 ± 0.2a0.8 ± 0.1a3.7 ± 0.3ab
    100% NPK + FYM2.0 ± 0.8b1.3 ± 0.1a0.3 ± 0.3ab3.5 ± 0.7ab
    FYM3.7 ± 1.3a1.0 ± 0.2a0.5 ± 0.5ab5.1 ± 1.9a
    Fallow2.1 ± 0.2b1.4 ± 0.7a0.4 ± 0.2ab3.9 ± 0.9ab
    Values in the same column followed by different letters are significantly different at p < 0.001, ± indicates the standard deviation values of the means.
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    Table 4.  Influence of tillage and nitrogen management on distribution of carbon fractions in soil[83].
    TreatmentsWSOC
    (g kg−1)
    SOC
    (g kg−1)
    OC
    (g kg−1)
    BC
    (g kg−1)
    POC
    (mg kg)
    PON
    (mg kg−1)
    LFOC
    (mg kg−1)
    LFON
    (mg kg−1)
    Depths (cm)
    0−1515−300−1515−300−1515−300−1515−300−1515−300−1515−300−1515−300−1515−30
    Tillage practices
    ZTR28.826.223.119.39.619.134.694.281342.8967.9119.5108.1194.7154.814.812.3
    ZTWR25.324.618.414.87.877.213.763.19981.1667.494.686.5120.5104.711.810.3
    RTR27.025.922.418.28.688.174.133.871230.2836.9109.797.8170.9144.913.711.6
    RTWR23.721.818.114.27.667.073.122.96869.4604.482.676.6107.197.39.78.6
    CTR26.124.421.817.48.497.963.823.481099.1779.498.489.3143.8115.912.810.9
    CT21.820.916.113.16.215.642.892.63617.5481.869.257.690.873.69.67.9
    Nitrogen management
    Control21.114.916.113.16.135.481.581.07709.7658.631.726.3123.9104.36.45.8
    80 kg N ha−128.321.217.814.76.466.162.461.75860.7785.668.456.2132.8116.17.66.9
    120 kg N ha−129.522.119.116.17.256.713.262.18952.2808.989.578.5150.6127.69.78.6
    160 kg N ha−130.223.120.818.27.757.283.822.661099.5823.896.883.4168.5145.710.29.8
    200 kg N ha−131.125.421.318.77.937.484.153.421153.1898.4103.997.3176.2152.911.710.6
    WSOC = Water soluble organic carbon, SOC = Total soil organic carbon, OC = Oxidizable organic carbon, BC =Black carbon, POC = particulate organic carbon, PON = particulate organic nitrogen, LFOC = labile fraction organic carbon, and LFON = labile fraction organic nitrogen. ZTR = Zero tillage with residue retention, ZTWR = Zero tillage without residue retention; RTR = Reduced tillage with residue retention, RTWR = Reduced tillage without residue retention, CTR = Conventional tillage with residue incorporation; CT = Conventional tillage without residue incorporation.
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    The soil characteristics such as plant available nutrients, microbial diversity and soil organic carbon transformation are dwindling on account of intensive cultivation under conventional tillage practices, therefore, demand relevant management approaches for soil and crop sustainability. Long-term application of organic amendments significantly increases soil properties by increasing plant available macro, micro, secondary nutrients and soil organic C, whereas the increase in organic C by INF application is, by and large, due to increment in organic C content within macro-aggregates and in the silt + clay compartments. The soil organic carbon and other plant available nutrients were significantly greater in conservation tillage systems as compared to conventional tillage (CT) that conservation approaches could be an exemplary promoter of soil productivity by modifying soil structure thereby protecting SOM and maintaining higher nutrient content. The mean concentration of different fractions of carbon MBN, PMN and soil respiration under integrated nutrient management treatments was higher as compared with to control. Therefore, the conjoint use of organic manures or retention of crop residues with inorganic fertilizers is imperative to reduce the depletion of SOC while sustaining crop production as a realistic alternative. Future research should focus mainly on the usage of organic and mineral fertilizers in conjunction with conservation tillage approaches to sustain the soil environment.

    The authors confirm contribution to the paper as follows: study conception and design: Dhaliwal SS, Shukla AK, Randhawa MK, Behera SK; data collection: Sanddep S, Dhaliwal SS, Behera SK; analysis and interpretation of results: Dhaliwal SS, Gagandeep Kaur, Behera SK; draft manuscript preparation: Dhaliwal SS, walia, Shukla AK, Toor AS, Behera SK, Randhawa MK. All authors reviewed the results and approved the final version of the manuscript.

    Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

    The support rendered by the Departemnt of Soil Science, PAU, Ludhiana, RVSKVV, Gwailor, CSSRI, Karnal, IISS, Bhopal, School of Organic Farming, PAU Ludhiana and Washington State University, USA is fully acknowledged .

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

  • Supplementary Table S1 Factors Affecting Medicinal Plants Regeneration.
    Supplementary Table S2 Plant hormones regulate the regeneration system of medicinal plants.
    Supplementary Table S3 Genes involved in regeneration.
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  • Cite this article

    Wang J, Zhou PH, Li CH, Liang YL, Liu GZ, et al. 2024. Progress on medicinal plant regeneration and the road ahead. Medicinal Plant Biology 3: e030 doi: 10.48130/mpb-0024-0026
    Wang J, Zhou PH, Li CH, Liang YL, Liu GZ, et al. 2024. Progress on medicinal plant regeneration and the road ahead. Medicinal Plant Biology 3: e030 doi: 10.48130/mpb-0024-0026

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Progress on medicinal plant regeneration and the road ahead

Medicinal Plant Biology  3 Article number: e030  (2024)  |  Cite this article

Abstract: Medicinal plants, which are valuable to human beings, play indispensable roles in various fields, such as health care, health promotion, and quality of life enhancement. They are not only pillars of traditional medicine but also valuable sources of modern pharmaceutical research and innovation. Although China has a rich variety of medicinal plants, in recent years, the drastic reduction in wild medicinal plant resources due to over-exploitation and over-utilization has affected the quality of Chinese herbal medicine. Therefore, the development of efficient in vitro regeneration culture technology for medicinal plants is particularly urgent. Here, the main regeneration pathways of medicinal plants are discussed, scientific progress of medicinal plant regeneration culture reviewed, and the main factors affecting the regeneration of medicinal plants analyzed, including the molecular mechanism of phytohormones in inducing the regeneration process, as well as the challenges faced by medicinal plant regeneration technology and directions for future development. Moreover, the challenges and future directions of medicinal plant regeneration technology are summarized, allowing us to find new ideas for the establishment of regeneration systems for rare and endangered medicinal plants, the screening of new regeneration-promoting drug molecules, and the preservation of traditional Chinese medicine (TCM) and its innovation.

    • Medicinal plant resources constitute the cornerstone of the sustained and healthy development of the traditional Chinese medicine (TCM) industry, as they not only treat diseases but also strengthen the human body's immunity and prevent the occurrence of diseases[1]. As an important country for the global production and use of medicinal plants[2], China has more than 13,000 species of medicinal plants, of which more than 200 commonly used Chinese herbal medicine have been planted on a large scale[3]. However, with the modernization of TCM and the expansion of the Chinese herbal medicine industry, the demand for Chinese herbal medicine has also increased annually. The rapid development of TCM, diet, health care, cosmetics, and other industries has led to the over-exploitation and large-scale use of medicinal plant resources, and many rare medicinal plant resources are facing a depletion crisis[2]. Therefore, the establishment of a regeneration system for medicinal plants is highly important to protect these valuable resources, especially endangered medicinal plants.

      To ensure the safety and efficacy of clinical drugs, it is essential to prioritize the development of high-quality Chinese herbal medicine. Biological breeding technology, especially the application of gene editing technology, is expected to lead the innovation of molecular breeding technology in TCM. Medicinal plant regeneration technology is key to achieve this goal[3]. Currently, the regeneration of medicinal plants is focused mainly on the application level, and systematic investigations and in-depth considerations of regeneration mechanisms and future development are still insufficient. The aim of this study was to elaborate on the regeneration pathways, influencing factors and molecular mechanisms of medicinal plants to provide new perspectives and ideas for the sustainable utilization of Chinese herbal medicine resources.

    • To achieve totipotency, differentiated cells must first undergo dedifferentiation, then redifferentiation. Dedifferentiated plant explants form healing tissues, which are transferred to differentiation medium, where morphogenesis is completed, resulting in shoot and root structures, which mature into complete plants.[4]. In vitro tissue culture of medicinal plants, plant regeneration is achieved mainly through somatic embryogenesis (SE), de novo organogenesis, and protoplast regeneration (Fig. 1).

      Figure 1. 

      Different pathways of plant regeneration. (a) Somatic embryogenesis: in the direct pathway, a somatic cell originating from an immature embryo is induced to form a somatic embryo, which then drives the development of the entire plant. In the indirect pathway, the explant is induced to initiate an embryonic callus, on which somatic embryos are formed. These embryos subsequently develop into shoots and roots. (b) De novo organogenesis: in the direct pathway, shoots and roots are induced directly on the stem with pre-existing meristems. In the indirect pathway, a pluripotent callus is produced around the wound in a leaf explant, with the formation of shoots and roots being subsequently induced. (c) Three methods of protoplast regeneration: one method involves the formation of a callus by protoplast differentiation, which is then induced to form shoots and roots, eventually differentiating into a plant. Another method involves differentiation from a protoplast to form a callus, followed by differentiation from a callus to an embryoid, and finally, the development of the whole plant. A direct method involves differentiating from a protoplast into an embryoid, which then develops into the whole plant.

    • SE is an important plant regeneration technique that allows somatic cells to undergo a series of developmental changes that culminate in the formation of embryo-like structures. This process reveals the totipotency of plant cells through embryogenic calli; they can dedifferentiate into embryonic stem cells and redifferentiate into complete plants[5,6]. This change in cell fate is usually realized under specific stress conditions, hormone induction (such as auxin) or gene expression modification, and has great potential for plant reproduction, genetic transformation and the protection of rare and endangered species[79]. The formation of somatic embryos can be achieved via two routes: direct induction from individual somatic cells or indirect induction through the healing of embryonic tissue[10] (Figs 1a & 2). The direct route involves the induction of embryoid formation from the epidermis, subepidermis, young embryos, cells in suspension culture, and protoplasts of explants. For example, Glycyrrhiza uralensis hypocotyls can be used as explants to directly induce embryoid formation on MS medium and successfully culture regenerated plants[11]. The indirect pathway is more common and begins with embryonic healing tissue induction, followed by the formation of pre-embryonic masses on the surface or inside of healing tissues, which then develop into somatic embryos. Under the right conditions, these embryos are capable of further developing into complete plants with roots and shoots[12].

      Figure 2. 

      Classification map of medicinal plants based on different medicinal parts and regeneration pathways. (a) Roots and rhizomes: arrangement of medicinal plants whose medicinal parts are roots and rhizomes. (b) Whole grass: arrangement of medicinal plants whose medicinal parts are the entire grass. (c) Fruits and seeds: arrangement of medicinal plants whose medicinal parts are fruits and seeds. (d) Flowers: arrangement of medicinal plants whose medicinal parts are flowers. Regeneration routes: the map is divided into three categories on the basis of different regeneration routes: the blue section represents medicinal plants regenerated through protoplast regeneration; the orange section represents medicinal plants regenerated through somatic embryogenesis; and the green section represents medicinal plants regenerated through de novo organogenesis. Note: All images are from Flora of China[13].

      SE in medicinal plants usually takes place in an indirect manner, where embryonic healing tissues are induced via different explants, which then differentiate to form embryoid bodies and eventually develop into plants[8] (Figs 1a & 2). For example, the regeneration of medicinal plants such as Picrorhiza kurroa[14], Panax notoginseng[15], and Eleutherococcus senticosus[16] has been achieved in this way. In addition, the hairy roots of plants such as Scrophularia buergeriana[17], and Panax quinquefolius[18] can also induce the formation of healing tissues in specific culture medium, subsequently forming embryos and regenerating new individuals.

      Somatic embryos produced via direct and indirect routes are morphologically similar but differ in culture time, susceptibility to genetic variation, and plant regeneration potential. Although the indirect route requires a longer culture time and is more susceptible to genetic variation, it is capable of producing large amounts of healing tissue, which increases the regeneration potential of plants[19,20]. In contrast, the direct route is more efficient at producing a limited number of regenerated plants. Therefore, the direct route is more appropriate when the goal is to regenerate a specific number of plants rapidly. However, the indirect route provides a better alternative for species from which explants are difficult to obtain or for which many regenerated plants are needed[21].

    • The organogenesis pathway is a regenerative process that does not depend on somatic embryos and is achieved through the differentiation of meristematic tissue centers, demonstrating the pluripotency of plant cells[22]. This regeneration mechanism allows plants to regenerate de novo root and/or de novo shoot in vitro or from damaged organs, a common phenomenon in nature. The processes of regenerating de novo shoots and de novo roots are referred to as de novo shoot organogenesis and de novo root organogenesis, respectively[23]. Although de novo organogenesis is simple to induce, with a high induction rate, it may lead to the formation of chimeras. Like SE, de novo organogenesis can be initiated directly or indirectly (Fig. 1b).

      The direct organogenesis pathway facilitates the rapid generation of regenerating plants from tissue organs such as stem tips, stems, metamorphic stems, and leaves of medicinal plants, eliminating the healing tissue induction stage and thus shortening the regeneration cycle[2] (Fig. 1b). This pathway is widely used in medicinal plants in China, such as Andrographis paniculata[24] Platycodon grandiflorus[25], Dendrobium nobile[26], and Schisandra chinensis[27], which are not only in high market demand but also maintain genetic stability during the culture process.

      The indirect organogenesis pathway involves a distinct healing tissue stage during regenerating plant culture; this pathway involves a wider selection of explants, including anthers, leaves, stem segments, petioles, and roots. The indirect pathway has relatively high amplification and transformation rates, but there are differences in the morphogenetic capacity of explants from different parts of the plant (Figs 1b & 2). For example, the flowering branches and inflorescences of Artemisia caruifolia are more effective than the leaves in healing tissue induction and shoot differentiation[28]. Previous studies have shown that the healing capacity of leaves from Ligusticum sinense is greater than that of stem segments and roots, whereas the healing capacity of leaves is similar to that of stem segments; roots are weaker in terms of shoot differentiation and rooting capacity. In general, the ability of plant spindles to differentiate and regenerate gradually decreases from top to bottom[29].

      In recent years, China has made remarkable progress in cultivating regenerated plant species through the organogenesis pathway, covering not only bulk medicinal plants such as the Asteraceae, Liliaceae, Labiatae, Araliaceae, Ranunculaceae, and Dioscoreaceae families but also slow-growing, endangered or progressively endangered medicinal plants such as Dendrobium officinale, Neopicrorhiza scrophulariiflora, Coptis teeta and Ginkgo biloba. At present, regeneration systems have been established for more than 200 medicinal plants[2] (Fig. 2).

      The essential difference between de novo organogenesis and SE is that the former does not involve the formation of somatic embryos. Both pathways involve direct and indirect methods of regeneration, but the indirect methods differ with respect to healing tissue characteristics. SE produces totipotent embryonic healing tissues, whereas de novo organogenesis induces pluripotent nonembryonic healing tissues[30,31] (Fig. 1a & b). Indirect de novo organogenesis may lead to genetic instability and somatic cell asexual lineage variation[32]. Direct organogenesis is a time-saving method but is not suitable for transgenic research because of the possibility of chimerism[33]. The development of somatic cell embryos for certain organs or tissues that readily induce de novo organogenesis is challenging. Therefore, a combination of both pathways is sometimes used to increase the frequency of plant regeneration in a particular species, either in the commercial market or in scientific research.

    • In addition to de novo organogenesis and SE, scientists have discovered the ability of individual cells to regenerate whole plants. As early as 1954, a method for single-cell healing tissue culture of Tagetes erecta was established[34]. Healing tissues in vitro from the phloem of Daucus carota roots were cultured, and single cells capable of undergoing multiple rounds of division and eventually developing into somatic embryos were obtained[35]. At present, plant protoplast regeneration culture and suspension culture techniques have been widely used for plants of several families, such as the Rosaceae, Boraginaceae, and Scrophulariaceae families, indicating great potential for application[36] (Fig. 2).

      In China, protoplast culture technology has been successfully applied to the culture of a variety of medicinal plants, including Gentiana Macrophylla, Peucedanum praeruptorum, Saposhnikovia divaricata, Salvia miltiorrhiza, Cercospora asparagi, Lycium chinense, Rhodiola rosea, and Codonopsis pilosula (Fig. 2). Protoplasts are used for plant regeneration through three main pathways: the differentiation of protoplasts to form healing tissues that then redifferentiate into plants; the direct differentiation of protoplasts into embryos that then develop into plants; and the differentiation of protoplasts to form healing tissues that then redifferentiate into embryos and eventually develop into plants (Fig. 1c). For example, many embryonic protoplasts were cultured from calli induced by cutting P. peucedanum seedlings, and whole plants were ultimately obtained[37]. Protoplasts were isolated from the hypocotyl callus of C. pilosula, and embryoids were obtained via shallow liquid culture and subsequently developed into complete plants[38]. Tissue culture was used to obtain tissue culture-generated seedlings of Rhodiola sachalinensis. The leaves of R. sachalinensis tissue culture-generated seedlings were hydrolyzed by enzymes to obtain protoplasts, and calli were obtained via shallow liquid culture. The calli differentiate into adventitious shoots and then develop into complete plants[39]. In addition, protoplasts have been isolated from S. miltiorrhiza plants via leaf enzymatic hydrolysis, and protoplast regeneration systems for S. miltiorrhiza have been established by targeting one or more sites with the sgRNA-Cas9 ribonucleoprotein (RNP) complex or a plasmid carrying CRISPR/Cas9 system genes[40].

      Notably, compared with other model plants and crops, research on the protoplasts of medicinal plants is still immature, and the technology needs to be improved. Therefore, further studies of regeneration culture, functional gene analysis and metabolite synthesis mechanisms of medicinal plant protoplasts are highly important to promote the sustainable and efficient development and utilization of medicinal plant resources.

    • During the in vitro regeneration culture of medicinal plants, the plant genotype is an important factor affecting regeneration capacity. There are significant differences in the response of plants of different genotypes to in vitro culture, a phenomenon that may be related to genetic differences and gene regulation[41]. For example, when stem segments were used as explants in S. miltiorrhiza f. alba and S. miltiorrhiza Bunge, 100% of S. miltiorrhiza f. alba stem segments developed shoots[42], whereas 85% of S. miltiorrhiza Bunge stem segments developed shoots (Supplementary Table S1)[43]. In addition, S. miltiorrhiza Bunge varieties from different regions, such as Shandong S. miltiorrhiza and Sichuan S. miltiorrhiza, present differences in regeneration systems, with Shandong S. miltiorrhiza having a relatively high shoot induction rate and Sichuan S. miltiorrhiza having a relatively high rate of healing tissue induction (Supplementary
      Table S1
      )[44]. When cotyledons of Astragalus membranaceus were used as explants, healing tissues were obtained from 90% of the cotyledons on healing induction medium, with an optimal shoot generation rate of 90%[45], whereas cotyledons of A. membranaceus var. mongholicus were used as explants, the rate of healing tissue induction was 25% on the same healing induction medium, with no shoot differentiation[46]. In studies of the tissue culture of Taraxacum mongolicum, the cultures of different types of T. mongolicum differ under the same conditions. For example, Menggu T. mongolicum and Liaodong T. mongolicum have relatively high frequencies of callus induction, at 85.3% and 76%, respectively, whereas Taraxacum ohwianum, Taraxacum coreanum and Republic of Korea T. mongolicum have relatively low rates of callus formation and severe browning (Supplementary Table S1)[47].

      In studies of G. uralensis, the highest frequency of in vitro regeneration (up to 44.7%) was observed when hypocotyls were used as explants, whereas the number of clumped seedlings was greater when cotyledonary stem segments and leaf-bearing stem segments were used as explants[11]. These findings suggest that there are significant differences in healing tissue induction among different genotypes and that the selection of suitable organs and materials is crucial for improving the induction rate and perfecting regeneration systems for medicinal plants. In practical applications, the regeneration efficiency of specific genotypes can be improved by optimizing the medium composition and culture conditions. For example, when calli of A. membranaceus were induced, the application of activated carbon and TCM extracts in improved MS medium significantly increased the induction rate (Supplementary Table S1)[45]. In addition, research progress on the tissue culture of G. uralensis has shown that the use of different concentrations of plant growth regulators (PGRs) and nitrogen sources has significant effects on callus induction and growth (Supplementary Table S1).

    • Exogenous hormones, especially PGRs, such as auxins and cytokinins, play key roles in plant SE and neoorganogenesis[48]. In vitro plant regeneration depends on the addition of exogenous hormones and the response to these hormones during tissue culture[49]. One study reported that the response of explants to PGRs occurs in three phases: first, the perception of phytohormone signals by explant cells, which induces dedifferentiation; second, specific cellular differentiation influenced by hormone homeostasis, which lays the groundwork for organ differentiation; and last, the completion of plant morphogenesis independent of exogenous hormones[50].

      Growth factors are determinants of SE, among which 2,4-dichlorophenoxyacetic acid (2,4-D)[51], a synthetic auxins, is a strong inducer of healing tissues and is widely used in many species, especially cereal crops and medicinal plants. The concentration of 2,4-D has a significant effect on the formation of healing tissues, with low concentrations promoting the formation of embryonic healing tissues, whereas high concentrations may inhibit their formation. In medicinal plants, 5–10 μM 2,4-D is often used to induce somatic embryos, but prolonged accumulation may have toxic effects on cells and increase the risk of somatic mutations (Supplementary Tables S1 & S2)[52].

      Other growth factors include indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA). NAA is often chosen for inducing roots differentiation and has a stronger effect than IAA and IBA do. However, the roots after NAA treatment are thicker and shorter and easier to break, whereas the roots after IAA treatment are weaker and readily degrade. In addition, the roots obtained by IBA treatment are more robust; therefore, many researchers have used a combination of NAA and IBA to optimize roots induction. For example, the roots induction rate of Angelica sinensis was highest at a hormone ratio of 2.0 mg/L IBA + 0.01 mg/L NAA, and the roots induction of Hemsleya chinensis was strongly induced at a hormone ratio of 2.0 mg/L 6-BA + 0.5 mg/L NAA + 1.0 mg/L IBA (Supplementary Tables S1 &S2)[5355].

      Cytokinins are also widely used PGRs in tissue culture, especially for de novo shoot induction and SE initiation. A proposed model of hormonal control of regeneration is widely used in the regeneration of explants. 6-BA, a commonly used cytokinin, is often used in conjunction with other hormones to induce callus differentiation and bulblet induction (Supplementary Tables S1 & S2).

      In addition to auxins and cytokinins, other plant hormones, such as abscisic acid (ABA) and gibberellin (GA), also affect plant regeneration[56]. For example, GA promotes the germination and differentiation of immature embryos, whereas ABA and other hormones, such as oleuropein steroids and abscisic acid, also induce healing in some species. GA can substitute for auxins or cytokinins in the formation of healing tissues (Supplementary Tables S1 & S2)[57,58].

      There are large differences in the phytohormone requirements for the regeneration of different genotypes and explants of medicinal plants; therefore, plant growth regulators suitable for different species need to be screened to achieve increased induction efficiency. The use of plant growth regulators is also widely used in the production of herbal medicines, but attention needs to be given to their effects on the effectiveness and safety of herbal medicines.

    • The ability of cells to initiate a regenerative program first needs to overcome specific tissue fate restrictions. Stress can effectively liberate cells from their fate constraints, resulting in the disruption or loss of intercellular communication. By dissecting and dissociating explant tissues and applying osmotic stress, cytoplasmic lysis, intercellular junction rupture, or cell death can be induced, resulting in the separation of living cells and the elimination of intercellular interactions. Isolated cells usually have thick cell walls and few or no intercellular junctions and exhibit cellular morphological features characteristic of stem cells, such as a large central nucleus and dense cytoplasm[6,59]. Owing to the loss of intercellular interactions, these cells may be excluded from fate restrictions imposed by neighboring cells and may lose positional information[60,61]. In contrast, cells in meristematic tissues are tightly connected by thin primary cell walls and intercellular filaments through which mobile transcription factors, signaling peptides, and phytohormones can regulate the proliferation and differentiation of surrounding cells[6264]. A study of stem tip-induced SE in Arabidopsis thaliana reported the immediate expansion of the expression of several genes related to stem meristematic tissue in the stress response, which may provide cells with the ability to shift their fate from shoot to embryo (Supplementary Table S1)[65].

    • The culture medium plays a crucial role in the isolated regeneration culture of medicinal plants, providing the necessary nutrients but also influencing the regeneration efficiency of plants through the regulation of different components. In tissue culture, MS medium is widely used as a basic medium because of its high nutrient content and is particularly suitable for the growth and development of the shoots and roots of medicinal plants. However, different species or tissues may require different culture medium[2,66]. For example, a study reported that the proliferation coefficient and differentiation rate of Lonicera macranthoides cultured on MS medium were greater than those of L. macranthoides cultured on other medium[67]. In addition, a study reported that 1/2 MS medium was significantly better than MS medium at promoting the growth of tissue culture-generated seedlings (Supplementary Table S1)[27].

      The carbon source is another key component of the culture medium that provides energy and regulates the osmotic environment for plant tissue culture. Commonly used carbon sources include glucose, sucrose, and maltose, which have important effects on the formation and maturation of somatic embryos[68]. Sucrose is considered one of the best carbon sources because of its ability to promote the growth and differentiation of explants[69]. In flax healing tissue culture, sucrose is used as a carbon source at an optimal concentration of 30 g/L (Supplementary Table S1)[70].

      Light and temperature are also important factors affecting the regeneration of medicinal plants in vitro. Light plays an inducing role in the growth and differentiation of cells, tissues and organs, but excessive light or an improper photoperiod may be detrimental to the formation and maintenance of healing tissues. For example, the oxidation of phenolic compounds under light conditions may lead to tissue browning and affect somatic embryo formation[71]. A proper dark culture time is beneficial for increasing the callus volume of perilla frutescens. The temperature affects the respiratory rate and metabolic reactions, and the optimum temperature for medicinal plants is generally approximately 25 °C (Supplementary Table S1); however, different medicinal plants, such as Linum usitatissimum, Scutellaria baicalensis, Asarum heterotropoides, and P. grandiflorus, need different temperature conditions to promote callus growth and organ differentiation.

      In the culture of medicinal plants in vitro, the physiological status and genetic differences of explants can be optimized for optimal regeneration by adjusting the composition of the culture medium. These methods include the selection of appropriate conditions, such as basic medium, carbon sources, photoperiods, and temperatures, to suit the specific needs of different medicinal plants (Supplementary Table S1).

    • Early studies on plant cell growth and development focused mainly on the role of plant hormones. However, since the end of the 20th century, with the development of molecular genetics and transcriptomic technologies, researchers have begun to pay more attention to transcription factors and the multiple signaling pathways and expression patterns they regulate. Recent studies at the molecular level have shown that plant cell dedifferentiation and morphogenesis depend on the orderly and correct expression of specific transcription factors[72]. These processes, including healing tissue formation, de novo shoot and root regeneration, and SE, are regulated by finely tuned hormonal and abiotic stress signals, in which cellular totipotent transcription factors play crucial roles[73].

      Epigenetic modifications, including DNA methylation and histone modifications, also play important roles in healing tissue formation and organ regeneration, especially in the restoration of somatic cells to pluripotent healing tissue cells induced by plant hormones and injury[74]. In addition, plant regulatory ncRNAs are essential for healing tissue induction because they influence gene expression and protein translation and participate in transcriptional and posttranscriptional regulation[75].

      Although studies on the molecular mechanisms of hormone regulation, epigenetic regulation and ncRNA regulation are rare in the field of medicinal plant regeneration, we can provide a theoretical basis and reference for future studies on the molecular mechanisms of medicinal plant regeneration by summarizing the regulatory mechanisms in other plants.

    • SE is a classical dedifferentiation process that allows differentiated somatic cells to revert to embryonic stem cells with totipotency, providing the basis for totipotency and regeneration in multicellular organisms. Recent studies have revealed that this process not only involves multiple transcription factors and hormonal signaling pathways but is also closely related to epigenetic regulation and the fine regulation of non-coding RNAs (ncRNAs) (Fig. 3).

      Figure 3. 

      Molecular mechanisms of somatic embryogenesis. The process of somatic embryogenesis is influenced by epigenetic regulation, transcription factors, and hormone signaling pathways. Epigenetic regulation, which includes DNA methylation (indicated by a pink shadow) and histone modifications (indicated by a green shadow), represses the access of transcription factors to gene-promoter regions, thereby inhibiting the expression of genes involved in somatic embryogenesis. Numerous transcription factors (indicated by a purple shadow) are involved in this regulatory network, where they also regulate each other and activate downstream auxin and cytokinin (CK) signaling pathways. Additionally, miR-165/-166 (indicated by a cyan shadow) are involved in regulating somatic embryogenesis.

      In terms of transcription factors, Plethora (PLT), BABY BOOM (BBM), Leafy cotyledon 1 (LEC1), LEC2, RWP-RK RWP-RK DOMAIN-CONTAINING 4(RKD4)/GROUNDED (GRD), At-hook motif containing nuclear localized15 (AHL15), WUSCHEL (WUS), FUSCA 3 (FUS3), and Abscisic acid insensitive 3 (ABI3) have been identified as key factors regulating SE (Supplementary Table S3). These transcription factors are important regulators that drive cell fate transitions and can be considered representative of totipotency-associated transcription factors[36].

      BBM and PLT, as members of the AP2/ERF family, play crucial roles in the growth and development of embryonic and root meristematic tissues in A. thaliana[76,77]. The ectopic expression of the BBM gene induced SE and regenerated seedlings without exogenous growth regulators in both A. thaliana and Brassica napus, highlighting the central regulatory role of BBM in the development of plant embryos[78]. In addition, this study further revealed the specific activation of the BBM gene during the transformation of plant somatic cells to embryonic cells and its precursor role in signaling pathways to promote cell differentiation and somatic embryo formation (Supplementary Table S3)[79].

      Transcription factors such as ABI3, FUS3 and LEC2 encode proteins containing the plant-specific b3 structural domain and belong to the AFL subfamily[80,81]. These proteins, together with the LAFL complex formed by LEC1, are involved in the activation of cellular allosteric transcription factors. BBM is located upstream of these transcription factors and can activate cellular allosteric transcription factors, such as LEC1, LEC2, and agamous-like 15 (AGL15), indirectly promoting the expression of auxins signaling factors such as YUCCA (YUC) and the AUX/IAA factor IAA30. BBM directly binds to the promoter regions of the YUC and tryptophan aminotransferase of Arabidopsis 1 (TAA1) genes, driving the upregulation of their expression, increasing auxin synthesis and promoting SE (Supplementary Table S3)[82,83] (Fig. 3).

      WUS homology box transcription factors also play key roles in the regulation of embryonic cell fate. In A. thaliana, WUS overexpression promotes somatic embryo production under hormone-free conditions and upregulates the expression of LEC1, LEC2, and AGL15 during SE[78]. WOX2 and WOX3, downstream targets of LEC2, are essential for somatic embryo formation. Moreover, WOX2/3 are essential for SE, but their overexpression is not sufficient to induce somatic embryo formation (Supplementary Table S3)[84] (Fig. 3).

      Wound-induced differentiation 1 (WIND 1), another APETALA2/ERF family transcription factor, is not directly involved in somatic embryo formation but plays an important role in healing tissue induction. WIND1 is located upstream of LEC2 during regeneration and is involved in cytokinin-specific responses rather than auxin biosynthesis and signaling through different hormonal pathways[9,84]. In particular, WUS represses negative regulators [type-A Arabidopsis response regulator (ARR) genes] of the CK response, whereas WIND1 stimulates the expression of positive regulators (type-B ARR genes) of the CK response (Supplementary Table S3)[72,85] (Fig. 3).

      Epigenetic regulation plays a key role in maintaining somatic cell identity and suppressing embryo-specific gene expression. DNA methylation and histone modification are important mechanisms that regulate gene expression and determine cell fate[86]. Mutation of methyltransferase 1 (MET1) affects the expression of the auxin efflux vector Pin-formed 1 (PIN1) and leads to abnormalities during SE[87]. The methylation levels of somatic embryogenesis receptor-like kinase (SERK), LEC2 and WUS in embryogenic healing tissues suggest a potential role for these genes in the regulation of SE[88]. In addition to DNA methylation, histone modifications, including methylation, acetylation and ubiquitination, also play important roles in regulating SE. A study reported that Polycomb repressive complex (PRC) 1 and PRC2 are required to establish and maintain stable epigenetic repression in response to developmental or environmental signals and that PRC1 and PRC2 repress the expression of embryo-specific genes, including LAFL, AGL15, WUSCHEL-RELATED HOMEOBOX 5 (WOX5), BBM, and PIN1[89]. In addition, PICKLE (PKL) plays an important role in preventing the generation of embryonic traits in somatic cells and is an epigenetic factor that plays a key role similar to that of PRC1 and PRC2[90]. A study further reported that PKL represses the expression of embryonic genes, including the LAFL genes, by promoting alterations in Histone 3 lysine 27 trimethylation (H3K27me3)[91]. In addition, histone acetylation regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) plays a key role in SE. The HDAC inhibitor tricosin A upregulates the expression of genes related to embryogenesis, including LEC1, FUS3 and ABI3 (Supplementary Table S3)[92] (Fig. 3).

      ncRNAs, including miRNAs and long-chain non-coding RNAs, also play a role in somatic cell embryogenesis. Specific miRNAs are involved in the regulation of SE through the modulation of phytohormone signaling pathways[74]. A study revealed that the regulation of RNA metabolism is essential for in vitro phytocellular dedifferentiation and that high levels of mini-nucleolar RNAs are required for in vitro cellular dedifferentiation and organogenesis in A. thaliana[93].

    • The formation of pluripotent healing tissues is a complex process that usually begins with the division of column sheath cells in the xylem pole; this process is similar to lateral root formation and involves the same molecular mechanisms[94] (Fig. 4). Auxin is a key hormone involved in the regulation of healing tissue formation and lateral root formation[95], which prompted us to hypothesize that some components of auxin signaling or downstream factors involved in lateral root formation may also play important roles in healing tissue regulation.

      Figure 4. 

      Molecular mechanisms of pluripotent callus formation. The ability of auxin to activate ARF7/19 is also regulated by epigenetic factors such as AXTR2 and JMJ30. Downstream of ARF7/19, LBD-bZIP59 and WRKY23 activate the expression of genes such as WOX5, PLT1/2, SHR, and SCR by removing bHLH041 and PLT3/7. The interaction between WOX5 and PLT1/2 enhances the expression of TAA1, leading to an increase in endogenous auxin levels in the callus and inducing the formation of pluripotent calli. The removal is indicated by dashed lines.

      In A. thaliana, auxin response factor (ARF)7 and ARF19, in conjunction with SOLITARY ROOT (SLR/IAA14), were initially identified as important regulators of lateral root formation, and they are also required for auxin-induced healing tissue formation[96]. When the function of ARF7/19 or SLR is disrupted or mutated, healing tissue formation is significantly inhibited[97]. In addition, the LOB-DOMAIN transcription factor lateral organ boundaries domain (LBD)16, LBD17, LBD18 and LBD29, which are downstream targets of the ARF7/19-IAA14 module, are likewise essential for healing tissue formation. The transcription factor bZIP59 synergizes with these LBD proteins, and growth factors promote cell fate transition by stabilizing bZIP59 and enhancing its interaction with LBDs[97]. Further studies revealed that LBDs form a heterodimeric complex with bZIP59, which directly activates, among other enzymes, FAD-BINDING BERBERINE (FAD-BD), which encodes a BBE-like enzyme involved in cell wall metabolism and is a direct target of LBD16 to promote lateral root emergence[98,99]. In transgenic seedlings overexpressing LBD or bZIP59, ectopic activation of the root meristem tissue regulators WOX5, PLT1, PLT2, SCARECROW (SCR) and SHORTROOT (SHR) (Fig. 4), which are essential for the maintenance of healing tissue pluripotency and subsequent neoplastic regeneration, was observed (Supplementary
      Table S3
      )[97].

      The physical interaction between WOX5 and PLT1/2 promotes the expression of the auxin biosynthesis gene TAA1, which is essential for maintaining the pluripotency of healing tissue cells[100]. In addition, the loss of function of the PLT3/5/7 genes does not affect the formation of healing tissues but hampers the ability of healing tissues to form preemergent cells in a subsequent regeneration program[101]. A. thaliana WRKY23 and bHLH041 act as transcriptional activators and repressors downstream of ARF7/19 and are responsible for the activation of root stem cytokines, which establish healing tissue pluripotency. WRKY23 directly targets and activates the transcription of PLT3/7, whereas the LBD-induced removal of bHLH041 represses the transcription of PLT1/2 and WOX5[102] (Fig. 4 & Supplementary Table S3).

      In addition to the above genes, the overexpression of BBM in different plant species also promotes cell proliferation and regeneration. For example, the overexpression of A. thaliana BBM in Nicotiana tabacum induces callus formation; the overexpression of GmBBM7 in soybean promotes the growth of calli and roots; and the overexpression of AtBBM in poplar leads to the formation of somatic embryos[73].

      The in vitro of healing tissue involves cell fate transitions and reprogramming at the genome-wide level. Epigenetic regulation plays an important role in auxin-induced healing of tissue. For example, Arabidopsis Trithoprax-related 2 (ATXR2) and JMJ30 activate LBD gene expression by regulating histone modifications, whereas the histone acetyltransferase GCN5 acquires pluripotency by catalyzing histone acetylation at the root meristem gene locus[103]. In addition, chromatin remodeling factors such as PKL and CURLY LEAF (CLF) are involved in the establishment of healing tissue pluripotency by controlling the expression of root stem cell regulators[64] (Fig. 4 & Supplementary Table S3).

      ncRNAs, such as miR160, also play key roles in healing tissue induction and plant cell dedifferentiation. miR160 and its target gene ARF10 are critical in the process of healing tissue formation in vitro (Fig. 4 & Supplementary Table S3). More prolific and faster healing tissue formation was observed in specimens with miR160-resistant forms of ARF10 (mARF10), whereas cell lines overexpressing miR160 had slower and fewer healing tissues[104].

    • In tissue culture, the transfer of healing tissues to a root-induction medium containing relatively high concentrations of auxin induces the development of new rooting organs. The inhibition of polar auxin transport blocks the rooting process, which highlights the key role of auxin in regulating the development of new root organs[105]. In addition, the YUC gene family (YUC1, YUC11, YUC8, and YUC9), which is involved in the biosynthesis of auxins, has been shown to inhibit the expression of WOX11 in receptor cells (Supplementary Table S3)[106].

      Recent transcriptomic, epigenomic and cell lineage analyses of healing tissues revealed similar genetic pathways for healing tissue formation and neoplastic root organogenesis. The de novo process of root organogenesis can be divided into two major steps: first, the transition from energetic to root-establishing cells, a process in which the expression of WOX11 is a hallmark event; and second, the transition from root-establishing to root-primary cells, marked by the expression of WOX5[107]. In the first step, auxin directly activates the expression of WOX11 and its homolog WOX12. WOX11/12 subsequently further promotes the expression of WOX5 and LBD16, and then LBD16 is responsible for activating the expression of WOX5, PLT1 and PLT2[108] (Fig. 5 & Supplementary Table S3).

      Figure 5. 

      Molecular mechanisms of de novo root organogenesis. Auxin, which is mediated by YUC, serves as a key regulatory factor that activates the expression of WOX11/12, ARF7/19, and PLT3/5/7. The translation products of these genes then directly or indirectly promote the expression of WOX5 and PLT1/2, which in turn induce the formation of de novo root organogenesis. The transcription factor EIN3 significantly reduces the frequency of new root organs by inhibiting the transcription of WOX11 and WOX5.

      The transcription factor EIN3 significantly reduces the frequency of newly rooted organs by repressing the transcription of WOX11 and WOX5. This finding is consistent with the observations that the activity of EIN3 increases with the age of the explant and that younger organs have greater regenerative capacity[109]. As mentioned previously, growth factors also induce the expression of PLT3, PLT5, and PLT7 and regulate the expression of downstream root meristem organization marker genes. In addition to the WOX11/12 and PLT genes, the auxin response factors ARF7 and ARF19 can target and activate the expression of LBD16 (Fig. 5 & Supplementary Table S3), which promotes expression during root regeneration.

      Although studies on the molecular mechanisms of epigenetic modifications and ncRNAs in the induction of de novo root regeneration are still relatively limited, further exploration in these areas will undoubtedly provide a deeper understanding. With further research, the elucidation of these regulatory mechanisms will help optimize plant tissue culture conditions, improve regeneration efficiency, and provide new strategies for plant biotechnology applications.

    • Cultivation of healing tissues in a cytokinin-rich medium induces continued cell division and proliferation mediated by cytokinin to form cell populations and promote subsequent differentiation, which marks the establishment of the stem cell ecological niche[110]. The maintenance of stem homeostasis is achieved through two main regulatory pathways, WUS–Clavata 3 (CLV3) and Shoot meristemless (STM)–cup-shaped cotyledon (CUC) (Fig. 6 & Supplementary Table S3), which are decisive factors in the early stage of stem cell ecological niche construction. The WUS gene begins to be expressed 2 to 3 d after tissue culture[111], and its initial expression is a hallmark of the establishment of shoot progenitor cells, which is the most critical molecular event in the process of de novo shoot organogenesis. WUS mutants completely lose their regenerative capacity, whereas WUS overexpression results in the ectopic formation of shoots, confirming the necessity of WUS in the regeneration of nascent shoots[112].

      Figure 6. 

      Molecular mechanisms of de novo shoot organogenesis. During the process of de novo shoot organogenesis, two pathways, the WUS-CLV3 pathway and the STM-CUC pathway, establish negative feedback loops and play critical regulatory roles. The WUS-CLV3 pathway is regulated primarily by DNA methylation, histone modification, and hormone signaling. Cytokinin (CK) activates the expression of type B ARRs, which in turn stimulates WUS expression, whereas type B ARRs repress YUC-mediated auxin biosynthesis. In the STM-CUC pathway, STM expression is promoted by CUC1 and CUC2, both of which are upregulated by PLT3/5/7, ESR1, ESR2, WIND1, and PIN1. Moreover, WUS and STM interact directly to activate CLV3 expression, suggesting that the two pathways converge and coordinate to control shoot regeneration.

      WUS promotes the expression of the encoded signaling peptide CLV3, which represses WUS expression through a negative feedback loop, serving as a negative feedback mechanism that plays a key role in maintaining stem cell populations. Similarly, the STM gene is expressed in stem meristematic tissues and represses the expression of CUC1 and CUC2, which in turn activates STM expression to maintain stem meristematic tissues[113]. The WUS–CLV3 pathway is regulated by DNA methylation, histone modification, and hormone signaling (Fig. 6 & Supplementary Table S3).

      The auxin and cytokinin signaling pathways jointly influence WUS expression. B-type response regulators (ARR1, ARR2, ARR10 and ARR12), as transcriptional activators of cytokinin signaling, activate the expression of WUS by directly binding to its promoter and inhibiting YUC gene-mediated auxin accumulation, which further promotes WUS expression[111]. In contrast, a-type response regulators (ARR5, ARR6, ARR7 and ARR15), negative regulators of cytokinin signaling, are regulated by b-type response regulators, which form a negative feedback loop to inhibit shoot regeneration by interfering with the function of b-type response regulators[114] (Fig. 6 & Supplementary Table S3). In addition, type B response regulators interact with the HD-ZIP III protein to form a transcription complex, which specifically activates the expression of WUS[110].

      In the STM–CUC pathway, a negative feedback loop between STM and CUC plays a key role in regulating neoplastic stem organogenesis. CUC proteins are essential for the establishment of the shoot progenitor system[77]. The CUC-induced polar localization of PIN1 determines shoot progenitor location, and the increased polarity of PIN promotes STM expression in the progenitor system[112]. In addition, PLT3, PLT5, and PLT7 upregulate the expression of CUC1 and CUC2 during shoot regeneration. These PLT proteins control shoot regeneration through a two-step mechanism: first, they activate the expression of PLT1 and PLT2 during pluripotent guaiac tissue formation to increase competence; second, they accomplish regeneration through the regulation of CUC[115]. Enhancer of shoot regeneration (ESR)1 and ESR2 act as upstream regulators of CUC genes during neoplastic stem organogenesis by directly binding to their promoters to activate expression. In addition, ESR1 expression is regulated by WIND1, linking wound signaling to shoot regeneration[116] (Fig. 6 & Supplementary Table S3).

      Both the WUS–CLV3 and STM–CUC pathways are required for stem cell development during de novo shoot organogenesis. Recent studies have reported that these two pathways are coordinated through direct interactions between the WUS and STM proteins. STM directly activates CLV3 expression by binding to the promoter at a site different from WUS. WUS–STM interactions enhance WUS binding to the CLV3 promoter and CLV3 transcriptional activation, suggesting that CLV3 is simultaneously regulated by WUS, STM, and WUS–STM complexes[117] (Fig. 6 & Supplementary Table S3).

      Epigenetic modifications can regulate gene transcription. The WUS locus is a site of DNA methylation and inhibitory histone modifications such as H3K27me3[111]. Under wild-type conditions, the WUS promoter is highly methylated; however, mutations in MET1, Chromomethylase 3 (CMT3), Domain rearranged methyltransferase 1 (DRM1) and DRM2 result in the deletion or reduction in DNA methylation in the regulatory region of the WUS promoter, which increases the expression of WUS and the shoot regeneration rate[114]. Neonatal shoot regeneration involves different histone modification sites in WUS, and the abundance of histone 3 lysine 9 acetylation (H3K9ac) and histone 3 lysine 4 trimethylation (H3K4me3) at the WUS locus increases during stem regeneration, whereas the abundance of the H3K9me2 repressor at the WUS locus decreases during stem regeneration[118]. HAC1 and Lysine-specific demethylase 1-like 3 activate WUS transcription and increase shoot yield[119]. The removal of H3K27me3 from the WUS locus appears to be cytokinesis dependent, and olomoucine delays the decrease in H3K27me3 levels at the WUS locus and induces WUS expression[111] (Fig. 6 & Supplementary Table S3).

      In addition, miR-156 targets SPL mRNA and decreases b-type ARR activity in an age-dependent manner[120]. In young explants, the expression level of miR156 inhibit SPL expression level, which increases b-type ARR activity and shoot regeneration capacity. Moreover, miR-165/-166 inhibits stem regeneration by splicing and decreasing the translation of the mRNA encoding the HD-ZIP III protein[121]. The Argonaute 10 (AGO 10) gene inhibits stem regeneration by repressing miR-165/166 activity (Fig. 6 & Supplementary Table S3).

    • Medicinal plants are important natural resources for humans; they can not only cure diseases but also enhance human immunity and prevent the occurrence of diseases. With the frequent occurrence of global public health crises and increasing attention given to life and health, TCM has ushered in a critical period of revitalization and development. Medicinal plant resources constitute the cornerstone for promoting the high-quality development of the TCM industry. According to preliminary statistics from the Fourth National Census of Chinese Medicine Resources, there are more than 13,000 kinds of medicinal plants in China, of which more than 200 commonly used bulk Chinese herbal medicine are cultivated on a large scale[122]. Strengthening the breeding of good seeds of local herbs and improving the quality of Chinese herbal medicines from the source are key to ensuring the safety, efficacy and stability of clinical medication. Biological breeding, especially the application of gene editing technology, will lead to changes in the molecular breeding technology utilized for TCM. However, research on the regeneration of medicinal plants has focused mainly on the application level; additionally, there is a lack of systematic and in-depth exploration of regeneration mechanisms and future development, and several challenges have limited the widespread use of medicinal plant regeneration.

      First, improving the quality of Chinese herbal medicines to ensure their clinical efficacy is a core issue of TCM resources. The development of the Chinese medicinal seed industry is key to ensuring the yield and quality of Chinese herbal medicine. The breeding of Chinese herbal medicine is at the stage of original domestication selection, hybridization and molecular breeding, and the molecular design of TCM is still in its infancy[123]. Recently, new varieties of Chinese herbal medicine have been cultivated, such as P. ginseng 'Xinkaihe No. 1' and S. miltiorrhiza 'chuan Danshen No. 1', all of which are obtained via conventional methods such as systematic selection and cross-breeding, and their breeding efficiency is low[124]. One of the main reasons for the relative lag in the development of the molecular breeding of medicinal plants compared with that of common crops is that medicinal plants generally suffer from poor regeneration and low genetic transformation efficiency, characteristics that are strongly influenced by species and genotypes[125].

      Second, explant browning is a major factor that hinders the growth and differentiation of medicinal plants. Browning refers to the activation of polyphenol oxidase in the explant culture process, which oxidizes phenolic substances into quinones, leading to browning of the culture medium and hindering the growth and differentiation of medicinal plants[126]. Different plant genotypes, sampling sites and physiological states are the main factors leading to browning. Therefore, preventing the browning of explants to ensure their normal growth and differentiation during the regeneration of cultured medicinal plants is crucial.

      Finally, the tissue culture systems themselves have several limitations. Tissue culture technology relies on the precise control of the concentration ratio of auxin and cytokinins, which play important roles in genetic transformation[55,125]. However, this process requires strict maintenance under aseptic conditions, growth medium with specific carbon sources and hormone ratios, and controlled environmental conditions with appropriate light and temperature. These conditions are demanding and require abundant resources and expertise, making the tissue culture process labor intensive and costly[127]. In addition, methods that rely on tissue culture are time-consuming and carry the risk of somatic cell asexual lineage variation, and different plant species and genotypes respond differently to tissue culture conditions, leading to wide variations in regeneration efficiency[19,128].

    • Enhancing the regenerative capacity of different species of medicinal plants requires a multifaceted approach. Key strategies include optimizing tissue culture systems, incorporating morphogenetic factors, exploring new ways to bypass traditional tissue culture, and using gene editing technology to improve plant cell regeneration efficiency.

      First, optimizing tissue culture systems is the basis for improving the regeneration ability of medicinal plants. Various factors in the tissue culture environment, such as the basic medium, carbon source, hormone concentration and environmental conditions (light intensity and photoperiod), profoundly affect regeneration efficiency. For example, MS medium is suitable for the proliferation and growth of A. paniculata shoots, with a proliferation rate of 83.3% and good growth[129]. However, five different basic medium (MS, 1/2 MS, MT, H and B5) were compared and optimized, and it was reported that 1/2 MS was better for the growth of A. paniculata seedlings. Finally, the effects of MS, N6 and Nistch medium on anther culture were compared and optimized, and it is reported that N6 medium was the most suitable for the culture of A. paniculata anthers.

      Morphogenetic factors are powerful catalysts for increasing the rate of regeneration by influencing key cellular reprogramming and differentiation pathways. Transcription factors such as WUS2 and BBM have a remarkable ability to stimulate somatic cells, inducing them to form embryos that subsequently develop into full plants. Furthermore, some transcription factors have been shown to promote plant regeneration when combined with their cofactors. For example, the fusion of Triticum aestivum GROWTH-REGULATING FACTOR 4 (GRF4) and its auxiliary factor GRF-INTERACTING FACTOR 1 (GIF1) has been shown to be very effective in increasing the regeneration speed and efficiency of T. aestivum, Secale cereale and Oryza sativa, transgenic GRF4-GIF1 plants were fertile without obvious defects. In addition, GRF4-GIF1 enhanced wheat regeneration in the absence of exogenous cytokinin, facilitating transgenic selection in the absence of selection markers[130]. The combination of these morphogenetic regulatory factors is particularly beneficial to plant species that are difficult to regenerate or have a long regeneration cycle.

      New methods that bypass traditional tissue culture have attracted significant attention because they have the potential to revolutionize crop regeneration. Techniques such as the flower dip method and the cut-and-dip budburst (CDB) system offer species-specific alternatives to transform seeds and direct root transformation. CDB delivery systems use Agrobacterium rhizogenes to inoculate explants, generating transformed roots that produce transformed shoots. A variety of plant species in multiple plant families have been successfully transformed through CBD, including two herbaceous plants (Taraxacum kok-saghyz and Coronalla varia), a tuberous root plant (Ipomoea batatas), three woody plants (Ailanthus altissima, Aralia elata, and Clerodendrum chinense), and three succulents (Kalanche blossfeldiana, Crassula arborescens, and Sansevieria trifasciata). The CDB method allowed efficient transformation or gene editing in these plants using a very simple explant dipping protocol, under non-sterile conditions, without the need for tissue culture. In addition, large numbers of plants might be able to be genetically modified using CDB. These methods simplify the genetic modification process and reduce time and resource consumption[131,132]. In addition, innovative gene editing strategies, such as the direct injection of WUS2 and IPT into plants along with gene editing reagents, have opened new insights for tissue culture-independent gene editing[133]. Viral vectors such as TRV and Barley Stripe Mosaic Virus (BSMV) enable heritable and DNA-free gene editing by efficiently delivering gene editing materials into germ cells. In planta particle bombardment (iPB) and nanoparticle technologies also have the potential to simplify transformation[134].

      Gene editing technologies have greatly improved the genetic improvement and regeneration efficiency of medicinal plants. CRISPR/Cas9 and other gene editing technologies improve plant cell regeneration[135]. Regeneration efficiency can be improved by gene editing, which promotes dedifferentiation and redifferentiation of plant cells. The CRISPR/Cas9 system allows precise gene modification, including single base substitution, and can be used to control the regeneration of medicinal plants and cultivate species with specific medicinal properties. The use of high-fidelity Cas9 mutants or optimized sgRNA designs will reduce off-target effects. The TnpB and IscB technologies, for example, offer a low risk of off-targeting, improving gene editing safety and specificity. Regeneration of medicinal plants with this method also reduces the need to modify non-target genes[136,137]. Gene editing has a wide range of potential applications in the regeneration of medicinal plants. In the future, these technologies will play a greater role in medicinal plant regeneration due to their continuous improvements and optimizations.

      These multifaceted strategies will expand the scope of crop regeneration, making them more accessible, efficient, and adaptable to different species and genotypes. Medicinal plant regeneration plays a key role in agricultural biotechnology, especially in combination with precision genome editing and synthetic biology concepts[11], and several promising directions have emerged that have the potential to overcome current challenges and revolutionize the field. By integrating genomics and high-throughput sequencing technologies, a deeper understanding of the genetic basis of regeneration efficiency is possible[138]. Pyramiding the multiple factors involved in regeneration can reveal new regulatory centers and interactions[139]. Exploring non-tissue culture methods can bypass species and genotype-dependent tissue culture processes for plant regeneration[131133]. Through the exploration of these future directions, regeneration research and the application of medicinal plants will contribute to the breeding of high-quality varieties of Chinese herbal medicine and the stock of germplasm resources, promote the establishment of regeneration systems for rare and endangered medicinal plants, provide new ideas for the screening of novel regeneration-promoting drug molecules[140,141], foster the intelligent production of Chinese herbal medicine, promote high-quality integration of the discipline of Chinese herbal medicine with modern science, and further promote the preservation and innovation of TCM.

    • In this work, two regeneration pathways of medicinal plants during SE and tissue culture, as well as the environmental factors and molecular mechanisms affecting these pathways, were comprehensively discussed. The information discussed provides valuable references for scientific research and technological development in this field. Although some progress has been made, the regulatory mechanisms of plant regeneration still need to be studied in depth. In vitro, the regeneration of plants is a complex process, and our understanding of this process is still relatively limited; more comprehensive and in-depth studies are needed to reveal the full picture.

      Although the regulatory networks involved in plant regeneration have been initially identified, there is a relative lack of research on these networks in medicinal plants, and how the players and signaling molecules within medicinal plants synergize to facilitate the various stages of regeneration is still unclear. Therefore, future studies should further explore regeneration mechanisms in medicinal plants.

      In tissue culture, traditional methods to improve the efficiency of plant regeneration rely heavily on altering external environmental factors. Future research should combine the understanding of molecular mechanisms with these traditional methods to optimize plant regeneration. Notably, this paper outlines regeneration control factors in nonmedicinal plants; however, whether medicinal plants have the same molecular mechanisms remains to be verified.

      Currently, rapid micropropagation and genetic transformation of many important crops and medicinal plants are still challenging. Therefore, the future direction of medicinal plant regeneration research may lie in the application of theoretical concepts of plant regeneration to agricultural practices to establish efficient regeneration systems and promote the development and industrialization of agricultural biotechnology.

      • This work was supported by the National Natural Science Foundation of China (82460743, 32170402), the Major Special Science and Technology Project of Yunnan Province (202304BI090009; 202403AK140082), Yunnan Characteristic Plant Extraction Laboratory (2022YKZY001), Yunnan Province Youth Talent Support Program (XDYC-QNRC-2022-0219), Program of Shanghai Academic/Technology Research Leader (23XD1423500), Organizational Key Research and Development Program of Shanghai University of Traditional Chinese Medicine (2023YZZ01).

      • The authors confirm contribution to the paper as follows: study conception and design: Zhao Y, Wang J; literatures collection and analysis: Wang J, Liang YL, Liu GZ; writing-original draft preparation: Wang J, Li CH, Zhao Y; writing-review & editing: Yang SC, Xiao Y, Zhao Y. picture preparation and drawing: Zhou PH, Li CH. All authors approved the final version of the manuscript.

      • Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

      • The authors declare that they have no conflict of interest. Dr. Ying Xiao is the Editorial Board member of Medicinal Plant Biology who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of Dr. Xiao and the research group.

      • # Authors contributed equally: Juan Wang, Pin-Han Zhou

      • Copyright: © 2024 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 (6)  References (141)
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    Wang J, Zhou PH, Li CH, Liang YL, Liu GZ, et al. 2024. Progress on medicinal plant regeneration and the road ahead. Medicinal Plant Biology 3: e030 doi: 10.48130/mpb-0024-0026
    Wang J, Zhou PH, Li CH, Liang YL, Liu GZ, et al. 2024. Progress on medicinal plant regeneration and the road ahead. Medicinal Plant Biology 3: e030 doi: 10.48130/mpb-0024-0026

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